Life without Albumin Encounters with Analbuminemia Andrew W. Lyon, PhD FCACB, DABCC. Department of Pathology and Laboratory Medicine, University of Calgary.

Life without AlbuminLife without AlbuminEncounters with AnalbuminemiaEncounters with Analbuminemia

Andrew W. Lyon, Andrew W. Lyon, PhD FCACB, DABCC.PhD FCACB, DABCC.

Department of Pathology and Laboratory Department of Pathology and Laboratory Medicine, University of Calgary &Medicine, University of Calgary &

Calgary Laboratory ServicesCalgary Laboratory Services

Boras, Sweden. May 2007Boras, Sweden. May 2007

Objectives:Objectives:

Review the pathophysiology of serum Review the pathophysiology of serum albuminalbumin

Review clinical laboratory methods of Review clinical laboratory methods of albumin determinationalbumin determination

Describe Analbuminemia and four cases Describe Analbuminemia and four cases found in Saskatoonfound in Saskatoon

Depict how common laboratory method of Depict how common laboratory method of serum albumin measurement confound serum albumin measurement confound the diagnosis of analbuminemia.the diagnosis of analbuminemia.

The History of ‘albumin’The History of ‘albumin’ 400400 HippocratesHippocrates: Foam on urine with renal failure.: Foam on urine with renal failure. 1500 1500 ParacelusParacelus: precipitated protein from urine with : precipitated protein from urine with

acidacid 1894 1894 KanderKander: Crystallized horse albumin: Crystallized horse albumin 1896 1896 StarlingStarling: Suggests role of albumen in : Suggests role of albumen in

maintaining circulation.maintaining circulation. 1926 1926 SvedbergSvedberg: Measures mass by ultracentrifugation: Measures mass by ultracentrifugation 1937 1937 TiseliusTiselius: Separated serum by electrophoresis: Separated serum by electrophoresis 1947 1947 KlotzKlotz: Studies how dyes bind to albumin: Studies how dyes bind to albumin 1950 1950 PetersPeters: Biosynthesis of albumin in liver slices: Biosynthesis of albumin in liver slices 1954 1954 BenholdBenhold: First report of analbuminemia, 2 cases.: First report of analbuminemia, 2 cases. 1957 1957 KnedelKnedel: Report of the genetic cause of : Report of the genetic cause of

bisalbuminemiabisalbuminemia

The History of ‘albumin’The History of ‘albumin’

1970 1970 KingKing: Studied tryptic fragments of albumin: Studied tryptic fragments of albumin 1975 1975 Brown / MelounBrown / Meloun: Amino acid sequence BSA & : Amino acid sequence BSA &

HASHAS 1979 1979 SargentSargent: Isolated the HAS gene: Isolated the HAS gene 1981 1981 LawnLawn: Genetic sequence of HAS cDNA: Genetic sequence of HAS cDNA 1986 1986 DugaiczykDugaiczyk: Complete HAS gene sequence: Complete HAS gene sequence

Mutation studiesMutation studies Crystallization studiesCrystallization studies Parenteral albumin utilization studiesParenteral albumin utilization studies

Physiological Roles of AlbuminPhysiological Roles of Albumin

Circulatory RoleCirculatory Role……….80% oncontic pressure……….80% oncontic pressure

Transport of metabolitesTransport of metabolites Bilirubin, calcium, fatty acids, bile acids, drugsBilirubin, calcium, fatty acids, bile acids, drugs Low affinity reservoir for thyroxine, vitamin DLow affinity reservoir for thyroxine, vitamin D

Sequestration of toxinsSequestration of toxins… delivery to the liver… delivery to the liver Bilirubin, carcinogens.Bilirubin, carcinogens.

Metabolic EffectsMetabolic Effects: enhances lipoprotein lipase: enhances lipoprotein lipase Miscellaneous EffectsMiscellaneous Effects: limits fibrin fiber : limits fibrin fiber

thicknessthickness

Clinical Utility of Serum Clinical Utility of Serum Albumin LevelsAlbumin Levels

Assess protein malnutritionAssess protein malnutrition

Renal diseaseRenal disease… nephrotic syndrome… nephrotic syndrome

G.I. pathologyG.I. pathology Liver disease… impaired synthesisLiver disease… impaired synthesis Hypoalbuminemia, intestinal edema, Hypoalbuminemia, intestinal edema,

diarrheadiarrhea

Methods of albumin Methods of albumin quantification quantification

TurbidometryTurbidometry: following acid or salt : following acid or salt precipitationprecipitation

Dye-binding methodsDye-binding methods Protein-error of pH indicating dyesProtein-error of pH indicating dyes

Protein electrophoresisProtein electrophoresis

ImmunoassayImmunoassay

Dye-binding MethodsDye-binding Methods High pH causes a High pH causes a colourcolour change: change:

High pHHigh pH

Dye-H Dye-H DyeDye-- + H + H++

Add albumin at Constant pHAdd albumin at Constant pH

AlbuminAlbumin

Dye-H Dye-H DyeDye---Albumin -Albumin

Applications: Urine dipstick, routine clinical chemistry.Applications: Urine dipstick, routine clinical chemistry.

Dye-binding MethodsDye-binding Methods

Urine DipsticksUrine Dipsticks: Urine Protein detection: Urine Protein detection Largely measures urine albuminLargely measures urine albumin False positive colour at high pHFalse positive colour at high pH Poor detection of Bence Jones proteins/ light Poor detection of Bence Jones proteins/ light

chains … as they don’t bind the dyes.chains … as they don’t bind the dyes. Serum AlbuminSerum Albumin

Bromcresol GreenBromcresol Green , , Bromcresol PurpleBromcresol Purple Short incubation times to improve specificityShort incubation times to improve specificity

Serum Protein Serum Protein ElectrophoresisElectrophoresisAlbumin (65%)Albumin (65%)

Globulins (35%)Globulins (35%)o Transthyretin / PrealbuminTransthyretin / Prealbumino Antitrypsin, Acid GlycoproteinAntitrypsin, Acid Glycoproteino Haptoglobin, Macroglobulin, Haptoglobin, Macroglobulin, o Transferrin, C3Transferrin, C3o Lipoproteins (VLDL, LDL, HDL)Lipoproteins (VLDL, LDL, HDL)o Missing: “Fibrinogen” Missing: “Fibrinogen” o Immunoglobulins ( IgG, IgA, IgM, IgD, Immunoglobulins ( IgG, IgA, IgM, IgD,

IgE)IgE)

LIVER

From:From: Immunofixation.Immunofixation. Killingsworth & Killingsworth & WarrenWarren

Calgary Laboratory Services

Serum Protein Electrophoresis

BisalbuminemiaBisalbuminemia Not particularly rare in Not particularly rare in

Canada! Often seen in Canada! Often seen in aboriginal peoplesaboriginal peoples

> 50 known Albumin > 50 known Albumin mutations.mutations.

Bisalbuminemia results Bisalbuminemia results from two copies of from two copies of different albumin different albumin genes, resulting in genes, resulting in different charges. different charges.

Albumin ImmunoassayAlbumin Immunoassay

Method usually reserved for urine or Method usually reserved for urine or CSF albumin determination: CSF albumin determination: ““microalbuminmicroalbumin””

Various immunoassay methodsVarious immunoassay methods: rate : rate nephelometry, nephelometry, nephelometry, nephelometry, turbidometry, radial turbidometry, radial immunodiffusion.immunodiffusion.

Objectives:Objectives:

Review the pathophysiology of serum Review the pathophysiology of serum albuminalbumin

Review clinical laboratory methods of Review clinical laboratory methods of albumin determinationalbumin determination

Describe Analbuminemia and four cases Describe Analbuminemia and four cases found in Saskatoon, SK, Canada.found in Saskatoon, SK, Canada.

Depict how common laboratory method of Depict how common laboratory method of serum albumin measurement confound serum albumin measurement confound the diagnosis of analbuminemia.the diagnosis of analbuminemia.

Our first case…Our first case…

Paul Meinert (pediatric resident) Paul Meinert (pediatric resident) complains “why are serum albumin complains “why are serum albumin levels in your lab fluctuating so much?”levels in your lab fluctuating so much?”

““What do you mean by fluctuating?”What do you mean by fluctuating?”

Variable levels of serum Variable levels of serum albumin over 6 months, albumin over 6 months, (7 (7

month old infant)month old infant)

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Method_1Method_2Method_3

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Impact of low albumin??Impact of low albumin??

Calcium - Alb

Adipose TissueFatty Acids - Alb

Bilirubin-Alb

T4 - Alb

Alb for Oncotic Pressure, to avoid edema

Patient #1Patient #1 Admitted to NICU as a newborn for hypoglycemia Admitted to NICU as a newborn for hypoglycemia

and perinatal asphyxia, mild tubular necrosis & and perinatal asphyxia, mild tubular necrosis & brain edema.brain edema.

Low serum albumin noted: Low serum albumin noted: 17 g/L17 g/L Normal 24 hr urine protein level.Normal 24 hr urine protein level.

Three admissions during first 6 months related to Three admissions during first 6 months related to respiratory distress and wheezing. respiratory distress and wheezing.

Nutritional status: Good. Nutritional status: Good. Gaining weight, growing, apparently normal liver Gaining weight, growing, apparently normal liver

function.function.

Patient #1Patient #1 At 6 months of age, still no explanation for the low At 6 months of age, still no explanation for the low

serum albumin (serum albumin (13 – 18 g/L13 – 18 g/L, routine chem). , routine chem).

Serum albumin was still lower by electrophoresis Serum albumin was still lower by electrophoresis ( (3-5 g/L3-5 g/L).).

TcTc9999-labelled albumin scan: negative for protein -labelled albumin scan: negative for protein loosing enteropathy. loosing enteropathy.

Clinical Biochemistry consult to review the results.Clinical Biochemistry consult to review the results.

AnalbuminemiaAnalbuminemia

Albumin (Dye-binding) : 10 – 17 g/L

Albumin (electrophoresis): 2 – 3 g/L

Albumin (Immunoassay) : < 0.01 g/L

Consistent with:Consistent with: AnalbuminemiaAnalbuminemiaA genetic lack of albuminA genetic lack of albumin

What is analbuminemia and what is the What is analbuminemia and what is the prognosis ?prognosis ?

Why did our routine laboratory methods Why did our routine laboratory methods detect 17g/L of albumin is there was NONE?detect 17g/L of albumin is there was NONE?

AnalbuminemiaAnalbuminemia

Serum albumin test results: very low.Serum albumin test results: very low.

Apparently a benign, recessive Apparently a benign, recessive inherited disorder: inherited disorder: elevated lipids and elevated lipids and globulins, 30% have lipodystrophy below waist, 30% globulins, 30% have lipodystrophy below waist, 30% mild ankle edema, low capillary blood pressure.mild ankle edema, low capillary blood pressure.

VERY RARE (approx. 42 reported VERY RARE (approx. 42 reported cases)cases)

Lipodystrophy in AnalbuminemiaLipodystrophy in Analbuminemia

Patient #1 Patient #1 continuedcontinued

Paul Meinert and I met with the staff Paul Meinert and I met with the staff pediatric GI specialist in Saskatoon: pediatric GI specialist in Saskatoon: Garth Bruce.Garth Bruce.

“ “ I thought it was analbuminemia. I I thought it was analbuminemia. I saw another child like this a few saw another child like this a few years ago.”years ago.”

AnalbuminemiaAnalbuminemia

Patient #2Patient #2

Admitted at 2 days of age with cellulitisAdmitted at 2 days of age with cellulitis Admitted 3 times during the first 6 Admitted 3 times during the first 6

months for respiratory distress / infection. months for respiratory distress / infection. Mild hyperbilirubinemia, mild ALP Mild hyperbilirubinemia, mild ALP

elevation and low serum albumin: elevation and low serum albumin: 10 – 15 10 – 15 g/Lg/L..

Nutritional status, weight gain & growth Nutritional status, weight gain & growth were normal. were normal.

Albumin by electrophoresis: Albumin by electrophoresis: 2 g/L2 g/L

Patient #3Patient #3

Admitted at 6 weeks of age with tetany Admitted at 6 weeks of age with tetany and prolonged diarrhea.and prolonged diarrhea.

Infant had low serum calcium and Infant had low serum calcium and magnesium levels. Tetany resolved on magnesium levels. Tetany resolved on magnesium administration.magnesium administration.

Low albumin levels were detected Low albumin levels were detected < 10 < 10 g/Lg/L

i.v. albumin was administered prior to i.v. albumin was administered prior to conducting serum electrophoresis. conducting serum electrophoresis.

AnalbuminemiaAnalbuminemia

A genetic lack of albuminA genetic lack of albumin

Why did our routine laboratory Why did our routine laboratory methods detect 17g/L of albumin is methods detect 17g/L of albumin is there was NONE?there was NONE?

Re-evaluation of Dye-binding Re-evaluation of Dye-binding Serum Albumin MethodsSerum Albumin Methods

Roche Diagnostics: Roche Diagnostics: BCGBCG method (rapid) method (rapid) Ortho Diagnostics: Ortho Diagnostics: BCGBCG method (slow) method (slow)

BCG: bromcresol greenBCG: bromcresol green Linearity of albumin methods with diluted Linearity of albumin methods with diluted

serum (constant albumin : globulin ratio)serum (constant albumin : globulin ratio)

Linearity of albumin methods with 100% Linearity of albumin methods with 100% human IgG or 100% human albumin. human IgG or 100% human albumin.

Comparison of serum albumin methods with diluted serum

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Why did our assays report the Why did our assays report the presence of albumin in presence of albumin in

patients with analbuminemia ? patients with analbuminemia ? Ortho Diagnostics Assay:Ortho Diagnostics Assay:

Reacts with globulinsReacts with globulinsGives a positive result in the absence of albumin.Gives a positive result in the absence of albumin.The assay did not report that albumin was below the The assay did not report that albumin was below the reportable limit.reportable limit.

Roche Diagnostics Assay:Roche Diagnostics Assay: Assay reported < 10 g/L in analbuminemiaAssay reported < 10 g/L in analbuminemiaAssay had no globulin interference.Assay had no globulin interference.

Serum Protein ElectrophoresisSerum Protein ElectrophoresisBaseline disturbances during densitometry lead to reports Baseline disturbances during densitometry lead to reports

of 2-3 g/L albumin.of 2-3 g/L albumin.

What was the serum albumin What was the serum albumin concentration reported in the concentration reported in the

reported cases of reported cases of analbuminemia?analbuminemia?

It depends on the albumin methods that were used

Our three patients with analbuminemia all appeared to have albumin present, according to the dye-binding albumin methods.

28 Cases of Analbuminemia: 28 Cases of Analbuminemia:

Levels of Albumin ‘detected’Levels of Albumin ‘detected’

A: Dye-binding methods

B: Salt Precipitation method

C: Protein Electrophoresis

D: Albumin Immunoassay

Awkward conclusions:Awkward conclusions:

Clinicians should consider a diagnosis of Clinicians should consider a diagnosis of analbuminemia (a genetic lack of albumin) analbuminemia (a genetic lack of albumin) even when the clinical laboratory detects even when the clinical laboratory detects serum albumin up to 17 g/L.serum albumin up to 17 g/L.

Albumin immunoassays and serum protein Albumin immunoassays and serum protein electrophoreses are capable of detecting electrophoreses are capable of detecting analbuminemia, but routine serum analbuminemia, but routine serum albumin assays are NOT. albumin assays are NOT.

Patient #3 Diagnosis following Patient #3 Diagnosis following i.v. albumin infusion.i.v. albumin infusion.

Establish a partnership with Monica Establish a partnership with Monica Galliano and Lorenzo Minchiotti, Univ. Galliano and Lorenzo Minchiotti, Univ. Pavia.Pavia. Isolation of genomic DNAIsolation of genomic DNA 14 exons were amplified by PCR14 exons were amplified by PCR Single-strand conformation polymorphism Single-strand conformation polymorphism

(SSCP) and heteroduplex analysis. (SSCP) and heteroduplex analysis. SequencingSequencing

Exon #3 SSCPExon #3 SSCP1 Control1 Control2 Mother2 Mother3 Patient3 Patient4 Control4 Control5 Analb Codogno5 Analb Codogno

Exon #3 Heteroduplex Exon #3 Heteroduplex AnalysisAnalysis6 Control6 Control7 Mother7 Mother8 Patient8 Patient9 Control 9 Control 10 Analb. Codogno10 Analb. Codogno

A: Control

B: Patient AT deletion

“Kayseri albumin”

‘ ‘ ‘ ‘

‘ ‘

‘‘AT’ deletion frameshift results AT’ deletion frameshift results in a stop condon and in a stop condon and

analbuminemia.analbuminemia.

Three cases of Three cases of analbuminemia…. Was this a analbuminemia…. Was this a

coincidence?coincidence? Dr. David Meyer, Dept Anthropology, Dr. David Meyer, Dept Anthropology,

Univ. Saskatchewan, Doctoral Thesis: Univ. Saskatchewan, Doctoral Thesis: Red Earth Crees 1860 – 1960 Red Earth Crees 1860 – 1960 Ethnogeographic and historical workEthnogeographic and historical work Near-complete pedigrees 1860 – 1970Near-complete pedigrees 1860 – 1970 Study of the DemeStudy of the Deme

Deme:Deme: a marriage isolate or a marriage isolate or universeuniverse

Red Earth and Shoal Lake Red Earth and Shoal Lake reservations had a common and reservations had a common and stable deme 1860 – 1960. stable deme 1860 – 1960.

Evacuation Photo: April 2007 Red Earth Reserve

Evacutation of Red Earth Reserve, April 2007

Red Earth and Shoal Lake Red Earth and Shoal Lake PopulationPopulation

DateDate PopulationPopulation In group marriageIn group marriage

1781 -17821781 -1782 0 0 smallpox smallpox epidemicepidemic

18501850 6 men6 men

1870 – 19001870 – 1900 187187 60%60%

1900 – 19301900 – 1930 220220 85%85%

1930 – 19601930 – 1960 391391 85%85%

20072007 15001500 ??

1971 Third Generation1971 Third Generation

75 Couples (married, 75 Couples (married, widows/widowers).widows/widowers). 8 marriages between first cousins.8 marriages between first cousins. 23 marriages between second cousins.23 marriages between second cousins. 35 marriages between relatives, > 235 marriages between relatives, > 2nd nd

cousin. cousin. 9 insufficient to establish kinship.9 insufficient to establish kinship.

There is likely a ‘founder effect’ There is likely a ‘founder effect’ responsible for the local incidence of responsible for the local incidence of analbuminemia at the Red Earth and analbuminemia at the Red Earth and Shoal Lake reserves.Shoal Lake reserves.

Future Investigations??Future Investigations??

September 2003: Patient #4September 2003: Patient #4

•29 year old native female from Red Earth, SK

– referred because of lower limb edema.

•History of presenting illness:History of presenting illness:–admitted to Nipawin hospital for pneumonia (July 2002) and right calf cellulitis–upon discharge the patient noticed that her legs were swollen–the family doctors consult note from June 2002 implies she has had a low albumin, large legs and lower extremities and an abnormal body habitus

And so the story continues…

Life without Albumin

AcknowledgmentsAcknowledgments Garth Bruce, Paul Meinert,Garth Bruce, Paul Meinert,

Robin Casey, Pat Blakely: Pediatrics,Robin Casey, Pat Blakely: Pediatrics,Jill Newstead, Sharon Card: Medicine.Jill Newstead, Sharon Card: Medicine.Univ. Saskatchewan.Univ. Saskatchewan.

Mark Salkie, Victor LaxdalMark Salkie, Victor LaxdalPathology, Univ. Saskatchewan.Pathology, Univ. Saskatchewan.

David Meyer, Anthropology, David Meyer, Anthropology, Univ. Saskatchewan.Univ. Saskatchewan.

Lorenzo Minchiotti, Monica Galliano & Lab,Lorenzo Minchiotti, Monica Galliano & Lab, Univ. Pavia.Univ. Pavia.

ReferencesReferences

Lyon AW, Meinert P, Bruce GA, Laxdal VA, Salkie ML. Lyon AW, Meinert P, Bruce GA, Laxdal VA, Salkie ML. Influence of methodology on the detection and diagnosis of congenital analbuminemia. Clin. Chem. 1998; 44: 2365-7. Clin. Chem. 1998; 44: 2365-7.

Galliano M, Campagnoli M, Rossi A, Wirsing von Konig CH, Lyon Galliano M, Campagnoli M, Rossi A, Wirsing von Konig CH, Lyon AW, Cefle K, Yildiz A, Palanduz S, Ozturk S, Minchiotti L. AW, Cefle K, Yildiz A, Palanduz S, Ozturk S, Minchiotti L. Molecular diagnosis of analbuminemia: a novel mutation identified in two Amerindian and two Turkish families. Clin Chem. 2002 ;48:844-9.. Clin Chem. 2002 ;48:844-9.

Newstead J, Card S, Lyon AW. Low serum albumin and abnormal Newstead J, Card S, Lyon AW. Low serum albumin and abnormal body shape in a young Canadian First Nations woman. Lab Med body shape in a young Canadian First Nations woman. Lab Med Volume 35, Number 6 / June 2004. Volume 35, Number 6 / June 2004.

For more details on analbuminemia, visit the Albumin Website: For more details on analbuminemia, visit the Albumin Website: http://www.albumin.orghttp://www.albumin.org