1. Life without Albumin Encounters with Analbuminemia
Andrew W. Lyon, PhD FCACB, DABCC.
Department of Pathology and Laboratory Medicine, University of Calgary & Calgary Laboratory Services
Boras, Sweden. May 2007

2. Objectives: Review the pathophysiology of serum albumin
Review clinical laboratory methods of albumin determination
Describe Analbuminemia and four cases found in Saskatoon
Depict how common laboratory method of serum albumin measurement confound the diagnosis of analbuminemia.

3. The History of ‘albumin’
400 Hippocrates: Foam on urine with renal failure.
1500 Paracelus: precipitated protein from urine with acid
1894 Kander: Crystallized horse albumin
1896 Starling: Suggests role of albumen in maintaining circulation.
1926 Svedberg: Measures mass by ultracentrifugation
1937 Tiselius: Separated serum by electrophoresis
1947 Klotz: Studies how dyes bind to albumin
1950 Peters: Biosynthesis of albumin in liver slices
1954 Benhold: First report of analbuminemia, 2 cases.
1957 Knedel: Report of the genetic cause of bisalbuminemia

4. The History of ‘albumin’
1970 King: Studied tryptic fragments of albumin
1975 Brown / Meloun: Amino acid sequence BSA & HAS
1979 Sargent: Isolated the HAS gene
1981 Lawn: Genetic sequence of HAS cDNA
1986 Dugaiczyk: Complete HAS gene sequence
Mutation studies
Crystallization studies
Parenteral albumin utilization studies

5. Physiological Roles of Albumin
Circulatory Role……….80% oncontic pressure
Transport of metabolites
Bilirubin, calcium, fatty acids, bile acids, drugs
Low affinity reservoir for thyroxine, vitamin D
Sequestration of toxins… delivery to the liver
Bilirubin, carcinogens.
Metabolic Effects: enhances lipoprotein lipase
Miscellaneous Effects: limits fibrin fiber thickness

6. Clinical Utility of Serum Albumin Levels
Assess protein malnutrition
Renal disease… nephrotic syndrome
G.I. pathology
Liver disease… impaired synthesis
Hypoalbuminemia, intestinal edema, diarrhea

7. Methods of albumin quantification
Turbidometry: following acid or salt precipitation
Dye-binding methods
Protein-error of pH indicating dyes
Protein electrophoresis
Immunoassay

8. Dye-binding Methods High pH causes a colour change: High pH
Dye-H Dye H+
Add albumin at Constant pH
Albumin
Dye-H Dye--Albumin
Applications: Urine dipstick, routine clinical chemistry.

9. Dye-binding Methods Urine Dipsticks: Urine Protein detection
Largely measures urine albumin
False positive colour at high pH
Poor detection of Bence Jones proteins/ light chains … as they don’t bind the dyes.
Serum Albumin
Bromcresol Green , Bromcresol Purple
Short incubation times to improve specificity

10. Serum Protein Electrophoresis
Albumin (65%)
Globulins (35%)
Transthyretin / Prealbumin
Antitrypsin, Acid Glycoprotein
Haptoglobin, Macroglobulin,
Transferrin, C3
Lipoproteins (VLDL, LDL, HDL)
Missing: “Fibrinogen”
Immunoglobulins ( IgG, IgA, IgM, IgD, IgE)
LIVER

11. From: Immunofixation. Killingsworth & Warren

12. Calgary Laboratory Services Serum Protein Electrophoresis

13. Bisalbuminemia
Not particularly rare in Canada! Often seen in aboriginal peoples
> 50 known Albumin mutations.
Bisalbuminemia results from two copies of different albumin genes, resulting in different charges.

14. Albumin Immunoassay
Method usually reserved for urine or CSF albumin determination: “microalbumin”
Various immunoassay methods: rate nephelometry, nephelometry, turbidometry, radial immunodiffusion.

15. Objectives: Review the pathophysiology of serum albumin
Review clinical laboratory methods of albumin determination
Describe Analbuminemia and four cases found in Saskatoon, SK, Canada.
Depict how common laboratory method of serum albumin measurement confound the diagnosis of analbuminemia.

16. Our first case…
Paul Meinert (pediatric resident) complains “why are serum albumin levels in your lab fluctuating so much?”
“What do you mean by fluctuating?”

17. Variable levels of serum albumin over 6 months, (7 month old infant)
<10

18. Impact of low albumin?? Bilirubin-Alb Adipose Tissue T4 - Alb
Fatty Acids - Alb
T4 - Alb
Calcium - Alb
Alb for Oncotic Pressure, to avoid edema

19. Patient #1
Admitted to NICU as a newborn for hypoglycemia and perinatal asphyxia, mild tubular necrosis & brain edema.
Low serum albumin noted: 17 g/L
Normal 24 hr urine protein level.
Three admissions during first 6 months related to respiratory distress and wheezing.
Nutritional status: Good.
Gaining weight, growing, apparently normal liver function.

20. Patient #1
At 6 months of age, still no explanation for the low serum albumin (13 – 18 g/L, routine chem).
Serum albumin was still lower by electrophoresis (3-5 g/L).
Tc99-labelled albumin scan: negative for protein loosing enteropathy.
Clinical Biochemistry consult to review the results.

21. Analbuminemia Albumin (Dye-binding) : 10 – 17 g/L
Albumin (electrophoresis): 2 – 3 g/L
Albumin (Immunoassay) : < 0.01 g/L

22. Consistent with: Analbuminemia
A genetic lack of albumin
What is analbuminemia and what is the prognosis ?
Why did our routine laboratory methods detect 17g/L of albumin is there was NONE?

23. Analbuminemia Serum albumin test results: very low.
Apparently a benign, recessive inherited disorder: elevated lipids and globulins, 30% have lipodystrophy below waist, 30% mild ankle edema, low capillary blood pressure.
VERY RARE (approx. 42 reported cases)

24. Lipodystrophy in Analbuminemia

25. Patient # continued
Paul Meinert and I met with the staff pediatric GI specialist in Saskatoon: Garth Bruce.
“ I thought it was analbuminemia. I saw another child like this a few years ago.”

26. Analbuminemia

27. Patient #2 Admitted at 2 days of age with cellulitis
Admitted 3 times during the first 6 months for respiratory distress / infection.
Mild hyperbilirubinemia, mild ALP elevation and low serum albumin: 10 – 15 g/L.
Nutritional status, weight gain & growth were normal.
Albumin by electrophoresis: 2 g/L

28. Patient #3
Admitted at 6 weeks of age with tetany and prolonged diarrhea.
Infant had low serum calcium and magnesium levels. Tetany resolved on magnesium administration.
Low albumin levels were detected < 10 g/L
i.v. albumin was administered prior to conducting serum electrophoresis.

29. A genetic lack of albumin
Analbuminemia
A genetic lack of albumin
Why did our routine laboratory methods detect 17g/L of albumin is there was NONE?

30. Re-evaluation of Dye-binding Serum Albumin Methods
Roche Diagnostics: BCG method (rapid)
Ortho Diagnostics: BCG method (slow)
BCG: bromcresol green
Linearity of albumin methods with diluted serum (constant albumin : globulin ratio)
Linearity of albumin methods with 100% human IgG or 100% human albumin.

32. Assay response to pure human Albumin

33. Assay response to pure human Ig G

34. Why did our assays report the presence of albumin in patients with analbuminemia ?
Ortho Diagnostics Assay:
Reacts with globulins
Gives a positive result in the absence of albumin.
The assay did not report that albumin was below the reportable limit.
Roche Diagnostics Assay:
Assay reported < 10 g/L in analbuminemia
Assay had no globulin interference.
Serum Protein Electrophoresis
Baseline disturbances during densitometry lead to reports of 2-3 g/L albumin.

35. Our three patients with analbuminemia all appeared to have albumin present, according to the dye-binding albumin methods.
What was the serum albumin concentration reported in the reported cases of analbuminemia?
It depends on the albumin methods that were used

36. 28 Cases of Analbuminemia: Levels of Albumin ‘detected’
A: Dye-binding methods
B: Salt Precipitation method
C: Protein Electrophoresis
D: Albumin Immunoassay

37. Awkward conclusions:
Clinicians should consider a diagnosis of analbuminemia (a genetic lack of albumin) even when the clinical laboratory detects serum albumin up to 17 g/L.
Albumin immunoassays and serum protein electrophoreses are capable of detecting analbuminemia, but routine serum albumin assays are NOT.

38. Patient #3 Diagnosis following i.v. albumin infusion.
Establish a partnership with Monica Galliano and Lorenzo Minchiotti, Univ. Pavia.
Isolation of genomic DNA
14 exons were amplified by PCR
Single-strand conformation polymorphism (SSCP) and heteroduplex analysis.
Sequencing

39. Exon # SSCP 1 Control 2 Mother 3 Patient 4 Control 5 Analb Codogno Exon #3 Heteroduplex Analysis 6 Control 7 Mother 8 Patient 9 Control 10 Analb. Codogno

40. ‘ ‘
‘ ‘
A: Control
B: Patient AT deletion
“Kayseri albumin”
‘ ‘

41. ‘AT’ deletion frameshift results in a stop condon and analbuminemia.

42. Three cases of analbuminemia…. Was this a coincidence?
Dr. David Meyer, Dept Anthropology, Univ. Saskatchewan, Doctoral Thesis:
Red Earth Crees 1860 – 1960
Ethnogeographic and historical work
Near-complete pedigrees 1860 – 1970
Study of the Deme

46. Deme: a marriage isolate or universe
Red Earth and Shoal Lake reservations had a common and stable deme 1860 – 1960.
Evacuation Photo: April 2007 Red Earth Reserve

47. Evacutation of Red Earth Reserve, April 2007

48. Red Earth and Shoal Lake Population
Date
Population
In group marriage
0 smallpox epidemic
1850
6 men
1870 – 1900
187
60%
1900 – 1930
220
85%
1930 – 1960
391
2007
1500 ?

49. 1971 Third Generation 75 Couples (married, widows/widowers).
8 marriages between first cousins.
23 marriages between second cousins.
35 marriages between relatives, > 2nd cousin.
9 insufficient to establish kinship.

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

51. September 2003: Patient #4
29 year old native female from Red Earth, SK
referred because of lower limb edema.
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

52. And so the story continues…
Life without Albumin

53. Acknowledgments
Garth Bruce, Paul Meinert, Robin Casey, Pat Blakely: Pediatrics, Jill Newstead, Sharon Card: Medicine. Univ. Saskatchewan.
Mark Salkie, Victor Laxdal Pathology, Univ. Saskatchewan.
David Meyer, Anthropology,
Univ. Saskatchewan.
Lorenzo Minchiotti, Monica Galliano & Lab,
Univ. Pavia.

54. References
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:
Galliano M, Campagnoli M, Rossi A, Wirsing von Konig CH, Lyon 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 ;48:844-9.
Newstead J, Card S, Lyon AW. Low serum albumin and abnormal body shape in a young Canadian First Nations woman. Lab Med Volume 35, Number 6 / June 2004.
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