Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1087 _____________________________ _____________________________ Urea and Non – Protein Nitrogen Metabolism in Infants With Special Reference to the Sudden Infant Death Syndrome (SIDS) BY MARY GEORGE ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001
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Urea and Non - Protein Nitrogen Metabolism in Healthy Infants
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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1087
Urea and Non – Protein NitrogenMetabolism in InfantsWith Special Reference to the Sudden
Infant Death Syndrome (SIDS)
BY
MARY GEORGE
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001
Dissertation for the Degree of Doctor of Medical Science in Anaesthesiology and IntensiveCare presented at Uppsala University in 2001
ABSTRACT
George, M. 2001. Urea and Non – Protein Nitrogen Metabolism in Infants. With SpecialReference to the Sudden Infant Death Syndrome (SIDS). Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1087, 43pp. Uppsala ISBN 91-554-5141-1.
A large amount of non – protein nitrogen, in the form of urea and ammonium, is present inhuman breastmilk; however its physiological role in the infant is as yet not fully understood.It has been hypothesized that an insufficient enteric metabolism of urea could play a role inthe sudden infant death syndrome (SIDS). This thesis was undertaken to study the entericmetabolism of non – protein nitrogen in healthy infants, in comparison with those who hadsuccumbed to SIDS.
Aerobic and anaerobic faecal microflora, were studied in healthy infants from birth to 6months of age. They were found to appear in faeces within 3 days of birth and were presentthroughout the first 6 months of life. The effect of nitrate, nitrite and nitric oxide on faecalurease activity was investigated and found to be inhibitory in action. The sigmoid faecalurease activity and sigmoid faecal urea content of SIDS infants were compared to those ofcontrol infants; significantly lower sigmoid faecal urease activity and greater sigmoid faecalurea content were found in the SIDS infants. The total number of SIDS cases occurring inSweden during the period 1990 through 1996 was analysed regarding geographical andseasonal distribution, in relation to the nitrate concentration in drinking water and changes inthe groundwater level. The northernmost parts of the country had its highest incidence whenthe rest of the country had its lowest incidence, and the occurrence of individual deaths wasassociated with the recharge of groundwater, which is known to increase its nitrate content.The effect of ingested ammonium on carbon dioxide production was determined in healthyinfants using the doubly labelled water technique. No change in carbon dioxide productionwas observed.
An insufficient enteric metabolism of urea in infants and peak or greatly varying nitrateconcentrations in drinking water are associated with the occurrence of SIDS. Ingestedammonium supplements in the given doses did not influence carbon dioxide production inhealthy infants.
Key words: Urea, non – protein nitrogen metabolism, SIDS, drinking water nitrateconcentration, carbon dioxide production.
Mary George, Department of Surgical Sciences – Unit for Anaesthesiology and IntensiveCare, University Hospital, SE – 751 85 Uppsala, Sweden
Printed in Sweden by Eklundshofs Grafiska, Uppsala 2001
There is no finer investment for any community than putting milk into babies.
Winston Churchill 1943
This thesis is based on the following papers, which are referred to in the text by their Romannumerals:
I Faecal microflora and urease activity during the first six months of infancy. George M, Nord CE, Ronquist G, Hedenstierna G, Wiklund L. Upsala J Med Sci 1996, 101: 233 - 250.
II Sudden infant death syndrome and nitrogen metabolism: further development of a hypothesis. Wiklund L, George M, Nord CE, Ronquist G, Saldeen T. Eur J Clin Invest 1998, 28: 958 - 965.
III Incidence and geographical distribution of sudden infant death syndrome (SIDS) in relation to content of nitrate in drinking water and groundwater levels. George M, Wiklund L, Aastrup M, Pousette J, Thunholm B, Saldeen T, Wernroth L, Zarén B, Holmberg L. Eur J Clin Invest Accepted for publication.
IV The effect of ammonium chloride ingestion on carbon dioxide production in infants determined by using the doubly labelled water technique. George M, Forsum E, Wright A, Coward WA, Wiklund L. Submitted for publication.
CONTENTS
INTRODUCTION 7
Urea metabolism and acid - base balance 7Sudden infant death syndrome (SIDS) 8Hypothesis 10
AIMS OF THE THESIS 12
SUBJECTS AND METHODS 13
Subjects 13Paper I and II Analyses of faecal specimens 13
Paper III Geographical and seasonal distribution of SIDS 17Paper IV Doubly labelled water technique (DLW) 18
RESULTS 22
Paper I 22Paper II 24Paper III 25Paper IV 27
DISCUSSION 29
Urea metabolism 29Breastfeeding 30Postmortem vitreous humor levels of urea nitrogen 31Preceding illnesses 31Helicobacter pylori infection 31Carbon dioxide production and total energy expenditure 32Smoking 32Prone position and body temperature 33Increased winter incidence of SIDS 34
CONCLUSION 36
Future research 36
ACKNOWLEDGEMENTS 38
REFERENCES 39
ABBREVIATIONS
2H Stable isotope, deuterium
18O Stable isotope
CO2 Carbon dioxide DLW Doubly labelled water
H+ Hydrogen ion
HCO3- Bicarbonate ion
NaCl Sodium chloride
NAD+ Nicotinamide adenine dinucleotide
NADH Dihydronicotinamide adenine dinucleotide
NH4+ Ammonium ion
NO Nitric oxide
SIDS Sudden infant death syndrome
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INTRODUCTION
As early as 1949 it was known that human breastmilk contains large amounts of non - protein
nitrogen in the form of urea and ammonium. (1) Non - protein nitrogen, half of which is made
up of urea nitrogen, accounts for 25 per cent of the total nitrogen in breastmilk. (2,3,4) The
physiological role of this urea and ammonium and its contribution to normal metabolism in
the infant is as yet not fully understood. In the adult, urea is considered to be a water soluble
relatively nontoxic waste product, which is excreted as urine through the kidneys.
Ingested urea, contained in breastmilk, is metabolized by urease - containing bacteria, which
are found in the intestines within 5 days of birth. (5,6,7,8) Currently available infant formula
feeds, which are similar to breastmilk in their high lactose content and low protein content,
were found to stimulate growth of intestinal Bifidobacteria but unable to suppress pathogenic
bacteria such as Clostridia and Klebsiella. (9) In adults, urea produced in the liver enters the
enterohepatic circulation and diffuses into the intestine, where about 20 per cent of the urea is
hydrolyzed by intestinal microflora into ammonia while the rest of it is excreted unchanged in
the urine. (10) The therapeutic use of antibiotics such as neomycin to abolish intestinal
bacterial action on urea in patients with impending hepatic coma is well established.
In the infant, the ammonia from dietary urea is transported to the liver where it is metabolized
once again to urea and excreted through the kidneys. Some of the nitrogen from dietary urea
is incorporated into body proteins. (11, 12, 13)
8
Urea Metabolism and Acid - Base Balance
Traditional view
Ingestion of protein results in the release of amino acids into the splanchnic circulation. The
deamination of amino acids, which is the removal of the amino groups from the amino acids
occurs mainly in the liver. The ammonium ion together with bicarbonate is synthesized into
urea via the urea cycle. Urea is then excreted through the kidneys in the urine. (14)
In the proximal tubular cells of the kidneys, deamination of glutamine yields 2 ammonium
ions and 1 α-ketoglutarate ion; the ketoglutarate ion is metabolized to glucose, or to carbon
dioxide and water producing 2 bicarbonate ions. Thus a bicarbonate ion is added to the blood
for each hydrogen ion that is excreted in the form of an ammonium ion. In the Loops of Henle
and in the medullary interstitium the concentration of the ammonium ion increases by means
of a countercurrent multiplication before being secreted into the collecting ducts. (15)
Alternative view according to Atkinson and Camien
Catabolism of protein produces a large amount of bicarbonate ion (base) rather than acid as is
generally believed. Bicarbonate ions are produced from the α carboxyl group of an amino
acid and from the carboxyl or carboxamide group of glutamate, aspartate, glutamine and
aspargine as a result of amino acid metabolism. Approximately 1 mol of bicarbonate ion is
produced from a daily consumption of 100 g of protein. Bicarbonate ions combine with
ammonium ions synthesizing urea. Synthesis of urea supplies protons, which titrate the large
amount of bicarbonate produced, thus maintaining acid - base balance.(16)
2NH4+ + 2HCO3
− NH2 − CO − NH2 + CO2 + 3H2O
9
Sudden Infant Death Syndrome (SIDS)
SIDS is defined as the sudden death of an infant or young child, which is unexpected by
history, and in whom a thorough post - mortem examination fails to demonstrate an adequate
cause of death.
Sudden and unexpected deaths in infancy have been described to occur as early as 500 B. C.
(17) Up until the early nineteenth century, overlaying of infants by adults was considered to
be the cause of such infant deaths. The mother or wetnurse sharing the infant`s bed was
forbidden; if death occurred, it was punishable by law in the middle ages, both in Sweden and
in other European countries.
In the nineteenth century, several theories were proposed and subsequently discarded. Such
was the fate of the theories known as the status thymico- lymphaticus (large thymus),
Table 1. Faecal urease activity expressed as ammonium ion formed (mmol/g faeces wetweight after 30 h incubation) ± SD in infants from 3 days to 6 months of age, using a low andhigh urea concentration, under aerobic and anaerobic preincubations.
Faecal urea content
The faecal urea content was found to be 5 - 15 µmol/g faeces wet weight from 2 to 6 months
of age, and approximately 40 µmol at 3 days after birth. (The water content of the faeces was
blood chemistry and remains stable for up to 30 hours after death. (49)
Preceding Illnesses
Gastrointestinal illnesses such as vomiting and diarrhea, are reported by Hoffman (23) and
others (24) to occur more frequently in the preceding 2 weeks in SIDS cases than in living
control cases; this difference was not found by others. (50, 51) A higher proportion of
antibodies to clostridial and enterobacterial toxins and a lower proportion of maternal
antibodies were seen in SIDS infants compared to controls suggesting that SIDS infants
succumbed to these infections because of an inadequacy in their immune response to these
toxins. (52) A change in the normal microflora to a preponderance of pathogenic bacteria
could explain the significant amounts of unmetabolized faecal urea seen in SIDS infants.
However, we found that faecal microflora in infants who had succumbed to SIDS were
similar to those found in normal infants. (Paper I and II)
Helicobacter pylori Infection
Helicobacter pylori infection has been proposed as a possible cause of SIDS (53) and this has
been supported by others. (54) It has been suggested that urease produced by H. pylori if
aspirated by infants, in whom the gastrooesophageal reflux is commonly seen, could on
reaching the alveoli react with plasma urea, to produce ammonia. This ammonia,
hypothetically, could be then absorbed systemically producing symptoms of ammonia toxicity
resulting in respiratory arrest. (53) On the other hand, absorption of ammonia through the
gastric mucosa enables it to be detoxified by the liver. Both H. pylori and Bifidobacteria are
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known to be urease producing and should be able to metabolize completely, the urea
contained in breastmilk and formula feeds. If SIDS is caused by a H. pylori infection, it does
not explain our finding of significant amounts of unmetabolized faecal urea in SIDS infants
compared to controls. (Paper II)
Carbon Dioxide Production and Total Energy Expenditure
In Paper IV no change in carbon dioxide production was found following ammonium
ingestion. The DLW technique was used to determine carbon dioxide production. As this is a
non - invasive technique not requiring the cooperation of the subjects, it is well suited for
investigations in infants. In a pilot study using the Datex monitor to measure carbon dioxide
production following ammonium chloride ingestion difficulties were encountered, as reliable
measurements were only possible in the resting or sleeping infant and this was not always the
case in the 90 minutes following ammonium ingestion.
Values of total energy expenditure (TEE) can be derived from carbon dioxide production
measurements determined by the DLW technique. In 1996 Butte et al (55) published a review
of available data of the TEE of infants, assessed by the DLW method, which revealed that
estimates of TEE during infancy tended to be lower when obtained by this method than from
estimates of dietary energy intake. An extensive data set published in 2000 (56) confirmed the
lower values of TEE, determined by DLW method compared to earlier values, which are used
in the recommendations for dietary energy by world health authorities such as the FAO. (55,
57) No such data has been published for Swedish infants. In Paper IV we produced these
data, which were found to be comparable to that produced by Butte et al, confirming that
present recommendations for dietary energy intake are in need of revision.
Smoking
Maternal smoking as a potential risk factor for SIDS has been reported as early as 1966 by
Steel et al, (58) and this has been confirmed by other researchers. (59, 60, 61) Passive smoke
33
exposure was also found to increase the incidence of SIDS, (62, 63) and a dose - response
relationship between a greater exposure to smoke and SIDS has been reported. (60, 63) Effect
of smoke on inflammatory mediators (64) and the presence of nicotine in breastmilk (65) may
contribute to the increased risk. Though several reasons have been postulated, no causal
mechanism has been elucidated. In Paper I we showed that nitric oxide, nitrate and nitrite
have an inhibitory effect on faecal urease, produced by enteric microflora. Nitric oxide
present in the ambient air of smokers, when inhaled by infants, could transform to nitrites
after dissolving in water, and then impair their enteral urease activity. Inhaled nitric oxide has
been demonstrated to be converted to nitrates and subsequently eliminated by the kidneys.
(66) Smoking mothers can be presumed to have increased nitrate levels in blood and
breastmilk. Inhaled nitric oxide and nitrates in breastmilk may together impair enteral urease
activity in the infant resulting in significant amounts of unmetabolized urea and consequently
decreasing the amounts of ammonium being transported to the infant liver. The protective
effect of breastfeeding was seen in nonsmokers and not in smokers. (63) Endogenous nitric
oxide production is known to increase during the neonatal period in normal term infants, and
then gradually decrease until the infant is 4 months old, when it reaches a plateau. Preterm
infants had greater nitric oxide productions than term infants. (67)
Prone Position and Body Temperature
Prone position, hyperthermia and overwrapping have been associated with an increased risk
of SIDS in several studies. (68, 69) Decreased ventilation as a result of heat stress has been
suggested as the common factor (70) leading to the final apnoea of SIDS. A newborn infant
with a low metabolic rate and a large surface area to mass ratio is prone to cold stress, while a
3 month old infant with a higher metabolic rate, lower surface area to mass ratio and better
tissue insulation is more prone to heat stress. (71, 72) Thus both cold and heat stress may
initiate or enhance hypoventilation especially in an infant with some degree of metabolic
alkalosis, which has been suggested to be present in SIDS infants. (34)
34
Increased Winter Incidence of SIDS
SIDS occurs most frequently in the winter months in both the northern and the southern
hemispheres. Infection, (73) overwrapping, and smoking in confined spaces have all been
previously suggested to contribute to the increased winter incidence. Peak levels of
environmental pollutants such as carbon monoxide, sulphur dioxide, nitrogen dioxide and
hydrocarbons have been shown to precede the increase of SIDS. (74) Animal studies have
demonstrated that nitrogen dioxide resulted in changes in the bronchial epithelium which
contributed to a susceptibility to infection. (75, 76) Prone sleeping partially accounted for the
increase in SIDS rates in winter but some unidentified seasonal factor was found to modify its
effect. (77)
In Paper III the seasonality of SIDS during 1990 through 1996 in Sweden was found to vary
according to the location of the infants` residences, with reference to the geographical
latitude. The incidence of SIDS was highest in the northernmost part of the country when it
was at its lowest in the rest of the country. The occurrence of individual deaths was associated
with the recharge of groundwater, which is known to increase its nitrate content. (78, 79, 80)
SIDS cases occurred when the groundwater level rose, mostly as a result of spring thawing or
autumn rains. No SIDS cases occurred in the northernmost parts of the country, when frost
penetration and snow prevented changes in the groundwater level. This is in agreement with a
study by Munro et al (81) who showed that infant mortality was more than 31 percent greater
in infants resident in waterlogged soils than on well drained soils. Change in the
environmental temperature rather than the exact temperature was found to be related to the
incidence of SIDS. (82) Greatest risk of SIDS was on the warmer days of winter when the
daytime average was 3oC greater than the preceding 30 days. (83) Thawing on the warmer
days following a colder spell with a resultant increase in water nitrate levels could be a
possible explanation.
35
CONCLUSION
1. Aerobic and anaerobic microflora appear in the faeces of healthy infants within 3 days of
birth and are present throughout the first 6 months of life. Faecal urease activity decreases
to a third while bacterial production of nitric oxide increases ten - fold during the same
period. Nitrate, nitrite and nitric oxide have an inhibitory effect on faecal urease activity.
2. An insufficient metabolism of enteric urea is associated with SIDS.
3. The seasonal distribution of SIDS varies widely in different parts of the country and is
associated with groundwater level changes. Peak or widely varying drinking water nitrate
concentrations is associated with the incidence of SIDS.
4. Ingestion of additional ammonium chloride, equivalent to at least two-and-a-half times the
normal amount found in breastmilk feeds, does not increase carbon dioxide production in
healthy infants.
Inadequate enteric urea and nitrogen metabolism in infants and peak nitrate levels in drinking
water are associated with the incidence of SIDS. Further evidence is required to demonstrate
if they play a causal role in SIDS.
Future Research
Several questions remain to be answered.
1. Do infants of smoking mothers have an impaired faecal urease activity and a high faecal
urea content compared to controls?
2. Are nitrate levels in drinking water in countries known to have a high incidence, greater
and more varying than those of countries with a low incidence?
3. Do infants at a high risk of SIDS have a metabolic alkalosis?
36
4. Does the addition of ammonium supplements to the feeds of low- weight infants and
infants with infections, increase their carbon dioxide productions?
37
ACKNOWLEDGEMENTS
This work was carried out at the Anaesthesiology and Intensive Care Unit, Department ofSurgical Sciences, Uppsala University Hospital, Sweden.
I wish to express my deepest gratitude to my mentor and supervisor Professor Lars Wiklund.His enthusism, expertise and generous help have been vital for the completion of this study. Isincerely appreciate his willingness at all times in promptly replying to my queries.
I am grateful to Elisabet Forsum, for sharing her knowledge and experience in the field ofstable isotopes, and as co- author.
My sincere thanks to Lars Holmberg, for unravelling the mysteries of epidemiology, and asco- author.
I wish to expres my gratitude to Gunnar Ronquist, Carl- Erik Nord, Andy Coward and AntonyWright, for analysing data, and as co- authors.
I wish to thank Göran Hedenstierna, Tom Saldeen, Björn Zarén, Lisa Wernroth, Jan Pousette,Mats Aastrup and Bo Thunholm for contributing their expertise to this study, and as co-authors.
My sincere appreciation for the Staff of the Paediatric Anaesthesia Section, The CentralIntensive Unit and the Anaesthetic Laboratory for their enthusiastic help and technicalsupport.
My deepest gratitude to the parents of all the infants who participated in the studies.
I wish to thank Siv Utterberg for invaluable administrative assistance.
My thanks to my colleagues at the Anaesthetic Department for their encouragement and foraccepting my absences from the clinical rota during my research weeks.
My heartfelt thanks to my friends, both far and near, for their enthusiastic support and genuineinterest in my scientific pursuits.
My warmest gratitude to my sister and brother and their respective families, for their lovingsupport. I also wish to thank my brother for finding solutions, to some of my computerproblems, at all hours of the day.
Financial support by the Uppsala University Hospital, the Marianne and Marcus WallenbergsFoundation and the Laerdal Foundation for Acute Medicine is gratefully acknowledged.
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