Co-ordinated Research Project - International Atomic Energy ...

XA0054915

NAHRES-51Vienna, 2000

Co-ordinated Research Project:

Comparative International Studies of OsteoporosisUsing Isotope Techniques

(CRP: E4.30.06)

Third Research Co-ordination MeetingSao Paulo, Brazil

24-27 August 1998

INTERNATIONAL ATOMIC ENERGY AGENCY

3 1 / 2 5

EDITORIAL NOTE

This document is not a formal publication of the International Atomic Energy Agency(IAEA) and the views expressed in it do not necessarily reflect those of the IAEA or ofgovernments of the Member States or organizations under whose auspices the work describedherein was carried out. In preparing some of the material in this document for reproduction, staffof the IAEA have mounted and paginated the original manuscripts and given some attention topresentation; otherwise, however, these manuscripts have not been edited by the IAEA. Theauthors are responsible for having obtained the necessary permission for the IAEA to reproduce,translate or use materials from sources already protected by copyrights. The use in thisdocument of particular designations of countries or territories does not imply any judgement bythe IAEA as to the legal status of such countries or territories, of their authorities and institutionsor of the delimitation of their boundaries. The mention of specific companies or of their productsor brand names does not imply any endorsement or recommendation on the part of the IAEA.

Co-ordinated Research Project:


(CRP: E4.30.06)

Third Research Co-ordination MeetingSao Paulo, Brazil

24-27 August 1998

NAHRES-51, IAEA, Vienna (2000)

A report prepared by the

Section of Nutritional and Health-Related Environmental StudiesDepartment of Nuclear Sciences & Applications

International Atomic Energy AgencyP.O. Box 100

A-1400 Vienna, Austria

Single copies of this report are available cost-freeon request from the above address

CONTENTS

SUMMARY REPORT

1. INTRODUCTION 1

2. PROGRESS TO-DATE 1

3. LOGICAL FRAMEWORK FOR THE CRP 1

4. GENERAL DISCUSSION - CORE PROGRAMME 2Representative sampling of study populations 2Number of subjects 2Reporting of DEXA measurements 2External QA/QC of the DEXA Measurements 2WHO questionnaire data 2Data evaluation by the CRL 3

5. GENERAL DISCUSSION - BONE ANALYSIS STUDIES 3

6. MAIN ACTIONS FORESEEN FOR COMPLETION OF THE CRP 4Lifetime of the CRP 4Participants' work plans 4The final report on the CRP 4Next meeting 5

Provisional timetable for completion of the CRP 5

7. ACKNOWLEDGMENTS 5

8. REFERENCES 5

APPENDICES

1. Participants 212. Agenda 233. Derivation of Standardised BMD using the European

Spine Phantom (E. V. McCloskey) 254. Protocol for Sampling and Analysis of Bone Specimens (N.K. Aras) 44

ANNEXES

COUNTRY REPORTS

1. Brazil (A. Borelli) 1-12. Canada (S.S. Krishnan) 2-13. Chile (6. Lobo) 3-14. China, Beijing (L Qin) 4-15. China (Y. Zhang) 5-16. Croatia (D. Dekanic)* 6-17. Hungary (A Balogh) 7-18. Philippines (MA LJm-Abrahan) 8-19. Russia (V.Y. Zaichick) 9-110. Singapore (K. Bose) 10-111. South Africa (S. Wynchank) 11-112. Turkey (N.K. Aras) 12-1

* Not present at the meeting (progress report submitted subsequently)

SUMMARY REPORT

1. INTRODUCTION

In 1994, the International Atomic Energy Agency (henceforth "the Agency") started a five-yearCo-ordinated Research Project (CRP) on Comparative International Studies of OsteoporosisUsing Isotope Techniques. The purpose and scope of this study are described in thedocumentation that has already been issued for this CRP [1-4]. A calendar of events is given intable 1.

The Third Research Co-ordination Meeting (RCM) for participants in the CRP - whichis the subject of the present report - was held in Sao Paulo, Brazil. The list of participants isgiven in appendix 1 and the agenda in appendix 2. The following report summarizes the mainconclusions of the meeting.

2. PROGRESS TO-DATE

Since the last RCM held in 1996, most participants have made good progress towards theobjective of measuring bone mineral density (BMD) in at least 25 subjects of both sexes in eachof the 5-year age ranges between 15 and 50 years (i.e. a minimum of 350 subjects in each studygroup). Some participants have also collected and analysed a number of bone samples, asspecified in the protocols for the CRP. Relevant statistics for both kinds of study are given intables 2-12. One centre (Croatia) was unfortunately not represented at the RCM due to theillness of the Chief Scientific Investigator (CSI).

One of the most important tasks within the CRP is to "normalize" the data from differentDEXA machines and study centres so as to allow a meaningful comparison of the BMDmeasurements between centres and study groups. To this end, a European Spine Phantom(ESP) has recently been circulating among the participating centres, who have reported theirresults to the Central Reference Laboratory (CRL) - Dr. McCloskey, UK. So far, the ESP hastravelled to the following centres: Canada, Turkey, Russia, Hungary, Croatia, China (Beijing,Shanghai), Singapore, Philippines and Brazil (during this meeting).

Further information on the progress of the work in individual countries was presented ineach country's report; these are reproduced in annexes 1-12. Information on projectperformance indicators (status as of the date of this meeting) is summarized in tables 2-12.

Central evaluation of the data has so far only been done for a few of the participatingcentres. A review of the information that is presently available and the conclusions that may bedrawn from it is given in appendix 3. It is interesting to note that there are some significantbetween-centre differences in spine BMD.

3. LOGICAL FRAMEWORK FOR THE CRP

The Agency's technical officer, Dr. Parr, explained that there is a new requirement on the partof the IAEA that all current CRPs should have a "logical framework which defines theobjectives, outputs and activities of the CRP together with some statements about theassumptions on which a successful outcome of the CRP might depend. For new CRPs, thislogical framework has to be defined at the time that the CRP is first proposed. However, thiswas not a requirement at the time that the present CRP was started. Accordingly, the logicalframework now has to be developed retrospectively.

1

Dr. Parr presented a draft version of the logical framework and, after some discussion andmodification, this was endorsed by the participants (Table 13).

4. GENERAL DISCUSSION - CORE PROGRAMME

Representative sampling of study populations

There is no change from previous recommendations. It was recognized that some centres havedifficulties with random sampling and low response rates. This issue will be discussed in the finalreport. To this end the "report writing team" (see later) will need to have access to a completedescription of (1) the population from which their subjects were drawn and (2) the method(s)used for selecting subjects from this population. Dr. McCloskey will provide some guidelineson the format for providing this information.

Participants should check that subjects chosen for study in the 15-19 year age group arenot the children of subjects from an older age group since this might introduce biases.

No. of subjects

There is no change from previous recommendations. 350 subjects in each study group (175females and 175 males) are still considered to be the minimum numbers, and all participantsaccept this as an attainable objective (many participants have already done this). Priority shouldbe given to achieving these numbers, in the first place, for females.

For some participants (e.g. Canada, Philippines) there are some financial constraints thatstill need to be clarified.

Reporting of DEXA measurements

There is no change from previous recommendations. However, a new requirement is that hipaxis length (HAL) should be reported for all new subjects. If possible, the same should also bedone retrospectively from the scans of subjects that have already been measured.

Additional software may be needed for obtaining this information from the scans.Normally, this is available cost-free. If helpful, the CRL and/or the IAEA would be willing toprovide a letter to support such a request from the scanner manufacturer.

All data should be reported to the CRL in an Excel file. Dr. McCloskey will specify thevariables to be reported and will also provide some guidelines on how to edit the data exportedfrom the scanner.

External QA/QC of the DEXA measurements

The European Spine Phantom (ESP) will continue to circulate (to Chile, South Africa, Turkeyand finally back to the UK).

WHO Questionnaire data

It was agreed that all centres will code their data using the Excel file format developed by Dr.McCloskey (those that did not yet have it were given a copy on a diskette during the RCM).This should be done for each and every subject individually. However, if this is not possible forsome reason, an alternative approach would be to report the data for a randomly selected sub-setof subjects, e.g. for 100 subjects instead of 350.

There was considerable discussion about the data on dietary intakes to be reported to theCRL. The recommendation from the previous meeting was repeated and accepted, namely that,for the purposes of central data evaluation, only the following parameters (intakes per day)should be reported: calcium, protein, fat, carbohydrate, and total energy. Each participatingcentre will continue to use the original WHO questionnaire (modified by each individual countryto account for special ethnic food items). The data so collected should be used by local dietaryexperts to derive the requested information on calcium protein, fat, etc.

It was recognized that the data so-obtained may be rather crude. Nevertheless, it wasaccepted by all participants that it is essential to try to obtain it. Validation of thereasonableness of the data so reported should be sought by comparison with any othercomparable data that may be available (for example, in Sao Paulo, the work on dietary studiesreported during the visit to IPEN).

Additional actions foreseen are:

• all participants will send to the CRL a copy of their locally-modified versions of theWHO questionnaires (with hand-written translations of just a few key words)

• Dr. McCloskey will provide (to IAEA, for onward transmission to the participants)a hard copy of his modified version of the WHO questionnaire (corresponding to thedata to be reported in the Excel spreadsheet).

Data evaluation by the CRL

The decision taken at previous RCMs that there should be a central evaluation of the BMD data,and associated parameters, was reconfirmed. The UK centre (Dr. McCloskey) will perform thisfunction. Dr. McCloskey will also provide a description of the kinds of data evaluation that heintends to do together with some suggestions for additional kinds of data evaluation that mightbe undertaken by individual participants.

All participants will be kept fully informed about the outcome of these evaluations, andnothing will be finalized or published without the full consent of all participants (see"Publications Policy" in the RCM-1 report [2] and the additional discussion on this topic below).

5. GENERAL DISCUSSION - BONE ANALYSIS STUDIES

At the previous RCM, it was tentatively decided that the main emphasis of the bone compositionwork should be given to the collection and analysis of specimens of iliac crest obtained eitherat biopsy or at autopsy. This decision was reconfirmed at the present RCM, However, it wasnoted that there are some differences in the details of the technical procedures followed at thedifferent centres. In order to provide the possibility of closer harmonization of these procedures,a technical protocol was prepared by Dr. Aras (appendix 4). Those participants who areengaged in bone composition studies agreed to follow this protocol as closely as possible.

It was further agreed that, at the time of preparing the final report on the CRP, there willbe a need for close coordination of the writing of this section of the report, and possibly also forsome central evaluation of the analytical data for minor and trace elements in bone. It wasforeseen that a small sub-group of those participants who are engaged in this work will need tobe identified. Dr. Aras was invited to be the main coordinator of the work of this sub-group.

The attention of participants was drawn to the newly published NAHRES report on traceelements in bone and teeth [5] (printed in 1999 but available in draft form at the time of theRCM).

6. MAIN ACTIONS FORESEEN FOR COMPLETION OF THE CRP

Lifetime of the CRP

All participants agreed that it will be impossible to finish the CRP within the presently approvedtimetable (by December 1998). A one-year extension (until December 1999) of the period forcompleting the work at individual centres was considered to be the minimum necessary.

The Agency's Technical Officer, Dr. Parr, undertook to do the necessary in-house workto seek authorization for such an extension with a view to renewing all contracts for one moreyear. In most cases this will mean from December 1998 to December 1999.

Further information on the timetable for completion of the CRP is given in table 14.

Participants' work plans

The participants were requested to revise their work plans in the light of the discussions that hadtaken place during the RCM. All current research contracts are due to be renewed for one yearby September 1998, which means that they will then all be approximately in phase with eachother. In addition to presenting their work plans, participants were requested to complete aform with information on "project performance indicators" covering the period from July 1998to September 1999. These work plans and performance indicators are given in tables 2-12.

The final IAEA report on the CRP

Many publications are expected to be produced as an outcome of this CRP. However, the firstpublication, including the central evaluation of the data, will be an IAEA report in the form ofa so-called TECDOC. This will comprise two parts:

Part A: A summary report on the whole CRP, including the presentation and discussionof the centrally evaluated data.

Part B: Individual final reports prepared by each of the participating centres. [IAEA willlater provide precise guidelines on the format, scope and contents on these final reports.]

A writing team was appointed for preparing the first draft of Part A. The team comprisesMcCloskey, Aras, Balogh and Parr (the tatter's role in doing this still needs to be clarified in viewof his impending retirement from the Agency).

There was some discussion about the "ownership" of the data produced by this CRP andimplications with respect to publication of the data.. Each participant, of course, is the "owner"of his/her own data. However, the central file combining the data of all participants belongscollectively to the IAEA and to all participants. Any participant may request a copy of the wholefile for whatever kind of study he/she may wish to undertake. The publication of the results ofsuch a study (i.e. based on the totality of the data) is subject to the constraints already acceptedas part of the publications policy for this CRP. [Quote. The preparation of such publications will,in the first place, be the responsibility of the Agency's technical officer. Such publications will

be co-authored by all CRP participants who have contributed in any meaningful way to theoutcome of the study.]

Next meeting

No additional Research Co-ordination Meeting (RCM) is presently foreseen for this CRP.However, it was agreed that an additional meeting for all participants would be highly desirable,if not essential, to discuss and obtain agreement on Part A of the final report, before it ispublished. It was further agreed that all participants will be invited to this meeting (target dateand location: March/April 2000 in Sheffield, UK). Dr. Parr undertook to try to secureauthorization and funding for such a meeting in the Agency's financial plan for the year 2000.

Provisional timetable for completion of the CRP

A provisional timetable (table 14) was drawn up for completion of the CRP, taking account ofthe various decisions summarized above. However, the Agency's technical officer, Dr. Parr,emphasized that there may be some difficulties keeping to this timetable in view of his pendingretirement and the time needed for the new incumbent to adjust to the duties of the post.

7. ACKNOWLEDGMENTS

All non-local participants joined in expressing their sincere appreciation of the warm hospitalityand efficient management of the local arrangements provided by their local host, Dr. AurelioBorelli. They also wished to thank Dr. Marina Vasconcellos for her help in arranging thescientific visit to IPEN on the third day of the RCM.

8. REFERENCES

[ 1 ] INTERNATIONAL ATOMIC ENERGY AGENCY, Comparative International Studiesof Osteoporosis Using Isotope Techniques (Report of an IAEA Advisory Group Meeting,Vienna, October 1992), IAEA, Vienna, NAHRES-14, 1993.

[2] INTERNATIONAL ATOMIC ENERGY AGENCY, Comparative International Studiesof Osteoporosis Using Isotope Techniques (Report on the First Research Co-ordinationMeeting, Vienna, December 1994), IAEA, Vienna, NAHRES-31, 1996.

[3] INTERNATIONAL ATOMIC ENERGY AGENCY, The Role of Trace Minerals inOsteoporosis (Report of an IAEA Consultants' Meeting, Vienna, December 1995), IAEA,Vienna, NAHRES-28, 1996.

[4] INTERNATIONAL ATOMIC ENERGY AGENCY, Comparative International Studiesof Osteoporosis Using Isotope Techniques (Report on the Second Research Co-ordinationMeeting, San Diego, October 1996), IAEA, Vienna, NAHRES-40, 1998.

[5] INTERNATIONAL ATOMIC ENERGY AGENCY, Minor and Trace Elements inHuman Bones and Teeth (prepared by G.V. Iyengar and L. Tandon), IAEA, Vienna,NAHRES-39, 1999.

Table 1. Calendar of events for the CRP up to the time of the present RCM

Date

1992: Oct

1994: May

1994:Nov/Dec

1994: Dec

1994: Dec

1995: Dec

1995: Dec

1996: Oct

1996: Oct topresent

1998: Aug

1998: Dec

Action

Advisory Group Meeting, Vienna, recommended the setting up of theCRP and defined its purpose and scope

CRP proposal approved by Agency's in-house committee

Research contracts and research agreements awarded to 12 participants

Author contract awarded to Iyengar, USA, for literature survey onminor & trace elements in human bone and teeth

First RCM, Vienna, Austria

Consultants' Meeting, Vienna, on the role of trace minerals inosteoporosis

Technical Contract awarded to UK (McCloskey) for QC and dataevaluation services

Second RCM, San Diego, USA

European Spine Phantom circulated

Third RCM, Sao Paulo, Brazil

Date presently foreseen for the end of the CRP

Table 2 COUNTRY, CSI: Brazil, Borelli A

PROJECT PERFORMANCE INDICATORS (status as of August 1998)

BMD measurements (age range 15-50 years)

Study group 1 (short description): Residents of Sao Paulo, hospital staff and relations (urban middle class)

Statistics

No. of subjects measured - male

No. of subjects measured - female

No. of measurements reported to CRL - male

No. of measurements reported to CRL - female

Group 1

100

100

0

0

WHO questionnaire data

Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

yes

yes

yes

Reported3

no

no

no

1. Collected on CSI's own questionnaire forms2. Coded by CSI into a computer file (Excel, Access) for use by the CRL3. Already reported by CSI to the CRL

Bone studies

Types of bone sample(s) collected:

No. of samples collected - autopsy:No. of samples analysed:Elements analysed (or to be analysed):

Work plan until the end of the CRP

rib, iliac crest

22 (rib)Br, Ca, Cl, Fe, Mg, Na, P, Rb, Sr, Zn

To increase the total number of BMD measurements to 350 (total) for the study groupTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection and analysis of 10 bone specimens for selected elements (as listed above)To complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

Table 3 COUNTRY, CSI: Canada, Krishnan SS



Study group 1 (short description): Urban group from Toronto

Statistics





Group 1

38

84

38

84


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

no

no

no

Reported3

no

no

no


Bone studies

Types of bone sample(s) collected: none


To increase the number of BMD measurements to at least 175 for both females and males in the studygroup (depending on availability of supplementary funding)To complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the evaluation of ray own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

8

Table 4 COUNTRY, CSI: Chile, LoboG



Study group 1 (short description): White, urban health centre (hospital, clinical) workers

Statistics





Group 1

64

167

0

0


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

no

no

no

Reported3

no

no

no


Bone studies



To increase the total number of BMD measurements to 350 (total) for the study groupTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

Table 5 COUNTRY, CSI: China, Qin Lin Lin



Study group 1 (short description): Northern Chinese (Han)

Statistics





Group 1

372

409

0

0


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded2

no

no

no

Reported1

no

no

no


Bone studies


No. of samples collected - biopsy:No. of samples analysed:Elements analysed (or to be analysed):


iliac crest

158Al, Ca, Cl, Co, Fe, Mg, Mn, Na, Sr, Zn

To maintain the total number of BMD measurements at 781 (total) for the study groupTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection and analysis of 30 bone specimens for selected elements (as listed above)To complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

10

Table 6 COUNTRY, CSI: China, Zhang Yuanxun



Study group 1 (short description): Urban Chinese (resident in Shanghai for > 10 years)

Statistics





Group 1

175

175

0

0


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

no

no

no

Reported3

no

no

no

1.2.3.

Collected on CSI's own questionnaire formsCoded by CSI into a computer file (Excel, Access) for use by the CRLAlready reported by CSI to the CRL

Bone studies


No. of samples collected - autopsy:No. of samples analysed:Elements analysed (or to be analysed):


femur

-25-25Ba, Ca, Cd, Cr, Cu, F, K, Mg, Mn, Na, Ni, P, Sr, Zn

To increase the total number of BMD measurements to >400 (total) for the study groupTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection and analysis of >55 bone specimens (including 10 iliac crest specimens) forselected elements (as listed above)To complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

11

Table 7 COUNTRY, CSI: Hungary, Balogh A



Study group 1 (short description): representative selection of Hungarian population

Statistics





Group 1

171

205

0

0


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded2

no

no

no

Reported3

no

no

no


Bone studies



To increase the number of BMD measurements to 450 (total, both sexes) for the study group

To complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection and analysis of 10-15 autopsy bone specimens for selected elements (provisionalplan - depending on local arrangements)To complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

12

Table 8 COUNTRY, CSI: Philippines, Lim-Abrahan MA



Study group 1 (short description): Urban residents of Metro Manila

Statistics





Group 1

56

58

56

58


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded7

yes

yes

yes

Reported3

yes

yes

yes

1. Collected on CSFs own questionnaire forms2. Coded by CSI into a computer file (Excel, Access) for use by the CRL3. Already reported by CSI to the CRL

Bone studies



To increase the number of BMD measurements to 350 (total, both sexes) for the study groupTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection of 10 iliac crest bone specimensTo complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

13

Table 9 COUNTRY, CSI: Russia, Zaichick V



Study group 1 (short description): Residents of Moscow

Study group 2 (short description): Residents of Obninsk

Study group 3 (short description): Residents of Obninsk (former Chernobyl cleanup workers)

Statistics





Group 1

166

168

166

168

Group 2

74

72

74

72

Group 3

47

0

0

0


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

yes

yes

yes

Reported3

yes

yes

yes

1.2.3.


Bone studies

Types of bone sample(s) collected:No. of samples collected - autopsy:No. of samples analysed:

iliac crest, femoral neck, rib, teeth420-200

Elements analysed (or to be analysed): Al, Ca, Cl, Co, Cs, Cu, Eu, Fe, K, Mg, Mn, Na, P, Rb, Sb, Se, Sr, Zn


To increase the total number of BMD measurements to 350 (total, both sexes) in each of study groups 1and 2To complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the analysis of 120 iliac crest specimens for selected elements (120 short-lived activations; 50long-lived activations)To complete the evaluation of ray own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

14

Table 10 COUNTRY, CSI: Singapore, Bose K



Study group 1 (short description): Residents of Singapore (ethnic Chinese)

Statistics





Group 1

204

318

0

0


1.2.3.

Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

no

no

no

Reported3

no

no

no


Bone studies



To increase the total number of BMD measurements to 350 (total, both sexes) in a second study group(Malays)To complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection of 30 iliac crest bone specimensTo complete the evaluation of ray own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

15

Table 11 COUNTRY, CSI: South Africa, Wynchank S



Study group 1 (short description): Residents of greater Cape Town area, European ethnic group

Study group 2 (short description): Residents of greater Cape Town area, mixed racial group

Study group 3 (short description): Residents of greater Cape Town area, African ethnic group

Statistics





Group 1

57

496

0

0

Group 2

99

480

0

0

Group 3

15

45

0

0


1.2.3.

Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded2

no

no

no

Reported3

no

no

no


Bone studies



To increase the total number of BMD measurements to at least 175 for each study group and sexTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection of 30 iliac crest bone specimensTo complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

16

Table 12 COUNTRY, CSI: Turkey, Aras NK



Study group 1 (short description): Randomly selected from among METU staff members,students, workers and their relatives

Statistics





Group 1

128

228

128

228


Type of data

Physical

Nutritional

Lifestyle

Collected1

yes

yes

yes

Coded1

yes

yes

yes

Reported3

yes

no

no


Bone studies


No. of samples collected - biopsy:No. of samples analysed:Elements analysed (or to be analysed):


iliac crest

1210~25 minor and trace elements

To increase to 175 the total number of BMD measurements for males in the study groupTo complete the coding of the BMD data and reporting to the CRLTo complete the coding of the WHO questionnaire data and reporting to the CRLTo report relevant QC and other technical data to the CRLTo complete the collection and analysis of 12 bone specimens for selected elementsTo complete the evaluation of my own centre's data and produce a final report on the work of my centre forinclusion in the final CRP report

17

Table 13 Logical framework for the CRP

Narrative Summary

Overall Objective:To make comparative measurements of bone mineral density(BMD) and bone composition of selected human subjects indifferent parts of the world (having different geographical, culturaland ethnic backgrounds) so as to obtain a better understanding ofsome of the factors that may play a role in the aetiology ofosteoporosisSpecific Objectives:• To identify study groups that are representative of the

populations of interest• To make comparable measurements of BMD in subjects of both

sexes across the age range 15-50 years• To determine the age range over which peak BMD is achieved

and sustained• To determine the elemental composition of selected bone

samples• To report relevant physical, nutritional and lifestyle data• To collate and evaluate the above data to meet the overall

objective

Outputs:• BMD measurements

i

«

• Bone composition measurements• Objective quality assurance evidence for the above data• Reporting of relevant physical, nutritional and lifestyle data• Individually and centrally evaluated data* Published reports

Activities:• Form networks of participants• Prepare agreed protocols• Establish and maintain co-ordinated research• Develop and apply appropriate quality assurance procedures• Collate and evaluate results• Publish results

ObjectiveVerifiable Indicators

N/A

• Agreement on the protocols tobe used

• Subjects recruited• Methods being implemented• Central Reference Laboratory

(CRL) identified and operational

• No. of subjects recruited, andBMD measurements made andreported

• No. of bone samples analyzedand results reported

• Availability of QA results• Availability of reports and

publications from the CRP

• 14 contracts awarded• AGM held 1992• RCM-1 held 1994; CM held '95• RCM-2 held 1996• RCM-3 held 1998• Reports on above activities

available• Other publications planned

Means of Verification

N/A

• Progress reports and RCMreports

• Progress Reports and RCMReports

• Other publications arisingout of the CRP

• The final publication

As above

Important Assumptions

N/A

• Participating countries have a sufficient rangeof differences in nutritional and lifestyle factorsthat might affect BMD and bone composition

• Subjects selected for study are sufficientlyrepresentative

• Suitable bone specimens can be obtained• National and IAEA and support is sufficient to

enable the research to be carried out• IAEA and CRL technical officers provide

appropriate technical support

As above +

• Suitable QA procedures can be developed andapplied

• Relevant physical, nutritional and lifestyle datacan be reported

As above +

• CRL able to complete its evaluation

• Content and quality of final report sufficient tojustify publication

Table 14 Provisional timetable for completion of the CRP

Date(s)

Sep 1998 to Sep 1999

First quarter of 1999

Jun 1999

Jan 2000

Feb 2000

Mar/Apr 2000

Apr 2000

Action(s)

All participants to ensure that they have adequate numbers of subjects forachieving the objectives of the core project.

BMD, HAL and WHO questionnaire data to be reported to the CRL.

The trace element working group to prepare a timetable for completion ofthe work on bone composition studies.

The writing team for part A of the final report to start its work.All CRP participants to receive instructions for preparation of their finalreports (part B).

First drafts of parts A and B due (to be copied and circulated to allparticipants).

Comments on part A to be submitted to the Agency and to the CRL.

Final meeting of all participants

Start of work to edit the final report for submission to the IAEAPublications Committee.Start of work by all participants to prepare other reports on the work ofthe CRP for publication in peer-reviewed scientific journals.

NEXT PAGE(S)left BLANK

19

PARTICIPANTS (in country/name order) Appendix 1

Dr. Aurelio Borelli *School of MedicineUniv. of Sao PauloAv. Dr. Arnaldo 455 / r.430901246-903 Sao Paulo, SPBRAZILtel: +55-11-3066-7467home: +55-11-822-4233fax: +55-11-3066-7250e-mail: ada9aasanet.c01n.br :aborelliadglnet.com.br

Dr. Hitiko SaikiIPEN/CNEN-SPRadiochemistry DivisionCP 11049 - CEP 05422-970Sao Paulo/SPBRAZILe-mail:mitiko3curiango.ipen.br

Dr. Marina B.A. VasconcellosIPEN/CNEN-SPRadiochemistry DivisionCP 11049 - CEP 05422-970Sao Paulo/SPBRAZILt e l : +55-11-211-6011fax: +55-11-816-9188e-mail:mbvascon3curiango.ipen.br

Dr. S. S. KrishnanMedical Physics LaboratoryToronto General HospitalRm. CCRW G-803200 Elizabeth StreetToronto, Ontario M5G 2C4CANADAtel: 416 340 4841home: 416 491 0626fax: 416 340 4707e-mail:kkrishnan3torhosp.toronto.on.

Dr. Gabriel Lobo, HeadNuclear Medicine DepartmentClinica IndisaAv. Santa Maria 1810Providencia, SantiagoCHILEtel: +56-2-225-3718fax: +56-2-737-8862

Dr. Qin Lin LinDepartment of IsotopeResearchInstitute of Clinical MedicalSciencesChina-Japan FriendshipHospitalYing Hua East Road, Chao YangDistrictBeijing 100029CHINAtel: +86-10-6422-1122-4441fax: +86-10-6421-7749home: +86-10-6427-2185e-mail:mecmai3mail.netchina.coin.cn

Dr. Zhang YuanxunLab. of Nuclear AnalysisTechniquesInstitute of Nuclear ResearchPOB 800-204Shanghai 201800CHINAte l : +86-21-5955-3998fax: +86-21-5955-3021e-mail: ylcui3fudan.ac.cn

Dr. Adam BaloghDept. of Obstetrics &GynecologyUniversity Medical School ofDebrecenPOB 37H-4012 DebrecenHUNGARYtel: +36-52-420545fax: +36-52-414577home: +36-52-327687e-mail: abalogh3obgyn.dote.hu

Dr. Mary Anne Lim-AbrahanPhilippine Society ofEndocrinology & MetabolismEndocrine Section, UP-PGHMedical CenterTaft AvenueManila 1000PHILIPPINEStel: +63-2-521-8450 x3230fax: +63-2-525-1062e-maiI:malim3freemail.Mebquest.com

Dr. V. Ye. Zaichick, HeadSection of INAA and XRFMedical Radiological ResearchCentreKorolyeva Str. 4Obninsk 249020Kaluga RegionRUSSIAN FEDERATIONtel: +7-8439-26497fax: +7-095-956-H40home: +7-8439-39065e-mail: pete3kaluga.ru

Prof. Kama I Bose, HeadDept. of Orthopaedic SurgeryNational University Hospital5 Lower Kent Ridge RoadSINGAPORE 119074tel: +65-772-4342 ext 4327fax: +65-773-2172e-mail: dosbosek3nus.edu.sg

Dr. S. UynchankMedical Research Council /Medical BiophysicsFrancie van Zijl DriveParowvallei, CapeP0 Box 19070 Tygerberg 7505SOUTH AFRICAtel: +27-21-938-0300fax: +27-21-938-0477e-mail:swynchan3eagle.mrc.ac.za

Prof. Dr. Namik K. ArasVice PresidentFatih UniversityBeylikduzu, B.CekmeceIstanbulTURKEYTel: +90(312) 872 6136Fax: +90(312) 872 6130e-mail: aras3fatihun.edu.tr

Dr. Neslihan Seckin **Fatih University HospitalBestepelerAnkaraTURKEYfax: +90 312 221 0375e-mail: Nesli4aHotmail.com

Dr. Eugene V. HcCloskeyWHO Collaborating Centre forMetabolicBone DiseaseUniversity of SheffieldMedical SchoolBeech Hill RoadSheffield S10 2RXUNITED KINGDOMtel: +44 114 271 2750fax: +44 114 273 9176home: +44 114 235 1309e-mai I:E.V.McCloskey3sheffield.ac.uk

Scientific Secretary

Dr. Robert M. Parr, HeadSection of Nutritional &Health-Related EnvironmentalStudiesIAEAP0 Box 100, Wagramerstrasse 5A-1400 ViennaAUSTRIAtel: +43-1-2060-21657fax: +43-1-20607-21657home: +43-1-3203977e-mail: R.Parr3IAEA.org

* Host** Observer


21

Appendix 2Third Research Co-ordination Meeting on


Sao Paulo, Brazil

24-27 August 1998

Agenda

Session 1: Monday, 24 August (Morning - starting 9:00 am) - Chair: A. Borelli

WelcomeAdoption of the agendaStatus report on the Co-ordinated Research Programmeand administrative arrangements for the meeting(ParrRM)

COUNTRY REPORTS (presentation of working papers)

CanadaChileChinaChina

Knshnan SSLoboGQin Lin LinZhangYuanxun

Session 2: Monday, 24 August (Afternoon) - Chair: SSKhshnan

COUNTRY REPORTS (presentation of working papers - continuation)

HungaryPhilippinesRussiaSingaporeSouth Africa

Balogh ALim-Abrahan MAZaichick VBoseKWynchank S

Session 3: Tuesday, 25 August (Morning) - Chair: A Balogh

COUNTRY REPORTS (presentation of working papers - continuation)

TurkeyBrazilUK

ArasNKBorelli AMcCloskey E

SEMINARS (see attached list)

Session 4: Tuesday, 25 August (Afternoon) - Chair: S Wynchank

SEMINARS - continuation

GENERAL DISCUSSION

General discussion on work plans for completion of the CRP

23

Session 5: Wednesday, 26 August: Visit to IPEN (whole day) - Chair: NKAras

Introduction to IPENOverview of some relevant activities at IPENLaboratory visitContinuation of discussion on work plans for completion of the CRP

Session 6: Thursday, 27 August (Morning) - Chair: EVMcCloskey

Final discussions

Report of the meeting

CLOSING OF THE MEETING

Thursday, 27 August (Afternoon)

No formal session is foreseen at this time. However, the Agency's Technical Officer will beavailable for discussions on any matters concerning individual research contracts.

LIST OF SEMINARS

1. Recent progress in bone mineral density studies including the role of phyto-oestrogens inbone metabolism (EV McCloskey)

2. The prevalence of osteoporosis among postmenopausal women admitted to a hospital inAnkara, and treatment results (N Seckin)

3. On the role of calcium and other mineral elements in osteoporosis (RM Parr)

24

Appendix 3

XA0054916

Derivation of Standardised BMD using the European Spine

Phantom

Dr EV McCloskey

WHO Collaborating Centre for Metabolic Bone Diseases

University of Sheffield Medical School

Beech Hill Road

Sheffield S10 2RX

August 1998

Sao Paolo, Brazil

25

Working paper Standardised BMD

Introduction

The primary aim of this study is to compare peak bone mass in a range of populations and to

examine the determinants of any differences which may be observed. Bone mineral density (areal

BMD) has been measured by densitometers manufactured by two companies, Hologic (Waltham,

USA) and Lunar (Madison, USA). Systematic differences exist between the manufacturers in

several stages of the scanning procedure (e.g. edge-detection, calibration) so that the normal ranges

produced are specific to each brand. To allow comparison of BMD between manufacturers (and

thus between centres) we have therefore undertaken a cross-calibration of die scanners in order to

derive a standardised BMD.

Methods

Phantom measurementThe cross-calibration has been made possible by measuring a standard phantom at all participating

centres. The phantom used is the European Spine Phantom (ESP, QRM Germany) which consists of

three semi-anthropomorphic "vertebrae" made of simulated cortical and trabecular bone. The

phantom is constructed so that the specified densities of die three vertebrae are 0.5, 1.0 and

I 5g/cm2 (Kalender 1992, Kalender ct al 1995). The measurement of the phantom was carried out

as specified in a protocol which was circulated with the phantom. Briefly, this required that the

ESP be scanned 10 times without repositioning and taking care to exclude air from the area

included in each scan. Results of the measurements were forwarded to the Sheffield centre for

confirmation and subsequent analysis.

Analysis and Calibration

At each centre, the mean and standard deviation of die measured BMD of each of the 3 vertebrae

were computed. The relationship between the actual densities of the vertebrae and the observed

densities were examined by simple linear regression (through the origin and also including a

constant) and non-linear regression. For the latter, the model used was that proposed by Pearson et

al (1995) and is shown below:

Observed density - a x jl - exp(-/? x Specified density)where

a = the asymptote and a * P is die slope when the specified density is small.

26


The standardised BMD (sBMD) for each centre can then be obtained by the following equation:

sBMD=5* b (a ) - S x ln(a - Observed density)where

5 = 1/p and In is the natural logarithm.

Adequacy of the calculation of standardised BMD was examined by reverse calculation of tlie

actual densities of the 3 vertebrae using linear and non-linear fits.

DXA results from study centresTo date, scan results from 5 centres have been received in Sheffield. These comprise Ankara,

Manila, Moscow, Obninsk and Toronto. At all centres apart from Toronto, data on standing height,

weight and BM1 has also been provided. Several centres have also provided data on dietary intake

of calcium, exercise etc but this has not been considered in the statistical analysis to date.

Statistical analysisData was usually entered into MS Excel and then transferred to SPSS (Ver 8.01 for Windows 95)

for analysis. The statistical approach has been kept simple at this stage as the datasets are not yet

complete. Differences between centres have been examined using general factorial analysis of

variance which also examined other variables including age-category, weight and height. The

analysis was carried out separately for each gender. The age categories were as follows: 15-19

years (I). 20-24 (2), 25-29 (3), 30-34 (4). 35-39 (5), 40-44 (6) and greater than or equal to 45 (7).

An upper age limit has nominally been set at 52 years for the analysis because only 1 centre has

substantial numbers of patients above 50 years of age (Canada).

Results

The process of ESP measurement is not yet complete. The centres that have been assessed to date

are shown in Table 1. It should be noted that only results from 3 ESP scans have been received

from Shanghai. It should still be possible to calculate a correction factor based on this number of

scans if no further scans are available.

Differences between centres/manufacturers

It is clear that there are major differences in apparent densities of the 3 vertebrae measured at the 10

centres to date. These differences are similar to those expected from previous experience witli

DXA technology (Pearson et al 1995, Genant et al 1994). For example, Lunar densitometers

systematically give higher readings than deusitometcrs manufactured by Hologic (Table 2, Figures

1-3). However, even within the same manufacturer, differences are observed between centres with,

27


for example, the low and medium density "vertebrae" having lower values in Beijing than at other

sites using Lunar equipment (Table 2, Figures 1-2).

Differences between observed and actual densities

In addition to obvious differences between centres/manufacturers, it is also evident that densities

produced by the DXA equipment differ from the actual values of the 3 semi-anthropomorphic

vertebrae. For example, the mean values of the low density vertebra are significantly higher than

die actual values in all centres apart from Canada It should also be noted that the slope of the

relationship between the actual density and the observed density differs between the manufacturers

so that at die high densities seen in the 3'd anthropomorphic vertebra, Lunar equipment tends to

overestimate the density whilst Hologic equipment shows significant under-estimations (Figure 4).

This is also consistent with previous observations (Pearson et al 1995).

Derivation of standardised BMD

Using each of the regression methods, the formulae produced were used to recalculate the actual

BMD values of the 3 ESP vertebrae (Table 3). All of the approaches showed close agreement with

die actual values, but linear regression is sub-optimal as residual plots demonstrate that the

distribution of residuals differs between the lower and higher density vertebrae (data not shown).

The best fit was obtained by using non-linear regression and die derived values for a and P for each

centre are shown in Table 4.

Standardised BMD at 5 centresThe number of subjects scanned at each centre is shown in Table 5 and the age distribution at each

of the 5 centres is shown in Figure 5. It should be noted that die numbers of subjects scanned

between the ages of 20 and 40 are relatively low at die Toronto site.

Following derivation of standardised BMD, it is clear diat differences exist between the 5 centres

assessed to date. Lowest values of sBMD are observed at Ankara and Manila (Figures 6 and 7,

Table 5) and die differences between these two centres and the odiers are statistically significant. It

is perhaps not surprising diat differences also exist in anthropomorphic indices between centres

with subjects in Manila showing significantly smaller heights than other centres, and in the case of

men, significantly lower weights (Figures 8-13). In females, body mass index was similar across

the 4 centres where this was analysed Differences between centres persist following adjustment for

body weight and height.

DiscussionThe analysis to date shows that there are significant between-centre differences in spine BMD in

this study It should be noted that the sBMD in the older age-groups in our study at Moscow are

28


comparable to those observed in the EVOS Study (Lunt et al 1997). The magnitude of the

difference between centres is approximately 1 population standard deviation between the centres

with highest and lowest BMD and may be associated with a 2-fold increase in fracture risk i f other

risk factors are assumed constant. It is also clear that the between centre differences will persist

after adjustment for anthropomorphic variables. The study merits a rapid collation of the remaining

data and decisions regarding publication policy

29


References

Genant HK, Grampp S, Gluer CC, Faulkner KJ, Jergas M, Engelke K et al. Universal

standardisation for dual X-ray absorptiometry: patient and phantom cross-calibration results. J

Bone Miner Res 1004.0:1503-14

Kalender WA, Felsenberg D, Genant HK, Fischer M, Dequeker J, Reeve J. The European Spine

Phantom: a tool for standardisation and quality control in spinal bone mineral measurements by

QCTandDXA. EurJRadiol 1995,20:83-92.

Kalender WA. A phantom for standardisation and quality control in spinal bone mineral

measurements by QCTand DXA: design considerations and specifications. Med Phys

1992:19:583-86.

Lunt. M. FeJsenbertrP, Adams J etal. Population-based sjeqgraphic variations in DXA bonedensity m Europe:Tne EVOSTStudy. OsteoporosisTnt I997f7:f7>89.

Lunt M, Felsenberg D, Reeve J et al. Bone density variation and its effects on risk of vertebral

deformity in men and women studied in thirteen European centres: The EVOS Study. J Bone

Miner Res 1907.12:1883-94.

Pearson J, Dequeker J, Henley M et al. European Semi-anthropomorphic Spine Phantom for die

calibration of bone densitometers: assessment of precision, stability and accuracy. The European

Quantitation of Osteoporosis Study Group Osteoporosis Int 1995,5:174-84.

30


Table 1

Centres at which measurements of the ESP have now been completed (as of 17lh August 1998).

Centres

Ankara

Beijing

Canada

Croatia

Hungary

Manila

Moscow

Obninsk

Shanghai*

Singapore

ESP apparently only measured 3 times (instead of 10)

31


Table 2

Mean±SD values of measured BMD (g/cm2) of the 3 ESP vertebrae at each centre.

* Only 3 measurements reported from this centre

Centre Manufacturer Low density Middle density High density

vertebra (L2) vertebra (L3) vertebra (L4)

ESP

Ankara

Beijing

Canada

Croatia

Hungary

Manila

Moscow

Obninsk

Shanghai*

Singapore

QRM

Hologic

Lunar

Hologic

Lunar

Lunar

Lunar

Lunar

Lunar

Hologic

Lunar

0.500

0.532±0.006

0.562±0.009

0.498±0.005

0.606±0.008

0.6!8±0.009

0.620±0.010

0.613±0.0II

0.6l9±0.008

0.543±0.004

0.613±0.006

1.000

0.966±0.009

1.066±0.0I5

0.941±0.005

I.I07±0.008

1.1 11±0.01 1

1O86±O.OI1

1.088±0.0I7

1.102±0.012

0.970±0.007

1.091±0.007

1.500

1.408±0.017

1.562±0.008

I.354±0.0I0

I.578±0.014

1.617±0.015

l.553±0.014

I.54U0.014

1.580±0.018

1.376±O.O18

1.550±0.015

32


Table 3

Adjustment of observed measurements to standardised BMD using 3 approaches on data obtained

from the centre in Toronto. The best fit was obtained using a slight curve defined by the model

Low Density

Actual

Observed

Linear Regression(through origin)

Linear Regression

Non-linear

Middle Density

Actual

Observed


Linear Regression

Non-linear

High Density

Actual

Observed


Linear Regression

Non-linear

Mean

.500

.498

.541

.494

.503

1.000

.941

1.022

1.012

.996

1.500

1.254

1.471

1.494

1.502

StdDeviation

.005

.005

.006

.005

.005

.006

.006

.006

.010

.011

.012

.013

33


Table 4

Centre-specific calibration factors for estimation of standardised BMD

Centre

Ankara

Beijing

Canada

Croatia

Hungary

Manila

Moscow

Obninsk

Shanghai

Singapore

5.2486

8.5840

5.4113

4.7586

5.6420

4.0540

.1.9295

4.5053

4.0834

0.2073

0.1336

0.1918

0.2677

0.2239

0.3197

0.3302

0.2864

0.3166

34

SEXM

00(Jl

CENTRE

Ankara

Canada

Manila

Moscow

Obninsk

SBMD

Valid N

N=93

N=40

N=56

N=I63

N=69

Mean

.967

1.080

1.052

1.118

1 131

StdDeviation

.112

.171

.197

168

150

HEIGHT

Valid N

N=93

N=0

N=56

N=I63

N=69

Mean

172753

165.536

178.294

177.623

StdDeviation

6.025

7.019

6.544

6.854

WEIGHT

Valid N

N=93

N=0

N=56

N=I63

N=69

Mean

71.871

62.313

77.785

77.986

StdDeviation

10 098

9.692

12.071

13.555

BV1I

Valid N

N=93

N=0

N=56

N=163

N=69

Mean

24.053

22 696

24.467

24698

StdDeviation

2.952

3.080

3.625

3.936

SEXF

Table 5Cmde means and standard deviations for sBMD at the spine and anthropomorphic variables at 5 study centres.

•

CENTRE

Ankara

Canada

Manila

Moscow

Obninsk

SBM1)

Valid N

N=I22

N=95

N=58

N=I6I

N=70

Mean

.985

1.113

1.011

1.068

1.095

StdDeviation

.126

.154

.180

.155

.133

Valid N

N=122

N=0

N=57

N=16I

N=7I

HEIGHT

Mean

162902

154.491

163.671

163.972

StdDeviation

6.241

6.541

5.808

5.297

WEIGHT

Valid N

N = 122

N=0

N=57

N=I6I

N=7I

Mean

59 500

56684

63.230

61.746

StdDeviation

9.530

13 191

11.378

9.302

Valid N

N=l 22

N=0

N=57

N=161

N=7I

BMI

Mean

22481

23.825

23.649

23019

StdDeviation

3.796

5.332

4.353

3.687

CO0 5 SEXF

•

^\GECAT

1.00

2.00

3.00

4.00

5.00

6.00

7.00

CENTRE

Ankara

Valid N

N=13

N=19

N=15

N=I6

N=17

N=20

N=22

Mean

.903

.999

1.006

990

1.052

974

903

Canada

Valid N

N=I7

N=3

N=8

N=8

N=6

N=1S

N=35

Mean

1.102

1.019

1.182

1.267

1.142

1.097

1.078

Manila

Valid N

N=8

N=8

N=8

N=8

N=7

N=9

N=9

Mean

.902

999

1 054

.990

1.051

1.066

.909

Moscow

Valid N

N=23

N=23

N=20

N=14

N=23

N=22

N=35

Mean

1 029

1.057

1.113

1.087

1.077

1.082

1.037

Obninsk

Valid N

N=S

N=8

N=ll

N=9

N=I3

N=8

N=I3

Mean

1.051

1.100

1.103

1.107

1.116

1 041

1.116

SEXM

AGECAT

1.00

J2.003.00

4.00

5.00

C..00

7.00

CENTRE

Ankara

Valid N

N=12

N=15

N=12

N=ll

N=I5

N=12

N=16

Mean

.900

1.005

966

1.041

.979

.965

.924

Canada

Valid N

N=2I

N=7

N=l

N=l

N=l

N=8

Mean

1.033

1113

1.052

816

1.282

1.119

Manila

Valid N

N=8

N=X

N=8

N=7

N=S

N=8

N=8

Mean

.915

1 076

1.024

1.077

1.070

1.043

1.045

Moscow

Valid N

N=26

N=I8

N=24

N=I9

N=I9

N=I9

N=36

Mean

.968

1.135

1.185

1 168

1.101

1.105

1 136

Obninsk

Valid N

N=8

N=I3

N=8

N=9

N=9

N=9

N=13

Mean

1.046

1.130

1.240

1.147

1.133

1.018

1 183

Table 6Mean sBMD by age category in women and men at each of the 5 centres. Please note the small number of subjects in some age groups

CDC\|

O

LOCD

CO-vl

Figure 1

Low density ESP vertebra

.62

.60

.58

.56

.54

.52

.50

.48

i t

- T

1 , . , P— ! ! ! ! j

N= 10 10 10 10 10 10 10 10 3 10

ankara Canada hungary moscow shanghaibeijing Croatia manila obninsk singapor

CENTRE

CO00

Q

00

O

LO

1.00

.90N =

Figure 2

Middle density ESP vertebra

10 10 10 10 10 10 10 10 3

ankara canada hungary moscow shanghaibeijing Croatia manila obninsk singapor

CENTRE

Q

CO

O0LO

COCD

1.30

Figure 3

High density ESP vertebra

N= 10 10 10 10 10 10 10 10 3 10

ankara Canada hungary moscow shanghaibeijing Croatia manila obninsk singapor

CENTRE

Figure 4

Linear regression of Lunar and Hologic machines1.81

1.6

1.4

1.2Q

OQ1.0

(D

(D(/)

O.4 .6

Slope=1.079 //

/s/

//

y

y

y ' Slope =0.956/

//

.8 1.0 1.2 1.4 1.6

CROATIARsq = 0.9982thru origin

ANKARARsq = 0.9987thru origin

Actual BMD

IO

LL.

LUCO

LU

~z.LJJ

oI —

roTO

a

"ETO

OOto

o

O

—Q-—H

I H

-e-

- H -

- - — Q - -

c:

_Q

O

CN

COCT>CMCM

COCMCMCD

oCN

CO

coCMCD

CO

i—

CO< 0

5CM;

148

00CO

198

8238

COT—

OO

ooCD

OO

LO

oo

ooco

ooc\i

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41

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42NEXT PAGE(S)

left BLANK

XA0054917

Appendix 4

PROTOCOL FOR SAMPLING AND ANALYSIS OF BONE SPECIMENS

Namik KAras

Fatih University, Istanbul, Turkey

The following sampling and analysis procedures are mostly based on the report of the IAEA Consultantsmeeting held in Vienna in December 1995 (NAHRES 28). Since then we have carried out extensivestudies on both sampling and analysis of bone by INAA and AAS. The followings are ourrecommendations for those who would like to do trace element studies within the CRP.

Collection Sites for Bone samples

Iliac crest of hip bone, and rib bone, are the most commonly used bone sampling sites for analysis.However, it is now proposed for the purposes of this CRP that rib bone is not the most suitable sampleto use. There are three main reasons for this:

• Rib bone has a highly variable composition.• It is metabolically not very closely related to the parts of the skeleton where measurements of

BMD are being made.• It is not usually available from living subjects with osteoporosis and would notjherefore, provide

reference values for biopsy studies.

The iliac crest of hip bone was chosen as the most suitable sampling site for several reasons:

• Local variation in the elemental concentration along the iliac crest is minimal• Iliac crest biopsies are commonly taken clinically on patients• The cortical part of the sanple is small (~2 mm) and can be separated easily from the trabecular

bone• The use of the trabecular part of the iliac crest for trace element analysis has the advantage of

reflecting rapidly changes in the composition of bone due to external parameters, includingmedication.

Biopsy studies, although in some ways more difficult than autopsy studies, becase of the need to obtainthe informed consents of the subjects, are potentially more useful than autopsy studies. Thereby manyproblems of postmortem migration of elements can be avoided and reliable datary and other data can becollected simultaneously.

Select the subjects among the patients undergoing orthopedic surgery due to any reason other thanosteoporosis. Follow an established protocol to obtain bone biopsies. Patients undergoing spgery shouldfill in the "OsteoporosisProject Questionnaire Form" including information on lifestyle variables, dieterintakes, the reason for surgery etc.

If possible, measure the bone mineral density (BMD) prior to removal of the biopsy sample. Howeverit may not possible to have BMD results on all the subjects because of difficulty of DEXA measurementafter an accident.

Sampling of Bones

• Bone biopsies from the iliac crest should be taken by a surgeon using a specially preparedtitanium tool or, if not available, with a stainless steel osteotome. 3-5 grams of bone obtainedfrom the anterior iliac crest are enough.

• Place them into pre-washed polyethylene bags and store at -20°C in a deep freezer.• Separate the cortical and trabecular parts of the bone carefully from eactother with an osteotome

A separation of the cortical from the trabecular part is necessary because some of the traceelements have a different (generally higher) concentration in trabeculabone than in cortical boneAlso, when the cortical substance is thin in the area being removedjhe sample consists almostsolely of trabecular tissue. This tissue, because bone remodeling in trabecular bone is miE active

44

than in cortical bone, has the advantage of reflecting more rapidly any changes irthe compositionof bone due to external parameters such as medication.

Preparation of Sample for Analysis

Soft tissues, muscle and fat should be removed mechanically with an osteotome as far as possible.

• Divide the sample into smaller parts. Forthe removal of possible surface contamination, use 5%citric acid solution. Place both cortical and trabecular parts of the same bone in a polyethylenebottle containing 50 mL 5% citric acid solution and ultrasonically clean for about 30 seconds.Instead of an ultrasonic shaker, a Soxhlet apparatus can also used. However it is more timeconsuming so an ultrasonic shaker is preferred.

• Remove the blood by placing the entire bone sample in 50 mL 5% glucose solution andultrasonically clean for about 60 minutes. This procedure could be repeated a few more times unia clear solution is obtained.

• Remove fat and muscle by placing the entire bone sample in 50 mL diethyl-ether for about 15minutes in the ultrasonic shaker and repeat the procedure once more.

• Freeze the bone sample in liquid nitrogen, in a polyethylene bag and then grind in a Tefloncontainer to produce a homogenous sample; then dry at 60°C for 24 hours.

• Weigh the cortical and trabecular parts separately. This is the final sample for analysis either byINAA or other techniques.

Please note that blood, muscle and fat can be removed much faster and easierif the separation processstarts immediately after the bone sample is obtained.

Quality assurance of the analysis

In order to assess the quality of analytical data being produced during the activation analysis, standardreference materials are used. There are only a fewreference materials for quality control of bone analysisThe National Institute of Standards and Technology issues the bone ash NISTSRM 140 and bone mealNIST SRM 1486. Of course one can use other appropriate SRMs for this purpose also.


45

XA0054918

302-E4-BRA-8188

STUDIES OF OSTEOPOROSIS IN URBAN RESIDENTS OF SAO PAULO,

BRAZIL, USING ISOTOPE RELATED TECHNIQUES.

Part of Coordinated Program: COMPARATIVE INTERNATIONAL STUDIES OF

OSTEOPOROSIS USING ISOTOPE RELATED TECHNIQUES.

CHIEF SCIENTIFIC INVESTIGATOR: DR. AURELIO BORELLI

Contracting Institute: School of Medicine (Faculdade de Medicina), Hospital das

Clinicas, University of Sao Paulo, Laboratory of Human Nutrition and Metabolic

Diseases (LIM/25) and, Radiochemical Division of the Institute of Nuclear and Energetic

Research (IPEN/CNEN-SP), Sao Paulo, Brazil.

1 -1

INTRODUCTION

This project is aimed at defining the peak of bone mass in an urban population chosen

randomly on account of the great miscigenation in Sao Paulo.

It is quite important to know the peak of bone mass and the factors that would interfere in

the amount of bone formed as they are valuable quides for future plans of osteoporosis

prevention.

In our previous meeting it was decided to increase the number of individuals studied from

210 to 350 and proceed with the evaluation of patient information according with the

questionnaire proposed.

In this report it is presented the results on bone mineral density of lumbar Ll-L4and neck

of femur in the total of 350 patients (175 women and 175 men) The complete data, as

well as the information obtained from the questionnaire will be sent in the future reports

as programed in the meeting of Sao Paulo..

1 -2

METHODS

The individuals were chosen from normal relatives of patients who have come to the

University Hospital for consultation, and Hospital workers and relatives. Subjects from

15 to 50 years old were distributed in 7 groups; each group with 25 individuals of each

sex.

A questionnaire with some modifications from the originally proposed by the CRP to

make it more aplicable to our conditions of life and nutrition , was filled in the day the

scan was performed covering details as milk ingestion, exercise, use of medicine which

could interfere in the metabolism of bone, presence of bone deformities in members of

the family and bone fractures.

Great variations from normal of milk ingestion or exercises were excluded. Secondary

causes of osteoporosis were rulled out clinically or through laboratory tests when

necessary.

LABORATORY METHODS

Bone density was measured with a densitometer 4500 A (DEXA) from Hologic. A

quality controle for the spine was done everyday and for the hip every week. The

variations of the standards were always within the accepted values.

1 -3

RESULTS

In Table 1-4 and Graphic 1-4 are presented the results of lumbar BMD (Li- L4) and neck

of femur from the total of 350 individuals as proposed. In this total it is included the

results already presented in the prior meeting and added to the 140 remaining in the

project to complete the total proposed.

The peaks of bone mass in this group of 350 brazilians from an urban area were the

following-

1 - The peak of hip BMD in women is in the 26-30 year old group:

2 - The peak of L1-L4 BMD in women is in the 36-40 year old group.

3 - The peak of hip BMD in men is the 26-30 year old group.

4 - The peak of L1-L4 BMD in men is in'the 31-35 year old group ' '

1 -4

Table 1

Patients123456789

10111213141516171819202122232425

AVGSD

16-20

0.8090.7610.9390.6970.6040.7300.7970.7060.8530.8790.6240.5520.8670.7920.6200.7510.6780.7890.6880.9820.6770.8110.7620.7750.597

0.7500.108

HIP

21-25

0.6.220.6760.7430.7220.9890.6630.5870.7180.6510.8300.7610.9020.6460.7710.8281.0100.8520.6460.5900.6000.7480.7570.6540.8440.764

0.7400.117

B.M.D. in

26-30

0.7160.7040.4870.8900.5570.6400.6590.7691.0880.6790.8700.7831.0630.7670.8170.7940.7480.6480.9660.9800.6820.7691.0961.0770.778

0.8010.167

women in

31-35

0.9090.7210.7110.6610.6230.6050.7800.8690.7220.6380.6780.6810.9430.8740.8670.8000.6430.8190.8150.7630.8330.7840.6420.8350.796

0.7600.097

different ages

36-40

0.7390.7080.6470.7320.9110.7810.9971.1671.0140.8020.7650.7220.8210.7820.8610.687 ,0.7800.7820.8440.7640.7620.8840.7440.6540.621

0.7990.123

41-45

0.9910.9420.7580.7320.9230.8800.8670.6380.9020.6160.7290.8590.7480.9140.5530.7850.6640.6870.9910.8970.6490.7240.8460.8410.921

0.8020.124

46-50

0.7500.5330.6020.6750.6380.9350.7550.7850.7690.6590.7600.7120.9270.8980.8450.9680.8010.7830.9010.8680.7101.0510.7270.8840.872

0.7920.123

1 -5

Graphic 1

HIP BMD IN NORMAL FEMALES IN DIFFERENT AGES P JM..n?i-

1.200

1.100

1.000CM

E 0.900

3 0.800

I 0.700CQ

0.600

0.500

0.400

i16-20 21-25 26-30 31-35 36-40 41-45 46^50

Age SMean+SD (n=175)

Table 2 "'

Graphic 2

CO

1.200

1.100

L1-L4 BMD IN NORMAL WOMEN IN DIFFERENT AGES

16-20 21-25 26-30 31-35

Age

36-40 41-45 46-50

0MEAN+SD(n=175)

Table 3

Patients

AVGSD

ages16-20

0.9590.7790.5710.5930.8630.9270.8460.8740.9050.9190.7350.9101.1281.0960.8040.9220.5330.6310.5330.6310.5840.7521.0051.1000.7840.689

0.8130.182

LI-L4 IN

21-25

0.7420.9790.5110.3970.9880.9690.7610.8900.9110.8640.8100.5961.1151.0740.9080.9190.9680.8890.7780.9440.6480.8740.7740.4560.6420.771

0.8150.183

NORMAL MEN

26-30

0.9311.0991.1190.9060.7560.6310.7300.5150.7910.6300.7910.6301.0321.0100.5410.6781.1370.8420.9070.8491.1961.0521.1791.2001.0060.784

0.8820.209

IN DIFFERENT AGES

31-35

0.7351.0620.7790.9740.8091.2191.1210.8790.9650.9330.8490.9331.084

-1.0980.760 '1.081 .1.0850.8560.7920.9420.8760.9640.9890.9971.0221.87

0.9720.219

36-40

0.9361.2501.1040.6831.0901.2161.1290.9840.6490.6760.9460.9420.9530.7280.8640.9180.8091.2671.3070.6500.3610.9970.9790.9970.8860.871

0.9300.221

41-45

1.1540.9331.0901.2161.1291.0691.0510.9470.7520.7830.7900.8560.8140.9200.8760.8480.7260.7450.9681.0421.2381.0690.8170.7710.8120.902

0.9350.153

46-50

0.9600.5560.5560.7871.2240.8060.8020.8450.9790.7180.9150.9212.6050.9841.0411.0341.1500.9960.9280.7260.9590.8270.8280.9840.9280.789

0.9560.370

1 -9

Graphic 3

L1-L4 BMD IN MEN IN DIFFERENT AGES1.400

1.300

1.200

1.100

16-20 21-25 26-30

i ' f. '• ;' ."'<.

36-40 41-45 46-50

HMean+/-SD(n=175)

Table 4

PATIENTS

123456789

1011121314151617181920212223242526

AVG.SD

AGE16-20

1.0970.9470.8211.0050.9361.0350.8510.5350.7160.6320.7590.7060.7390.8280.7960.6440.7050.6900.6510.7640.8530.57.907000.5380.5501.131

0.7770.168

21-25

^•0".7790.3590.2800.9681.0060.7500.7800.7860:9830.7550.4960.7650.7520.9180.8620.8620.5570.690

- Q.5U0.7300.7890.9030.7460.7290.9930.9§8;

0.759: 0.190

HIPBMDIN

26-30

0.7150.7450.6730.7050.7540.5420.4390.9851.036 .0.6660.5460.8960.8740.9311.1180.7970.7431.281i:i551.1970.9930.8940.7710.9210:9311.071

0.861 i0.211

MEN IN1988

31-35

0.6460.8420.8301.1551.1740.7230.6790.9700.6701,1710.9010.7470.8240.9360.8270.6310.9980.6971.1020.8980.8420.9930.8050.6680.7720.781

0.8570.165

DIFFERENT

36-40

0.7770.8850.8870.7100.7050.9480.9420.7330.9430.8790.8050.7210.7740.9370.6310.9980.7320.7950.8030.7560.5920.9570.8550.5910.5751.288

0.8160.155

AGES

41-45

0.7120.9251.0050.8210.6290.6310.8320.9210.8650.6840.6540.7250.6811.0460.7680.5680.7220.3920.6880.6970.8710.9050.6970.9050.8030.811

0.7680.144

46-50

0.8630.9450.76T0.7340.8390.8110.9271.2090.8610.8910.8940.7230.8671.0301.0981.2481.1780.5940.7820.9570.9640.9190.7000.9840.7780.884

0.9020.158

1 -11

Graphic 4

1.200

1.100

1.000

0.900oS 0.800

| 0.700

0.600

0.500

0.400

HIP BMD IN NORMAL MALES IN DIFFERENT AGES

mJ / .

/ / ' ' ' • • I! t

16-20 21-25 26-30 31-35 36-40 41-45 ^ 46-50Age 0 Mean+/-SD (n=182)

PLANS FOR THE FUTURE

1. Prepare the complete data to be submitted according the spread sheet presented in the

Sao Paulo meeting.

2. Measure the length of the neck of the femur (we have not yet received the necessary

software).

3. Analyze and prepare the data from the questionnaire.

4. 4. After approval of the project by the Comission of Ethics of the University Hospital

we will proceed with the collection of bone samples from the autopsy room and the

neutron analysis as was presented in our first report.

1 -13

XA0054919

Instrumental Neutron Activation Analysis of Rib Bone Samples

and of Bone Reference Materials

M. Saiki,' M. K. Takata,1 S. Kramarski,' and A Borelli,2

'institute de Pesquisas Energeticas e Nucleares, 1PEN-CNEN/SP, RadiochemistryDivision, PXXBox 11 049, CEP 05422-970, Sao Paulo, SP, Brazil

2 Fat: de Mediciiia-i/SP.'Av. Dr Arnaldo 455, CEP 01246-903, Sao Paulo, SP, Brazil

ABSTRACT

The instrumental neutron activation analysis method was used for the

determination of trace elements in rib bone samples taken from autopsies of accident

victims. The elements Br, Ca, Cl, Cr, Fe, Mg, Mn, Na, P, Sr, Rb and Zn were

determined in cortical tissues by using short and long irradiations with thermal neutron

flux of the IEA-Rlm nuclear reactor. The reference materials NIST SRM 1400 Bone

Ash and NIST SRM 1486 Bone Meal were also analyzed in order to evaluate the

precision and the accuracy of the results. It was verified that lyophilization is the most

convenient process for drying bone samples since it does not cause any element losses.

Comparisons were made between the results obtained for rib samples and the literature

values as well as between the results obtained for different ribs from a single individual

and for bones from different individuals.

Index Entries: Rib bone; neutron activation analysis, bone reference materials,

trace elements

INTRODUCTION

In the last few years, there has been an increasing interest in determining trace

elements in biological tissues in order to elucidate their roles in human beings as well

as to diagnose diseases. Trace element studies have also been undertaken in bones

since they are deposits of essential and toxic elements. Controlling the mineral content

of bones can be a good indicator for the detection of different diseases, as

osteoporosis.

1 -14

Osteoporosis is one of the primary metabolic and degenerative bone diseases

characterized by a low mineral bone mass although the remaining bone is normal.

Today at least 10 million of individuals in Brazil aged more than 60 are suffering from

this disease. Salmon and Strause(l) studied the role of trace minerals in osteoporosis

and confirmed that adequate amounts of dietary Ca maintains optimal bone mineral

density (BMD) in post menopausal women and Cu, Mn and Zn are essential to the

maintenance of BMD.

Analyses of trace elements in bones are very rare as they are examples of

biological samples presenting difficulties for obtaining representative specimens for

chemical analyses. In the case of humans, collecting samples generally presents

problems because of medico-legal implications.

The determination of trace elements in bone samples have been carried out using

several techniques(2) including in vivo and in vitro nuclear methods (3,4).

In this work, the instrumental neutron activation method was developed in order

to analyze preliminary results of trace elements in ribs. The precision and the accuracy

of the results were also evaluated by analyzing the reference materials NIST SRM

1400 Bone Ash and NIST SRM 1486 Bone Meal.

MATERIALS AND METHOD

Sample Collection and Preparation.

Human rib bone samples were obtained from autopsies of accident victims

performed at the Institute of Forensic Medicine of the Universidade Mogi das Cruzes,

SP. The samples were wrapped in polyethylene foils and stored in a freezer until they

were treated for analysis. The ribs were cleaned free of connected soft tissues

(periosteum) and were washed with distilled water to remove the blood. The cortical

tissues were broken up into small pieces and then lyophilized for analyses.

Instrumental Neutron Activation Analysis

Aliquots of about 200 mg of sample weighed in polyethylene envelopes were

irradiated in the IEA-Rlm nuclear reactor along with the synthetic standards of the

elements. The synthetic standards were prepared by pipetting the elemental solutions

1 -15

<oO

onto pieces of Whatman No. 41 filter paper. These standard solutions containing one

or more elements were prepared from standard solutions provided by Spex Chemical.

Two procedures were used for the irradiations. Irradiations of 1 min at the pneumatic

rabbit station with thermal neutron flux of about 1012 n cm"2 s'1 were used for

determining the elements Ba, Ca, Cl, Mg, Mn, Na, P and Sr: Longer irradiations of 8

hours under thermal neutron flux of 1012 n cm-2 s'1 were used for Ba, Br, Ca, Cr, Na,

Fe, Rb, Sr, Sc, and Zn determinations. After appropriate decay times, the gamma

activities of the samples and the elemental standards were measured using a EG & G

Ortec hyperpure Ge detector with a resolution 0.90 keV for 122 gamma-ray of 57Co

and 1.98 keV for 1332 keV of wCo. This detector is connected via TRUMP card to a

microcomputer with EG & G Ortec software - MAESTRO. For P analyses, the beta

activity of 32P was measured in a Geiger Muller detector. The gamma spectra were

processed using VISPECT software. The comparative method was used for calculating

the content of the respective elements. The radioisotopes used in this study were:l39Ba, 13IBa, 82Br, 49Ca, 47Ca, 38C1,51Cr, 5SFe, 27Mg, 56Mn, 24Na, 86Rb, 87mSr, 85Sr, 46Sc

and 65Zn.

Analysis of Certified Reference Materials

The certified reference materials NIST SRM 1400 Bone Ash and NIST SRM

1486 Bone Meal were analyzed in the same conditions as in the analyses of bone

samples. The concentrations of reference materials were evaluated on a dry weight

basis determined, as recommended in their respective certificates. The following values

(in percent) of weight loss were found for correcting the final results. 0.26 for Bone

Ash and 2.71 for Bone Meal

RESULTS AND DISCUSSION

Elemental concentrations obtained in rib samples are presented in Table 1 along

with literature values. These results indicated a good precision with relative standard

deviations varying from 3.8 to 14%. The less precise results wer obtained for Fe

probably due to the rib contamination by blood containing high levels of this element.

Besides, most results obtained for human ribs are within the range values for healthy

individuals reported in the literature.

1 - 16

Table 1. Elemental concentrations in cortical rib bone from healthy individuals

Results in ug/g, dry weight, unless otherwise indicated

Element ;

Br

Ca, %

Cl

Fe

K

: ; M § ' %

Na, % ,.

P,%

Rb

Sr

Zn

X M ± sM

0.83 ±0.23

20.5 ±0.8

547 ±183

18.7± 14.6

843 ± 200

., . 0.30 ±0,04

0.46 ±0.07

9.47 ± 1.5

1.33 ±0.43

100.4 ±9.9

91.1 ± 14.3

This work

Range

0.55- 1.27

19.4-21.7

217-716

3.4-44.4

529- 1182

0.23-0.37

0.37 - 0.56

7.4- 12.1

0.84-2.10

84-113

78- 120

Ref(5)

XM ± SM

4.1 ±4.0

20.0 ±4.1

23 ± 11

: 0,2$.+ 0.04

0.54 ±0.10

8.8 ±2.2

2.1 ±3.0

62 ± 18

180 ±44M ± SM - Arithmetic mean and standard deviation

1 -17

Table 2. Comparison of elements in two different ribs from a same individual and ribs

from two different individuals. Results in ng/g of dry weight, unless otherwise

indicated

Element

Br

Ca, %

Cl

Fe

•K , . , ; , ,

Mg,% . ;„•!..

Na, %

P,%

Rb

Sr

Zn

Individual

Rib 1

0.81 ±0.06

19.4 ±0.5

708 ±37

21.3 ±2.7

... .»• 836 ±75

:i! „ . 0.31 ±0.01 ..

0.422 ± 0.003

8.7±O.l

1.26 ±0.70

100+10

81.8±0.7

No. 1

Rib 2

1.05 ±0.88

21.0±0.1

647 ±21

13.4 ± 1.8

831 ±70

. 0.29 ±0.01

0.51 3± 0.007

9.1 ±0.1

1.05 ±0.88

84 ±13

83.8 ±0.7

Individual No. 2

Rib 3

0.77 ±0.06

21.2 ±0.4

508 ±19

14.0 ±3.9

1182 + 212

0.350 + 0.016

0.37 ±0.006

9.1 ±0.1

2.10±0.24

113 ± 9

83 ± 1

Table 3. Concentrations of elements in reference material NIST SRM 1486 Bone Meal

Element

Ba, ug/g

Ca, %

Cl, ug/g

Fe, ug/g

Mn, ug/Kg

Mg, ug/g

Na, ug/g

P,%

Sr, ug/g

Zn, ug/g

n

6

9

7

6

6

5

7

5

10

6

This workX ± s

251 ±29

26.0 ±1.8

194 ±15

89 ±12

1008 1 ± 106.6

4530 ± 206

4508 ±241

11.5 ± 0.9

233.0 ±26.1

133 ± 8

Sr

11.7

7.0

7.8

13.4

11.5

4.5

5-4

7.5

11.2

6.2

Ref. (6)

26.50 ±0.24

99 ± 8

1000(N)

4060 ± 19

5000 (N)

12.30±0.19

264 ±7

147 ± 16n - number of determinations

Table 4. Concentrations of elements in reference material NIST SRM 1400 Bone Ash

Element

Ba, \ig/g

Ca, %

ci, ng/g

Cr, ng/g

Fe, ig/g

Mn, iag/Kg

Mg, ng/g

Na, ig/g

P, %

Sb, ^ig/Kg

Sc, ^g/Kg

Sr, ^g/g

Zn, ng/g

n

6

8

6

5

6

5

5

4

4

4

5

8

6

This workX ± s

231 ±18

36 ±2

211 +21

2.9 ±0.5

597 ±27

17.3 ±0.7

6384 ± 300

4925 ± 287

17.1 ±0.6

558 ±41

65.9 ±7.8

224 ±19

168 ±11

Sr

• 7.7

5.9

10,3

17.0

4.2

4.0

4.7

5.8

3.4

7.4

11.9

8.3

6.4

Ref.(7)

38.18000± 0.00000

660 ± 27

17 N)

6840±137

6000 (N)

17.91010.179

249 ±7

181 ±3

1 -19

The short irradiation procedure has the advantage of presenting a quick

response for the analysis of several elements. Even though there is no spectral

interference of bremsstrahlung, this irradiation condition does not allow the analysis of

elements such as Br, Fe, Rb, Sc and Zn. The lyophilization was the most convenient

process for drying the sample since it does not cause any loss of elements. Samples

prepared by calcination showed loss of Br and Cl. Comparison of the results obtained

for different ribs from a single individual as well as from different individuals did not

show a significant difference. These results are presented in Table 2.

Tables 3 and 4 present results obtained in the reference materials NIST SRM

1400 Bone Ash and NIST SRM 1486 Bone Meal, respectively. In these Tables are also

presented certified values, for comparison. The mean values found in the analysis of

reference materials are in good agreement with those reported by NIST. The relative

errors obtained are lower than 11.7%. The precision of the results was satisfactory for

most elements with relative standard deviations varying from 3.4 to 11.8%. The less

precise results were obtained for Fe in Bone Meal with relative standard deviation of

13.4% and for Cr in Bone Ash with relative standard deviation of 17%. The results

obtained for Ba, Cl, Cr, Mn, Na, Sb and Sc in these reference materials constitute a: • • •- .7 ! , 7 . | ? . I 7 7 .

contribution for their certification.

Results, obtained in this work confirm that the INAA is a suitable method for

bone analysis due to its multi elemental character, absence of a destruction step and its

quality results, , . ,.... , ;

ACKNOWLEDGMENTS

To FAPESP and, CNPq from Brazil for financial support and to Joao R. Ferreira

Batalha from the. Facujdade de Medicina da Unjversidade de Mogi, das Cruzes, SP for

his valuable help in sample collection.

REFERENCES. i • ; : . . 1

1. P D. Saltman and L.G Strause, The role of trace minerals in osteoporosis. J. Am.

Coll. Nutr. 12,384-389(1993).

U20

2. F. A. EI-Amri and M. A. R. El-Kabroun, Trace Element concentrations in human

bone using instrumental neutron activation analysis. J. Radioanal. Nucl. Chem.,

Articles, 217, 205-207 (1997).

3. V.Y. Zaichick, Instrumental neutron activation and X-ray fluorescent analysis of

human bone in health and disease J. Radioanal. Nucl. Chem. Articles, 179, 295-303,

(1994).

4. S Krishnan, W. C. Sturtridge, M Krishnan and R. Y. Qureshi, Comparison of the

determination of body calcium by in vivo neutron activation analysis (IVAA) and

energy X-ray absorptiometry (DEXA). J. Radioanal. Nucl. Chem. Articles, 216, 33-36

(1997)

5. L. Samudralwar and L.D. Robertson. Determination of major and trace elements in

bones by simultaneous PIXE/PIGE analysis. J. Radional. Nucl Chem. Articles, 1, 259-

267(1993). . , .

6. National Institute of Standard and Technology. Certificate of Analysis, Standard

Reference Material I486, Bone Meal, December, 1992.

7. National Institute of Standard and Technology. Certificate of Analysis, Standard

Reference Material 1400 Bpne Ash, December, 1992.

1 -21

XA0054920

COMPARATIVE INTERNATIONAL STUDIES OF OSTEOPOROSIS USINGISOTOPE TECHNIQUES

CONTRACT NUMBER Research Agreement Number 8189/CF

TITLE Working Paper for the Third Research Co-ordination Meeting on Comparative InternationalStudies of Osteoporosis Using Isotope Techniques.

DATE/PLACE August 24-7, 1998 at Sao Paulo, Brazil

INSTITUTE General Division, The Toronto Hospital, MedicalPhysics Laboratory, 200 Elizabeth Street, Toronto,Ontario, Canada M5G 2C4 and the University ofToronto, Toronto, Ontario, Canada.

CHIEF INVESTIGATOR S. Krishnan

2 - 1

COMPARATIVE INTERNATIONAL STUDIES OF OSTEOPOROSIS USINGISOTOPE TECHNIQUES.

S. Krishnan

Medical Physics Laboratory, General DivisionThe Toronto Hospital200 Elizabeth StreetToronto, Ontario, CanadaM5G2C4.

ABSTRACT

Over 200 volunteers were recruited through Newspaper advertisements and other meansto measure the BMD of the Lumbar Spine and Femoral Neck of normal healthy Canadiansfrom the Toronto Area. AJl these people were carefully interviewed by trained scientists ormedical personnel with respect to their medical history, life style and habits. Persons withhistories that will affect bone mineral status were excluded from the study. The resultingstudy population consisted of 162 persons.

The data was used to determine the age or the range of ages at which peak BMP isattained in this normal population, t he Measurements were made using a DEXAinstrument made by Hologic Inc., Model 1000W.

1. BACKGROUND

Osteoporosis is a major public health problem around the world especially among thewestern countries. It is particularly common among post-menopausal white women.Among the variables which determine low bone mass among the elderly are the age, sex,ethnicity/race and the geographical origin of the subjects.

According to the decision made at the Second CRP at San Diego, USA during 7-10October 1996, the objective of this study is the collection of BMD data of lumbar spineand femoral neck of the study population. In this work Urban Canadian population for theToronto area was chosen as the subjects. Both males and females were studied in the agegroup from 15 to over 50 years. As discussed in the previous CRP it was not practical orpossible to obtain subjects through truly random sampling. The subjects were derived by avariety of means but mainly through advertisements in the Media, newspapers and hospitaland university bulletin boards. AJl the subjects were critically interviewed and those withcertain exclusion criteria were removed from the study. The exclusion criteria usedincluded the following:

2 - 2

.. Moderate or severe scoliosis (as found visually or in the DEXA scan)

.. Known chronic illness for greater than 3monts duration

.. Known chronic use of any medication other than dietary/vitamin supplements

.. Previous low trauma fractures

.. Greater than one month immobilization

.. Over-exposure to toxic metals or irradiation

Excessive consumption of alcohol or cigarettes was not an exclusion criteria but it wasnoted.

OC/OA

Routine QC/QA procedures were followed throughout this study. The spine phantom wasmeasured everyday and ensured that the BMD measured was within accepted limits. TheESP phantom was measured to enable cross comparison of date with other country groupsin this study.

RESULTS

The data obtained are given in the following tables and figures. For Canadian females, itappears that the peak bone mass is reached around 33.5 years of age for the lumbar spineand the femoral neck. With males the peak BMD appears at two ages for bothmeasurements. The first peak is around 23 years and the second peak appears at around44.7 years.

2 - 3

FIGURE 1. DAILY SPINE PHANTOM MEASUREMENT OVER ONE YEAR.

£i.86l(-

fl.BSl

wl.B41

l l . 8 3 1

£1.821

Ml. 811

8.981-

1AR

> S o-o-- --am.-a - .-1

IJ

I I I IAPR MAV JUM JUL

9~ "oa%"

SEP OCT NOU DEC

Reference Ualues•pine phantom 8284

PlOAT) — X • B Z l o SfPIS'cri<*

S.D. = B.BB38 grns/cm2U n i t * = ± 1 . 5 S.D.

Plotn =

nean =S.D. =

cv =

Statist ics2 6 11. B227 gns//cn2B.B846 sns/cm2B.45x

TORONTO GENERAL HOSPITAL

HOLOGIC

2 - 4

TABLE 1. BMD MEASUREMENT OF ESP PHANTOM

16-Oct-96

0.9530.9460.9480.9420.9480.95

0.9490.9430.9470.948

Mean 0.947SD 0.003

2 - 5

TABLE 2. BMD MEASUREMENT OF FEMALES

AGE GROUP

15-19 years

20-24 years

25-29 years

30-34 years

35-39 years

40-44 years

45-49 years

50-54 years

55-59 years

AGE

17.3 (0.883)

23.3(1.762)

27.8(1.548)

33.5(1.27)

37.7(1.96)

43.6(1.283)

47.9(1.493)

52.9(1.576)

57.8(1.738)

n

17

3

8

7

7

19

23

12

23

LS BMD

1.03 (0.094)

0.96 (0.077)

1.097(0.123)

1.136(0.13)

1.104(0.216)

1.022(0.103)

1.014(0.129)

0.997(0.133)

0.892(0.155)

FNBMD

0.888 (0.064)

0.861 (0.18)

0.894 (0.128)

0.891 (0.044)

0.861 (0.164)

0.792 (0.109)

0.785 (0.135)

0.757 (0.081)

0.706(0.114)

2 - 6

TABLE 3. BMD MEASUREMENT OF MALES

AGE GROUP

15-19 years

20-24 years

25-29 years

35-39 years

40-44 years

45-49 years

50-54 years

55-59 years

AGE

17.5 (0.729)

23 (0.716)

25.3

36.8

44.7(1.697)

46.7(1.662)

52.6(1.061)

58.2 (2.54)

n

22

7

1

1

2

5

2

3

LSBMD

0.993(0.156)

1.038(0.13)

0.989

0.784

1.147(0.047)

1.029(0.153)

0.996 (0.07)

0.93 (0.06)

FNBMD

0.978 (0.173)

0.995(0.186)

0.847

0.814

0.868 (0.036)

0.782 (0.165)

0.724(0.117)

0.748 (0.017)

2 - 7

Females '

MeanAge

iiJy23.327.833.537.743.647.952.9

Females

MeanAge

17.323.333.537.743.647.952.957.8

FIGURE 2.

MeanLSBMD

1.0300.96

1.0971.1361.1041 0221.0140.997n flQ?

VARIATIOf*

V200

t 100

O 1 000

CO 0900

0 800

17.3

i

i

MeanFNBMD J

0.8880.8610.6910.8610.7920.7850.7570.706

1 000 i

0 900,

E o.eooCD

0700

06O0173

—

OF BMD WITH AGE IN FEMALES

I

F Normal LS BMD Plot

_ x ^*——*-—-•.

- • — >

233 278 33.5 37.7 43.6 47.9 S2.9 57.8

Age

F Normal FN BMD Plot

233 335 37.7 43.6 47.9 S2.9 57.8

Age

2 - 8

MeanAge

17.523.025.336.8447j46.752.6

MeanAge

17.523.025.336.844.746.752.6

MeanLSBMD

0.9931.0380.9890.7641.1471.0290.996

MeanFNBMD

0.9780.995,0.8470.8140.8680.782,0.724

:IGURE 3. VARIATION OF BMD Wl'm AGE IN MALES.

M Normal LS Plot

1.300

1 200

1.100

§ 1.000,a:

0.900

0.800

0.700

. • : • . . : . • : . • • • • • : ' • • • : « : : . : • : : : H : p « | i ;

••:': :,:;r:::;":. =iS-. •: .r::fei!Ki:g;ls^^fe^-

• "^™h "-••••:--^.\4:jSl i ...Jis~"J'^^~~~>~^"':"::&• 1 :n j igte|?>-3 i S i ; - \ ::«:;ls/ "'" mJSi£l: •

iiliiil^/il " -^230 25.3 36.8 44

ASe

i

!

M Normal FN BMD Plot

1050

1000

0.950Q2 0 90003

0 850

o

o o 00

50

" • " — ~ —

-„-—^jSjsjp,^; ** -* •^y-w-y ~

\ „ ~ ^ ^ = #

\ •£•-:

17.5 23.0 25.3 36.6 44.7 46.7 52Age

1

1

.6

2 - 9

XA0054921

PROGRESS REPORT CONTRACT N° 8190/RB

Studies on osteoporosis in Chile using Isolope - related Iccli/iiqucs.

Dr. G. LoboDra. T. PnlmaSr. E. Cortes

INSTITUCION : Clinica INDISAAvda. Santa Maria 1810Providcncia - SantiagoCHILE

AGOSTO, 1998

3 - 1

INTRODUCTION

According to the objectives stated in the last coordination meeting, Bone mass density(BMD) measurements in men and women belonging to a healthy Chilean population, werecontinued. The selection of individuals was made in the manner described in previousreports, using the same exclusion criteria and using the modified WHO questionnaire.

We present in this report the results of BMD measurements performed from the beginning ofthis study until May 1998 A statistical analysis of the data is presented

MATERIALS AND METHODS

A. Equipment for BMD measurements.

Hologic QDR 1000 Bone densitomcter

In - house spine phantom for quality control Measurements with this phantom gavea C.V. of 0 30 - 0.40 during the period the proyect was performed

B. Materials

A total of 231 individuals were studied, 64 men and 167 women in the age range 15 - 50years old, all belonging to the white race. Their anthropomorphic characteristics arc shownin table 1A y IB

TABLA IA. ANTHROPOMORPHIC CHARACTERISTICS OF POPULATION STUDIED.

RACE

WEIGHT (Kg)

HEIGHT (cm)

BM1 (K£/cm2)

AREA SPINE (cm2)(L1-L4)

AREA FEMORAL (cm2)NECK

LENGHT OF FEMORALNECK (cm)

n

WOMEN

While

Mean i SD

GO. IX* 11.8

157 9 i 5.8

24 1 ±4.40

54 92 i 4.75

4 92 10.33

9.59 ± n.XS

167

MEN

While

Mean ± SD

74.4 i 12.9

170.4 i 6.1

25.5 ± 3.95

63.2 ± 5 94

5 61 if) 44

10 31 ± 0 88

64

DiTcrcucc

Mean A SE

14 2 i 1.75

12 0 i 0 86

1.40 ± 0.6

8.34 ± 0.75

0 69 ± 0.054

0 82 ±0.003

103

DM1 Body mass Index.

3 - 2

TABLA IB. VARIATION COEFFICIENT OF THE MEAN VALUES OFBODY SIZE AND BMD IN THE POPULATION STUDIED.

WEIGHT (Kg)

HEIGHT (cm)

BM1 (Kg/m2)

SPINE AREA (cm2)

FEMORAL NECKAREA (cm2)

LENGHTOF FEMORALNECK (cm)

BMD SPINE

BMD FEMORAL NECK

MEN

17.3

3.5

154

9.3

7.9

8.5

13.5

N.I

WOMEN

19.6

3 6

1R 2

8.7

6.78

V 2

1 1 5

12 1

C. Methods

BMD was measured in Lumbar Spine (LI - L4) and in the femoral neck region (right andleft). All data were collected in floppy diskettes for processing.

Curves for BMD vs. Age for spine and both femoral necks, for women and men,were plotted

- These curves were compared with those obtained from the Hologic data base.

- A correlation between BMD and weight, height, body mass index (BMJ) and scannedarea in spine and femoral neck, in men and women, was made

- The statistical analysis proposed by Prentice et al. (1) was applied to our data

- The statistical tests used were the following:

1 ) Statistical correlation between the curves obtained and the theoretical curves.

2 ) Multiple regression analysis for association between variables

3 ) Descriptive stalislics

EXCLUSION CRITERIA

Moderate to severe scoliosis

Known chronic illness (past or present) ofmore than 3 months duration

Known chronic (>1 month) use ofany medication, other than dietary / vitaminsupplementation.

Previous low trauma fracture

3 - 3

COMPARISON BETWEEN HOLOGIC DATA AND DATA OF THISSTUDY

1 Comparison of BMD according to age and sex (Table 2,3, 4,6,7) ( Figura 1 y 2).

TABLA 2. nONE MINERAL DENSITY ACCORDING TO ACE fN NORMAL WOMEN IN g/cm2.

AGE RANGE

J5-2O

21-25

26-30

.11-15

36-40

41-45

46-50

TOTAL

»

19

2.1

24

29

31

21

19

166

LUMBAR SPINE

Mean ± SD

0.9556 1 0.1042

1.0068 ± 0 1056

0.9772 ± 0.06775

0.9987 ± 0 . 1 0 8 5

0,9724 ± 0 .1306

0 9S89 ± 0.1461

0.9692 ± 0 . 1 3 2 4

0.98.14 ± 0 , 1 1 3 6

FEMORAL NECK

Mean

0 S4260

0 S645

0.7933

0 8349

0 773 1

0 7817

0 7957

08111

±SD

i. 0 0S34

i 0 1102

±0.0844

i 0.0892

± 0.0960

± 0 1016

±0 ,1106

±0.1002

TABLA 3. BONE MINERAL DENSITY ACCORDING TO AGE IN NORMAL MEN IN g/cm2.

AGE RANGE

15-20

21-25

26-30

31-35

36-40

41-45

46-50

TOTAL

n

8

4

14

13

15

8

1

G3

LUMBAR SPINE

Mean ±SD

0814 i. 0.122

1 050 ± 0.068

0.9908 ± 0.1298

1.0604 ± 0,1385

1.0138 ± 0 . 1 6 ) 5

0.9936 ± 0,1003

1 0169 ±0

1,0078 ± 0.1370*

FEMORAL NECK

Mean ± SD

0.9343 i. 0.0915

10401 ± 0 042

0 8804 ±0 156

0.9251 i (I 1169

0.959 ± 0 , 1 5 6 7

0.8731 ± 0 . 1 2 6 5

0.8.159 ± 0

09131 ±0 1288*

3 - 4

TABLA 4. COMPARISON OF BMD OF RIGHT AND LEFT FEMORAL NECK.

MEN

WOMEN

LEFT FEMORAL NECK(LFN)

0.9049

o so v;

RIGHT FEMORAL NECK(RFN)

0.9009

OH 160

P VALUE

0.703 (NS)

0.0002 (S)

i

There is agreement between curves, both for spine and femoral neck, in men andwomen

2 The age range in which peak bone mass remains constant was established Thisrange is 23 - 27 years in all groups (Table 5).

TABLA 5. AGE RANGE OVER WHICH PEAK BMD IS MAINTAINED (YEARS)

WOMEN MEN

SPINE

FEMORALNECK

23-27 (1.0068 g/cm2)

23-27(0.8645 g/cm2) 23-27 (1.0401 g/m2)

3 - 5

TABLE 6A. COMPARISON OF THE IIOLOGIC REFERENCE CURVE VSOBTAINED CURVE FOR SPINE IN WOMEN

HOLOGIC REFERENCE CURVE

AGE RANGE

15-1617-1819-2223-272S-3233-3738-424.3-47•18-5253-58

MEAN

1 0101 0151 0191 040i 0471 04]1 024(1 9990.9C70 892

SD

0 10 10 10 10 I0 !0 10 10 10 I

OI5SF.RVF.D CURVE

AGE RANGE

15-1617-18

19-2223-2728-3233-373S-4243-4748-52

TOTAL

MEAN

0 9560.9510.9721.0070.9770.9990.9720.9890969

0.983

SD

0.10400960.0990.106O.OGS0.1090.1310 !460 132

0 114

Frcq.

3313232429

312119

166

STATISTICAL SIGNIFICANCE

ACE RANGE

15-1617-1819-2223-2728-3233-3738-4243-4748-52

P Value for (hemean

0.400.330 0 70.07

< 0,0001*0.010.030.540.92

F \;ilue forIlit SD

0 560 9 7

0.430.61

<0001*0 5 50 150 0 80 24

' Differences .ire -.laiiMically sigjiific-nnt nficr bonferroni correction for Multiple comparisons

3 - 6

TABLE 6B. COMPARISON OF THE HOLOGIC REFERENCE CURVE VSOBSERVED CURVE FOR SPINE IN MEN


ACE RANGt

15-1617-IXfy-2223-2728-3233-3738-4243-474X-5253-58

MEAN

! 011 051 0911.0911 091

091091OCX

.053038

SD

0 110.110.110.110.110,110 110.110 110.11

OHSEKVED CURVE

ACE RANCE

15-1617-1819-2223-2728-3233-373X-42•13-474R-52

TOTAL

MEAN

1 0530815! 0431 05109911 0601 0140 99-1! 037

1 008

SD

0 0000.12200860.0680.1290 1390 1620.1000 000

0 137

Frcq.

I434

1413158

1

63

.STATISTICAL SIGNIFICANCE

ACE R A N G E

15-1617-1S19-2223-2728-3233-3738-424 3-4 748-52

P Value for Ilicmean

-

0,030 380 3200080.410 07006

P value forthcSD

-

0.620 82050 560.320 020.87

3 - 7

FIGURE N° 1. COMPARISON OF THE HOLOGIC REFERENCE CURVE INSPINE VS OBSERVED CURVE.

WOMEN

ti:11

•W-

I i 10 15 ^

MEN

J

15 ?fl JJ 30 3J *0 '5 3-0 5! 00 «J JO 15 tO 13VF.ARS

3 -8

TABLE 7A. COMPARISON OF THE ROLOGIC REFERENCE CURVE VSOBSERVED CURVE FOR FEMORAL NECK IN WOMEN


AGE RANGE

15-2223-27

28-323.3-3738-42

•13-47

48-5253-58

MEAN

089508940 8860 87108500 8260 7970 766

SD

0 10,10 10,10 10 10 10 1

OISSERVED CURVE

ACE RANGE

15-2223-2728-3233-3738-4243-4748-52

TOTAL

MEAN

0.8430865079308350 7730 7820 795

0811

SD

0 0830.1100.0840.0890.0960.1020 111

0.100

Frcq.

19232429312119


ACE RA.NCE

15-2223-2728-3233-3738-4243-4748-5253-58

P Value for themcnn

0 0080.150

< 0 0001'0 01

< 0.0001*0.050.78

—

P \ aluc Tor(he SD

0.260.450.190.140.480.910.48

—

;uc slalislicall) t after Kmfcnoni cuircclion foi Multiple comparisons.

3 - 9

TABLE 7B. COMPARISON OF THE HOLOGIC REFERENCE CURVE VSOBSERVED CURVE FOR FEMORAL NECK IN MEN


ACE R A N C E

15-2223-2728-3233-3738-4243-47•18-5253-58

MEAN

0 9790 9580 93609150.S940 8730 8510 SKI

SD

0 10 10 I0 10 10 10 10 1

I

OBSERVED CURVE

ACE RANGE

15-2223-2728-3233-3738-4243-4748-52

TOTAL

MEAN

09341.0400 8800.92509060 8730.836

U VII

SD

0.0910.0420 1160.1)70.1570.1270 000

0 125

t'rcq.

84

14

1315S1

63


ACE RANGE

15-2223-27

28-32.13-37

38-4243-4748-5253-58

P Value for themean

0 170 020 070 870 880 94

P \ aluc for(lie SD

0 6 30 110 890 890 050 61

-

3- 10

FIGURE N° 2. COMPARISON OF THE UOLOGIC REFERENCE CURVE INFEMORAL NECK VS OBSERVED CURVE.

WOMEN

1 IT

i f .it

»C J$ S3 ^ 60 i i 10 71 13 IS

MEN

i-H

3- 11

3. The distribution of Z and T values for the population studied was established accordingto the Hologic data base (Table 8).

A predominance of negative values was observed This was true for spine, both in menand woman, but only observed for women in fermoral neck.

This indicates a tendency to lower values of BMD in the population studied withrespect lo the Ilologic data base

TAHLA 8. VALUES OF Z, AND T SCORES IN NORMAL MEN AND WOMEN.

WOMEN

MEN

WOMEN

MEN

VALUE OK 7. SCORE INNORMAL MEN AND WOMEN

SPINE Z SCOREMC;III i SD

-0.3.144 ± 1 (1451(65% |-1 S)

-0,6465 i 1.22';y(70 % [-] S)

VALUE OF T SCORE INNORMAL MEN AND WOMEN

SPINE T SCOREMean ± SD

1 5-50 years 20-10 years

-0.6001 ±1.02 -0.5X65 i 0X3 (S)

-0.8358 ± 1.191 -0.5884 i 1.03 (S)

(HOr.OCIC DATA BASE)

FEMORAL NECK 7. SCOREMean ± SD

-0 5224 ± 1,0157(68% |-1 S)

0.0213 ± 1.202(48%|-]NS)

(IIOLOGIC DATA BASE)

FEMORAL NECK Z SCOREMain ± SD

15-50 vcars 20-30 years

-0.V04 ± 1 01.1 -0.4416 ± 1 05 (S)

-0 5S3O i l 198 -0 4308 i 1 14 (NS)

3- 12

STUDY OF THE CORRELATION OF BMP WITH THE INDICATORSOF CORPORAL SIZE.

WeightHeightBMIScanned area Spine and Femoral neck

a) Lumbar spine

A statistically significant correlation was found between BMD oflhc lumbar spine with thefollowing parameters:

Scanned areaWeightHeightBone Mass Index (BMT)

MEN WOMEN

A I cm2 area => A 0.0076 g/cm2 A 0 0066 g/cm2A 1 kg weight => A 0 0037 g/cm2 A 0.004 g/cm2

b. FcmorMl necks

A statislistically significant correlation was found between BMD ofboth femoral necks withthe following parameters:

I. Weight2 Body mass index (BMI)

No correlation was found with height or scanned area for men or women.

1 kg => A 0 002 g/cm2 for both men and women

3 - 13

APPLICATION OF TITE METHOD PROPOSED DY PRENTICE ET AL m .

Evaluation of the proportionality between the area considered in the calculation of BMD andBMC (Bone mineral content).

A regression between the logarithm of the area and the logaritlun of BMC is performed. Ifthe slope of the regression curve is significantly different from " 1 " , this means that thenormalization with respect lo area docs not correct adequately other factors which arcrelated to corporal size.

Slope of (lie regression curve

Women MenSpine Fern Neck Spine Fern. Neck1.35 (p<0.0001) 0 81 (NS) 1 48 (p<0 0001) 0.87 (NS)

SUMARY OF THE MAIN FINDINGS OF THIS STUDY

1. It has been difficult lo find individuals which prescnl lhc characteristics necessary lo beincluded in this study This has been specially true for men in all age ranges, and moreso in the extreme age ranges. The number of individuals in these ranges need to beincreased.

2 The anthropomctric parameters of the population studied show homogeneous variationcoefficients in men and women The lowest variation coefficients are found for heigh!and scanned areas. Variability between men and women is also significant, with itslowest value for the area of the femoral neck.

3. Plots of BMD vs Age both Hologic and observed data were in agreement However, asignificantly predominance of negative Z score was found specially in Spine in. Thismight indicate that the Chilean population has a tendency to lower BDM values thanthose incorporated into the Hologic data base.

4. Peak bone mass is reached and maintained in the third decade of life in the Chileanpopulation

5. BMD was higher in men than in women. Women shows higher BMD in theright femoral neck.

6. Body sizerelated paramaters have a significant and positive con-elation with BMD.This might be due to:

a) The fact that normalization for bone area is not enough to correct for the influence ofIridimcnsional bone size, which in turns depends on corporal size or others body sizerelated factors (body composition) (2,3).

b) Greater body size is actually associated with higher BMD

7 Body sizerelated parameters affect spine BDM differently than femoral neck BMD.Spine BMD seems to be influenced to a higher degree by bone size (bone area) and itsvariations might not reflect true changes in BMD. Femoral neck BMD is moredependent on weight and its variation might reflect true changes in BMD due to apreatcr mechanical load.

B°\ 3-14

CONCLUSIONS

I. Because of the influence of body size - related parameters in (he values of BMD obtainedwith conventional desintometers, it is necessary to consider them both in the clinical andepidemiological interpretation of BMD measurements.

2 It is necessary to validate locally the data bases provided by the manufacturers of bonedesintometers This implies that the anthropomorphic parameters for the individualsincluded in those data bases must be known and they should be compared with theparameters of the local population.

3. In a comparative study such as (his, the influence of anthropomorphic and perhaps bodycomposition characteristics of the population studied should be globally evaluated,before drawing conclusions about BMD in these populations May be intcrcsing toconsider all, BMC, BMD and Bone Area to determine wheler real change occur in bonevariables in the populations studied and to analize the relation between this variables inthe different countries.

REFERENCES

1 PRENTICE ETAL

Uncritical use of bone mineral density in absorptiometry may lead to size-related artifactsin the identification of bone mineral determinants.

Am J Clin Nutr 1994 60: 837-42.

2. ALBALA ET AL.

Obesity as a protective factor for post menopausal osteoporosis

International Journal of Obesity 1996 20: 1027-1032.

3. VAN LOAN ET AL

Effect of weight loss on mineral content and bone mineral density in obese women.

Am J Clin Nutr 1998 67:734-38.

3 - 15

XA0054922

NO.8191/RB

Studies of Osteoporosis In The Northern China,

using Isotope-Related Techniques

China-Japan Friendship Institute of Clinical Medical SciencesQin linlin, Nov, 1994- July, 1998

Authors: Qin Linlin, Chief Scientific InvestigatorChen Jinbiao,Ma Haibo, Zhang Wei,Ge Chonghua, Xiao Yaxia,Bao Ande

(China-Japan Friendship Institute of Clinical Medical Sciences)

Li Zirong, Li Zhongshi, Zhang Nianfei, Yue Debo(Dept. of Othopedics, China-Japen Friendship Hospital)

Tian Weizhi, Ni Bangfa, Nie huilin(China Institute of Atomic Energy)

4 - 1

—. Age of PBM and variation of BMD1. Sampling subjects and physics, lifestyle factor

Target population of this investigation is healthy person in north China, aged 15-50, the

"northern" means: someone is born and grew up in north, furthermore, parents are "northerner".

Selection of subjects was done by community sampling, subjects have been selected by random

from China-Japan Friendship Hospital and some social organizations closing to the hospital.

Recruited subjects rilled our questionnaire according to the WHO's one, then the quesnnaires

were checked. Rejected participants were no northern healthy persons.

1005 subjects were recruited in this study, 901 were healthy among them, furthermore 845 were

aged 15-50 years old. 821 were healthy Han ethnic person in northern China, 781 were aged 15-50

years old among them . Among the 184 surplus subjects, 66 were healthy Han ethnic person in

south China(50 aged 15-50 years old), 72 had history of fracture euthenics. 32 had chronic disease,

14 were euthenics.

Many physics and lifestyle factors were included in our questionnaire, such as high, weight,

smoking, drunking, exercise level etc. The results were listed in the table 1.

Table 1 The result of physical, lifestyle characteristjcs( x + s)

Age(years)

Height(cm)

Weight(kg)

BMI'

symptom1

menarche age(year)

menopausal age(year)

bear children

lactation

cigarette(count/day)

smoke history(year)

liquor

liquor history(year)

tea(g/day)

tea history(year)

exercise level

in teenager'

in the last year"

in the last five years*

calcium(mg/day)

Male (409)

32. 1 ± 10.0(372)

172.8 ± 5.6(372)

68.8 ± 10.2(372)

23.0 ± 3. 1(372)

0.02 ± 0. 13(372)

11.7 ± 8 . 7(152)

12.2 ± 7. 1(152)

46.0 ± 58.8(106)

10.4 + 5.8(106)

10.2 ± 68.4(184)

11.2 ± 6.7(184)

1.2 + 0.6(372)

0. 9 ± 0.6(372)

0. 5 ± 0.5(372)

392. 1 ± 214.0(178)

female(372)

32.4 ± 10.0(409)

161.4 ± 4.5(409)

56.2 ± 8.0(409)

21.6 ± 3.0(409)

0.03 ± 0. 17(409)

13.6 ± 1.5(409)

44.5 ± 4.2(8)

1. 1 ± 0.3(231)

6. 9 ± 7.4(162)

9. 0 ± 8.0(4)

5 ± 3.6(4)

19.6 ± 10. 1(12)

11.8 ± 9.5(12)

2. 3 ± 1.3(120)

9. 1 ± 5.2(120)

1. 1 ± 0.5(409)

0. 9 ± 0.5(409)

0. 3 ± 0. 5 (409)

372.2 ± 175.8(170)

* BMI=weight/(height/100)

$ classical variable, 0 — no symptom , 1 — having symptom

* classical variable, compared with age-matched person : 0 — low, 1 — same, 2 — high

* classical variable, if exercise per week is over 3 hours, il is 0 ; if not it is 1

4-2

The absorbing dietarys of partly population were investigated . The method of"review of diet in the last 3 days" was used in this study, 348 subjects were investigated.The nutrient composition of diet were calculated according to the food compositiontable. The results were listed in the table2 .

Table 2 The dietary survey result (x±s)

male (n= 178) female (n=l 70)age (years)height (cm)BMIenergy (Kcal/day)protein (g/day)fat (g/day)carbohydrate (g/day)Ca (mg/day)P (mg/day)Mg (mg/day)Fe (mg/day)Zn (mg/day)Cu (mg/day)Mn (mg/day)Se (mg/day)

32.9± 10.7173.315.323.5±3.0

2266.8±502.276.6+17.172.3± 26.2

327.4185.3392.11214.0949.1+213.2284.01 67.3

19.0+5.611.613.0

1.710.95.6+1.5

45.61 13.8

28.91 10.4161.6+4.154.7+ 7.4

1474.41336.654.9+ 16.048.5+ 17.3

203.9153.3372.21 175.8709.41210.6210.8159.5

13.214.67.91 2.51.5+1.93.811.3

32.5+ 10.4

BMI=height/( weight/100)2

2.The quality control(QC) of BMD and measured results2.1 System Quality Assurance727 measurements of standard phantom were carried out during investigation from

June 1995 to April 1998, the CV(%) is less then 0.60. The accuracy and precision ofthe instrument were monitored regularly at least 1 per month, using spine phantom ,which is provided by Lunar company. The results of 42 measurements show thecoefficient variable(CV%) of BMD, bone mineral content(BMC) and area is 0.44, 0.50and 0.39. Measurement of 10 subjects was repeated in the same day, precision in vivowas calculated according to method reported by Larnach et alf", the result showscoefficient variabIe(CV%) of measurement repeatedly at spine lumbar, neck, Ward'striangle and troch are 0.75, 0.47, 1.14 and 0.72, respectively.

10 measurements of ESP were carried out on November 6, 1997, the results ofBMD and CV% at the secondary, third, forth and total spine lumbar(L2, L3, L4 andL2-4)are in table 3.

The same data analyzed by difference operator is different'21, so same operatoranalyzed all of data.

4 - 3

Table 3 the measurement results of ESP

L2L3L4L2-4

BMCX + s , g5.518±0.07010.686±0.18115.753±0.28332.022±0.277

CV%1.271.691.800.87

AreaX±s,cm2

9.818+0.10810.021±0.14010.132±0.15029.970±0.214

CV%1.101.401.480.72

BMD% ± s , g / cm2

0.563±0.0101.066+.0.0151.564±0.0091.069±0.003

CV%1.781.440.580.25

2.2 The results of BMD

Using DXA (Lunar, DPX-L, !.31 version), BMD of lumbar, hip, total body (Partly) was

measured. The result list in tables 4,5.

Table 4. The BMD result of 372 healthy male population by age and site (x± s, g / cm2)

age (year) Total body AP spine Neck Ward's Troch

15—19

20—24

25—29

30—34

35—39

40—44

45—50

1.146+.0.098

1.242+0.061

1.193±0.080

1.158±0.065

1.206±0.073

1.213±0.089

1.181±0.099

1.177±0.138

1.228±0.129

1.135±0.106

1 139±0.127

1.161+0.149

1.176±0.142

1.117±0.129

1.052±0.130

1.122+0.144

1.012±0.127

0.973±0.112

0.952+0.131

0.970+0.103

0.908±0.107

0.959+0.137

1.050±0.160

0.935±0.140

0.882+.0.144

0.851±0.160

0.847±0.132

0.765±0.124

0.881±0.127

0.943±0.122

0.853±0.120

0.834±0.115

0.827±0.120

0.841+0.112

0.808±0.101

Table 5. The BMD result of 409 healthy female population by age and site (x± s, g /cm2)

age (year) Total body AP spine Neck Ward's Troch

15—19

20—24

25—29

30—34

35—39

40—44

45—50

1.108±0.072

1.123+.0.072

1.152+.0.074

1.143±0.065

1.162±0.071

1.160±0.064

1.171±0076

1.109±0.

1.128±0.

1.170±0.

1.197±0.

1.183±0.

1.199±0.

1.180±0.

107 0.923±0.104

118 0.922±0.113

120 0.937±0.112

114 0.931±0.118

135 0.922±0.108

109 0.905±0.114

132 0.901±0.115

0.909±0.128

0.915±0.139

0.918±0.131

0.884±0.140

0.856±0.123

0.842±0.136

0.838±0.148

0.770±0.114

0.764±0.103

0.784±0.107

0.753±0.106

0.761+0.102

0.772+0.111

0.798±0.126

The results show that the age of forming peak bone mass is about 25 years at all sits in

male, about 30 years in female, and after that bone mass will keep stable. The PBM of

male is higher than female.

4 - 4

3.The multi-variable analysis of BMD with influencing factorsThe results of multivariate regressive analysis for BMD with physics and lifestyle are listed in

table 6,7.

Table 6- The multivariable regression results for male BMD: hysics .lifestyle characteristics

Variableage<25year

(ge (ye«r)

weight (Kg)sum of liquor1

exercise in teenager1

age>25year•ge (year)

beigbt (cm)

weight (Kg)symptom'

sum of cigarette'

Totalcoef0.0110.005

.

0.034

0.004

-0.000

bodyP

0.0020.000

0.027

0.000

0.006

APcoef.0.0090.006

-0.0010.079

——0.004

-0.119-0.000

spine

P0.0390.0000.0350.000

0.0000.0230.043

Neckcoef.0.0110.003—

0.069

-0.005—0.003——

P0.0180.037

0.001

0.000

0.000

Ward'scoef.0.0140.003—0.068

-0.009-0.005

0.004—*—

P0.0080.029

0.003

0.0000.0060.000

Trochcoef.0.0090.004—0.050

-0.003—0.004——

P0.0250.002

0.009

0.001

0.000

note: 1. sum of liquor = liquor (g/day) x history of liquor (year)

2. classical variable: compared with age-matched person, 0—low, 1—same, 2—high.

3. classical variable: symptom, 0—no, 1—yes.

4. sum of cigarette = cigarette (count/day) x history of smoking ( year )

Table 7. The multivariable regression results for female BMD: physics , lifestyle characteristics

Variableage<30year

age (year)beigbt (cm)

weight (Kg)

age at menarche(year)

average lacatioo1

exercise in teenager

age>30year•ge (year)

weight (Kg)age at menarcbe(year)

average lacation1

exercise in teenager2

TotalCoef0.006—0.005

-0.021—0.036

—0.003———

body

P0.003

0.0000.004

0.010

0.000

APCoef.0.0060.0040.009

-0.0170.0100.034

-0.0030.005

-0.0160.0030.028

spine

P0.0060.0300.0000.0140.0900.023

0.0270.0000.0010.0070.041

NeckCoef.,—0.0040.005———

-0.0040.004—.——

00

0.0

p

.028

.001

005.000

Ward1

Coef.

0.005—,——

-0.0050.005

——

0

0.0

s TrochP Coef. p

.002 0.007 0 000

——

.002 —.000 0.005 0.000

——

note: 1. average lacatin =lacation (month) /bear children

2. classical variable: compared with age-matched person, 0—low, 1—same, 2—high.

The results show that weight is correlated with BMD of both sexes at every site The

exercise levels of teen-ager also have interplay with BMD at most sites. The drinking and

smoking also have some influence in male; age of menarche and average time of lactation

have some influence to BMD in female. Certainly, age have importance influence.

The results of single regressive analysis for BMD show when weight increases 10Kg,

BMD of total body and lumbar spine will increase 0.0390mg and 0.0319mg in male,

0.0528mg and 0.0695mg in female.

4 " 5

The relationship between exercise and BMD was further analyzed according todifference exercise level of teen-ager. The levels of low, medium and high were separatedaccording to comparing with same teenager. The results show that the more of exerciselevel, the higher of BMD. The BMD of low and medium exercise level compare with highthere are significant difference in male before formation of PBM . The result preliminaryshows exercise level of teen-ager has important influence to forming of PBM.

There are 152 subjects having history of smoking in male, only 4 in female, so therelationship between smoking and BMD was further analyzed according to differencesmoking level in male. The outcome is similar with the result of multivariate analysis. TheBMD value is correlated reversed with smoking at total body and lumbar spine; there issignificant difference after modified with weight and age.

There are 106 subjects having history of drinking in male, only 12 in female, the malewas divided into two groups according to whether drinking or not. The BMD between nodrinking and drinking has no significantly difference, except lumbar spine; maybe theinfluence of drinking to BMD is small.

The average time of lactation = lactation (month)/bear children. Both lactation and bearchildren have no significantly influence, but average time of lactation have significantlyinfluence at lumbar spine during analyzing the interplay of multivariate on BMD. Forsubjects (>30 years old) the single-factor regression formula shows BMD will increase0.0035g/cm2 at lumber if average time of lactation increase 1 month.

4.Femur axis length (FAL) and Hip axis length (HAL)The FAL and HAL were measured using automatically analysis software supported

by Lunar Company. The results listed in table 8.

Table 8 The HAL and FAL results by age (x±s, mm)

Age(year)

15—1920—2425—2930—3435—3940—4445—50total

N22171513171817

119

MaleHAL

109.98±6.55108.89±5.57108.69+6.59111.25±5.86112.82±5.46108.63±6.50112.51 ±5.58110.36±6.13

N48485251495448

350

FAL95.82±3.7395.75±4.1696.05±5.7198.07±4.3797.58±4,9596.49+5.0497.88±4.8696.81±4.79

N27272728223329193

FemaleHAL

97.30+.4.1896.33±4.5497.94±4.8197.28±4.8096.92±6.3299.28+4.8798.04±4,9997.66±4.93

N50525266506351

384

FAL87.39±4.86.65±4.87.40±3.87.30±3.86.95±3.89.09±487.38±4.87.49+4.

5641794695114513

Because HAL was asked to measure after second CRM, so some persons no HALfor the restrict of scan region in 753 sample population. The correlation and single-factorregression between HAL and FAL was carried out in 312 subjects, who have both HALand FAL, HAL correlated significantly with FAL(r=0.88-0.81).

4 - 6

_ . The trace element analysis of iliac crest

1. preparation of bone specimensThe bone specimens of iliac crest were from orthopedic surgery on traffic accident

victims. Total 12 and 19 samples of cortical and trabecular age range of 25-51 (mean

age of 35 years old) in male were collected, and 1 of cortical of 74 year old in man

was collected. 3 and 4 samples of cortical and trabecular age ranged of 23-42 (mean

age of 36.3 and 34.3 years old) in female, and 3 and 4 of cortical and trabecular age

50-73 (mean age of 64.3 and 63.3 years old) in old woman were collected. According

to the report of IAEA Consultants' Meeting, the compact cortical and trabecular parts

of the bone were separated with an tool, then both cortical and trabecular parts of the

same bone were placed in 50ml 5% citric acid for about 30 seconds in an ultrasonic

shaker. Blood were removed by placing sample in polyethylene bag containing 50ml

5% glucose solution, shaking for about 60 minutes, remove the solution and repeat

the procedure six times. Fat and muscle were removed using 50ml ether and shaking

for 15 minutes, the procedure was repeated twice, the samples were dried at 80° for

three hours and stored in polyethylene bags in a refrigerator prior to trace element

analysis.

2. The results of INAAThe samples were radiated into reactor , the neutron flux is 5 x 1013n/cm2.s.

Because reactor have trouble, The part samples were radiated in the reactor of small

power of 7x 1011n/cm2.s neutron flux and the elements of long radiation haven't been

obtained in time. The reference material NIST SRM 1486 was used as QC. The

primal analytic of Neutron Activation Analysis results were listed in table 9.

Table 9 Average results of bone samples of iliac crest By NAAM

Mali Ca Mg Mn Zn AJ Fe Sr Na CO

1* COrtical 2.09X10' 2.13X1O3 3.24X10' 0.97X10* 1.25X10* 2.66X10"5 1.08X10' 4.75X10"3 2.52X10"

Trabecular i .98xio ! 2.07x10' 5.04xio7 1.13x10- i.37xios 6.97x10* i.05xio* 3.90x10' 6.45x10*

2* cortical 2.08x10'2.63xio3 5.22xio3

Female

1 * COrtical 2.24X10' 2.23X10J 1.66X10' 8.47X1O5 9.80X1O6 6.86X1O5 1.20X10"1 5.24X1O' 1.17X1O7

Trabecular 2.07x10' 2.01x10° 6.00x10' 6.22x10' 9.53X106 5.12x10* 1.10x10" 4.25x10' 0.67x10'

2* cortical 1.92x10-'2.19x10-' i.oexio* 9.8ixio4 5.53x10* 6.91x10* 8.osxio5 4.20X103 3.32x10"

Trabecular 1.66x10' i.87xioJ 3.76x10' 7.53x10* 6.36x10* 7.40xio5 5.77xio5 3.43xioJ 4.50x10*

* 1- group of normal control, 2- group of old people

4 - 7

3

1. We will continuously collect bone specimen of iliac crest in normal female and oldhealth subjects, then the results of two groups of normal contract and old man andwoman will be compared.

2 The blood samples of 150 subjects had been collected and stored in -70°refrigerator for gene analysis. More blood sample will be continuously collected andvitamin receptor alleles and other relevant genetic markers will be studied.

Reference1. Larch TA, Boyd J, Smart RC, et al. Reproducibility of lateral spine scans using

Dual Energy X-ray Absorptiometry. Calcif Tissue Int, 1992, 51:255-258.2. Qin Linlin, Chen Jinbiao, et al. The research on measuring procedure of dual-

energy X-ray absorptiometry in determining the bone mineral density. Atomicenergy science and technology. 1996, 30(3). 230-235.

4 - 8

XA0054923

WORKING PAPER

1. Contract Number: 8192/R2

2. Title of Project: Studies of Osteoporosis in Shanghai, China,Using Isotope Related Techniques

Part 1: Trace element research in OsteoporosisPart 2: Bone mineral density measurement

Part of Coordinated Programme: Comparative InternationalStudies of Osteoporosis Using Isotope Techniques

3. Research Institute: Shanghai Institute of Nuclear Research,Academia Sinica

4. Chief Scientific Investigator: Zhang Yongpeng

5. Starting Date of Current Contract: Sep. 1997

5-1

/\oo

Determination of inorganic elements in iliac crests of rabbitsbyNAA

Zhang Yuanxun Zhang Yongpeng Li deyi Zhuang guisun(Shanghai Institute of Nuclear Research, The Chinese Academy of Sciences, Shanghai 201800)

Wang Zhixing Xia Jun(Department of Orthopedics, Ruijin Hospital, Shanghai Second Medicine University, Shanghai

Institute ofTraumatology and Orthopedics, Shanghai 200025)

AbstractThis paper deals with die osteoporosis model of New Zealand rabbits in order to

study the functions of inorganic elements for women patients with osteoporosis. Thethirty cases of iliac crest are collected and determined using Neutron ActivationAnalysis (NAA) technique. The results show mat the Fluorine concentrations inexperimental group are higher man those in control group and the Magnesiumconcentrations are lower significantly (P<0.001), but the concentrations of K, Na, Ca,Ba, Mn, Sr are not remarkable difference. Combined with the serum biochemicalmarkers of bone formation and resorption, the physiological functions of some metalelements in bone are also discussed.

Keywords: Osteoporosis, Inorganic elements, Neutron Activation Analysis

1. Introduction

The human skeleton is composed of organic matter, inorganic matterand water. In addition to Ca, P, Na and K classified as macroelements,inorganic matter also includes Mg, Mn, Fe, Cr, Ni, Cu, Zn, Se and Sr,which are essential elements of human body. These elements areenveloped in bone and combined with other ions, therefore, trace elementsare closely related to the bone structure^.

In recent years elderly peoples are increase rapidly at a three percentfor every year in China. Up to 2000 years, the old peoples over sixty yearswill break through four hundred million. Osteoporosis is a commondisease occurring among elderly peoples special for menopause women. Ithas a high incidence and easily causes other secondary affection andsymptoms such as fracture, ostealgia. It is seriously harmful to elderlypeople's health. With the coming of old aged society, the prevention andtreatment of common diseases in old age should be paid more attention.

The relationship between Ca, P metabolism and osteoporosis is wellknown to us, but the relationship between other inorganic elements in the

5-2

bone and osteoporosis is reported less. In order to study the relationshipbetween trace elements in the bone and osteoporosis, we made a rabbitmodel with osteoporosis and observed the change of trace elements in theiliac crest of rabbit using neutron activation analysis (NAA). Combinedwith the determination of biochemical indicators in serum, we can furtherstudy the change pattern between inorganic elements and biochemicalparameters, also provide scientific basis for osteoporosis prevention andtherapy.

2. Material and method

2.1 Experimental animalNew Zealand rabbits, female, body weight about 3 kg, in total of 30

rabbits are provided by experimental animal center of Songlian, ShanghaiSongjiang county.

After feeding seven months, both control group and experimentalgroup are divided at random. In control group, 21 samples of iliac crest arecollected and dried for keeping. In experimental group, both ovaries of 9rabbits are taken off by operation and these rabbits are fed for anothereight months at normal conditions. Measured bone mineral density ofmodel rabbits, we can confirm that these rabbits are suffering fromosteoporosis. Then killed them and collected iliac crests for keeping untilpreparation.

2.2 Sample preparationTo keep contamination and the loss of elements from the samples to

a minimum, instruments used for the collection of specimens and theirhanding prior to the analysis were acid-cleaned and washed with highpurity water.

First, we weighed each bone sample before put them into crucibles.Then charred these samples in oven at 400°C for 4 hours. Later pulverizedcharbone to fine powder using a glass stick. Each powdered sample(lOOmg) was weighed and put into polythene bag, which was sealed in asmall special bottle for NAA.

2.3 Experimental determinationThe determination was performed using Slowpoke reactor and NAA

set up in Shanghai Analysis Survey Center*2*. Two groups of samples weresuccessively placed in a pneumatic transfer rabbit system and irradiatedfor 60 sec at a thermal neutron flux of 5xl0 n n/cm2 sec in the reactor. Theradiation activities were measured by using an Ortec Ge(Li) detector

5 -3

having a resolution of 2.0 Kev of ^Co-ray. The signals from detector werecoupled to a 8192 multichannel pulse height analyzer equipped with PCcomputer system. Some results such as net peak area, concentration anderror were printed out automatically based on an NAA computer program.

Analytical quality control is an essential part of the analyticalprocedure. The contents of all elements were quantified by using preparedchemical standard method and the procedure was checked by analyzingstandard reference material Bone Meal-1486 provided by NationalInstitute of Standard Technique (NIST), USA.

3. Results

3.1 The ratio of dry and wetThe ratios of dry and wet, carbide and dry, carbide and wet for iliac

crests of rabbits are listed in the table 1. We can see that the ratios incontrol group are 7 % -14 % higher than those in the experimental group.It means that bone mineral substance is lost obviously and spongysubstance of bones is increased.

Table 1 The ratio of dry and wet in iliac crest samples

Wet wcight(g)Dry weight(g)Carbide weight(g)Dry /WetCarbide / DryCarbide / Wet

Control group( n=21 )

2.01 ± 0.291.46 ±0.220.76 ± 0.160.73 ± 0.070.52 ± 0.040.38 ±0.04

Experimental group( n=9 )

2.51 ±0.171.71 ±0.210.82 ±0.150.68 ± 0.070.48 ± 0.050.33 ± 0.05

3.2 Concentration of inorganic elementsTable 2 shows the results of inorganic elements in rabbit iliac crest

by NAA. Based on the T test, it is demonstrated that the concentration of Felement in the experimental group is higher than that in the control group( PO.001) and the concentration of Mg in the experimental group is lowerobviously, compared with the control group ( PO.001). While theconcentrations of Na, Ba, Mn, Sr and K are not significantly different. Thedifferences of elemental concentrations are mainly from the results ofchanges due to osteoporosis.

5 - 4

Table 2 The results of inorganic elements in rabbit iliac crest by N AA (pg/g)

Elempnt

CaFMgNaBaMnSrK

X±a

331790 ±33500 (21)991± 3875274 ± 6467594 ± 815

108 ± 591.026 ±0.18215 ±49

1763 ±500

(21)(21)(21)(7)(21)(20)(21)

X±a

341280 ± 22580(9)2075 ±267 (9)4438 ±194 (9)8106 ±545 (9)

111+38 (2)1.060 ±0.16 (9)

205 ± 61 (9)2012 ± 610 (9)

0.77357.6118 <0.0013.7720 O.0011.87500.15080.48320.47821.4772

3.3 Determination of biochemical parametersBesides inorganic and trace elements, we also collected the serum

samples from rabbits before and after operation. Three kinds ofbiochemical indicator ( acid phosphatase, alkaline phosphatase and bonespecific alkaline phosphatase ) in serum are measured and results aresummarized in table 3. We can see that the activities of three serumphosphatase in control group are higher than those in the experimentalgroup. The differences are very remarkable ( PO.OOl ). The reduce rate ofactivity in alkaline phosphatase is obviously higher than that in bonespecific alkaline phosphatase(3).

Table 3 Three serum phosphatase activities in New Zealand rabbitsbefore and after operation U/L

Before operation

Acid PhosphataseAlkaline PhosphataseBone SpecificAlkaline Phosphatase

19.06 ± 5.4620.06 ± 8.2014.04 ± 1.67

8 months afteroperation

11.04 ±4.1014.60 ± 4.64

4.44 ±2.10

Reduce rate%

42.0827.2268.38

Significance

<0.001<0.001O.OOl

4. discussions

There have only been a few reports in the literature about traceelements in bone. A method in common use is Atomic Absorption

5 -5

Spectroscopy (AAS). Spark Source Mass Spectroscopy has beendeveloped to improve sensitivity. More than 30 elements can bedetermined, and both accuracy precision can be as good as ±10%.Secondary Ion Mass Spectrometry (SIMS) can measure the elementalconcentrations at the levels of normal and micro quantities with excellentresolution, but can not determine the elements simultaneously^. NeutronActivation Analysis (NAA) is well investigated as a nuclear analyticaltechnique of high sensitivity. The method measures low concentrations ofseveral elements simultaneously in biological tissues. This is leading to arapidly growing interest in trace element content and distribution inhuman organ tissues.

Magnesium is present in bone in macromineral quantities,comprising 0.5-1 % of bone ash. Magnesium influences both matrix andmineral metabolism in bone, and magnesium depletion causes cessation ofbone growth, decreased osteoblastic and osteoclastic activity, osteopenia,and bone fragility. Magnesium prevents bone fragility by destabilizinghydroxyapatite crystals, and acts synergistically with ATP to stabilizeamorphous calcium phosphate and prevent hydroxyapatite formation(5).Sojka's study shows that a group of menopausal women were givenmagnesium hydroxide to assess the effects of magnesium on bone density.At the end of the 2-year study, magnesium therapy appears to haveprevented fractures and resulted in a significant increase in bone density^.The conclusion is that magnesium therapy in postmenopausal womenwarrants further study certainly appears valid.

Our experiment result shows the fluorine concentration in modelgroup is more than that in control group significantly, and the overfluorine have obvious effect on the metabolism of cartilage organize andblood biochemistry. Fluorine is human nutrition's necessary microelementand human body tissue's composition. It have particular affinity to bonetissue and affect bone growth. Some research reported(7) that over fluorinecause parathyroid growled, and PTHS ( parathyroid hormone secretion )increased, but it's mechanism isn't clear now. It is necessary to furtherstudy on fluoride's using.

The determination of serum phosphatase activities in New Zealandrabbits before and after operation is very widespread and gives valuableinformation about the bone formation. The rate of bone loss aftermenopause has been shown to be significantly correlated with the rate ofbone turnover measured by the markers. This correlation becomes apotentially useful screening system in the risk assessment ofpostmenopausal osteoporosis. During anti-resorptive therapies, the valuesof the markers are reflecting the actual decrease in bone degradation.

5 - 6

Thereby, the monitoring of biochemical markers during therapy is a usefultool in the management of osteoporosis in order to increase the complianceof the women and assure to a safe and effective therapy.

This work is part of Scientific Foundation by Chinese Academy of sciences, also a partof Co-ordinated Research Programme supported by IAEA.

References

1 Ahlgren L, Mattsson S. Phys Med Biol, 1979; 24: 1362 Cheng YuandL, Zhuang Guisheng, Wang Yinsong et al. The Journal of

Trace Elements in Experimental Medicine, 1988; 1: 193 Wang Zhixing, Xia Jun, Yang Qinming. Shanghai Experimental

Animal Science, 1996; 16(2): 794 Zhang Yuanxun, Zhang Yongping, Tong Yongpeng et al. J. Radioanal.

NucLChem., 1996; 212: 3415 Blumenthal NC, Berts F, Posner AS. Calcif Tissue Res 1977; 23: 2456 Sojka J.E. Nutrition Reviews, 1995; 53(3): 717 Bratter P. Trace element analytical chemistry in medicine and biology,

Volume 2, New York; New York Academic, 1983; 5558 John H, Beattie K, Avenell A. Nutrition Research Reviews, 1992; 5:

1679 Yasuhivo Suzuki. Tohoku J Exp Med, 1979; 129: 327

5 - 7

Part 2: Bone Mineral Density Measurement

2.1 Measurement of the European Spine Phantom for quality control

IntroductionFor the comparison of bone mineral density across the CRP centres

the European Spine Phantom (ESP) provided by Dr. Eugene McCloskeywas transited from Beijing to Shanghai during Feb. 1998.

Measurement of the ESPESP measurement is performed in Shanghai 6th people's hospital

using QDR-2000 Dual Energy X-ray Absorptiometry (DEXA) importedfrom USA Hologic Co. The standard daily calibration of the scanner hasbeen carried out using a spine phantom provided by manufacturer. Thesame scanning technician operates the machine in an identical way topatients during the CRP.

Fig 1 is a long-term stability of QDR during three years. Themeasurement results obtained during more than thirty months show thatthe rate of change is ±0.02%, up to a high precision. The table 1 shows thescan parameters of the QDR-2000 for this ESP. In Fig 2-3, we can seethree detail print out reports on the scanner. Table 2 is a brief report,which contains area, BMC and BMD for each of the three 'Vertebrae".

According to the IAEA arrangements, the ESP measurement wasperformed and then delivered to Dr. Bose in Singapore.

2.2 Bone density measurement

The bone density in selected residents is measured in Shanghai 6thpeople's hospital using QDR-2000 DEXA The positions of bone densitymeasurement are: (a) in the lumbar spin© region ( L1-L4, LI, L2, L3, L4 )and (b) at the femoral neck ( neck, troch, inter, total, ward's ). A spineprovided by Hologic Inc is used to control quality of the measurements. Upto now, the BMD measurements of total 350 subjects have beenperformed. All results are shown in table 3.

5 - 8

/1O6

Table 2. The results of European Spine Phantom measurement

Region

LlL2L3

TOTAL

LlL2L3

TOTAL

LlL2L3

TOTAL

Est.Area(cm2)

8.4110.2711.3830.06

8.2610.4211.0529.74

8.4610.4511.3230.24

EstBMC(grams)

4.549.95

15.4329.92

4.5110.0415.3229.87

4.6110.2115.7130.53

BMD(gms/cm2)

0.5390.9691.3560.995

0.5460.9641.3861.005

0.5450.9771.3871.010

5 - 9

Table 3. The results of BMD measurement in 360 subjects across the age range 16-60 (M±SD)

Group

M-1

M-2M-3M-4M-6

M-6M-7

F-lF-2F-3

F-4F-6

F-6F-7

L1-L4

0.960

0.941

0.9740.972

0.9200.939

0.962

0.9250.9330.974

1.0201.014

0.9740.911

LI

0.9010.8690.9410.9280. 9180.8880.910

0.8400.8480.8790.9080.9090.8840.820

L2

0.903

0.928

0.968

0.972

0.928

0.948

0.978

0.911

0.920

0.970

1.020

1.001

0.961

0.893

L3

0.979

0.971

0.9770.9900.9480.969

0.970

0.9670.9701.0011.069

1.0361.0230.944

L4

0.968

0.960

0.991

0.9790.9380.968

0.990

0.9600.9600.992

1.0871.069

1.0400.939

Neck

0.9100.8760.8880.8360.7990.7940.796

0.8190.7880.7500.8010.8680.8100.733

Troch

0.7480.7100.8910.6800.6430.6400.690

0.6820.6400.6300.6280.6680.6280.689

Inter

1.113

1.090

1.079

1.063

1.064

1.037

1.080

1.030

1.012

0.991

0.997

1.077

1.011

0.969

Total

0.9860.939

0.933

0.9110.8910.882

0.930

0.9000.8480.8420.8660.910

0. 8680.820

Ward' 6

0.830

0.788

0.808

0.7280.6640.660

0.660

0.7600.7690.716

0.7110.7800.710

0.696

* : M (Male) -groups , F (Female) -groups.

£1.871e11.861

£ 1.641

£1.831

a,Ml.021u

£3

"' l .BBl

B.991

Fiji.I I I 1 I I I I I I I I I I I I I I I | 1

rate of change = -B.00X per year (•/"- 0 . 0 2 x )

1 I I I I I i I i ) i ) i ) ) ) i i i i r i i i i i i i i i i t i i i i |F M A H J J A S 0 N D J F K A H J J A S O N D J F ti A M J J A S 0 N D J1 9 9 5 1 9 9 6 1 9 9 ?

Reference Ualuesspine

neanS.D.

1imits

phantom= 1.8388= B.BB56= il .5x

81541gns''cn2gns/cn2

jf mean

Plotn =

mean =S.D. =

cu =

Statistics6381.8345 gms/cn2B. 0843 gns-^cnZB.42>:

SHANGHAI &th PEOPLE'SHOSPITAL (2386)

HOLOGIC

E

\ 1.861

il.851Q

B1.841

= 1.031

M1.021

ii.enE3•^l .BBl

B.991

1 1 1 1 i i 1 1

_

-

-

rate of changel l 1 l 1 1 1 lF M A M J J A S

1

1f,

1 1 1 i 1 1

-8.00X peri i i I iN D J F n

1 1 1 1 1 1 1 1 1 1 1 I 1 1 t 1 1 1 1 I I I

-

' • _

-

-

-

year (••- 8.82x)I I I 1 I I i I 1 1 1 I I I I 1 I I 1 I 1M ,T J A S 0 M D J F M A I 1 J J A S 0 M D J

Reference Ualuesspine

nean :phantom 111541

= 1.83B8 sms/cn2S.D. = B.B8S6 gns^cn2

limits = i l . 5 x of mean

Plotn =

nean =S.D. =

cu =

Statistics638 rn1.8345B.BB43B.4Zx

= 23gms/'cn23^5/01^2

SHAHGHAI 6 th PEOPLE'SHOSPITAL (2386)

HOLOGIC

5-11

L7*SHRNGHRI 6th PEOPLE'S HDSPITRL

k = 1 211 dB = 112 9(1 8BBH) 6.424

mm

•Fob 12 13.52 1998 C113 x 961Ho logic QDR-2008 (S'N 2386)Array Spine Medium V4-57A:i

YB212981E Thu Fob 12 13:48 1998Name: European Spine PhantomComment: for normal valueI.D.: Sex: FS.S.W- - - Ethnic:ZIPCode: Height: cmScan Code.' Height: kgBlrthDato: y / Ago:Physician: ZHOUImage not for diagnostic use

TOTAL BMD CU FOR LI - L4 1.8*

C.F. B.993 8.988 1.888

Region Est.Area Est.BMC BMD(cm2> (grams) (gms-'cmZ)

LI 8.41 4.54 8.539L2 10.27 9.95 8.969L3 11.38 15.43 1.356

TOTAL 30.B6 29.92 0.995

HQLOGIC

SHRNGHRI 6th PEOPLE'S H05PITRL

1 212 dB = 112 7(1 8BBH) 6 416

•Feb 12 13:57 1998 C113 x 89]Ho logic QDR-2880 CS/N 2386)Array Spine Medium U4.57A:!

V021Z981F Thu Feb 12 13:54 1998Name: European Spine PhantomComment: for normal valueI.D.: Sex: FS.S.S: - - Ethnic: ;ZIPCode-. Height: enScan Code: Weight: kgBirthDate: / / Age:Physician: ZHOUImage not for diagnostic use

TOTAL BMD CU FOR LI - L4 1.8*

C.F. 8.993 8.988 1.800

Region Est.Aroa Est.BHC BMD(cm2) (grams) (gms/cm2)

LI 8.26 4.51 0.546L2 IB.42 18.04 0.964L3 11.05 15.32 1.386

TOTAL 29.74 29.87 1.B85

HOLDGIC

5-12

Fij 3.SHRNGHfll 6 t h PEOPLE'S HOSPITRL

= 1.Z14 dB = 112.3<1.BBBH) 6.416

•Feb 1Z 14:81 1998 [113 x 89]Ho logic QDF-ZB8B CS/N Z386)Array Spine Medium U4.57A:l

VBZ1Z9BZB Thu Feb 12 13:58 1998Namo: European Splno PlianlonComment: for normal valueI.D.: Sex: FS.S.8: - - Ethnic:ZIPCode: Height: enScan Code: Uolght: kgBirthDate: / / Age:Physician: ZHOUInage not Tor diagnostic use

TOTAL BHD CU FOR LI - L4 l.Bz

C.F. 0.993 8.980 I.8BB

Region Est.Area Est.BHC BHD(cnZ) (grans) Cg

LI 8.46 4.61 8.545L2 18.45 IB.21 8.977L3 11.32 15.71 1.387

TOTAL 38.24 38.53 1.81B

HOLOGIC

Hologic QDR 2000 - 7.IOCPatient: European Spine PhantomScan type: A Lumbar Spine Scan # None

Room for 4 76 scansThu, Feb 12, 1998 13 :54

-Select Scan Parameters-6.50 X 0.05 Coll.208 lines249 samples/line

Length Of Scan (cm) 10

Width Of Scan (cm) 10.772

Line Spacing (cm) 0.1003

Point Resolution (cm) 0.0862

** DEVICE READY **Press F10 to begin X-RAY scan

140/702.0 mA

kVpavg.

177 seconds50 Hz

5-13

XA0054924

Contract No: 8193/R1

STUDIES OF OSTEOPOROSIS IN CROATIA USING ISOTOPERELATED TECHNIQUES

Institute for Medical Research and Occupational Health

Principal investigator: Darinka Dekanic Ozegovid

The counterpart was not present at the meeting (progress report submitted subsequently)

Research period covered by report: October 1997 - July 1998

6-1

DESCRIPTION OF THE RESEARCH

Pilot study was held to evaluate bone stiffness in children andadolescents in two districts of Croatia which, according to previousknowledge, differ in calcium intake. The relationship between habitual caldumintake and bone stiffness was determined. It was difficult and expensive toorganise a transport of Istrian children to Zagreb in order to perform x-raysabsorptiometry. Therefore, bone stiffness was determined with a portableultrasound system.

Bone mineral density (BMD) measurements were continued in subjectsof both sex, across the age range 20-50 years from the central district of thecity of Zagreb. Subjects were randomly selected from the demographic list.

METHODS

Bone stiffness was determined on calcaneus of the undomlnant foot,using portabile sonometer "Sahara", which was borrowed from the HoJoglccompany. The measurements are expressed as Quantitative Ultrasound index(QUI) and also as an estimate of the bone mineral density (BMD; g/cm2).The calibration of the sonometer was performed daily.

All children and adolescents, were questioned about diet hfstory, by thedietitian. The energy value of the food (10) and the daily intake of proteins,fats, carbohydrates (g) and calcium (mg) were calculated.

In order to establish a national reference database, bone mineraldensity (BMD, g/cm2) of the lumbar spine (L2-L4) and of the left femoral neckof randomly choosen subjects, aged 20-49 years, was measured by dual x-rayabsorptiometry (Lunar DPX).

6-2

RESULTS

Bone mineral density (BMD), body mass index (BMI), energy value ofthe food and daily intake of proteins, fats, carbohydrates and calcium in maleand female children and adolescents from Zagreb and Pazin are shown onTable 1 and Table 2. Comparing children from the two regions there weresignificante differences in energy value and daily nutritive intake. Small boysfrom Pazin had significantly lower intake of proteins (p<0.0001), fats(p<0.05), carbohydrates and calcium (p<0.0001) comparing to children ofthe same age and sex from Zagreb. Their BMD was also lower but notsignificantly.Small girls from Pazin also had significantly lower intake of proteins, fats(p<0.0001), carbohidrates (p<0.05) and calcium (p<0.001) but their BMDwas not much lower comparing to girls from Zagreb. Adolescent boys fromZagreb had significantly higher calcium intake (p<0.01) and their BMD washigher too, although not significantly. Although adolescent girls from Zagrebhad higher calcium intake than gtrls from Pazin, their bone mineral densityalso did not differ significantly.

Bone mineral density in male and female subjects in 5 year agegroups Is shown on Table 3. The mean BMD values did not differ significantlybetween five-year age groups in both sexes. In women there was anapparent decline In BMD after 40 years. Conclusions are limited in menbecause of the relatively small number of subjects in each age group.

CONCLUSIONS

There is a tendency of BMD fall after the age of menopause in women.I t appears that peak bone mass in women is obtained in the mid- 20s and Inmen at 30 years of age.The general conclusion about the trends observed inthe population will be given after collection of all data.

Calcium intake has an influence on peak bone mass, which becomesevident at the adolescent age. Children who had higher calcium intake had ahigher bone mass, although that difference was not statistically significantThat finding was more apparent in males. The calcium intake in Zagrebchildren was higher, so was the energy intake, but the mean body mass Indexwas identical, which indicates higher physical activity in children from Zagreb.Besides the calcium intake, the role of energy value of the food and habitualphysical activities seem to be important in bone maturation.

6-3

Table 1 . Bone mineral density (BMD), body mass Index (BMI), energy valueof the food and daily intake of proteins, fats, carbohydrates and calcium Inmale (A) and female (B) children and adolescents from Zagreb

A. Males

NAge (years)BMI (kq/rrr)BMD (g/crrr)Energy value of the food (kJ)Proteins (g)Fat (q)Carbohydrates (g)Calcium (mg)

Males519.4 (8-10)17.9 (14,0-29.8)0.512 (0.377-0.710)9844.67 + 2022.1786.00 + 24.1983.75 + 23.55319.35+101.171219.15 + 517.62

3515.8 (15-18)21.6 (16.6-31.4)0.556 (0.389-0.983)11139.27 + 3591.87106.01 + 44.88103.91 + 39.14330.26 ± 102.651308.79 ± 522.05

B. Females

NAge (years)MenarcheBMI (kq/nV*)BMD (o/cm2)Energy value of the food (kJ)Proteins (q)Fat (g)Carbohydrates (g)Calcium (mg)

Females489.3 (8-10)017.3 (13,7-24.3)0.496 (0.336-0.686)8945.09 ± 1994.5780.26 + 23.9282.45 + 33.08235.97 + 97.911006.99 ± 426.48

7115.7 (15-17)12.5 (10-16)20.8 (14.9-30.1)0.554 (0.296-0.896)11100.17 ± 3231.8798.02 + 29.56108.94 + 49.14421.86 + 132.251408.74 ±618.25

6 - 4

Table 2. Bone mineral density (BMD), body mass index (BMI), energy valueof tiie food and daily intake of proteins, fats, carbohydrates and caldum inmale (A) and female (B) children and adolescents from Pazin

A. Males

NAge (years)BMI (kg/m2)BMD (g/cm2)Energy value of the food (kJ)Proteins (g)Fat (g)Carbohydrates (g)Calcium (mq)

Males498.9 (8-10)17.2 (14,5-21.2)0.518 (0.392-0.642)7483 + 1919.8368.39 ± 15.5472.22 + 24.99216.58 + 55.31829.73 ± 274.53

5116.3 (15-18)20.6 (17.2-32.6)0.532 (0.343-0.768)11962.75 + 4275.8598.18 + 34.69122.62 + 103.10395.64 + 223.541015.44 + 396.46

B. Females

NAge (years)MenarcheBMI (kg/m2)BMD (q/cm2)Energy value of the food (k3)Proteins (g)Fat (g)Carbohydrates (g)Calcium (mg)

Females488.7 (7-10)017.1 (13.3-25.8)0.501 (0.395-0.689)6523.28 + 1324.5857.73 + 13.7960.01 ± 14.52196.85 + 45.91723.59 ± 265.43

5716.1 (15-17)12.6 (10-15)22.2 (15.8-28.3)0.547 (0.363-0.942)8983.45 + 3198.1871.28 + 32.3578.16 + 30.29283.44 + 106.69870.56 ± 355.98

6-5

/14&

XA0054925

PROGRESS REPORT AND PLANS FOR THE COMPLETION

a (i) CRP, iAEA Contract Number: 8194/R1(ii) Project Title:

STUDIES OF OSTEOPOROSIS WITHIN THE DEBRECEN REGIONALOSTEOPOROSIS PROGRAM (DROP) IN HUNGARY USING ISOTOPE RELATEDTECHNIQUES

Part of IAEA Coordinated Research Programme:

COMPARATIVE INTERNATIONAL STUDIES OF OSTEOPOROSIS USING ISOTOPETECHNIQUES

(iii) Institute where research is being carried out

DEPARTMENT OF OBSTETRICS AND GYNECOLOGY,UNIVERSITY MEDICAL SCHOOL OF DEBRECENH-4012 DEBRECEN, PO Box 37HUNGARY

(iv) Chief Scientific Investigator: Dr. Adam Balogh

v) Time period covered: 1 December 1994 - 31 July 1998 (not all data takenduring the period have been included)

Starting date of current contract: 1 December 1994Planned duration: 5 years

7-1

A A'}

b. Description of research carried out

Aim of the research. The aim of the research carried out within the above CRP wasduring a four-year period devoted to measuring bone density in the lumbar spine andthe femoral neck in 15-50 year old normal subjects of both sexes, and studying thebone composition of selected bone biopsy and autopsy specimens. These activitieswere planned in conformity with the guidelines issued for this coordinated researchprogramme.

Agreements concerning major parts of the research. Major cornerstones of the researchprogramme were agreed upon during the first meeting of the investigators in Vienna,12-15 December 1994 and updated in San Diego, 7-10 October, 1996. An importantrecurring issue was in these meetings, how the target population will be defined andwhich investigations were likely to be performed during the subsequent years. It hasalso been agreed that bone biopsies and subsequent histomorphometry and traceelement analysis would be delayed within the study time frame until agreement on theuniform procedures will be achieved. The issue of questionnaires concerning nutritionalstatus and habits as well as daily physical activities and wellbeing of the subjects alsoleft uncertainties and neeeded final wording. The dual X-ray absorption (DXA) bonedensitometry as one of the most prominent part of the study has been agreed upon,open the agreement concerning the preferred type of densitometer, and the cross-calibration. Collection of deep frozen serum samples have been agreed upon, the siteand type of assays remained to be determined.

During the time passed since initiation of the programme we could finally join theEuropean Prospective Osteoporosis Study (EPOS) although some aspects of theparticipation are still unclear. Starting it in our Center would be beneficial for the IAEAproject, since there are close similarities between the two studies, especially in the useof bone densitometry. Major differences are however in the different target populations:the EPOS aims examining older age groups (40-80), the overlap being 10 years only.Of course, the IAEA CRP includes more explicitly isotope related methods, e.g. thedetection and quantification of trace elements.

Due to the access to a standard population in our centre, in our study we haveconcentrated on the bone densitometry and the nutritional calcium questionnaire, aswell as the lifestyle and physical activity questionnaire validation.

Recruitment of subjects for the study. The referendum and residence (street) registerswere not possible to access. Instead, we could use the Health Service Register whichfully covers the population of Debrecen town, total population of 230,000. During thepast 3 and half year 376 persons were examined by DXA (LUNAR DPX-L), a total of205 women and 171 men (AP spine and proximal femur). The data are in file and readyfor final analysis. We have called from the all age groups. Originally we aimed to have50 subjects from each of the 7 age groups (5 year strata) but failed to complete it.

/M87-2

The WHO questionnaire was used for lifestyle data collection an we used a separateform for the assessment of calcium intake. Both have been adapted and validated inHungarian population. The Quality Assessment of the DXA was made using daily QAroutine as it is written in the Operating Manual. Beyond this we have performed monthlyAluminium spine phantom measurements, as well as weekly Hologic anatomic spinephantom assays.

c. Results

Bone densitometry

For the 205 women BMD results of the AP spine (L1-L4) and the proximal femur were asfollows:AP spine L,-L4: 1.139 +/- 0.16 (S.D.) g/cm2, Femoral neck: 0.925 +/- 0.089 g/cm2 (S.D.)

For the 171 men corresponding values were 1.214 +/- 0.15, and 0.985 +/- 0.103 g/cmz

(S.D.)

Breakdown of the results to 5-year age strata is enclosed separately (page 12).

The WHO Osteoporosis Project Questionnaire has been used in 110 persons, likewisea separate Calcium intake questionnaire. These questionnaires became necessary tovalidate in our local population in order to ensure identical interpretation of thequestions and possible answers since until now there were no such internationallyaccepted forms to use in Hungarian osteoporosis studies. The data however are onlyon the questionnaire sheets, need to be fed into the computer for further analysis.

d. ConclusionsMean DXA bone densitometry results appear to be slightly lower than a typicalEuropean standard e.g. used by Lunar as population reference database originatingfrom Germany, however it is unlikely that the difference will be statistically significant.Further number of measurements will make it possible to establish our own regionaldatabase for comparison. This would be especially usefor for the population stratusbelow 20 years of age. Additionally, the Questionnaires fulfil similar task giving achance to take regional Hungarian data on the variables included in the questionnaires.

At the time of this report there is a debth of our centre concerning some components ofthe research plan. It seems however realistic to catching up by the end of the 5 yearresearch plan with the most important components of the original proposal. Some of theimbalance of the detailed work can be rectified only during the planned extra yearbeyond the completion of the 4 calendar year calculated from the start-up date of 1December 1994. This is proposed according to the original study plan.

7-3

/IAH

The work plan for the rest of the year 1998, and for a proposed extension until 1December 1999 (without extra fininacial support from IAEA) contains further extensionof our population based study of male and female subjects of Debrecen town andsurrounding region (county) aged 15-50 years, 7 age strata, further 100 persons,continuing the list of the 376 persons sofar participating in this study. As an extensionof the densitometry we aim to measure retrospectively the hip axis length and/orfemoral axis length in the study persons. Furthermore, continuing collection of bloodsamples will be done during this period to assess bone metabolic markers of thisnormal population.

Methods of performing the above procedures will be: DXA bone densitometry usingLUNAR DPX-L densitometer. The same instrument will serve as tool for hip axis lengthmeasurements. The software of the machine allows this measurement in the ManualAnalysis routine. We may consider further options such as to do spine morphometryusing our new Hologic QDR 4500, although comparison with the Lunar results will becomplicated. A possible way of solving this problem is to select a group of thepopulation sample and do densitometry + morphometry on this sample. Bodycomposition assessment could be part of the study. Finally, the calcaneal ultrasoundbone assessment is another option. Collection of blood samples from persons notreceving bone-affecting treatment in order to assess osteocalcin and/or bone specificalkaline phosphatase blood levels and in a smaller population sample urinary levels ofmetabolites indicating bone resorption (deoxy-pyridinoline crosslinks, or crosslaps)could be desirable addition to the densitometry study. The delayed bone compositionanalysis from traumatology samples done by PIXE method would be a high preferencepart of the last year.

List of referencesa. Balogh A, Bettembuk P, Margitai B, Bacsko G: The role of bone densitometry in theearly detection of osteopoporosis within the Debrecen Regional Osteoporosis Program(DROP) in Hungary. Osteoporosis International 1996, 6 Suppl. 1,129 (abstr.)b.Balogh A., Bettembuk P.: HRT and prevention of osteoporosis: Risk assessment andpractical advice Eur. J. Obstet. Gynecol. Reprod. Med. 1997, 71, 189-191c. Balogh A, Bettembuk P, Jakab A, Bacsko Gy: Hormone replacement therapy (HRT)

for postmenopausal osteoporosis (abstract) Acta Obstet Gynec Scand 1997, 76 Suppl167, 61d. Bettembuk P, Balogh A: Quantitative imaging ultrasound of the os calcis. Evaluationof a new method. J Bone Mineral Res 1997, 12 (Suppl. 1), S387

AIP7-4

THE DEBRECEN REGIONAL OSTEOPOROSISPROGRAM (DROP)

LIFETIME TODAY:

LOCATION:COUNTY

POPULATION SIZE:

COMBINED)POPULATION TYPE:

ACTIVITIES:

OVER 6 YEARS (SINCE APRIL 1992)

DEBRECEN TOWN AND HAJDU-BIHAR

HUNGARY230,000 (DEBRECEN)550,000 (DEBRECEN + COUNTY)

URBAN & RURAL, EQUAL NUMBERS

STUDYING BMD IN PRIMARY ANDSECONDARY OSTEOPOROSISEARLY DIAGNOSISMALE OSTEOPOROSISEDUCATION (UNDER- ANDPOSTGRADUATE)DRUG RESEARCH

MULTIDISCIPLINARYHEALTH CARE FUNCTIONHIGHER REFERRAL CENTEREPIDEMIOLOGY & CLINICAL RESEARCHDRUG STUDIES

FUNCTIONAL UNITS (DROP): OUTPATIENT CLINICS AND WARDSBONE DENSITOMETRY UNIT

LABORATORY FOR BONE METABOLICMARKERS (PERSONNEL, INSTRUMENTS)

PROGRAM TYPE:

7-5

/12-

DEBRECEN REGIONAL OSTEOPOROSIS PROGRAM1996-1998

OSTEOPOROSIS DRUG STUDIES INITIATED (INTERNATIONAL,MULTICENTER)Raloxifene 7 Strontium / Na alendronate / PTH / Alpha- Hydroxy D vit.(last 2 in preparation)

FUCTIONING AS A REGIONAL CENTER OFTHE NATIONAL OSTEOPOROSIS PROGRAM OF HUNGARY

RUNNING OUTPATIENT CLINIC FOR OSTEOPOROTIC MENOPAUSALWOMEN

USING A HOLOGIC QDR 4500 BONE DENSITOMETER (NOV 1996)AND AN ULTRASOUND BONE IMAGING SYSTEM (UBIS) (NOV 1996)POSSESS A SAHARA ULTRASONIC BONE ANALYZER (JUNE 98)

PATIENT TURNOVER:

1996 4,350 PATIENTS 5,480 SCANS

1997 5,103 PATIENTS 5,672 SCANS

1998 3,356 PATIENTS (31JULY)COLLABORATION: INSTITUTE OF PUBLIC HEALTH

THE COCHRANE COLLABORATIONNATIONAL OSTEOPOROSIS PROGRAMEPOS

7 -6

RECRUITMENT FOR THE STUDY IN DEBRECEN

TOTAL POPULATION (E.G. REFRENDUM LIST) WAS NOT AVAILABLE

HEALTH AUTHORITIES POPULATION LIST AND ADDRESSES WEREUSED

RANDOMISATION BY AGE STRATA

LETTER TO 500 PERSONS AGED 15-50 (MALE : FEMALE RATIO: 1)

81 REPLIED NO

43 DID NOT ATTEND WITHOUT NOTICE

376 PERSONS ATTENDED

WOMEN: 205

MEN: 171

7 - 7

416

AV*

METHODS OF INVESTIGATION

AP SPINE DXA BONE DENSITOMETRY (LUNAR DPX-L)

FEMUR DXA BONE DENSITOMETRY

OSTEOPOROSIS QUESTIONNAIRE: LIFESTYLE, ACTIVITY (WHO)

NUTRITIONAL QUESTIONNAIRE (CALCIUM INTAKE ASSESSMENT)

VITAL SIGNS ASSESSMENT (BW, HEIGHT, BP, PULSE RATE)

LABORATORY FOR HEALTH ASSESSMENT

7 - 8

RESULTS OF THE STUDY

SEX DISTRIBUTION: 54.5 % WOMEN45.5 % MEN

DXA RESULTS: LUMBAR SPINE (W) L, - L4: 1.139 +/- 0.16 g/cm2

LUMBAR SPINE (M) U - U- 1.214 -/- 0.15 g/cm2

FEMORAL NECK (W) 0.925 +/- 0.089 g/cm2

FEMORAL NECK (M) 0.985 +/• 0.103 g/cm2

ATTENDED: 376 SUBJECTS OF THE 500 INVITED (75.2%)

AGE DISTRIBUTION: 15-50, INCREASING NUMBERS BY AGE

LIFESTYLE QUESTIONNAIRE: LOW LEVEL OF PHYSICAL EXERCISE(PAST AND PRESENT)

SIGNIFICANT CO-MORBIDITY IN THE UPPER AGE STRATA

CALCIUM INTAKE: 62% OF THE SAMPLE DID NOT TAKE THEAMOUNT OF CALCIUM WHICH CORRESPONDS TO THE RDAFIGURES.

7 - 9

/US'

DXA RESULTS: MEAN BMD BY AGE AND SEX

FEMALE MALE

L1-4 Femur L1-4 Femur

[g/cm2]

15-19 1.010 0.842 1.018 0.886(15/12)

20-24 1.184 0.945 1.236 0.982(25/15)

25-29 1.210 0.948 1.254 1.045(30 / 22)

30-34 1.176 0.950 1.258 1.048(30 / 22)

35-39 1.162 0.936 1.252 1.001(30 / 24)

40-44 1.130 0.928 1.245 0.968(35 / 36)

45-50 1.102 0.926 1.238 0.963(40/40)

7-10

CONCLUSIONS

INVITATION BY LETTER FOR POPULATION BASED STUDIESYIELDED SUFFICIENT NUMBERS OF SUBJECTS

THE PROJECTED AGE SPECIFIC NUMBERS MAY NOT BESUFFICIENT TO SHOW TRENDS OVER 15-50 YEARS

FEMALE AND MALE NORMAL DATA SHOW LESS THAN EXPECTEDVARIATION

IN THE YOUNGEST AGE STRATUM INSUFFICIENT DATA COLLECTED

LAST YEAR OF THE STUDY WILL NEED INTENSIVE DATACOLLECTION

7-11

/ill-

PLANS FOR 1998-1999

COMPLETING THE FULL NUMBER OF SCREENING PROCEDURES(I.E. 50 PER AGE STRATA OF 5 YEARS AND SEX)

ESTABLISHING REGIONAL / NATIONAL REFERENCE DATABASE OFBMD VALUES. AT LEAST DUBLE NUMBER OF SUBJECTS OF THEPRESENT PLAN SHOULD BE CONSIDERED

COLLECTION OF BONE SAMPLES FOR TRACE ELEMENT ANALYSISAND PERFORMIND THE PROCEDURE (PIXE)

COLLECTION OF BLOOD SAMPLES FOR BONE METABOLICMARKERS

HIP AND FEMORAL AXIS LENTGH MEASUREMENTS TO BECONTINUED, AS WELL AS VERTEBRAL MORPHOMETRIC DXA ATLEAST IN A SMALL SAMPLE OF THE STUDY POPULATION

CORRELATING CALCANEAL ULTRASOUND PARAMETERS (SOS,BUA) WITH BONE MINERAL DENSITY

PERFORMING BODY COMPOSITION ANALYSIS AT LEAST IN ASMALL SAMPLE OF THE STUDY POPULATION

ANALYZING AND PUBLISHING LOCAL RESULTS IF SUITABLE ANDAGREED UPON BY IAEA

COMPARING LOCAL RESULTS TO THOSE COMING FROM OTHERREGIONS

7-12

XA0054926

AGENCY RESEARCH CONTRACT NUMBER: 8195/RB

TITLE OF PROJECT

"DETERMINATION OF PEAK BONE MASS DENSITY AND COMPOSITIONIN LOW INCOME URBAN RESIDENTS OF METRO MANILA USING ISOTOPETECHNIQUES

Part of Coordinated Programme:

COMPARATIVE INTERNATIONAL STUDIES OF OSTEOPOROSIS USINGISOTOPE TECHNIQUES

CONTRACTING INSTITUTE

PHILIPPINE SOCIETY OF ENDOCRINOLOGY AND METABOLISMENDOCRINE MEDICAL RESEARCH LABORATORYUP-PGH MEDICAL CENTERTAFT AVE., MANILA, 1000PHILIPPINES

j CHIEF INVESTIGATOR

MARY ANNE LIM-ABRAHAN, MD

8- 1

PROGRESS REPORT

1. DESCRIPTION OF THE RESEARCH CARRIED OUT

The work described in this paper is a continuation of the first phase of the study,which is the determination of the peak bone mass density among residents of MetroManila using dual energy x-ray absorptiometry. However, it also aims to correlate sex,body mass index, nutritional factors, physical activity and lifestyle to peak bone mass andthus attempts to explain any discrepancies in peak bone mass density to that seen in othercountries.

2. METHODOLOGY:

This is a cross-sectional study where subjects were randomly chosen from amonghospital companions of the Philippine General Hospital, a tertiary care government-owned hospital, and St. Luke's Medical Center, a tertiary private hospital. The hospitalcompanions of every 10th admission to the hospital were chosen and a consent wasobtained prior to inclusion in the study.

Patients were 15-50 years old, male or female with no history of chronic illness,chronic infection nor chronic intake of drugs such as steroids. Ten (10) cc of blood wereextracted from the patient, preferably in the fasting state; five (5) of blood was used forbiochemical studies such as RBS, creatinine, alkaline phosphatase, calcium, phosphorous,sodium and potassium, while the other five (5) ml was stored in an ordinary freezer forfuture studies.

They were then sent to St. Luke's Medical Center for dual energy x-rayabsorptiometry (DEXA) done on the Lunar DPXL machine. Measurements were doneon the anteroposterior spine (L2-L4 level) and on the femoral neck. Quality assurancewas done prior to each bone density analysis using the phantom provided by Lunar Co.Afterwhich, the patients were interviewed as to their diet by a food day recall, physicalactivity, and lifestyle using a Filipino version of the WHO questionnaire on osteoporosis.

3. RESULTS:

A total of 69 subjects were gathered for this study year, (35 females and 34 males),with 4-6 subjects per age range of 5 per sex. Bone density measurements were performedon the lumbar spine at the L2-L4 level and on the femoral neck. Additionalmeasurements on the hip were done at the trochanter and at the Ward's triangle. Forpurposes of analysis, the measurements on the L2-L4 and on the femoral neck wereincluded. These were then scattered against age for each sex.

For females, the mean weight and height were 54.56 kg + 19.84 kg and 152.65 cm +10.43 cm, respectively. The body mass index was 26.31 g/m2 + 21.11 g/m2. The totalcaloric intake was 1506.75 kcal + 498.225, while the total calcium intake per day was388.02 gm + 227.22. The highest bone mass density was seen in the 30-34 year ear oldage group (1.35g/cm2) while the lowest BMD was observed at the 45-5Oyear old agegroup. However, the mean bone mass density was highest among the 40-44 year old agegroup (1.315 g/m2) widi the lowest mean BMD occurring in the 15-19 year old agegroup. At the level of the femoral neck, the bone density was observed to be highest

8-2

among the 40-44 year old age group (1.07 g/cm2). Most of the patients were in theirchild-bearing age and only three patients were menopausaL

Most males included in this study were smokers and alcoholic beverage drinkers.The mean height and weight were 167.62 cm + 6.5 and 64.69 kg + 8.82, respectively.The mean body mass index was 23.14 g/cm2+ 3.04. The total caloric intake was 1947.57kcal ± 663.597 and of the total intake, calcium accounts for 409.79 g + 289.309g. Thehighest BMD at the L2-L4 level was 1.91 g/cm2 seen at 20-24 years old. At the femoralneck, the highest observed BMD was 1.37 g/cm2 in the 30-35 year old age group.

Food frequency listing was done to determine the variety of food consumed by thesubjects. Lifestyle factors such as exercise were also noted.

The results in this report were then included in the preliminary report done in 1994for further statistical analysis especially in the questionnaire data

4. DISCUSSION:

For females, although the highest bone mass density was observed at 35-39 year oldage group, the gradual rise in bone mass prior to this age group was not observed. Therewas only a slight fall in bone mass density after age thirty five. One patient at the 40-50 year old age group had a bone density of 1.39 g/cm and this was attributed to herphysical activity.

For males, the peak bone appears to be achieved earlier at 20-25 years old at bothsites of interest (L2-L4 level and femoral neck). One subject in the 20-24 year old was avarsity basketball player in school (1.37 g/cm2 and 1.44 g/cmz at the L2-14 level andfemoral neck, respectively). The lowest bone mass at the L2-L4 level was 0.35 g/cm2 in a15 year old who smokes daily, drinks alcohol regularly and is not involved in any form ofsport or exercise. Like in females, there is a gradual decline in bone mass density afterreaching its peak but the levels of the 40-44 year old age group but do not approximatethose of the 15-19 year old age

The bone mass density curve of Filipinos as a function of age cannot be wellillustrated (Figures 1-4). Although the peak bone mass density was achieved at 30-39years old, the mean bone mass density at the L2-L4 level of the spine and at the femoralneck among females was highest at 40^44 years old, with a mean BMD= 1.261 g/cm2 and1.089 g/cm , respectively. For males, the highest mean bone mass was observed at 30-35 years old (mean BMD= 1.261 g/cm2) at the lumbar spine. Similar results wereobserved at the level of the femoral neck

5. SUMMARY:

Peak bone mass density at the L2-L4 level among females in this series is achieved at35-39 years old while it appears to be achieved at an earlier age in males. Similar resultsare observed at the femoral neck. The gradual rise in bone mass until it reaches a peak asobserved in previous studies is not very well clearly defined in this study. After reachinga peak bone mass density, there is also a gradual decline in bone mass but the values ofthe 45-50 year old age group do not approximate the levels observed in the 15-19 yearold age group.

Data in this phase of the study need to be subjected to more statistical analysis. Dietand lifestyle correlation will also be done on the final analysis.

8 - 3

6. SIGNIFICANT DEPARTURE FROM THE CONTRACT

The second phase of the study which should have included trace element analysis,was not done due to lack of manpower to do the atomic absorptiometry for trace elementanalysis. Moreover, lack of resources and financial constraints hinder us from doing thetrace element analysis.

The Philippine Nuclear Research Institute proposes the use of X- ray Fluorescencefor trace element analysis in bone, and this may come out cheaper than the recommendedmethodology. We have decided to forego the trace element analysis for the meantimeuntil such time that we are advised regarding the method to use.

7. PUBLICATIONS:

a) Poster Presentation: 15th Annual Meeting of the Japanese Society for Bone andMineral Research. July 23-26, 1997. Omiya Sonic CityBuilding, Omiya, Saitama, Japan.

b) Oral Presentation: 9th Congress of the ASEAN Federation of EndocrineSocieties: "Advances in ASEAN Endocrinology". December3-6,1997, Shangri-la Hotel, Singapore.

c) Publication: Philippine Journal of Internal Medicine, January 1998.

8 - 4

Figure 1. BONE MASS DENSITY AT THE LEVEL OF L2-L4 IN FILIPINO MALES

20-24 25-29 30-34 35-39AGE GROUPS (years)

40-44 45-50

00

Figure 2. BONE MASS DENSITY OF THE FEMORAL NECK IN FILIPINO MALES

15-19 20-24 25-29 30-34

AGE GROUPS (years)

35-39 40-44 45-50

CD

oo

1.6 -

1.4

1.2

2

z 0 8

LUQ

UJ

O0.6ffl

0.4

0.2

£3

3?

15-19

Figure 3. BONE MASS DENSITY AT THE LEVEL OF L2 TO L4 INFILIPINO FEMALES

S5

00

20-24 25-29 30-34 35-39

AGE GROUPS (years)

I40-44 45-50

Figure 4. BONE MASS DENSITY OF THE FEMORAL NECKIN FILIPINO FEMALES

1.2-1-

15-19 20-24 25-29 30-34 35-39

AGE GROUPS (years)

40-44 45-50

COI

CO

Appendix 2RESULTS-

PROGRESS REPORT

NAME OF PATIENT

1. Tomas Robosa2. Gerry Hinayon3. Ulysses Obsequio4. Frederick Idiesca5. Tom AJdrich Robosa

I

Age/Sex

15/M18/M18/M18/M

19/M

6. Christopher Maglalang 22/M7. Paulo Maglapid8 Lyle Dwen Leonaldo9. Levy Deocares10. Arvin Gonzales

11. Sandriel Jacob12. Enrique Bulalacao13. Marius Leviste14. Joselito delà Cruz15 Nicolas Flores

J16. Dionisio Buenaflor17. Juanito Paulino18. Raymond Ulep19. Nilo Molino20. Fernando Policarpio

I21. Rolando Ligón22 Miguel Castro23. Gerry Matías24. Eric Lupisan25. Edgar;Vrceo

26. Jing Flores27. Edgardo Cuesta28. Justino Lopez29. Edbert Julia

130. Abundio Obsequio31. Enrico Campos32. Antonio delà Cruz33. Serafín Castillo34. Abraham Tantingco

22/M __j22VM23/M25/M

26/M26/M26/M26/M28/M

28/M29/M32/M32/M34/M

35/V35/M36/M36/M37/M

40/M41/M42/M42/M

47/M47/M48/M49/M

50/M

I

Ethnic.

FilFilFilFilFil

FilFilFilFilFil

FilFilFilFilFil

FilFilFilFilFil

FilFilFilFilFil

FilFilFilFil

FilFilFilFilFil

Weight

4946496254

6559575470

6667577069

5672727265

7870656163

807965 .74

65806061

67.5

Height(cmj_

160164162 j167170

177173161162176

166167165169177

162170177172165

L 188163165169163

157162165167

169168165160176

BMI

_M!LL1917192220

2120

2123

2424212522

2125

L 2 3

2424

23L 26

242124

3230 J

2427

2328222422

Total Cal(kcal)

2454195619239723778

14181707233717191966

20361849375916811903

2077180528732881949

207519541464

1330.52213

u 2607183715281369

21021986104914741186

Calcium(gm)

265343313120809

438 i274~^332232266

457248548681199

228517682257202

372245264325356

596112271544

2873001660298892

12-L4 ^(g/cm)

I0.0340.9351.121.19

0.955

1.141.371.11.21103

1.080.9421.16

,_ 1.391.19

1.031.911.221.16

.985 E4

0.9991.071.131.161.25

0.9451.121.251.26

1.180.9551.18

0.8821.31

Fern Neck(g/cm)

0.910.9481.011.16

0.969

0.9461.44

0.8941.130.99

1.051.05

L 1-161.12

0.979

0.9671.01

0.9611.1

0.848

1.040.9581.07

0.8751.02

0.8641

0.9841.12

1.070.9520.8660.8980.991

8 - 9

Appendix 1RESULTS -

PROGRESS REPORT

Name of Patient

1. Amelia Obsequio2. Joly Jill Idiesca3. Evangeline Ragojo4. Jalen f antingco5. Desiree Domingo

6. Winelyn Lamerá7. Evelyn déla Cruz8. Maricel Tumbocon9. Analyn Panrtonal10. Sheila Bulaiacao

11. Cecille Caro12. Elizabeth Tantingcc13. Margarita Pación14. Jennifer Ramirez

15. Maritess Jaime16. Zeny Lauron17. Gina Ferrer18. MaLuzGuanzon19. Felecitas Galanida

|20. Chita Matunog21. Tita de Leon22. Maribel Co23. Felina Masadao24 Isabel Galimba

25. Lilian Rodriguez26. Elva Lina Rayos27. Rosalinda Julia28. Mary Ann Jamison29. Rhodora Domingo30. Victoria Obsequio

31. Estrella Casería32. Consejo Amador33. Lourdes Tantingco34. Jeanny Badong35. Salvación Atún

Age/Sex

~ 1 7 / F ~18/F18/F19/F19/F

23/F j23/F23/F24/F24/F

25/F26/F27/F27/F

30/F30/F32/F32/F34/F

35/F37/F37/F38/F39/F

41/F41/F42/FA5944,44/F44/F

45/F46/F47/F48/F48/F

I

Ethnic.

FilFilFilFilFil

FilFilFilFil ,Fil

Fil ,FilFilFil

FilFilFilFilFil

FilFilFilFilFil

FilFilFilFilFilFil

FilFil jFilFilFil

Weight(Kg)

4966454351

45 ,40454649

4680 ,52

i _ 6 5 _ 4

45585967

6358494539

5264747 2 .5050

5648586668

Height(cm)

141157140170

^ 151

149148

154151 ,

158i_ 166 i' 153 I

159

158150176

u 158153

15815715015146

162 J

156148163 j144155

158155159155160

BMI(g/m)

2427231522

2018201921

19173421

26I 20

192427

25242220145

202638272420

2220232727

Total Cal_J!«*')

217716451213Í5291312

^678.51543774

24711768

86411016551565

22941261136814801543

10941101229416462002

14362113134268313211514

11791709239815762087

Calcium(gm)

13770244650236

67821286663170

157228407378

238248500316494

401653238294673

987773281106172512

320563 j368382548

L2-L4(g/cm)

1.041.14

11.18

0.898

1.181.091.050.861.21

1.061.211.191.06

1.110.9731.051.141.06

1.241.031.3

0.954

1.161.27

, 1.281.061.021.04

0.8751.39

0.9980.8360.97

Fern Neck(g/cm)

0.9860.9360.8431.02

0.713

1.040.8560.8390.6850.927

0.9251.011.010.91

0.9960.7220.9870.9790.892

0.7310.8950.8240.9540.731

1.051.07

0.9850.9990.7270.922

0.750.9390.7710.8520.824

8- 10

XA0054927

Agency Research Contract No 8196/RO

Progress Report (Sept. 1996 - Aug. 1998) for

STUDIES OF OSTEOPOROSIS IN RUSSIA USINGISOTOPE-RELATED AND OTHER TECHNIQUES

As Part of Co-ordinated Programme

Comparative International Studies of Osteoporosis UsingIsotope-Related Techniques

(Presented at a Meeting in Sao Paulo, Brazil, 24-27 August 1998)

Medical Radiological Research Centreof Russian Academy of Medical Sciences

Obninsk 249020, Russia

Chief Scientific Investigator: Dr. Vladimir ZAICHICK

9-1

Part I: DXA BMD IN NORMAL RUSSIAN SUBJECTS AGED 15 TO 55

INTRODUCTION

There exists a direct interdependence between bone strength and bone mineral density

(BMD). According to the present concept, good bone health in later years is primarily

determined by the attainment of a sufficient high BMD during early adulthood. The higher is

the level of peak BMD (1), the older is the age when peak BMD is maintained (2), and the

lower is the rate of BMD decrease (3), the less is a probability of senile osteoporosis

development. Those three parameters depend on both sex, ethnicity and geographical origin of

the subjects over the age range from 15 to 50 years.

The aims of the study were:

- To investigate BMD age dynamics of the total body, spine and femoral neck (total body,

spinal and femoral BMD) in health women and men aged 15 to 55, residents of a large

industrial centre (Moscow)

- To investigate BMD age dynamics of the total body, spine and femoral neck in health women

and men aged 15 to 55, residents of a small city (Obninsk) situated in the rural area, 100 km

south-west far from Moscow

- To compare between Moscow and Obninsk data and determine a regularity of BMD age

dynamics for the Russian population

- To compare between BMD Russian results and those obtained for the US Reference

Population (LUNAR)

SUBJECTS AND METHOD

Examined were 480 healthy Russians (240 women and 240 men) aged 15 to 55, of which

334 Moscow citizens (168 women and 166 men) and 146 Obninsk citizens (72 women and 74

men). Both examined population groups were randomly selected. Each subject was asked to

complete a questionnaire and to make a decision about his individual peculiarities, race and

nationality, health status, mode of life, diet, moving activity, social status, etc. Examined were

not subjects of the following exclusion criterion:

9 - 2

- Moderate or severe scoliosis

- Known chronic illness (past or present) of >3 months duration

- Known chronic (>1 month) use of any medication other than dietary/vitamin supplementation

- Previous low trauma fracture

- Prolonged immobilization (>1 month)

- Over-exposure to toxic metals or irradiation

BMD measurements were performed at the Central Institute of Traumatology and

Orthopedics (Moscow) and Medical Radiological Research Centre (Obninsk) using dual

energy X-ray absorptiometry (LUNAR DPX-L Densitometer).

BMD of femoral neck (right and left), spine (anterior-posterior, L2-L4),and total body

was measured in each studied subject. Prior to DXA, the height, arm circumference and span

as well as weight of the individual was assessed. Daily calibration of the densitometers were

performed according to a standard protocol as described by the supplier of the instruments.

Data reproducibility was controlled by weekly measurement of LUNAR Spine Phantom. The

accuracy of obtained data was confirmed by the results of European Spine Phantom

measurement (WHO Collaborating Centre for Metabolic Bone Diseases, Sheffield, UK).

RESULTS

There was found no statistically reliable difference between mean values of the weight,

height, body composition (fat, lean mass) and BMD for the appropriate age studied groups of

Moscow and Obninsk citizens aged 20 to 49 (Tables 1 and 2). This ensured unification of the

results obtained for both population groups including those of 15 to 19 years and 50 to 55

years which were less representative by the examined subjects.

Mean values of some body parameters including femoral, spinal and total body BMD for

the healthy Russians aged 15 to 55 are shown in Tables 3 and 4. Figures 1, 2 and 3 represent

data about estimation of peak BMD level, the age at which peak is attained and the rate of

BMD decrease.

Comparison between BMD results for the US Reference Population (US RP) and those

obtained for the Russian population is given in Table 5.

9 - 3

/I if4

DISCUSSION

Female femoral peak BMD is probably attained by 18 years, although BMD level of 15-

year girls averages that of female adults. There were no significant changes in femoral BMD up

to 50 years inclusive. The mean level at the age between 18 and 50 makes up 1.005 g/cm2 that

is 2.5% higher than the level typical of the US RP. A maximum spinal BMD is attained by 18

to 20 years. At the age of 18 to 50 the level of spinal BMD remains the same to average 1.200

g/cm2 (1.200 g/cm2 for the US RP). A maximum total body BMD is also attained by 18 to 20

years to remain previous up to 50 years inclusive. On the average, the level of total body BMD

amounts to 1.175 g/cm2 at the age of 18 to 50 that is 4.4% higher than the level typical of the

USRP.

Male femoral peak BMD is attained by 20 to 25 years. Then BMD decreases uniformly

to be 16% reduced by 55 years. On the average, male BMD at the age of 20 to 45 is 4.7%

higher than the level of the US RP. A maximum spinal and total body BMD is probably

attained by 25 to 30 years, although at the age of 20 to 50 BMD changes are insignificant to

average 1.245 g/cm2 and 1.275 g/cm2 respectively (1.240 g/cm2 and 1.220 g/cm2 for the US

RP).

CONCLUSION

Mean values of spinal and total body BMD for the Russian population are very close to

those obtained for the US RP by the level, value of standard deviation and stability of the age

between 20 and 45 for both women and men.

The mean level of femoral BMD in the Russian men aged 20 to 45 is 4.7% higher that

that in the Americans, although the dynamics of BMD decrease with age is almost the same to

be about 0.45% per year.

Mean values of female femoral BMD in the Russians and Americans aged 20 to 45 are

almost the same. The rate of femoral BMD decrease of the Russian women does not exceed

0.09% up to 50 years, while that of the US women aged 20 to 45 is 2-fold higher to make up

about 0.18% per year.

9 - 4

Part m BONE MINERAL LOSSES IN THE YOUNG MALE ADULTSWHO TOOK PART IN THE CHERNOBYL CLEAN-UPOPERATIONS

INTRODUCTION

Idiopathic osteoporosis in the male adults aged 20 to 50 is a rarely found disease

[Franke, Runge, 1987]. Following the Chernobyl disaster study, Dr. V. Zaichick assumed

idiopathic osteoporosis to be likely developed in the young male adults who took part in the

clean-up operations. The assumption was based on possible importance of the following

factors in the illness etiology:

- Thyroid and parathyroid irradiation by incorporated iodine radionuclides

- Body intoxication with Pb, Cd and other toxic elements either used during clean-up

operations or being construction material constituents of the destroyed nuclear reactor

- Chronic emotional stress caused by radiophobia

- External total body irradiation

Despite substantiated assumption, all the efforts of National and International Programs

during the after-disaster period were mostly aimed at diagnostics and treatment of thyroid and

blood diseases. No attention was paid to osteoporosis problems of cleaners.

The first study on calcium metabolism of cleaners was carried out by Medical

Radiological Research Centre (MRRC) only in 1992, six years after the disaster. Estimated

data of hormonal status and major electrolytes (including calcium) in blood, saliva, and hair of

some cleaners showed noticeable changes in calcium metabolism which indirectly testified to

bone mineral losses [Zaichick et al., 1994; Zaichick et al., 1995]. It should be noted that in the

mid 1970s a unique system of the equipment for in vivo neutron activation analysis of calcium

in the foot, hand, and spine was developed in our Centre [Zaichick, 1993]. However, the

application of this equipment for direct estimation of bone mineral status of cleaners was

impossible for the lack of financing and comparatively high radiation dose in the process of

examination (about 1 cSv).

In 1997 MRRC purchased a LUNAR DPX-L Densitometer to determine bone mineral

density (BMD) based on dual energy X-ray absorptiometry (DXA). The present study includes

BMD measurements of men who took part in the Chernobyl clean-up operations.

9 - 5

SUBJECTS AND METHOD

A group of 47 male adults aged 30 to 46 was randomly selected in the course of a

regular preventive examination performed by MRRC every year for the Chernobyl cleaners,

citizens of Obninsk and Kaluga Region. Each examined subject at the age between 19 and 35

has been involved in the clean-up operations at different time since early May 1986 until late

January 1990.

BMD measurements were performed at MRRC Tomography Department using DXA

(LUNAR DPX-L Densitometer). BMD of femoral neck (right and left) and spine (anterior-

posterior, L2-L4) was determined for each studied subject. Prior to DXA, both height and

weight was assessed. Daily calibration of the densitometer was performed too according to the

standard protocol as described by the supplier of the instruments. Data reproducibility was

controlled by a weekly measurement of the LUNAR Spine Phantom. The accuracy of obtained

data was confirmed by the results of the European Spine Phantom measurement (WHO

Collaborating Centre for Metabolic Bone Diseases, Sheffield, UK).

The results obtained collaboratively by MRRC and Central Institute of Traumatology

and Orthopedics for the Russian reference population were used as BMD control values. The

population includes 188 randomly selected healthy men, citizens of Moscow and Obninsk aged

20 to 50.

Conventional computer programs and Student t-test were used for statistical data

processing.

RESULTS

Individual values of spinal and femoral BMD in cleaners are given in Figure 4.

The values of spinal and femoral BMD averaged 7.4% (p<0.001) and 7.6% (p<0.001)

respectively lower than the normal levels (Table 6).

Following T and Z criteria to estimate BMD levels, the bone state was specified as

osteopenia and osteoporosis in 27 and 3 examined cleaners respectively (Table 7 and 8).

9-6

DISCUSSIONr

A decrease in spinal and femoral BMD lower than the level of Mean-S.D. was found in

30 of 47 cleaners and could be classified as osteopenia (27) and osteoporosis (3). Since a

group of male adults for the purpose of BMD analysis was selected randomly in the course of a

regular annual preventive examination of the Chernobyl cleaners, 64% of subjects has been

assumed to have noticeable bone mineral changes by 1997. Because over 0.5 million of young

men was involved in the Chernobyl clean-up operations, social importance of this medical

problem is rather evident.

An indirect assessment of different etiologic factor contribution enables to draw a

conclusion about minimum importance of external total body irradiation with the dose up to

23 cSv. It follows from the lack of some interdependence between possible osteopenia or

osteoporosis development and the level of absorbed dose in this range (Fig. 5).

Among the cleaners with osteopenia and osteoporosis, 78% of subjects took part in the

1986 clean-up operations. Possible significant intoxication of the 1986 cleaners may be caused

by iodine radionuclides induced in the destroyed nuclear reactor active zone. Further,

intoxication may also arise from the exposure to cadmium and other "specific" elements,

constituents of reactor construction materials. Particular attention should be paid to the lead

which was thrown off the helicopters in considerable amount (about 10,000 tons) into the

destroyed reactor active zone for the purpose of gamma-radiation protection. Bulk of the lead

has evaporated to penetrate to the cleaners' lungs in the form of air aerosols. Even six years

after the disaster, the level of lead contamination was so high that there was found a lead

increase in the blood of rural workers from the tough radiation control zone and territories

adjacent to the Chernobyl NPP [Dobrovolsky et al., 1993].

Thyroid-incorporated iodine radionuclides irradiate both thyroid parenchyma and a

parathyroid gland. Different radiation doses and radioresistance levels of the thyrocytes, C-

cells, and parathyroid cells can cause an imbalance of the thyroid hormones, calcitonin, and a

parathormone [Bayraktar et al., 1990; Fujiwara et al., 1994]. It is know that these hormones

are main regulators of calcium metabolism [Franke, Runge, 1987].

Heavy metals such as Cd and Pb may affect the bone both directly, increasing bone

resorption, and indirectly, disturbing function of the kidneys, endocrine system and intestine

[Yashiki et al., 1975; Kozlovskaya et al., 1990; Stark et al., 1993; Peretz, 1996].

9 - 7

/Iks-

The most common factor responsible for osteoporosis development in cleaners may be

chronic emotional stress caused by radiophobia. A huge amount of young men, who have no

professional knowledge about the level of possible negative influence following small dose

radiation and, thus, who have no proper psychological training, were involved in the clean-up

operations. A post-disaster unprecedented flow of mostly pseudo-scientific frightening

information transmitted through the TV, radio, newspapers, and books favored the

development of stable, distinctly pronounced radiophobia.

The results of many researches name a chronic stress as one of the possible reasons of

osteoporosis development. Emotional stress, like a general adaptive body response, arises from

the influence of nervous and neuroendocrinal systems which stimulate hypothalamus and

secretion of hypophysial and adrenal complex. An endocrinal component action adds to a long-

term activity increase of many, if not all, body systems. The long-term activity of the stress

agent can result in the stable metabolic changes of bone major minerals, such as Ca, P, and Mg,

water-electrolyte metabolism, breakdown of the immune system, reserve of vitamins and vitally

essential trace elements (Zn, Fe, Cu, Mn, F, Sr, Se) playing an important part in the bone

formation [Sabbot et al., 1972; Papadopulos et al., 1977; Shkhinek, 1987; Gribauskas et al.,

1988; Singh et al., 1991; Stratakis et al., 1995].

Alcohol should be noted as one of the factors of BMD decrease. For some reason, there

is an assumption of mostly foreign researchers concerning an extremely excessive alcohol

intake by the cleaners. Our examination showed that the level of alcohol intake by the cleaners

under study was the same as that in the control group of male adults. In addition, according to

the data available [Feitelberg et al., 1987], even chronic alcoholism can not cause so great

BMD losses which we found in the present study.

Neither effective preventive measures nor treatment is practicable without information

about basic etiologic factors of osteoporosis in the young adult cleaners. Modern methods

make it possible to perform precise studies of endocrine and immune status, to assess the state

of water-electrolyte, mineral and trace element metabolism, and to estimate retrospectively the

levels of the Chernobyl cleaners' intoxication with Pb and Cd. Unfortunately, for 11 years past

since the disaster, neither national, nor international sources have been found to finance such

studies. Also, there is no promising prospect to receive any financial support on the part of the

Russian Ministry for Healthcare in the near future.

9 - 8

Part m. NAA OF MAJOR AND TRACE ELEMENTS IN THE HUMANSAMPLES OF BONE, TEETH, AND HAIR

INTRODUCTION

The main aim of this research portion was to search for interrelations between contents

of major and trace elements in the iliac crest, femoral neck, rib, teeth, and hair. A finding of

such interrelations would clear the ways for the development of new methods for diagnostics

of the bone state.

MATERIALS AND METHODS

Based on the Supplementary Program, for August 1998 autopsy samples were taken

from 82 people (36 women and 46 men) died a sudden death (accidents, murders, suicides,

drowning, acute alcohol intoxication). The died had neither chronic, nor systematic diseases.

The distribution of the died within the age groups is given in Table 9. Autopsy samples

included those of bone (femoral neck, iliac crest, and rib), teeth, and hair.

In our studies we proceed from the fact that for the present there are no methods

available for separation between mineral and organic bone components without great

disturbance of chemical element native distribution and composition. Thus, chosen was a

method for sampling and sample preparation which minimized element losses of bone samples,

to our mind, and eliminated possible sample contamination. Conventional surgical instruments

were used for sampling. Samples were put in polyethylene containers, frozen and then

transported to the analytical laboratory. Instruments made of titanium and plastic were used to

clean samples from muscles, connective tissues and blood. Bone sample edges interacting with

generally used surgical instruments were cut with a titanium knife. Dental enamel was

thoroughly wiped twice with alcohol-washed gauze wad to avoid contamination related to

stomatologic pincers. After cleaning, samples of the bone and teeth were weighed both by an

ordinary way and when dipped into bidistilled water to estimate wet mass and sample density.

Then samples were frozen and freeze dried. Right after lyophilization, samples were weighed

again to assess a water content using sample dry and wet mass ratio. Both compact and

trabecular bone were taken from dried samples of femoral neck and iliac crest using a titanium

knife. Teeth were separated into a crown and a root. Hair samples were taken and washed

according to the IAEA recommendations.

9 - 9

Using INAA with short-lived radionuclides, contents of Ca, Cl, K, Mg, Mn, Na, P, and

Sr were determined in the bone, teeth and hair samples. At present, the analysis of all collected

samples is coming to an end. Then data will be finally processed. In the present paper, as an

example, there given results of the progress statistical data processing on 112 samples (14

women and 42 men) of both compact and trabecular iliac crest using INAA with short-lived

radionuclides.

INAA with long-lived radionuclides was used to assess contents of Ag, Co, Cs, Eu, Fe,

Hg, Rb, Sb, Sc, Se, Sr, and Zn in the bone and teeth samples. The amount of analyzed samples

was limited (50) because of an unpredictably many-times increase in the cost of sample

irradiation in the reactor channel (this technique requires 100 to 120 hour duration of

irradiation). The present paper shows the results of statistical data processing on 24 samples of

the iliac crest and 22 samples of tooth crown using INAA with long-lived radionuclides.

The quality of the results on estimation of the element contents in bone samples using

methods we developed for the INAA with short- and long-lived radionuclides was checked by

analyzing both IAEA H-5 bone reference materials (Animal Bone) and NIST SRM 1486 (Bone

Meal). The description of the INAA methods and obtained results on estimation of major and

trace elements in bone reference materials was given in the paper presented at BERM-7

Conference held on 21-25 April 1997 in Antwerp.

For statistical data evaluation, standard programs were used to estimate the mean,

standard deviation (S.D.), standard error of the mean (S.E.) and correlation coefficient (r). To

evaluate the reliability of the difference between means, a parametric t-test was used.

RESULTS

The age changes in concentrations of Ca, Cl, K, Mg, Mn, Na, P, and Sr found by using

INAA with short-lived radionuclides for both compact and trabecular bones of intact female

and male iliac crest are presented in Table 10 and Table 11. Concentration ratios of these

elements in compact and trabecular iliac crest of healthy men with respect to the age are shown

in Table 12. Correlation coefficients calculated for concentrations of Ca, Cl, K, Mg, Mn, Na,

P, and Sr in both compact and trabecular iliac crest of healthy men are given in Table 13.

Contents of 12 trace elements in the iliac crest (total) and tooth crown of healthy men

based on the data of the INAA with long-lived radionuclides are shown in Table 14. The age

dynamics of Fe, Zn, and Sr in the iliac crest and Zn and Sr in the tooth crown are presented in

9-10

Table 15. Table 16 includes correlation coefficients calculated for Sr in the iliac crest and tooth

crown for different age ranges.

CONCLUSION

Maximum concentrations of Ca, P, and Mg in the female compact iliac crest are shown

for the age between 15 and 20. No age dynamics of these major mineral concentrations is

found for the trabecular iliac crest tissue (Table 10). Maximum concentrations of Ca, P, and

Mg occur in both male compact and trabecular iliac crest at the age of 15 to 30 (Table 11).

The compact iliac crest is more saturated with Ca, P, Mg, Sr, and Na in comparison with

trabecular one at the age of 15 to 55 (Table 12). All these elements are the basis of bone

hydroxiapatite or connected with it. It is interesting to note that Mn concentration in the

compact bone is higher than that in trabecular. These results contradict the idea of higher

trabecular saturation with trace elements. The ratio between K concentration in the compact

tissue and that in trabecular decreases with age. If the value of this ratio is close to 1 at the age

of 15 to 20, then it is 0.5 for the age between 45 and 55. The change of the ratio value is

related to the decrease in compact K concentration. Because K is an intracellular electrolyte,

the decrease in its concentration may indicate a reduce in the bone cellular component. On the

contrary, Cl is mainly an extracellular electrolyte. Therefore, the age dynamics of this

electrolyte found in both compact and trabecular bones is evidence of appropriate

redistribution of extracellular space.

A clear correlation between concentrations of major bone minerals such as Ca, P, and

Mg does not cause any surprise (Table 13). A correlation between major minerals and elements

such as Sr, Na, and Mn is more expressive. This correlation indicates availability of the direct

relation between Sr, Na, Mn and bone hydroxiapatite crystals. A correlation between

trabecular K and Cl confirms interrelation between cellular and extracellular volumes.

The method developed for the INAA with long-lived radionuclides provides estimation

of 12 trace element concentrations in the iliac crest and tooth samples (Table 14). However,

with sample mass of about 50 mg and time of spectrometric measurement of not more than

3 h, only three elements were determined in each studied sample (Zn, Sr, and Fe). The study of

the age dynamics of these element concentrations (Table 15) showed that there was a tendency

to increasing with age in the iliac crest Sr and to decreasing with age in the tooth crown Sr. At

young ages (15 to 35), there is a clear interdependence between Sr concentration in the tooth

crown and that in the iliac crest (Table 16).

9-11

The present data analysis of major and trace elements in the bone and tooth samples is of

preliminary nature. The final conclusion will be drawn after completion of statistical processing

of data obtained by INAA of the bone, tooth and hair samples.

9-12

REFERENCES

1. Bayraktar M., Gedik O., Alcalin S., Usman A., Adalar M., Telatar F. The effect of radioactiveiodine treatment on thyroid C-cells. Clin. Endocrinol., 1990, 33, 625-630.

2. Dobrovolsky L., Vitte P., Belashova I., Andrusishina I., Dudko I. Blood lead monitoring studies inChernobyl region in 1992. In: Trace Elements in Man and Animals - TEMA-8. Verlag MediaTouristik, Gersdorf, 1993, 868-869.

3. Feitelberg S., Epstein S., Ismail F., D'Amanda C. Deranged bone mineral metabolism in chronicalcoholism. Metabolism, 1987, 36, 4, 322-326.

4. Franke J., Runge H. Osteoporose. Diagnose, Differentialdiagnose und Therapie unterBerücksichtigung der Natriumfluorid-Behandlung. VEB Verlag Volk und Gesundheit, Berlin,1987,315 p.

5. Fujiwara S., Sposto R., Shiraki M., YokoyamaN., Sasaki H., Kodama K., Shimaoka K. Levels ofparathyroid hormone and calcitonin in serum among atomic bomb survivors. Radiât. Res., 1994,137. 96-103.

6. Gribauskas P.S., Kushleikaite M.Y., Goshtautas A.A. Emotional stress effect on the thrombocyteaggregation, contents of Zn, Cu, Mn, Ca and Mg in serum, erythrocytes and hair of healthysubjects with different types of the behavior. Fisiologiya cheloveka, 1988, J4, 5, 857-862 (inRussian).

7. Kozlovskaya A.G., Yagubov A.S., Makovetsky V.D. Ultrastructural changes in the white ratthyroid C-cells following experimental cadmium injection. Gigiena i sanitaria, 1990, 5, 41-43 (inRussian).

8. Papadopulos J.S., Papadopulos G., Douka Th., Varónos D.D. Dauerstress und Osteoporose. Med.Welt, 1977,28,882-885.

9. Peretz A. Trace elements and bone metabolism. In: Therapeutic Uses of Trace Elements. PlenumPress, New York, 1996, 271-276.

10. Sabbot I., McNew J., Hoshizaki T., Sedgwick C, Adey W. Effect of a 30 day isolation stress oncalcium, phosphorus and other excretory products in the unrestrained chimpanzee. AerospaceMedicine, 1972, 43, 2, 142-148.

11. Shkhinek E.K. Interrelations between endocrine and immune systems under stress. In: EndocrineSystem and Unfavorable Factors of the Environment. Leningrad, 1987, 261 (in Russian).

12. Singh A., Smoak B.L., Patterson K.Y., Le May LG., Veillon C , Denster P.A. Biochemicalindices of selected trace minerals in men: Effect of stress. Am. J. Clin. Nutr, 1991, 5_3_, 1, 126-131.

13. Stark C , Kahrmann B., Walzel E. Histomorphologische Befunde an Knochen und Nieren vonbleiexponierten Ratte. In: Mengen-und Spurenelemente. 13. Arbeitstagung. Friedrich-Schiller-Universität, Jena, 1993, 81-88.

14. Stratakis CA., Chrousos G.P. Neuroendocrinology and pathophysiology of the stress system. In:Stress. Basic Mechanisms and Clinical Implications. Annals of the New York Academy ofSciences. Vol. 771. New York, 1995, 1995, 1-18.

15. Yoshiki S., Yanagisawa T., Kimura M., Otaki N., Suzuki M., Suda T. Bone and kidney lesions inexperimental cadmium intoxication. Arch. Environ. Health, 1975, 30, 11, 559-562.

16. Zaichick V. The in vivo neutron activation analysis of calcium in the skeleton of normal subjects,with hypokinesia and bone tissue diseases. J. Radioanal. Nucl. Chem., Articles, 1993, 169. 2, 307-316.

17. Zaichick V., Lyasko L.I., Dubrovin A.P. The main electrolyte and trace element metabolism in theChernobyl clean-up workers. In: Radiobiological Consequences of Nuclear Accidents. Moscow,1994, Part 2, 311.

18. Zaichick V., Tsyb A., Bagirov S. Neutron activation analysis of saliva: Application in clinicalchemistry, environmental and occupational toxicology. J. Radioanal. Nucl. Chem., Articles, 1995,195. 1, 123-132.

ASA

CO

20-2425-2930-3435-3940-4445-49

Table 1Some parameters (MeaniS.D.) of examined healthy citizens of Moscow (1) and Obninsk (2)

Agerangeyears

n

1 2

Ageyears

1 2

Weightkg

1 2 1WOMEN

Heightcm

2

Fatkg

1

Body

2

compositionLean mass

kg1 2

232014232219

812814812

21.7±1.327.1±1.332.5±1.636.7+1.641.6±1.447.0±1.4

21.8+1.226.511.432.111.137.211.542.6+1.346.911.3

58.318.564.6+12.066.018.161.0110.366.819.672.1113.9

56.617.159.5+8.759.115.862.619.370.816.269.7+9.4

16515166171651516216162+616117

166141671516115164161611516116

19.7+7.423.1111.423.8+7.621.518.725.3+8.028.4110.2

16.4+8.215.5+7.220.714.621.716.929.0+5.027.016.4

37.1+3.239.215.338.912.138.115.139.013.241.5+4.0

38.713.39.113.37.314.37.813.39.1+3.40.113.

MEN20-2425-2930-3435-3940-4445-49

182519201923

138912812

22.111.726.1+1.032.111.237.511.442.3+1.547.211.4

21.8+1.227.510.932.0+1.537.211.741.611.247.211.7

78.8111.275.2+10.480.9+11.481.5+10.682.6+11.379.519.1

78.219.580.9111.876.118.475.6111.481.6118.487.1+15.1

179+5179+617918176181791717714

180+6181+81751517817174+817615

16.4+9.914.118.520.118.819.118.218.8+8.918.916.8

14.2+5.518.918.116.516.116.117.315.7+9.024.619.5

58.4+5.658.0+5.558.816.461.418.363.2+7.157.417.2

58.6+5.59.514.54.713.56.515.60.5110.59.517.

Table 2Bone mineral density (Mean+S.D.) of examined healthy citizens of Moscow (1) and Obninsk (2)

Agerangeyears

20-2425-2930-3435-3940-4445-49

20-2425-2930-3435-3940-4445-49

n

1

232014232219

182519201923

2

812814812

138912812

Total

1

1.13010.0901.173+0.0751.16210.0741.17110.0821.18510.0761.21610.069

1.28810.1001.29110.1041.29410.0681.27910.0931.28210.0921.27510.059

body

2

1.14710.0911.14210.0651.15910.0531.20610.0671.16910.0491.19510.058

1.22210.0791.26610.0821.26610.0541.24810.0811.24310.0751.29710.078

111111

111111

1

.15410.

.20710.

.18310.

.17310.

.18110.

.21410.

.22510.

.28310.

.25410.

.21610.

.19910.

.24810.

Spine

146107115131100127

130156144175120112

Bone mineralL2-L4

2WOMEN1.21410.1101.21210.1251.23210.099.23110.099.130+0.072.23610.148

MEN.24310.125.345+0.082.260+0.102.20010.173.15110.126.31210.146

density, g/cm2

right1

1.036+0.1361.00110.1160.99310.1710.98610.0960.98510.1481.01510.112

1.193+D.1241.19310.1781.12510.1451.08810.1421.04410.1151.06510.081

Femoral

2

1.02610.1730.967+0.1130.97110.0961.02510.1000.921+0.1220.99210.087

1.13910.1181.069+0.1131.07010.1251.026+0.1271.03910.1641.04110.118

neck

1

0.99810.1401.00610.1250.995+0.1470.99610.1070.98010.1311.03410.103

1.188+0.1381.19810.1661.12810.1571.08510.1481.05310.1371.07510.087

left2

1.02510.170.98110.100.96910.101.03810.090.953+0.100.96110.06

1.105+0.101.08210.131.10210.121.03210.131.03510.141.071+0.10

Table 3Some body parameters and bone mineral density (MeaniS.D.) of healthy Russian teenagers

CO

a>

Ageyears

151617181920

151617181920

n

666877

8105657

Heightcm

163±4164±5168+4168+4166±3165+5

179+12178±518117176±8173+5180±4

Weightkg

50.814.754.0±5.656.5+7.554.5±3.858.9±8.260.716.6

62.0111.364.3111.660.811.366.017.965.215.0

81.3111.4

BodyFatkg

14.013.715.0+3.116.414.114.1+6.517.216.821.6+5.5

11.018.67.717.45.9+2.911.8+7.69.9+1.9

18.5110.4


kgGIRLS

34.7+2.135.713.337.512.937.9+3.939.612.839.4+3.3

BOYS48.418.352.917.951.413.254.016.949.915.258.514.8

Total body

1.06610.0741.044+0.1001.10310.1051.165+0.0781.190+0.1191.11810.113

1.10910.0851.142+0.0851.13210.0801.140+0.0581.12710.1081.26710.092

Bone mineralSpine L2-L4

1.10810.1341.02310.1531.12310.1281.20110.1461.279+0.2111.10710.188

1.03910.1311.10510.1061.11210.1301.08110.1151.15910.1461.226+0.124

density, g/cm2

Femoralright

1.054+0.1370.951+0.1671.030+0.1481.11010.1531.05510.1591.008+0.212

1.11610.1561.12510.1321.06010.1291.06210.0791.10410.1641.19610.125

neckleft

1.05910.1151.025+0.3420.98910.1511.12410.1231.091+0.2170.99210.206

1.07810.1531.14810.1681.04810.1341.08410.0841.12810.1671.19210.177

CO

Table 4Some body parameters and bone mineral density (MeaniS.D.) of healthy Russian

female and male adults

Agerangeyears

n Ageyears

Heightcm

Weightkg

BodyFatkg


kgWOMEN

Total bodyBone mineral

Spine L2-L4density, g/cm2

Femoral neckright left

20-2425-2930-3435-3940-4445-4950-55

31322237303117

21.7±1.326.911.432.4±1.436.9+1.541.9±1.447.011.453.112.3

16514166161641516316162161611616415

57.918.162.7111.063.517.961.619.867.818.971.1112.266.6113.5

18.817.620.2110.622.716.722.017.726.317.427.418.625.219.5

37.513.339.114.638.313.038.114.639.013.140.913.740.915.5

1.13410.0891.16210.0721.16110.0661.18410.0781.18110.0691.20810.0651.14810.094

1.16910.1391.20910.1121.20110.1101.19610.121.16710.095.22310.134.06610.182

1.03410.1440.98810.1140.98510.1461.00010.0980.96810.1421.00610.1020.94610.125

1.00510.140.99710.110.98510,131.01110.100.97710.121.00610.090.97010.13

MEN20-2425-2930-3435-3940-4445-4950-55

31332832273513

22.011.526.511.132.011.337.311.542.111.447.211.553.012.1

1801518016178+717718178181771417817

78.6110.476.6110.879.4110.679.3111.182.3113.482.1111.987.217.1

15.818.615.218.618.918.117.917.917.918.920.818.223.814.7

58.415.458.415.357.5+6.059.517.762.418.258.117.160.013.8

1.26010.0961.28510.0991.28510.0641.26810.0891.27110.0881.28210.0661.24910.085

.23210.126

.29810.143

.256+0.1301.210+0 1711.18510.1221.27010.1261.18810.134

1.17010.1221.16310.1711.10710.1391.06510.1381.04310.1281.05710.0940.99910.124

1.15410.131.17011.651.11910.141.06510.141.04810.131.07410.091.00010.10

Table 5Bone mineral density of both US Reference Population and healthy Russians aged 18 to 50

toI

00

BoneRussian Reference Population US Reference Population

Age, years

Range

BMD, g/cm2

Mean+S.D.

BMD decrease,%/yearMean

Age, years

Range

Young adults aged 20-45 (LUNAR)Age matched (LUNAR)

BMD, g/cm2

Mean+S.D.*

BMD decrease,%/yearMean**

Total BodySpine L2-L4Femoral neck

Total BodySpine L2-L4Femoral neck

18-5018-5018-50

20-5020-5020-45

1.175±0.0801.200±0.1251.005+0.125

1.275±0.0851.245+0.1401.120+0.120

WOMEN-0-0

<0.09MEN~0-0

-0.45

20-4520-4520-45

20-4520-4520-45

1.125+0.8001.200+0.1200.980+0.120

1.220+0.0801.240±0.1201.070+0.120

-0-0

-0.18

-0-0

-0.41

Table 6

Mean values (Mean±S.D.) of some body parameters and BMD in both examined cleaners and healthy male controls

Parameter Cleanersn=47

Controlsn=188

t-test

P<

co

CD

Age, years

Weight, kg

Height, cm

Spinal BMD (L2-L4), g/cm2

Femoral BMD (right), g/cm2

Femoral BMD (left), g/cm2

39.2±4.3

77.0±11.0

174±7

1.153±0.131

0.994±0.133

1.012±0.124

35.0±9.0

79.7±11.4

178±7

1.245±0.140

1.085±0.125*

1.085±0.125*

-

-

-

0.001

0.001

0.001

* For the age of 39.2 years, an average age of the examined cleaners

too

Table 7

Distribution of the cleaners with osteopenia (n=27) depending on the affected bones (values of T or Z criteria in the rangebetween -1.0 and -2.5)

Bone Amount of cleaners

Femoral neck (right or left) 1

Femoral neck (right and left) 5

Spine (L2-L4) 10

Spine (L2-L4) and Femoral neck (right or left) 2

Spine (L2-L4) and Femoral neck (right and left) 9

Table 8

Values of T and Z criteria for the spine and femoral neck of cleaners with osteoporosis

CO-21

Name

O.E.

P.V.

G.V.

Age,years

42

43

44

Spine,L2-L4

-2.85

-1.57

-1.17

T criterionFemoral neck,

right

-2.76

-2.17

-3.26

Femoral neck,left

-2.16

-2.55

-1.96

Spine,L2-L4

-2.55

-1.25

-0.37

Z criterionFemoral neck,

right

-2.67

-1.54

-2.25

Femoral neck,left

-2.07

-1.92

-0.95

Table 9Amount of died people from whom autopsy samples of bone (femoral neck, iliac crest, rib), teeth and hair were taken

(for August 1998)

Age range (years) Female Male_____ _ _

21-25 3 5

26-30 3 5

31-35 5 7

36-40 4 5co

M 41-45 6 8ro46-50 5 5

51-55 5 6

Total: 36 46

Bone

Table 10Contents (Mean+S.D.) of chemical elements in the iliac crest (compact or trabecular bone) of healthy women

with respect to the age (u.g/g dry tissues)

Age(years)

n Ca(%) Cl K Mg Mn Na Sr

to

15-20 19.0±3.5 941±31 439±232 2275±632 0.364±0.089 5292+625 8.56±1.47 941+706

Compact

26-30

31-35

36-40

2

2

3

18.3±6.7

18.910.2

15.9+1.0

875+28

681±281

596±158

509±3

55O±373

295+34

19281549

19191437

1514+412

0.295+0.143

0.290+0.009

0.235+0.019

4477+1623

44531617

32151595

7.

7.

5.

1411

5510

8911

.31

.03

.31

3071144

115167

1241127

46-50 2 16.217.0 11721571 5841174 2100+502 0.35910.038 50091662 6.6412.04 3411149

15-20 5 13.712.1 1301+107 1392+518 21161178 0.34810.117 4232+908 6.0410.92 5581414

26-30

31-35

2

2

12

9.

.310

1+3.

.8

4

19611755

1254+301

10611899

145011096

22331142

1675+647

0.27310

0.223+0

.065

.169

4459183

3789+42

5

4

.8010.21

.05+1.60

218136

5631640

36-40

46-50

11.8+2.7 16031561 11181251 17131150 0.233+0.106 43041532 5.2711.34 3411160

17.1+7.9 11731491 1585+693 2387+839 0.301+0.026 5488+1933 7.7813.62 684+694

r°Table 11

Contents (Mean+S.D.) of chemical elements in the iliac crest (compact or trabecular bone) of healthy menwith respect to the age (u.g/g dry tissues)

Bone Age (years) n Ca(%) Cl K Mg Mn Na Sr

ro

15-20 15.0±3.2 953±8 1340+1150 22911808 0.33+0.07 44591414 7.27+0.39 408+300

21-25

26-30

31-35

Compact 36-40

41-45

46-50

51-55

15-20

21-25

26-30

Trabecular 31-35

36-40

41-45

46-50

3

6

6

6

8

5

5

3

3

6

6

6

8

5

18.812.

17.7±6.

18.4+4.

15.014.

15.311.

17.0±3.

18.9+3.

13.7±3.

11.1±3.

12.3±6.

10.412.

10.0+2.

11.4+4.

11.813.

4

1

0

0

6

3

5

8

7

2

9

6

5

3

13301700

7101240

9751169

7551206

965+360

848+228

1127+565

1075+162

10061331

11941212

1089+60

12991435

11961427

13541354

9971168

687+196

609+341

6701432

5981258

497+273

8381427

10171510

14701314

1313+499

9211403

13031516

11601752

10771243

22231893

218211046

20651383

1425+513

1835+281

1656+306

19651758

2132+932

1257167

18061450

15621295

14921273

15971519

15611492

0.4310.18

0.3910.13

0.3810.10

0.2210.07

0.2810.08

0.32+0.08

0.3910.01

0.31+0.08

0.2910.03

0.40+0.24

0.2510.07

0.2410.08

0.31+0.17

0.3310.34

5936+1227

500011430

513711033

3951+1420

45651237

4646+798

55361422

4603+1346

47731818

4176+1751

35171487

35711820

398911359

4244+1143

7.3610

7.5912

6.7911

6.52+1.

6.1410

6.9411

7.8111

5.77+1

5.1711

7.3713

4.6811

4.6311

5.3212

5.72+2

.16

.23

.51

881

.66

.36

.12

.53

.53

.70

.25

.37

.39

.26

314189

7091585

3321219

252198

416197

332+225

4091119

5201477

1921119

3481185

177182

401+302

3681191

5561423

51-55 10.913.2 18181765 17291772 17621436 0.3110.08 41011841 4.7911.10 2951114

toOi

Table 12Concentration ratios (Mean+S.D.) of the element contents in compact and trabecular iliac crest of healthy men with respect to the age

(based on dry tissue)

Age (years)

15-20

21-25

26-30

31-35

36-40

41-45

46-50

51-55

n

3

3

6

6

6

8

5

5

CaVCa1

1.18+0.46

1.65±0.76

1.58±0.78

1.95±0.86

1.6010.64

1.5510.67

1.57+0.64

1.7910.30

C17C1'

0.90210.151

1.2510.36

0.60510.219

0.89610.148

0.60110.152

0.87410.398

0.70710.422

0.60510.101

K7K'

1.1810.42

0.86410.183

0.64510.263

0.65010.337

0.52610.213

0.69710.486

0.44310.165

0.51010.267

MgTMg1

1.1310.27

1.6610.50

1.2510.69

1.3810.39

0.99+0.38

1.2710.46

1.1810.50

1.20+0.66

MnTMn1

1.1110.19

1.38+0.35

1.81+1.85

1.61+0.71

1.0710.57

1.3111.03

1.7211.16

1.3410.33

1

1

1

1

1

1

1

1

NaTNa'

.0110.22

.1710.47

.2810.48

.5010.40

.19+0.66

.2610.39

.18+0.43

.3910.25

P7P'

1.3410.

1.4310.

1.2710.

1.6110.

1.5510.

1.3910.

1.35+0.

1.6710.

42

50

76

75

69

62

47

30

1

2

2

2

0

1

0

1

Sr7Sr'

.2610.84

.12+2.22

.69+2.91

.0911.69

.9710.65

.3810.59

.9410.68

.4610.42

Go

Table 13Correlation coefficient (r) of the element contents in compact and trabecular iliac crest of healthy men (based on dry tissue)

Ca Cl K Mg Mn Na Sr

Compact tissue

CaClK

MgMnNaPSr

10.153-0.0950.587*0.607*0.785*0.876*

0.356***

10.252

0.382**0.309***0.327***

0.0790.041

10.344***0.288***

0.2010.1350.108

10.679*0.642*0.587*

0.318***

10.811*0.535*

0.319***

10.744*0.404**

10.332***

Trabecular tissue

CaClK

MgMnNaPSr

10.1410.1340.764*

0.434**0.822*0.716*

0.412**

10.608*0.1560.137

0.330***0.122

0.271***

10.305***

0.1880.399**

0.2490.204

10.432**0.640*0.657*

0.406**

10.458**0.740*

0.259***

10.625*

0.428**1

0.436**

p<0.001p<0.01

*** p<0.05**

Table 14Contents of some trace elements in the iliac crest and teeth of healthy men based on the data of neutron activation analysis with long-lived

radionuclides (|!g/g dry tissue)

COI

AgCoCsEuFeHgRbSbScSeSrZn

RangeRangeRangeRange

MS±S.D.(S.E.)RangeRangeRangeRangeRange

MS±S.D.(S.E.)MS±S.D.(S.E.)

Iliac crest

n=24

<0.00090-<0.10<0.00005-0.0429<0.00020-0.512<0.00002-0.0021

213±130 (26)<0.0017-0.011<0.090-2.52

0.00030-0.117<0.00002-0.0026

<0.0037-2.1229±182(37)

67.9±21.3(4.3)

Tooth(crown)

n=22

<0.0010-<0.040<0.00006-0.0098<0.00020-<0.009<0.0003-0.00164.7±3.1(1.2)*

<0.00010-0.0011<0.058-<1.87

<0.00020-0.035<0.00003-0.0004<0.0040-<0.070

166±149(32)183±170(37)

* „ =n = 7

Table 15Age dynamics for contents (Mean±S.D.) of Fe, Sr, and Zn in the iliac crest and teeth of healthy men based on the data of

neutron activation analysis with long-lived radionuclides (jig/g dry tissue)

Object ofstudy

ElementAge (years)

Fe

Sr

Zn

Sr

15-30

223±147(60)

199±126(51)

66.1±9.9(4.0)

225±137(56)

31-40

135±87(33)

134±56(21)*

58.0±14.0(5.3)

163±221(83)

41-50

227±167(59)

310*204(72)*

76.5±26.3(9.3)

148±85(35)

51-55

237±52(30)

300±347(200)

71.6*35.8(20.7)

91.4±25.8(14.9)

CO

tooo Iliac crest

Tooth (crown)Zn 126±32(14) 185± 122(46) 129±30(13) 138*27(16)

p<0.05

Table 16Correlation coefficient ( r ) of Sr in the iliac crest and tooth crown of healthy men (based on dry tissue)

Age (years) F^35 36^55 15-55

_ o.87* O08 0.25CDi

^ * p<0.001

BMDg/cm2

1.40

1.J0

1.20

1.10

1.00

0.90

Women, total body

•

—

-

:

—

. •

- - r ~ 1. -A— .

» Ti

L. __ i_ ._ J

4f

1L

15 20 25

1

30

i

35Age, years

i

40!45

i

50

i

55

BMD_g/cm2

1.40

1.30

1.20

1.10

1.00

0.90

Men, total body

r r T

• /

T

15

1

20

1

251 - -

30

1

35

Age, years

—i

40 45

I

50

i

55

Fig. I. Total body DXA BMD of healthy Russian women (n=240) and men (n=240)

9-30

BMDg/cm2

1.40

U 0

1.20

1.10

1.00

0.90

Women, spine

I .

-1 1 1 1 1 I—

15 20 25 30 35 40

Age, years45 50 55

BMDg/cm2

1.40

1.30

1.20

1.10

1.00

0.90

Men, spine L2-L4

-T-

15 20 25 30 35 40Age, years

45 50 55

Fig.2. Spinal DXA BMD of healthy Russian women (n=240) and men (n=240)

9-31

g/cm2

1.20

1.10

1.00

0.90

0.80 .

0.70

Women, femoral neck

I T II

-•

T

— ——

T

• — I •

1

T

1

15 20 25 30 35 40Age, years

45 50 55

BMD „g/cm2

1.30

1.20

1.10

1.00

0.90

0.80

Men, femoral neck

15 20 25 30 35 40Age, years

45 50 55

Fig 3. Femoral DXA BMD of healthy Russian women (n=240) and men (n=240)

9-32

BMD n spine L2-L4

g/cm2

!.4O

I.JO

1.20

1.10

1.00

0.90

• •_* . .

20 25 30 35 40 -15

Age, years50

BMDg/cm2

1.30

1.20

1.10

1.00

0.90

0.80

femoral neck

Mean+S.D.

Mean

Mean-S.D.

Mean-2S.D.

Mean-2.5 S.D.

Mean+S.D.

Mean

Mean-S.D.

Mean-2 S.D.

Mean-2.5 S.D.

20 25 30 35 40 45

Age, years50

Fig. 4. DXA BMD of examined cleaners (n=47)9 - 3 3

-p

CO

80

60

40

20

J all examined cleaners

cleaners with osteopenia and osteoporosis

• / z

up to 10 cSv between 11 and 23 cSv

Fig. 5. Distribution of cleaners by the level of radiation dose

XA0054928

Agency Research Contract No. 8197/RB

STUDIES OF OSTEOPOROSIS IN SINGAPORE USINGISOTOPE RELATED TECHNIQUE

Department of Orthopaedic SurgeryNational University of Singapore

Lower Kent Ridge RoadSingapore 119074

Chief Scientific Investigator: Professor Kamal BoseProgress Report: June 1997- June 1998

10-1

/in

IntroductionOsteoporosis is one of the common medical problems facing an aging population. It isprojected that the number of hip fractures occurring worldwide will increase from 1.66million in 1990 to 6.26 million by the year 2050 (1). Many studies have shown a strongrelationship between low bone density and increased fracture risk. The World HealthOrganization (WHO) has proposed a threshold of bone mineral density (BMD) basedon peak bone mass, with osteoporosis denoting a value of BMD that is 2.5 standarddeviations or more below the peak bone mass (2). The interpretation of bone mineraldensitometry results for a particular individual relies on valid reference data from arepresentative population sample. Recent studies have suggested that apart from bonedensity, there are racial differences in hip axis length. A longer hip axis length isassociated with an increased risk of fracture (3). The hip axis length of Asian and blackwomen were reported to be significantly shorter than white women (4). Japanesewomen were found to have a shorter hip axis length and a lower hip fracture incidencethan American women despite lower bone density values (5).The main objectives of this study was to a) determine the age of peak bone density ineach study group, b) quantify differences in bone density as functions of age and sex.The hip axis length was also measured.

MethodsThe recruitment of subjects for this study was done through the hospital staff and theirfamily as well as through an advertisement in the local media. Normal healthy subjectsfrom the three major ethnic groups i.e Chinese, Malays and Indians , of both sexes ,aged 15 -50 years were recruited. All subjects completed a questionnaire whichdetailed their medical history. The exclusion criteria include;• moderate or severe scoliosis• known chronic illness (past or present) of more than 3 months, eg renal, liver or

thyroid disease• known chronic use of any medication eg. steroids, estrogen or fluoride• previous low trauma fracture• prolonged immobilization ( > 1 month)

A total of 204 males and 318 females who met the initial criteria were recruited (Table1). Height and weight of the subjects were measured and the body mass index (BMI)calculated using body weight in kilograms divided by the square of height in metres.The subjects were interviewed by the same technologist using the WHO questionniareand included details on lifestyle factors such as food intake, exercise and medication.Bone mineral density (BMD) was determined using a LUNAR DPX-L (Lunar Corp.,Wisconsin, USA) at the lumbar spine and femoral neck. The BMD of the spine wasanalysed in the region L2-L4 while analysis of the hip gave BMD values for the femoralneck, trochanter and Ward's triangle. The hip axis length (HAL), defined as the lengthbelow the lateral aspect of the greater trochanter through the femoral neck axis to theinner pelvic brim was also measured. This was done by placing the software rulerparallel to the femoral neck axis with the ends of the ruler positioned at the boneedges.A dedicated quality assurance programme was done throughout the period of the studyto ensure instrument consistency and accuracy of results. Daily calibration wasperformed by the system's internal calibration system. Daily scans of a phantom was

10-2

also performed to ensure consistent and reliable readings. The European SpinePhantom (ESP) has also been scanned.The results are given as mean + SD. Student's t test was performed to test differencesbetween the male and female groups, and between the three ethnic groups.

ResultsI) Sex differences

Table 2 summarizes the anthropometric and BMD data for the males and females.The male subjects were significantly taller and heavier than the female subjects.However, there was no significant difference in body mass index of males and females.There was no significant difference in lumbar spine BMD of males and females.However, femoral neck BMD of males was significantly higher than females.In males, peak BMD in both the lumbar spine and femoral neck occurred in the agegroup 25-29 years (Table 3). There was a linear regression of BMD with age in males.In females, peak BMD occurred in the age group 35-39 years (Table 4).There was no significant difference in peak spine BMD between the sexes (1.170 ±0.118 g/cm2 for males versus 1.224 ± 0.135 g/cm2 for females). However, males werefound to have significantly higher peak density in the femoral neck ( 1.076 g/cm2 formales versus 0.969 g/cm for females; p < 0.01). Males attained the peak BMD in theage group 25-29 years compared to the females where the peak BMD was attained adecade earlier.Hip axis length was moderately correlated to height in both males ( r =0.500) andfemales (r= 0.545) but not correlated to weight and body mass index. The hip axislength in males was significantly longer in males than in females ( 10.92 cm in malesversus 9.76 cm in females).

II) Racial differencesThe data was also analysed according to the 3 major ethnic groups. Among the males,there was no racial differences in height, weight, spine or hip BMD. However, Chinesemales had longer hip axis length than the Indian and Malays (Table 5). Similarly ,Chinese females had significantly longer hip axis length (p < 0.05) than Indian orMalay females although there was no difference in height. Chinese females also hadsignificantly lower body weight ( p < 0.01). There was no significant difference in bothlumbar spine and hip BMD among the 3 races (Table 6).

III) Lifestyle factorsNone of the females interviewed had any history of smoking. In males, only 15% of thesubjects smoked at least a pack of cigarettes a day and over 70% of this cohort wereMalays. 17% of males had at least a glass of milk everyday, while in females, only 13%drank milk regularly. According to a survey done by the Ministry of Health (7), the meanintake for calcium for men and women is 567 mg and 448 mg respectively (Table 7).Calcium intake was found to be highest among the Indians for both men (714 mg) andwomen (506 mg). The RDA for calcium for adults for all age and sex groups is 500 mg.49.8% Chinese men and 70.7% Chinese women have calcium intakes less than theirRDAs.Most of the subjects interviewed led sedentary lifestyle with no regular exercise. Only25% males and 17.6% females did regular weight bearing exercises at least once aweek.

10-3

4)5-

ConclusionsFrom the data collected so far, there was no significant differences in bone densityamong the three ethnic groups in both sexes. A recent study in Singapore however,showed that Chinese had higher hip fracture incidence compared to the Malays orIndians (6). Mean calcium intake among the Chinese was also the lowest among theraces. The hip axis length in Chinese was found to be significantly longer and this couldpartly explain the racial differences in hip fracture rates.In comparison to the western population, Singapore females had significantly shorterhip axis length (9.8 ± 0.51 cm) compared to American women (10.5 ± 0.62 cm).

Other related work performedOur department is involved in a multi-centre study involving Singapore, Philippines,Thailand and Malaysia to determine the risk factors for hip fracture, using a casecontrol design: dietary status, load bearing activites, cigarette smoking, alcholicbeverages, low body mass index and role of medical diseases. This one year studystarted in August 1997. Patients over 50 years of age with an incident hip fracture dueto minimal trauma were interviewed. Patients were recruited from five majorGovernment/Restructured Hospitals in Singapore. Controls, over 50 years of age wasselected within the age and sex strata.A total of 303 females and 126 males with hip fractures were recorded in a year. Therewere more than twice as many hip fractures in females than in males (ratio 2.4:1).We are also doing another study with the University of Tokyo, Japan to look at theenvironmental factors regulating the relationship between osteoporosis and hipfractures. This study involves Singapore, Japan, Hong Kong and Pakistan.

References

1. Cooper C, Campion G, Melton LJ III. Hip fractures in the elderly: a worldwideprojection. Osteoporosis Int 1992; 2:285-2892. Kanis JA, Melton LJ III, Christiansen C. Perspective : the diagnosis of osteoporosis. JBone Miner Res 1994;9:1137-11413. Faulkner KG, McClung M, Cummings SR. Automated evaluation of hip axis lenghthfor predicting hip fracture. J Bone Miner Res 1994:9(7): 1065-704. Cummings SR, Caulet JA, Palermo L, Ross PD, Wasnich RD, Black D, Faulkner KG.Racial differences in hip axis length might explain racial differences in rates of hipfractures. Study of Osteoporotic Fractures Research Group. Osteoporosis Int1994;4(4):226-295.Nakamura T, Turner CH, Yohsikawa T, Slemenda CW, Peacock M, Burr DB, MizunoY, Orimo H, Ouchi Y, Johnston CC Jr. Do variations in hip geometry explain differencesin hip fracture risk between Japanese and white Americans? J Bone Miner Res1994:9(7):1071-6.6. Lee ST. Osteoporosis and the incidence of hip fractures in Singapore. Proceedings,Third Asian Symposium on Osteoporosis, 1992, pp 6-107. Food Consumption Study 1993. Food & Nutrition Department. Ministry of Health,April 1994

A16 10-4

Table 1. Number of subjects in the five year age group

Age group15-1920-2425-2930-3435-3940-4445-50

Males26283027333129

Females27273047475090

Table 2. Anthropometric and bone mineral density data for the male and femalepopulation

nHeight (cm)Weight (kg)

Body mass indexHip axis length

Lumbar spine BMD (g/cm2)Femoral neck BMD (g/cm2)

Males204

170.1 ±6.3*68.2 ±10.3*23.53 ±3.3010.92 ± 0.49*1.135 ±0.1341.014 + 0.142*

Females318

158.5 ±5.257.0 ±9.8

22.69 ±4.09.76 ±0.51

1.187 ±0.1270.939 ±0.111

* Significant difference at p < 0.01

10-5

Table 3. Characteristics of study variables of males in 5 year age group

Agegroup(years)15-1920-2425-2930-3435-3940-4445-50

Height(cm)

173 ±6.7176 + 6.3173 ±5.7168 + 6.9170 ±4.8170 ±6.4168 ±4.9

Weight(kg)

53 + 10.768 ± 9.167 ± 9.566 ±10.571 ± 8.571 +12.569+ 9.1

Body massindex(BMI)

17.8 + 2.221.9 ±3.122.5 ± 3.223.1 ±2.624.7 ± 3.024.6 ± 3.724.4 ±3.1

Hip axislength (cm)

11.12 ±0.1111.19 ±0.5510.93 + 0.6110.88 ±0.5010.85 ±0.5710.86 ±0.4010.91 +0.35

Lumbar spineBMD (g/cm2)

1.120 ±0.0591.160 ±0.1031.170±0.118"1.165 ±0.1041.162 ±0.1211.144 ±0.0951.147 ±0.131

Femoral neckBMD (g/cm2)

1.028 ±0.0701.074 ±0.1181.076 ±0.122"1.055 ±0.1341.052 ±0.1160.989 ±0.1240.975 ±0.119

" peak BMD

Table 4. Characteristics of study variables of females in 5 year age group

Agegroup(years)15-1920-2425-2930-3435-3940-4445-50

Height(cm)

164160160159159157158

±±±±±±±

11.26.56.34.64.45.43.9

Weight(kg)

50545456575859

±±±±±±±

7.28.98.8

10.99.6

10.59.2

Body massindex(BMI)

18.7 ±2.321.2 ±3.521.1 ±2.921.9 ±4.322.8 ±3.923.8 ±4.523.7 ±3.7

Hip axislength (cm)

9.779.689.759.699.689.739.89

+ 0.94±0.59±0.56± 0.45^±0.48+ 0.49±0.48

Lumbar spineBMD (g/cm2)

0.9921.1741.1711.1881.2241.2131.210

±0.112±0.137±0.117±0.131±0.136"±0.117±0.117

Femoral neckBMD (g/cm2)

0.909 ±0.1380.940 ±0.1290.946 ±0.1370.959±0.113

0.969 ±0.125"0.957 ± 0.0900.938 ± 0.088

peak BMD

Ate 10-6

Table 5. Anthropometric, BMD and hip axis length in Chinese, Malay and Indian males

nAge

Height (cm)Weight (kg)

Body mass indexLumbar spine BMDFemoral neck BMDHip axis length (cm)

Chinese194

33.6 +9.0170.8 ±6.167.1 ±10.922.96 ± 3.521.127±0.1100.997 ±0.12111.03 ±0.46+

Malay73

37.0 ± 7.7169.2 + 5.469.8 ± 9.3

24.28 ± 3.041.155 ±0.1621.042 ±0.15010.79 ± 0.48

Indian51

37.3 ± 8.3169.8 ± 8.769.1 ±9.9

23.95 ± 2.831.121 ±0.1461.014 ±0.19010.79 ±0.52

+ significant difference at p < 0.05

Table 6. Anthropometric, BMD and hip axis length in Chinese, Malay and Indianfemales

nAge

Height (cm)Weight (kg)

Body mass indexLumbar spine BMDFemoral neck BMDHip axis length (cm)

Chinese194

38.2 ±9.4159.2 ±4.855.2 ±7.5

21.64 ±2.951.199 + 0.1310.937 ± 0.0939.84 ± 0.53++

Malay73

36.3 ±8.3157.1 ±4.860.4 ±11.624.73 ±4.811.181 ±0.1130.931 ±0.1389.63 ± 0.44

Indian51

35.3 ± 8.3159.3 ±6.360.5 ±12.423.63 ± 4.751.149 ±0.1270.959 ±0.1269.65 ±0.53

significant difference at p < 0.01

Table 7. Mean energy and nutrient intakes in males and females

Energy intake (kcal)Protein (g)

Fat (g)Carbohydrates (g)

Iron (mg)Ca (mg)

Sodium (mg)Vitamin A (ug)

Males223285.474.6300.6

165673920642

Females170967.457.7228.612.64482959605

10-7

RESEARCH CONTRACT 8406/RB

Progress Report Pec . 1994 - Jul. 1998) for XA0054929

(TO BE PRESENTED AT SAO PAULO, BRAZIL AT THE RESEARCHCORDINATION MEETING 24-27 AUGUST 1998)

STUDIES OF OSTEOPOROSIS IN SOUTH AFRICAUSING ISOTOPE-RELATED AND OTHER TECHNIQUES

As Part of Co-ordinated Programme

COMPARATIVE INTERNATIONAL STUDIES OF OSTEOPOROSISUSING ISOTOPE-RELATED AND OTHER TECHNIQUES

1. ESSENTIAL INFORMATION

(a) Contract number: 302-E4-SAF-8406B5-SAF-23144

(b) Title of Project: Studies of Osteoporosis in South Africa usingIsotope-related and other techniques.

(c) Locations where the research is done:Academic Hospitals of the Medical Faculty, University of CapeTown, Cape Town, South Africa (Groote Schuur and Red CrossChildren's Hospital) andNational Acceleration Centre, Faure, Western Cape, South Africa

(d) Chief Scientific Investigator: Dr S Wynchank

METHOD AND SUBJECTS

Daily dietary intake information for calcium, protein, magnesium, zinc,copper, manganese and flourine, was sought for those subjects for whom bonemineral density (BMD) were obtained. These are urban Africans (male andfemale, aged 16-55 years), those of mixed racial origin and European all withinthe same age range. Relevant South African literature was studied, inparticular two review documents (1,2).

The measurements presented here were made with a Hologic (Model 1000)osteodensitometer, located in the Department of Nuclear Medicine of theUniversity of Cape Town. The data presented in this report are BMDmeasurements in 1546 normal urbanised female subjects and 216 similar malesof various ethnic groups, who live in the greater Cape Town area. Theyrepresented the three main ethnic groups who live near to Cape Town in theWestern Cape. Of them 1371 females and 216 males are aged between 16 and55 years. Details of the height, mass and of relevant social and medical historywere recorded for each subject. Ethnic groups, sex, age ranges and totalnumbers of the subjects are given in Table 1.

11 -1

st&o

BMD in g/cm2 was measured at the following sites:

(a) In the bodies of lumbar vertebrae numbers 1, 2, 3, 4, (denoted LI, L2, L3,L4) and the mean BMD value was obtained.

(b) In the left greater trochanter.(c) In the left intertrochanteric region.(d) In the left femoral neck.(e) In the left triangle of Ward.

3. RESULTS

Results will be given below for the mean BMD of LI, L2, L3 and L4 and thetotal hip BMD. (The latter is the mean BMD for femoral neck, trochanteric andintertrochanteric regions.) These representative results (means and standarddeviations [SD]) indicate the trends observed. Data for females are given inTables 2 and 4 (lumbar and hip data respectively) and similarly for males inTables 3 and 5.

Two principal sources (1, 2) of dietary intake information provided part of therequired information. This is given in Tables 6-10. Information for copperand fluorine is regrettably unavailable.

4. DISCUSSION

Since the previous report a large number of additional females have beenmeasured and, for the first time, male subjects also. There remains a lack ofdata from male African subjects and also data for the youngest subjects. Thenon dominant side was chosen for the measurements, this of course normallybeing the left.

Beyond the usual age of the menopause there is a slight fall in BMD forfemales. Data from African subjects is clearly not significantly lower that thatof the other races, in spite of contrary expectations resulting from lessprivileged social and economic circumstances. Statistics continue to be betterfor European and Mixed Racial Origin subjects, than for Africans. The SDs areof course of little significance when calculated for very few data in a group,but when numbers exceed 10 or 20 the SD, when considered in conjunctionwith the tabulated means, shows that there is little difference in BMD betweenthe three ethnic groups.

It still remains to be determined what are the relative contributions of geneticand dietary influences to differences in BMD. Further BMD studies, especiallyin Africans, preferably accompanied by investigations of dietary and otherrelevant habits will be necessary to answer such questions. In the ruralsetting, the African diet differs from that of the other groups. But there isevidence to show that such differences are becoming greatly reduced with therapidly increasing urbanisation being experienced in South Africa and in mostother African countries. (Such evidence arises both from dietary surveys andfrom incidence of the burden of life-style associated diseases in urbanisedAfricans.)

A&A11 -2

There is a striking difference in intake of Protein, Mg and Zn for men andwomen with male consumption being consistently higher. The statistics forthese intake data result from measurements on several hundred individuals.Such differences between dietary intakes of the sexes is less marked forpersons of mixed racial origin than for the other two groups. However, thesedifferences in dietary intake between the sexes are not reflected in anysignificant differences in values of BMD, whether for hip or spine. A finalpoint that is important for analysis of any relation between dietary intake andBMD, is that our data for BMD of African men arises from small numbers(never more than 3 in any 5 year age group).

We are very pleased to be able to contribute to this CRP and trust data fromthe particular ethnic groups which comprise our subjects will allow ourcontribution to be of use to you.

5. ACKNOWLEDGEMENTS

The research team wishes to express its gratitude to Mmes Linda Bewerungeand Ann Langley for valuable assistance in making BMD measurements, toMs Patricia Josias for meticulous preparation of this manuscript, and to DrChris Seebregts and Mr Paul Selby for aid with the data reduction andanalysis. Dr Petra Wolmarans is thanked for invaluable assistance in locatingdietary intake data.

6. REFERENCES

1. The nutritional status of South Africans: A review of the literature from1975-1996. Vorster HH, Oosthuizen W, Jerling JC, Weldman FJ, BurgerHM. Health Systems Trust, Durban, 1997.

2. Food habits, dietary intake and health of older coloured South Africans,Charlton K, Wolmarans P. HSRC/UCT Centre for Gerontology, Universityof Cape Town, Observatory, 1995.

11 "A&L

Table 1 The subjects studied to date.

Ethnic GroupEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originAfricanEuropeanMixed originEuropeanMixed originEuropeanEuropeanEuropeanEuropean

-

European

Age range(Vr)

16-20

21-25

26-30

31-35

36-40

41-45

46-50

51-55

56-60

61-65

66-7071-7576-8081-8586-9091-95

FemalesN931292113767756706

1071081186976

172114132151269452

582

313033-1

Malesn1212531016381421319215222821228143

11 -4

Table 2A Mean lumbar (L1-L4) vertebral bone mineral density (g/cm2) inEuropean women

Age range(vr)

16-2021-2526-3031-3536-4041-4546-5051-5556-6061-6566-7071-7576-8081-8586-9091-95

Table 2B

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-5556-6061-65

Table 2C

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-55

n9

293756

10786

1722154558313033-1

Lumbar vertebral (L1-L4)Mean0.950.980.961.021.001.041.021.020.900.880.810.840.801.20

-1.34

BMD in stem2

SD0.090.220.140.110.120.120.110.120.140.120.190.110.070.31

--

Mean lumbar (L1-L4) vertebral bone mineraldensity (g/cm2) in women of mixed racial origin

n321677010897114126

22

Lumbar vertebral (L1-L4)Mean0.940.980.950.980.971.001.031.000.880.96

BMD in gfcm2

SD0.100.160.100.120.120.120.140.140.070.14

Mean lumbar (L1-L4) vertebral bone mineral density(g/cm2) for African women

n1

L 176

116

139

Lumbar vertebral (L1-L4)Mean0.870.971.051.031.051.05 _j0.970.99

BMD in g/cm2

SD--

0.140.110.100.020.130.12

11 -5

Table 3A Total hip bone mineral density (g/cm2) in European women

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-5556-6061-6566-7071-7576-8081-8586-9091-95

Table 3B

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-5556-6061-65

Table 3C

Age range(yr)

16--2021-2526-3031-3536-4041-4546-5051-55

n9293756

107861722154558313033-1

Total hip BMD in g/cm2

Mean0.900.920.940.940.940.930.920.930.830.820.800.770.720.86

-0.91

SD0.120.130.130.100.110.110.100.100.100.120.100.110.190.08

--

Total hip bone mineral density (g/cm2) in womenof mixed racial origin

n3216770

10897

11412622


Mean0.900.870.940.940.930.970.960.950.791.05

SD0.120.160.100.090.100.110.110.100.010.14

Total hip bone mineral density (g/cm2) forAfrican women

N1176116

139

Total hip BMD in a/cm2

Mean0.841.060.971.040.940.940.921.02

SD--

0.160.090.160.110.140.15

11 -6

Table 4A Mean lumbar (L1-L4) vertebral bone mineraldensity(g/an2) in European males

Age range(vr)

16-2021-2526-3031-3536-4041-4546-5051-55

n1210813158

28

Lumbar vertebral (L1-L4) BMD in stem2

Mean0.960.850.981.011.040.981.051.00

SD-

0.060.140.130.150.150.130.12

Table 4B Mean lumbar (L1-L4) vertebral bone mineraldensity (g/cmz) in males of mixed racial origin

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-55

n25161419222114

Lumbar vertebral (L1-L4) BMD in g/cm2

Mean0.860.961.021.041.040.970.940.98

SD0.030.200.140.140.120.130.090.18

Table 4C Mean lumbar (L1-L4) vertebral bone mineral density(g/cm2) for African males

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-55

n13322223

Lumbar vertebral (L1-L4) BMD in g/cm2

Mean0.910.990.870.960.980.900.940.93

SD-

0.140.050.040.230.090.190.07

11 -7

Table 5A Total hip bone mineral density (g/cm2) in Europeanmales

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-55

n12108

1315828


Mean0.931.071.070.910.971.030.990.92

SD-

0.260.070.10

L 0.100.150.110.09

Table 5B Total hip bone mineral density (g/cm2) in males ofmixed racial origin

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-55

n25161419222114


Mean0.900.99

[ 0.981.020.990.950.930.98

SD0.020.100.130.100.190.100.100.14

Table 5C Total hip bone mineral density (g/cm2) forAfrican males

Age range(yr)

16-2021-2526-3031-3536-4041-4546-5051-55

n|_ 1L 3

3

L 2

2223


Mean0.821.050.900.931.060.810.930.99

SD-

0.190.110.030.15

--

0.09

11 -8

Table 6A Daily calcium dietary intake of South Africans(means and standard deviations (SD)) in mg.These intakes were measured with the 24 hourrecall method.

Age Group (yr)GenderChildren. 2-5.9Children, 6-10.9Boys, 11-15.9Girls, 11-15.9Men, 16-24.9Women. 16-24.9Men, 25-64.9Women, 25-64.9

WhiteMean SD n

62EL613742613

1033618737560

360400441347528386396349

2 6 ,12

163164124121

Urban BlackMean SD n

354404418407570335

352 i 515399 369

201238277244457223450253

1761347475861

459594

ColouredMean SD n552 388617

LJ63290500518481367

369294186336331

4324

1731675853

344 288214 301

IndianMean SD n

384556474310

275315335211

185140254257

Rural blackMean SD n320302309325

343

320278256231

274

11847

^140160

95

Table 6B Daily calcium dietary intake (mg) of SouthAfricans obtained using methods other than 24hour recall.

Age Group (yr)GenderChildren, 0-1.9Children, 2-5.9Children, 6-10.9Boys, 11-15.9Girls, 11-15.9Men, 16-24.9Women, 16-24.9Men, 25-64.9Women, 25-64.9

WhiteMean SD n963807

803991917

859277

451571306

93942

3117323

Urban BlackMean SD n442539

967732

399236

677633

1753746_j

5674

ColouredMean SD n1454

_ 899

478437

2053434

237306

17135

1614

IndianMean SD n

582795

991.5765

511281

498372

43596

126128

Rural blackMean SD n

390

655

205

270696 | 371

681

6189

11-9

Ate

Table 7A Daily protein dietary intake (in grams) of SouthAfricans using the 24 hour recall method.

Age Group (yr)GenderChildren, 2-5.9Children. 6-10.9Boys. 11-15.9Girls. 11-15.9Men, 16-24.9Women, 16-24.9Men, 25-64.9Women. 25-64.9

WhiteMean SD n

47.8^60.283.462.7

102.763.197.4

59

17.624

31.522.338,225.238.6

22

2612

163164124121352399

Urban BlackMean SD n40.945.352.9

_54.27049

78.156.5

16.128.520.2 j22.1

3836

43.528.2

17613

1151755861

459684

ColouredMean SD n57.1

7762.359.3

83__72_

82.158.1

3230.5

2823.8

2825

35.626.9

4324

2953005853

288301

IndianMean SD n

68 j68

73.157.169.5

47

2723

32.126.238.225.1

9897

185140254257


4055.457.156.2

83

2430

27.229.8

60

118118140160

95

Table 7B Daily protein dietary intake (in grams) of SouthAfricans, using methods other than 24 hourrecall method.

Age Group (yr)GenderChildren, 0-1.9Children, 2-5.9Men. 16-24.9Women, 16-24.9Men. 25-64.9Women. 25-64.9

WhiteMean SD n

43.760.3162

27.112.817.7

82.8 26.7120

86.250.127.1

93942

1534726623

Urban BlackMean SD n31.547.6

10371.2

20.613.2

60.433.3

1753746

5674

ColouredMean SD n56.4

60

82.865.8

6018.4

23.627.4

17135

1614

IndianMean SD n30.5

62108

83.2

17.618.1

3634.0

43596126128


I32.8112

75.870.3

11.437.2

27.626.9

68117

6189

11 -10

Table 8A Daily magnesium dietary intake (in mg) ofSouth Africans using the 24 hour recall method.

Age Group (yr)GenderChildren, 2-5.9Children, 6-10.9Boys, 11-15.9Girts. 11-15.9Men. 16-24.9Women, 16-24.9Men, 25-64.9Women. 25-64.9

WhiteMean SD n

207206j

370.9222312218

7861

15183.8115

93.8

2612

124121259399

Urban BlackMean SD n165.6

218

287199326206

80.5143

14594

72996

1313

5861

383481


263229236168

104125

9775

11169.8

4324

5853

2888301

IndianMean SD n

256212270207

10196

11688.3

10769

254257


270257254

434

999892

152

4"8f

io:-

96

Table 8B Daily magnesium dietary intake (in mg) ofSouth Africans using methods other than the 24hour recall method.

Age Group (yr)GenderChildren, 0-1.9Children, 2-5.9Men, 16-24.9Women, 16-24.9Men, 25-64.9Women, 25-64.9

WhiteMean SD n151249

254474302

82.879.5

9216792

93942

318023


109226

503313.7

52.158.9

252146

1753746

5674


263228

20684.4

101111

17135

1614

IndianMean SD n112266386287

59.781.6129

93.5

43596126128


179

440374

51.6

223137

681

6189

11 -11

AHo

Table 9A Daily zinc dietary intake (in mg) of SouthAfricans using the 24 hour recall method.

Age Group (yr)GenderChildren. 2-5.9ChildrenJ>-10.9Bovs. 11-15.9Girls, 11-15.9Men. 16-24.9Women, 16-24.9Men, 25-64.9Women. 25-64.9

WhiteMean SD n

12_,9

14.29^

14.28.4

64.b

64.26.93.7

163164124121352399


5.76.1

98

9.96.8

10.97.5

2.73.6

55

6.95.19.95.8

131347475861

383481

ColouredMean SD n

911.1

97.5^

11.710.111.27.7

6.25.4

6^4.55.54.36.34.7

4324

173167

5853

288301

IndianMean SD n

9.87.69.86.4

7.85.77.34.6

10769

254257


98.47.6

44.9

4

47140160

— • HI —

Table 9B Daily zinc dietary intake (in mg) of SouthAfricans using methods other than the 24 hourrecall method.

Age Group (yr)GenderChildren, 0-1.9Children. 2-5.9Men, 16-24.9Women, 16-24.9Men. 25-64.9Women, 25-64.9

WhiteMean SD n

78.5

9.216.512.2

5.33.2

3.37.44.3

93942

31L 173

23


4.46.8

14.69.7

3.21.8

7.14.17

1753746

5674

ColouredMean SD n

9.18.6

10.48.5

9.82.7

3.54.3

17135

1614

IndianMean SD n

5.28.8

16.612.8

4.42.76.26.4

43596126128


4.7

11.911.3

1.5

5.04.23

681

6189

11 -12

Table 10A Daily copper dietary intake (in micrograms) forelderly South Africans (aged > 65 years) ofmixed racial origin.

Age Group (yr)GenderMen, > 65Women, > 65

ColouredMean SD n

1.51.3

0.90.8

96104

11 -13

/ti-L

XA0054930

(Presented at the second RCM on Osteoporosis, Sao Paulo, Brazil, August 24-27,1998)

Contract #Tur-8198

STUDY OF OSTEOPOROSIS THROUGH THE MEASUREMENT OF BONE MINERAL DENSITYAND TRACE ELEMENTS

N.K. Aras1*, G.Yilmaz1, S.Alkanl, F.Korkusuz2, M.Ungan2, L.Kuscu3, Y.Laleh3, F.EksiogluJ,B.Sepici4, U. Gunel5

1 Department of Chemistry, Middle East Technical University, Ankara, Turkey2 Medical Center, Middle East Technical University, Ankara, Turkey3 Duzen Laboratories, Ankara, Turkey4 Ankara State Hospital, Department of Orthopedic Surgery, Ankara, Turkey5 Numune State Hospital, Department of Orthopedic Surgery, Ankara, Turkey

*Chief Scientific Investigator.Present Address: Fatih University, Beylikduzu, Buyukcekmece, Istanbul, Turkey

I. Introduction

The main purpose of this study was to establish a relation, if any, between bone mineral density, BMD, of thehealthy Turkish population of the ages between 15 and 50 with social and demographic information, familyhistory of fractures, personal and inherited characteristic, smoking and alcohol habit, history of fertility, levelof physical activity, food consumption especially trace elements and other variables. Most of these relationswere discussed in the last RCM in San Diego, CA, October 7-10,1996. Since then we have concentrated ourwork on more BMD and trace element measurements in bone.

2.Bone Mineral Density Measurements

Bone mineral density measurements were performed at the Ankara Duzen Laboratory with DEXA, HalogicQDR-2000 and QDR-4500 machines. The spinal column (L1-L4) and right hip region (femoral neck,trochanteric region, intertrochanteric area and ward triangle) of the individual were evaluated in the anteriorposterior and lateral directions. Prior the DEXA, the height, arm span and weight of the individual is assessed.Daily calibration of DEXA equipment is performed according to a standard protocol as described by thesupplier of the instrument. The QDR-4500 is also calibrated with he phantom sent by Dr. Eugene Me Closkey,Sheffield University. Bone mineral density results were evaluated according to the age groups. Two hundredand fifteen (93 male and 122 female) subjects' BMD results have been measured with this system up to now.Results of the measurements and age distributions are given in Table 1 for female and male groups.

We recently purchased a new machine, LUNAR DPX and placed at the Medical center, Middle East TechnicalUniversity. Unfortunately we were not able yet to test this machine with the phantom supplied by Dr. E. MeCloskey. However, daily calibration is performed according to standard protocol as described by LUNAR Co.As done before, subjects were randomly selected among the university students, staff members and employers.The results of 106 female and 35 male subjects are given in Table 2. The Hologic and Lunar results are alsorepresented in Fig. la and Fig. lb respectively. As seen the maximum BMD corresponds to ages 30-40.

12-1

Table 1. Bone Mineral Density results of Female and Male subjects measured with Halogic Machine

FemaleAge

15-1920-2425-2930-3435-3940-4445-49Male15-1920-24 ^25-2930-3435-3940-4445-49

n1319

15

1617

2022

1215

1211

15

1216

BMI19.3 ±1.720.0 ±1.5213 ± 3.12l.4±2.224.4±3.623.7±3.925.5±4.2

21.1 ±2.421.6±2.923.4±2.125.3 ±3.224.4 ±2.825.4 ±2.525.4 ±2.7

Weight (kg)54 ±655 ±557 ± 1156 ±6

65 ±1061 ±1166 ±9

64 ±965 ±1071 ±9

79+1271 ±874 ±9

74 ±10

Hejght (cm)167 ±7165 + 5164 + 6161 ±4163 ±6160 + 7161 ±7

174 ±5168 ±5175+8177 ±7171 ±6171 ±4170 ±5

L1-L4896 ± 85981 ±90987 ± 80__j974 ± 78

1027 ±119958 ±140948 ±126

892 ±104914 ±71

952 ±1061018 ±102963 ± 62

950 ±107913 ±115

L2-L4783 ±112827 ±126815 ±102790 ± 96804 ± 95795 ± 126783 ±113

857 ± 94914 ±71870 ±101845 ±119798 +104792 ±132779 ± 102

TotalL J 7 3 ± 96

911 ±86864 ±110

L 849 ±122898 ± 122849 ±144857+105

943 ± 96813 ±118967 ±1591009 ±99958 + 95977 ± 96940 ±112

Table 2. Bone Mineral Density result of Female and Male Subjects with LUNAR DPX Machine

FemaleAge

15-1920-2425-2930-3435-3940-4445-4950-54Male15-1920-2425-2930-3435-3940-4445-49

n1510

4

16

17

16

22

32

9

23

1

5

2

5

BMI

20.0 ±2.620.6 ±2.722.4 ±1.521.3 ±2.1

22.1 ±2.625.5 ±3.725.7 ±4.4

26.5 ±4.0

20.6 ±2.120.9 ±1.024.0 ±4.5

33.6

26.1 ±2.122.3 ±0.1

26.0 ±4.3

Weight (kg)53 ±553 ±761 ±956 ±557 ±666 ± 1365 ±11

71111

64 ±861 ±874 ±16

97

81146411273 ±19

Height (cm)

163 ±7160 ±516419163 ±6161±5161 ±5160 ±5163 ±5

177 ±5170 ±7175 ±5

170

176 ±8169116166+10

L2-L41072 ±1011194 ±2981234 ± 1961169 ±1421093 112012801116113211301103 + 140

115411631146 ±38311361 145

1223

125211281137 + 24611381131

Neck1015 ±107923 11181126 + 181926 ±100915 ±1129801118925 1179916 + 106

1128H661388

8821120

958 ± 781043 ±379958 1194

Total1006 ±10793011201146 ±175963 ±120939 ±1071042 ±1231009 ±130956+121

112711131467

8921138

997 ± 729641216991 ±219

12-2

1050

1000

950

i •« 900 -! = i: t !

850 -i

800 --

• r - i

a'

1'JS•c

iIj

11|2

I

1?,

a

|

15-19 20-24 25-29 30-34 35-39 40-44 45-49

DMalc Q Female

1403

1200

woo

B 800

400

2 »

10-14 15-19 2D-24 25-3 30-34 35-39 40-H 45-P

__*#

• Mb Oftmfc

Figure la. Figure lb.BMD vs. Age for both male and female subjects measured by Hologic(la) and Lunar(lb) machines

2. Trace Element Studies

Bone is an example of a biological sample that presents numerous difficulties in obtaining a specimen forchemical analysis This problem is acute when investigations are to be conducted to determine total skeletalcontent of a given analyte. First of all, the basic process of sampling bone can be a very difficult task. Withhumans, this problem is compounded due to medico-legal implications. Also, the question of which particularbone qualifies to be a representative sample of the skeleton as a whole is a debatable point. Even if someassumptions are made to answer this question, the sub-compartments of a bone sample, namely cortical,trabecular and the marrow and their relative proportion and significance in relation to bone as an organ,present intricate situations in making definitive decisions. Therefore, it is not surprising that reliable chemicalcomposition data, particularly for minor and trace elements, are scarce (Iyengar et. al., 1997). This is alsosubstantiated by the fact that there are only a couple of certified bone reference materials available forvalidating analytical methods In this study, as agreed in previous RCM, iliac crest bone is selected as thesample.

3.1 Sampling of Bone Biopsies

Bone biopsies were obtained according to protocol presented in earlier RCM. Twelve persons were selected,four female and eight male, ages distributed between of 15 and 50 The information about these subjects isgiven in Table 3. Soft tissues, muscle and fat were removed according to established protocol. They were driedand powdered These samples were subjected to Instrumental neutron Activation analysis, ISE for fluorideanalysis and AAS Details of these studies are given below.

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3.2 Instrumental Neutron Activation Analysis

Samples and standards were prepared for irradiation in a laminar flow cabinet Quartz tubes were used tocontain the bone samples irradiated in the reactor Bone samples were subjected to irradiation using both TngaMark II Reactor at Istanbul Technical University and MIT II Reactor at Massachusetts Institute of

Technology Samples were irradiated at the Triga Mark II with a thermal neutron flux of 8*10 " n/ cm secfor 2.5 hours. Collection of the gamma rays coming from the samples were carried out twice, 7 and 20 daysafter the end of irradiation at the Nuclear Chemistry Laboratory, Middle East technical University, METU,with HPGe detector. At MIT, samples were irradiated for 6 hours at a flux of 8x10'^ n/s.cm- and gamma rayscoming from the samples were counted twice, after 8 and 82 days of cooling Since bone samples contain large

amount of P, it was necessary to reduce the intense bremsstrahlung of P, which appreciably interferes withmeasurements up to the 0.7-0.8 MeV gamma rays For its oppression 0 8 cm thick Plexiglas was placedbetween detector and the sample.

Table 3. Information about the persons from whom bone biopsies were taken

Name

HB

UT

SDZK

SeDMDCSAA

YG

FD

NO

AD

Sex

F

FFF

M

M

M

M

M

M

M

M

Age

17

30

47

50

15

25

29

31

36

40

41

43

Wt

(kg)

58

93

75

80

85

57

65

64

Height(cm)

160

168

150

170

170

L J 7 3

160

175

BMD (g/cm2)(L1-L4)

1.094

1.1800891

1.301

1.3640.9210.9101.347

(Hip)Neck^

0.9540.756

1.119

0.7880.9241.017

(Hip)Ward

0.8910.593

1.268

0.6460.7710.825

(Hip)Trock

0.8810.635

0972

0.6290.9150.814

Reason of Surgery

Solitaire bone cyst of the calceneusFracture of the left femurPseudoarthrosis of the left UlnaPseudoartrosis of the radius and ulnaMal-union of the right femurTibial FractureMal-union of the left femurGiant cell tumor of the wristPseudoartrosis of the humerusRadius-Ulna fractureRadius-Ulna fractureFracture of the left ulna

Quality assurance of measurements: In order to assess the quality of analytical data being produced duringthe activation analysis, standard reference materials were used. There are only a few reference materials forquality control of bone analysis. The bone ash NIST SRM 1400 and bone meal NIST SRM 1486 are issued bythe National Institute of Standards and Technology. At METU, Bone Meal and Orchard Leaves NIST 1571were used as a standard reference material. Unfortunately only Mg, K, Ca, Sr, Zn and Fe values are certified inSRM Bone Meal. Mauerhofer et al., 1996 studied bone meal and determined the concentration of few otherelements such as Ba, Br, Mn and Cl. The agreement between the certified and measured values was very good.We used these results in our calculations. For the irradiation of the samples and blanks, two referencematerials, one of which was in the center and the other at the edge of the rabbit was used At MIT, IAEAHuman Diet Standard H-9 and urban paniculate material NIST-1648 were used. The average concentrationsand associated standard deviations of the observed elements in five cortical and five trabecular bones measuredat MIT are given in Table 4 and Table 5 respectively. Errors are estimated by taking into account bothstatistical errors from counts and the errors given in standard reference materials elemental concentrations.

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Table 4 Minor and trace element concentrations in cortical parts of iliac bone samples*

Elements

Na(g/kg)

Ca(g/kg)

K(g/kg)

Sr(mg/kg)

Fe(mg/kg)

Zn(mg/kg)

As(mg/kg)

Se(mg/kg)

Rb(mg/kg)

Mo(mg/kg)

Cd(mg/kg)

Ba(mg/kg)

Ce(mg/kg)

Cr(mg/kg)

Hg(mg/kg)

Br(mg/kg)

Sc(ng/kg)

Co(u.g/kg)

Sb(u.g/kg)

La(|ig/kg)

SeD-C, M15+

3.5 ±0.2

247 ± 7

1.6

83 ±3.0

56 ± 8

55 ±7

0.23 ±0 02

0.32 ±0.26

1.1 ±0.109

0.27

4.6 ±0.4

0.31 ±0.090.4

2.2± 0.10.81 ±0.04

3.0 ±0.8

46 ± 12

16

25

CSC, IVI29

4.6 ±0.3

237 ±5

5.2

101 ±6

81 ±26

76 ± 10

0.52

077

0.84 ±0.51

1.4

2

6.8 ±0.4

0.65 ± 0.03

0 48 ±0.23

2.3 ±0.1

0.54 ± 0.02

2.6 ±0.9

83 ± 16

7 ± 6

30 ±9

YG-C, M36

7.2 ±0.5

218±4

2.1 ±0.1

62 ±3.4

62 ± 14

120 ±20

0.27

0,77 ±0.67

1.0 ±0.6

0.35 ±0.29

1.7±0.6

-

0.64

1.2 ±0.3

3.2±0.1

1.5±0.1

6.6 ± 1.5

65 ±31

25

140 ± 10

HB-C, F17+

2.7 ±0.2

198 ±3

3.3

74 ±2.5

180 ± 10

62 ±8

0.37 ±0.04

-

1.2

1.2

0.36 ±0.06

4.5 ±0.2

0.45 ±0.02

0.9±0.17

l.9±0.1

0.69 + 0.02

5.3 ± 1.3

26 ± 10

22 ±2

120 ± 10

ZK-C, F50

6.7 ±0.4

-

-

-

210±20

110 ± 20

1.2

0.83 ±0.51

2.4

3.3

0.89 ±0.52

-

0.29

1.3

4.5 ±0.2

-

8.1 ±2.3

130 ±30

60

54 ± 14

Avg * ± Std

4.5 ±2.0

225 ± 22

3.0± 1.6

80± 16

95 ± 58

78 ±29

0.35 ±0.13

0.62 ±0.26

1.0 ±0.2

1.0 ±0.5

1.1 ±0.9

5.3 ± 1.3

0.51 ±0.16

0.8-0.4

2.4 ±0.6

0.9 = 0.4

5 x 2

55 ± 25

18 i 8

79 ±60

* The results of ZK are not included in the average calculations (see text). + M15 male, age 15, F17 female,age 17

These results are belong to apparently health people not in the state of osteoporosis but are classified as normalbone samples Noticed that the minor and trace element concentrations of all the subjects, except ZX are withina range when t test is applied at 95% confidence level. When bone sample from ZK was arrived to ourlaboratory for trace element analysis, it was noticed that they are in dark color. It was difficult to completelyseparate fat and blood Also note that, most of the trace element values for this sample are significantlydifferent than the others. Therefore trace element results from ZK were not included in Table 4 and 5, inoverall average calculations

The average ratio of concentration of elements in trabecular to cortical bones is 0.96 ± 0.52 and median being0.97 Although for few elements the ratio is considerably different than one, this indicates that there is no majordifferences in terms of trace element concentrations between cortical and trabecular bones

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Table 5. Minor and trace element concentrations in trabecular parts of iliac bone samples*

Elements

Na(g/kg)

Ca(g/kg)

K(g/kg)

Sr(mg/kg)

Fe(mg/kg)

Zn(mg/kg)

As(mg/kg)

Se(mg/kg)

Rb(mg/kg)

Mo(mg/kg)

Cd(mg/kg)

Ba(mg/kg)

Ce(mg/kg)

Cr(mg/kg)

Hg(mg/kg)

Br(mg/kg)

Lu(jig/kg)

Scfcg/kg)

Co(ng/kg)

Sb(jig^g)

La(ng/kg)

SeD-T, M15+

3.5 ±0.2

186±5

1.2

134 ±2

280 ± 20

88 ± 12

0.14 ±0 07

0.66

16

0 8

0.54

1.4 ±0.2

0.54

0 58 ±0.16

1.3±0.1

1.2 ± 0.1

7

3.0 ±0.9

62 ±20

12

19±5

CS-T, M29

5.0 ±0.3

224 ±4

1.1

85.0 ±2.8

37

97± 13

0.2

0.60 ±0.43

2.8

1

0.28

3.2 ±0.2

0.74

2.3 ±0.3

2.3 ±0.1

0.24 ±0.01

9.4

4.4±1.7

64 ±23

3

43 ±8

YG-T, M36

3.7 ±0.2

157±5

0.76

41.8 ± 1.8

34 ± 10

53 ±7

0.15 ±0.2

0.76

L L 6

0.82 ±0.18

0.013

0.23 = 0.1

0.84 ±0.17

2.1=0.1

7.8 ±0.4

6.7= 1.4

5.5 ± 1.8

55 ±21

16

17

HB-T, FI7 +

2.9 ±0.2

183 ± 6

1.2

57.0 ±2.3

110 ± 10

53 ±7

0.23

0.40 ±0.32

0.38

1

0.082 ±0.077

3.4 ±0.4

0.51

0.76 ±0.2

2.2 = 0.1

1.1 ±0.1

8

4.1 ± 1.2

47± 17

20

12 ±6

ZK-T, FSO

4.9 ±0.3

-

2.6 ±0.2

-

1100± 100

110 ± 20

0.27 ±0.09

0.25

1.3

1.1

0.13

-

0.47

6.8 ±0.3

2.6 ± 0.1

-

9.1

6.1 ± 1

97± 16

54 ±8

85 ±8

Avg* ±Std

3.8 ±0.8

188 ± 27

1.1 ±0.2

79 ±41

115± 115

73 ±23

0.18 ±0.04

0.61 ±0.15

1.6± 1.0

0.9 ±0.1

0.23 ±0.24

2.7 ± 11

0.50 ±0.21

1.1 ±0.8

2.0x0.5

2.6 ±3.5

7.8 ± 1.2

4.3 ± 1.0

57 ±8

13±7

23 ± 14

* The results of ZK are not included in the average calculations (see text). * Ml5: male, age 15, F17: female,age 17

3.3 Fluoride Analysis

Fluoride is incorporated into bone mineral by exchange with appetite OH. Fluoro apatite is less soluble thanhydoxyapatite and would be expected therefore to facilitate mineral deposition. Therefore fluoride analysis isvery important in bone studies. Ion selective electrode, ISE, is used in this study.

Two types of wet digestion system, acid bomb and microwave, were tested for the dissolution of bone samplesand microwave system was found most convenient. Three replicate samples each about 100 g from the samebone sample were dissolved separately, cooled and then the sample solution was transferred to a 50 mLpolyethylene beaker. The use of proper Total Ionic Strength Adjusting Buffers, T1SAB, which is used toprovide a constant ionic strength, is most important in ISE studies. Five TISAB solutions were examined tofind the best one suitable for the determination of fluoride in bone matrix. The TISAB-II (85g sodium nitrate,68g sodium acetate, and 92.4g sodium citrate diluted to 1 L at pH 5.5) is found best for our purposes(Covington et a! , 1980)

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In order to asses the quality of analytical data being produced during the fluoride electrode measurements,standard reference material, SRM 1486 Bone Meal, is used The fluoride value is not certified but given as 800mg/kg in SRM 1486. Its value is found as 820±47(n=46) by Mauerhofer by INAA. Our result is 912±54(n=4)The results of fluoride concentrations in cortical and trabecular bone samples are given in Table 6.

Table 6 The fluoride concentrations tn cortical and trabecular bone samples (mg/kg)

Female

HBUTSD7K

Female AvsMaleSeDMDrsAAYGFDNOAD

IVfaleAvs

Avg ± StdCortical703+4

816 + 2877.5 + 74

578 ± 107581+770Avs> + Std

309 + 9493 + 56706 + 18731 + 20

1144 + 105974 + 78687+ 103589? 38704 -767.

Avg ± StdTrabecular

931 +427.25 + J577? + 38655+7

646 J: 302AVP + Std388 + 144R3 + 751078 + 50950 + 751550 + 3973? +31698 + 35641 +12

809 + 368

3.4 Atomic Absorption Spectrometry (AAS)

For the AAS measurement, ground and homogenized bone samples were dissolved in nitric acid. For thispurpose, several experiments were carried out by using fresh animal bones for the selection of the most suitableacid combination and dissolution procedure: Nitric acid solutions with different concentrations were tried; Thebest result was obtained with 1+1 (v/v) HN03. Several microwave programs were tried and the best result wasobtained with 80% power and 8 minutes time period.

The determination of copper by INAA is not easy because of low concentration in blood and also not verysuitable nuclear properties of copper isotopes. Copper determinations in bone were carried out by FAAS, ICP-AES or PIXE methods in the literature (Robertson et al., 1994, Yoshinaga et al., 1985, Hisanaga et al., 1992)GFAAS was used for the determination of copper since the concentration of the copper was too low to bedetected by FAAS The first stage was the preparation of an aqueous solution containing only copper at areasonable concentration This concentration was selected as 10 u.g/L after experimenting with 25 u.L of 5, 20,40 |og/L solutions. Temperature program was made up of five Iwic steps; drying, thermal pretreatment,ashing, atomization and cleaning. In order to obtain best results for any particular analysis, each of thesestages had to be carefully optimized (Table 7). Standard addition method was used to obtain copperdetermination

Determination of Ca, Na, K, Mg and Zn by FAAS: Since the concentrations of these elements in bone arehigh enough, FAAS was used for their determination. The blank, standard and samples were aspirated intoflame directly for Mg and Ca Since the concentrations of these elements are high, calcium and magnesiumwere diluted to 500 and 200 fold respectively. Calcium was determined by means of nitrous oxide-acetyleneflame Depression of calcium response in air-acetylene flame may be observed because of the formation ofstable salt of phosphorous in bone. To eliminate this effect nitrous oxide- acetylene flame was used forcalcium. For Na, Zn and K micromjection by using Teflon accessory was performed. NIST SRM 1486-Bone

12-7

Table 7 Temperature programs for copper determinations

Phase

DryingPretreatment

Ashing

Atomization

Cleaning

Temperature (°C)(Cu solution)

110

190

900

2100

2600

Temperature(°C)(Bone Meal )

110

190

1200

24002600

Time(s) j

30.05.0

20.0

3.0

2.0

Ramp(°C/s)

50

Commands

TC RD

TC

meal and one of the aqueous standards were measured to achieve the similar conditions for each day Theabove conuiuuns were applied to eight fresh animal bones. In this experiment, cortical and trabecular partswere not separated from each other, that is, die results refer to whole bone. When the precision of the analysisis considered (Table 8), %RSV values are not low as expected. Especially, in the case of Zn, it reaches up to10.5%. We also faced this problem when measurmg human bone, as will be discussed later On the other hand,%RSD for Cu is very low, only 3 16% Applying, Q-test at 95% confidence level to determine whether anyresult will be rejected or not, it is seen that all the values are acceptable for each elements at this confidencelevel

Table 8 Concentrations of the elements in anima! bone (n=8)

MeanMedianGeomean%RSD

Ca(g/kg)161±13

163

161

8

Na

5.6310.175.635.623.00

Mg(g/kg)

5.53±0.405.615.527.23

K(g/kg)

3.18±0.2I3.203.176.56

Zn(mg/kg)84.818.9

84.3844

10.5

Cu(mg/kg)

4 8010.154.604.603.16

Similar experiments were carried out for NIST SRM 1486 Bone Meal. By applying student's t-test at %95confidence level for AAS and INAA, AAS and certified values, separately, we found no significant differencebetween them (Table 9).

Table 9. Comparison of concentrations of elements in NIST SRM 1486-Bone meal

Element

Ca(g/kg)Na(g/kg)K(g/kg)Mg(g/kg)Zn(mg/kg)Cu(mg/kg)

This work(AAS)281 ±6

5.2210.160.3710.054.41+0 15

160+81.10 + 0.05

Mauerhofer and Porte,1997(INAA)

268.61 1.15.710.4

0 394 10.0524.62 +0.23

149 +37.6

NIST SRMCertified value

266

5*

0.414.66147

0.8** These values are not certified, but given as information values

The results obtained for human studies are given in Table 10 and Table 11. Except a few cases, the results arethe average of 3 replicate experiments.

Zoo 12-8

Table 10.Concentrations of the elements in cortical bone determined by AAS

Sample(C)AA(n=3)HB(n=3)UT(n=2)MD(n=3)SeD(n=2)YG(n=3)CS(n=2)AD(n=3)SD(n=3)ZK(n=7)N0(n=2)FD(n=2)Average

Ca(g/kg)21I±I8156±20I88±6175 ±7

255 ± 17206 ± 16202 ±12214 ± 10200 ± 20186 ±10185 ± 14174 ±18196±27

Na(g/kg)4.57 + 0.104.20 ±0.226.67 ±0.353.41+0.174.80 ±0.138.35±0.335.35±0.128.51+0.215.74+0.226.53±0.538.07+ 0.205.83 + 0.155.96±1.73

K(g/kg)

3.94 + 0.393. 29+0. 162. 86 ±0. 132 04 + 0 10I. 99 ±0.082. 73 ±0. 102. 83+0. 072. 84 ±0 093 . 2 9 + 0 .075.71 ±0. 13I .23±0. 083.91 ± 0 . 0 53.0S± 0.66

Mg(g/kg)2.24 + 0 121.92±0.17

2.14 + 0.210.97 ±0.102.35±O.ll2.37±0.222.54±0.191.79 ±0 .191.63 ±0.231.70 ±0 16

0.39 ±0.132.37+0.131.87±0.59

Zn(mg/kg)_118 + 961 ±5112 + 46 9 + 476 ±5109 ±488 ±375 ±490 ±5

L 82 ±6U 3 ± 6180 ±698±30

Cu(mg/kg)2.58+0.094.08±0 183.58±O.2O7.00+0.193.83±0.054.22±0.052.27±0.035.05±0.015.0810.179.11±0.035.75±0.178.91±0.025.12±0.03

Table 11. Concentrations of the elements in trabecular bone determined by AAS

Sample(T)AA(n=3)HB(n=3)UT(n=2)MD(n=2)SeD(n=3)YG(n=3)CS(n=2)AD(n=2)SD(n=3)ZK(n=2)NO(n=2)FD(n=2)Average

Ca(g/kg)192 ±23184 ±6184 ±11181 ± 10271 ±83225 ± 9209 ±6189 ±9178 ±20185 ± 112 1 3 ± 8182 ± 17201±37

Na(g/kg)

4.94 ±0.16

3.82+0.306.92 ±0.453.04±0.168.42±0.393.89± 0.115.31+0.137.08±0.232.91±0.096.16±0.435.47±0.195.29 ±0.215.27±1.73

K(g/kg)

1.5120. 211.91 ± 0 . 0 8

1. 68 ±0. 101. 14 ±0.661. 11 +0.370. 87 ± 0.111.53 ±0.092. 99 ±0. 141. 37 ±0.051.99 + 0. 131.26 ±0.084. 21 ±0.09

1.80±0.61

Mg(g/kg)

1.7910.142.55±0. 151.9 0+0.282.14±0.221.82 ±0.141.9510.192.50±0.151.54 ± 0.191.31 ±0.151.73 ± 0.171.90±0.192.14 ± 0.181.8S±0.42

Zn(mg/kg)

101 ±557 ±4118±581 ±699 ±562 ±4104 ±474 ±388 ±65 8 + 5113+6141+690±25

Cu(mg/kg)

4.57 ±0.044.18±0.093.04±0.0210.3+0.1

5.46±0.094.12±0 162.53+0.033 18±0 014.80±0.037.68±0.075.50±0.076.86±0.075.18±3.0r'

Table 12 summarizes the result of 12 subjects and their trabecular/cortical ratios Except K, the ratios arearound 1.00, indicating that at least in terms of trace element concentrations, there is no difference between twoparts of the bone. Unfortunately, there are only two studies we found in literature, which give both cortical andtrabecular parts. As seen in Tablel2, Robertson (1992) analyzed rib bones both trabecular and cortical partsThere is exceptionally good agreement between his and our results. They also found similar trabecular/corticalratios. Grynpas (1992) obtained also similar results

Ratio of the concentration of the elements to calcium concentrations: In order to investigate therelationships between the concentrations of trace elements, it was convenient to look at the ratio ofconcentrations of the elements to calcium concentration This type of comparison gave somewhat meaningfulresults for the case of Na, K, Mg, Zn and Cu. As calcium concentration increases, sodium concentration inboth cortical and trabecular parts, decreases. That means, there may be a substitution between sodium andcalcium For potassium, a decrease in potassium concentration is detected as calcium concentration increases

12-9

ZO4

Table 12 Comparison of the trabecular/cortical ratios of the 12 human iliac crest bones and the valuesobtained by Robertson et al, 1992.

ElementCafe/kg)Na(g/kg)K(g/kg)Mg(g/kg)Zn(mg/kg)Cu(mg/kg)

This

Cortical196

5.962.841.8796

5.03

work

Trabecular2015.191.501.8590

5.09

Robertson

Cortical2005.20

-2.61825.10

et al,1992

Trabecular1905.20

-2.7144

4.81

This work

RatiofTVC)1.020.870.530.990.941.01

Robertsonetal,1992

Ratio(T/C)0.951.00

-1.040.790.94

for cortical bone but for trabecular bone a direct relationship is observed. For magnesium, the ratio is almostconstant with the increase of calcium concentration for cortical bone. For trabecular part, as calciumconcentration increases, magnesium concentration decreases, which may show the substitution of Mg for CaFor zinc, a similar relationship with magnesium case is observed The only difference is, in the case zincconstancy in ratio with an increase of calcium concentration is detected for trabecular bone, and a decrease inzinc concentration is observed with the increase of calcium concentration for cortical bone. For copper anincrease in concentration with the increase of calcium for cortical part is detected. For trabecular part, decreasein copper concentration is observed with the increase of calcium concentration.

References

Covmgton, AK., Ion-Selective Electrode Methodology, CRC Press Inc., 2nd ed., Vol. 2., (1980) pp 79-80

Hisanaga, A., Hirata, M., Tanaka, A, Ishinishi, N, Eguchi, Y, Variation of Trace Metals in Ancient andContemporary Japanese Bones, Biological Trace Element Research, 22 (1989) 221-231

Iyengar, V., Tandon, L., Minor and Trace Elements in Human Bones and Teeth, Private Comm., 1997

Mauerhofer and N. Porte, University of Mainz, Germany Private Communication, 1996

Samudralwar, DL, Robertson JD. J.Radional Nucl.Chem., Articles, 169 (1993) 259-267.

Yoshinaga, J., Suzuki, T., Morita, M., Sex and Age Related Variation in Elemental Concentrations ofContemporary Japanese Ribs The Science of The Total Environment, 79 (1989) 209-221

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