Life without Albumin Encounters with Analbuminemia

1. Life without AlbuminEncounters 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 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 #1 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. 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. What was the serum albumin concentration reported in the reported cases of analbuminemia? Our three patients with analbuminemia all appeared to have albumin present, according to the dye-binding albumin methods. 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 #3 SSCP1 Control2 Mother3 Patient4 Control5 Analb CodognoExon #3 Heteroduplex Analysis6 Control7 Mother8 Patient9 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

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??