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
Objectives:
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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.
The History of ‘albumin’
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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
The History of ‘albumin’
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1970
1975
1979
1981
1986
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King: Studied tryptic fragments of albumin
Brown / Meloun: Amino acid sequence BSA & HAS
Sargent: Isolated the HAS gene
Lawn: Genetic sequence of HAS cDNA
Dugaiczyk: Complete HAS gene sequence
Mutation studies
Crystallization studies
Parenteral albumin utilization studies
Physiological Roles of Albumin
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Circulatory Role……….80% oncontic pressure
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Transport of metabolites
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Sequestration of toxins… delivery to the liver
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Bilirubin, calcium, fatty acids, bile acids, drugs
Low affinity reservoir for thyroxine, vitamin D
Bilirubin, carcinogens.
Metabolic Effects: enhances lipoprotein lipase
Miscellaneous Effects: limits fibrin fiber thickness
Clinical Utility of Serum Albumin
Levels
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Assess protein malnutrition
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Renal disease… nephrotic syndrome
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G.I. pathology
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Liver disease… impaired synthesis
Hypoalbuminemia, intestinal edema, diarrhea
Methods of albumin quantification
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Turbidometry: following acid or salt precipitation
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Dye-binding methods
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Protein-error of pH indicating dyes
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Protein electrophoresis
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Immunoassay
Dye-binding Methods
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High pH causes a colour change:
High pH
Dye-H
Dye-
+ H+
Add albumin at Constant pH
Albumin
Dye-H
Dye--Albumin
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Applications: Urine dipstick, routine clinical chemistry.
Dye-binding Methods
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Urine Dipsticks: Urine Protein detection
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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
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Bromcresol Green , Bromcresol Purple
Short incubation times to improve specificity
Serum Protein Electrophoresis
Albumin (65%)
Globulins (35%)
o
o
o
o
o
o
o
Transthyretin / Prealbumin
Antitrypsin, Acid Glycoprotein
Haptoglobin, Macroglobulin,
Transferrin, C3
Lipoproteins (VLDL, LDL, HDL)
Missing: “Fibrinogen”
Immunoglobulins ( IgG, IgA, IgM, IgD,
IgE)
LIVER
From: Immunofixation. Killingsworth & Warren
Calgary Laboratory Services
Serum Protein Electrophoresis
Bisalbuminemia
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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.
Albumin Immunoassay
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Method usually reserved for urine or CSF
albumin determination: “microalbumin”
Various immunoassay methods: rate
nephelometry, nephelometry,
turbidometry, radial immunodiffusion.
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.
Our first case…
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
Paul Meinert (pediatric resident) complains
“why are serum albumin levels in your lab
fluctuating so much?”
“What do you mean by fluctuating?”
Variable levels of serum albumin
over 6 months, (7 month old infant)
18
Method_1
Method_2
Method_3
16
14
12
g/L
<10
10
8
6
4
2
0
a
b
c
d
e
f
g
Impact of low albumin??
Bilirubin-Alb
Fatty Acids - Alb
Adipose
Tissue
T4 - Alb
Calcium - Alb
Alb for Oncotic Pressure,
to avoid edema
Patient #1
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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.
Patient #1
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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.
Analbuminemia
Albumin (Dye-binding) :
10 – 17 g/L
Albumin (electrophoresis):
2 – 3 g/L
Albumin (Immunoassay) : <0.01 g/L
Consistent with:
Analbuminemia
A genetic lack of albumin
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What is analbuminemia and what is the
prognosis ?
Why did our routine laboratory methods detect
17g/L of albumin is there was NONE?
Analbuminemia
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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.
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VERY RARE (approx. 42 reported cases)
Lipodystrophy in Analbuminemia
Patient #1
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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.”
Analbuminemia
Patient #2
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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
Patient #3
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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.
Analbuminemia
A genetic lack of albumin
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Why did our routine laboratory methods
detect 17g/L of albumin is there was
NONE?
Re-evaluation of Dye-binding
Serum Albumin Methods
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Roche Diagnostics: BCG method (rapid)
Ortho Diagnostics: BCG method (slow)
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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.
Comparison of serum albumin methods
with diluted serum
Observed Albumin g/L
30
25
Roche
20
Ortho
15
10
5
0
0
10
20
30
Target Serum Albumin g/L
40
70
Roche Serum Albumin g/L
Ortho Serum Albumin g/L
Assay response to pure human Albumin
60
50
Ortho
40
30
20
10
0
0
20
40
60
100% Human Albumin g/L
80
70
60
50
Roche
40
30
20
10
0
0
20
40
60
100% Human Albumin g/L
80
12
Roche Serum Albumin g/L
Ortho Serum Albumin g/L
Assay response to pure human Ig G
Ortho
10
8
6
4
2
0
0
20
40
100% Human Ig G g/L
60
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Roche
0
20
40
100% Human Ig G g/L
60
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.
Our three patients with analbuminemia all appeared
to have albumin present, according to the dyebinding albumin methods.
What was the serum albumin
concentration reported in the
reported cases of analbuminemia?
It depends on the albumin methods that were used
28 Cases of Analbuminemia:
Levels of Albumin ‘detected’
A: Dye-binding
methods
B: Salt Precipitation
method
C: Protein
Electrophoresis
D: Albumin
Immunoassay
Awkward conclusions:
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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.
Patient #3 Diagnosis following i.v.
albumin infusion.
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Establish a partnership with Monica
Galliano and Lorenzo Minchiotti, Univ.
Pavia.
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Isolation of genomic DNA
14 exons were amplified by PCR
Single-strand conformation polymorphism
(SSCP) and heteroduplex analysis.
Sequencing
Exon #3
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
‘
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A: Control
B: Patient AT deletion
“Kayseri albumin”
‘
‘
‘AT’ deletion frameshift results in a
stop condon and analbuminemia.
Three cases of analbuminemia….
Was this a coincidence?
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Dr. David Meyer, Dept Anthropology, Univ.
Saskatchewan, Doctoral Thesis:
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Red Earth Crees 1860 – 1960
Ethnogeographic and historical work
Near-complete pedigrees 1860 – 1970
Study of the Deme
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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
Evacutation of Red Earth Reserve, April 2007
Red Earth and Shoal Lake
Population
Date
Population
1781 -1782
In group marriage
1850
0 smallpox
epidemic
6 men
1870 – 1900
187
60%
1900 – 1930
220
85%
1930 – 1960
391
85%
2007
1500
?
1971 Third Generation
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75 Couples (married, widows/widowers).
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8 marriages between first cousins.
23 marriages between second cousins.
35 marriages between relatives, > 2nd cousin.
9 insufficient to establish kinship.
There is likely a ‘founder effect’ responsible
for the local incidence of analbuminemia
at the Red Earth and Shoal Lake reserves.
Future Investigations??
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
And so the story continues…
Life without Albumin
Acknowledgments
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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.
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: 2365-7.
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. 2002 ;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.
For more details on analbuminemia, visit the Albumin Website:
http://www.albumin.org