Homocysteine
The role of
Holotranscobalamin
inHomocysteine
Vitamin B12
in Health and
metabolism and
Mexico November 2010
diagnosis of Vitamin
B12 deficiency.
Disease
Topics
o Metabolism of Vitamin B12
o Symptoms and diagnosis of Vitamin B12 deficiency
o Laboratory tests for assessment of Vitamin B12 status
o Current state and unmet needs
o Role of Holotranscobalamin
Cobalamin (Vitamin B12)
An essential nutrient
(must be obtained from
the diet).
Found in meat, fish and
dairy products.
Needed for producing
red blood cells and for a
healthy nervous system.
Lack of B12 can cause
tiredness, dizziness and
reduced sense of taste.
C63H88CoN14O14P
Absorption and transport of Vitamin B12
Protein-bound Cobalamin
Cbl.P
Cbl
Cobalamin in food released during peptic digestion
HoloHC
DUODENUM
STOMACH
IF
IF.Cbl
Gastric parietal cells secrete Intrinsic Factor (IF)
80%
HoloH
C
IF.Cbl
Gastric parietal cells secrete Haptocorrin (HC)
HoloHC
Pancreatic protease HC
degrade HoloHC
LIVER
HCl, pepsin
HC
HoloTC
IF
IF.Cbl
TC
HoloTC
Brush border receptors for IF
Only HoloTC is taken up by cells
enterocyte
20%
HoloT
C
TERMINAL ILEUM
BLOOD
HoloTC
TISSUE CELLS
Vitamin B12 uptake
Cobalamin
Holotranscobalamin
(Biologically active)
Cobalamin
binding
proteins
Around 20% of circulating B12
Holohaptocorrin
(Biologically inert)
Around 80% of circulating B12
Hcy
Met
Methyl-B12
Adenosyl-B12
MMA
Specific receptormediated cellular uptake
Development of B12 deficiency
Normal
HoloTC
MMA
Homocysteine
Serum B12
Erythrocytes
MCV
Hb
B12 depletion
B12 deficiency
Normal B12
status
Early serum
depletion
Cell
depletion
Damaged
metabolism
Clinical
damage
I
II
III
IV
V
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Low
Normal
Normal
Normal
Normal
Normal
Normal
Low
Normal
Normal
Normal
Normal
Normal
Normal
Low
Elevated
Elevated
Low
Normal
Normal
Normal
Adapted from Herbert V, Nutrition Science as a continually unfolding story: the folate and vitamin B12 paradigm
American Journal of Clinical Nutrition 1987; 46: 387-402
Low
Elevated
Elevated
Low
Macrocytic
Elevated
Low
At risk groups
Patients at risk of B12 deficiency fall under 2 main categories:
1. Insufficient intake
o Vegetarians and vegans (lack of cobalamin in diet)
2. Reduced absorption
o Pernicious anaemia
o Elderly (age-related, often asymptomatic atrophic gastritis)
o Patients with intestinal disease or after gastric surgery (loss of
intrinsic factor)
o Patients on long term therapy with proton pump inhibitors and
histamine receptor antagonists (inhibition of gastric secretion of acid)
Prevalence of Vitamin B12 deficiency
o In Canada, approximately 5% are B12-deficient (1)
o In the USA, 3-6% of adults are estimated to have vitamin B12
deficiency (Total B12 <148pmol/L) (2) (3)
o In the UK, B12 deficiency (Total B12 ≤150pmol/L) is estimated at 5%
of those aged 65-74y and 10% in those aged ≥75y (4)
o Marginal depletion (Total B12 of 148-221pmol/L) is more common,
occurring in >20% of those aged >60y (2) (3)
(1) Macfarlane et al Am J Clin Nutr 2011;94:1079-1087
(2) Pfeiffer et al Am J Clin Nutr 2005;82:442-50
(3) Pfeiffer et al Am J Clin Nutr 2007;86:718-27
(4) Clarke et al Age Ageing 2004;33:34-41
Diagnosing Vitamin B12 deficiency
There is no gold standard test for determining B12 deficiency
and no general consensus on diagnosis of deficiency.
• Medical History
• Physical exam
• Blood tests
• Blood Cell Counts (CBC)
• Haemoglobin (Hb)
• Biochemical tests
•
•
•
•
•
•
Total Serum B12
MMA
Homocysteine
Anti-Parietal Antibody
Anti-Intrinsic Factor Antibody
Serum Gastrin
Assessment of B12 deficiency – symptoms
Symptoms are diffuse and can easily be overlooked:
•
•
•
•
•
Weakness
Pale skin
Shortness of breath
Chest pain
Neurological symptoms
• Ataxia
• Paraesthesia of hands and feet
• Diminished perception of vibration and position
•
Neuropsychiatric symptoms
• Confusion and memory disturbances
• Cognitive decline
•
Headache
Assessment of B12 deficiency – haematological signs
Haematological tests can be misleading:
•
Low Haemoglobin (Hb) and/or macrocytosis have traditionally been
indications of B12 deficiency.
•
Absence of macrocytosis in B12 deficiency may also be encountered
in patients with concurrent iron deficiency.
•
>25% of patients with neurological manifestations of B12 deficiency
have a normal haematocrit or Mean Corpuscular Volume (MCV) or
both*.
•
Elevated MCV is not specific and in most cases is related to other
conditions including alcoholism, liver disease or drug use.
* Lindenbaum et al N Eng J Med 1988;318:1720-1728
Laboratory Tests
Four biochemical tests are routinely available for assessment of Vitamin
B12 status:
o
Total Serum B12
o
Methylmalonic Acid (MMA)
o
Homocysteine
o
Holotranscobalamin (HoloTC or Active-B12)
Assessment of B12 deficiency – laboratory tests
Total Serum B12
o
Measures total, not bio-active B12.
o
Levels <148 pmol/L (200 pg/ml) are generally considered indicative of deficiency.
•
•
o
Proportion of patients with levels below 148pmol/L have no clinical or biochemical evidence of
deficiency (1)
Neuropsychiatric and metabolic disturbances can occur with B12 levels well above 148pmol/L (1),(2)
Significant indeterminate zone above 148pmol/L.
•
•
Total B12 discriminates poorly between 75-300pmol/L (3)
Further testing is required for levels 125-250pmol/L (4)
o
Estimates suggest up to 45% of B12 deficient subjects would be missed if only
relying on Total B12 as a screening test (5).
o
Large variation between laboratories and methods due to pre-analytical steps
(1)
(2)
(3)
(4)
(5)
Green R et al., Baillieres Clin Haematol., 1995; 8: 533-6
Lindenbaum J et al., New Eng J Med., 1988; 318: 1720-8
Snow CF Arch Intern Med., 1999 ;159 :1289-98.
Hvas AM et al., Ugeskr Laeger., 2003; 165: 1971-6.
Hermmann W et al Curr. Drug Metab. 2005; 6 : 47-53
Assessment of B12 deficiency – laboratory tests
Methylmalonic acid (MMA)
o Functional marker of intracellular cobalamin metabolism.
o Can be elevated despite normal cobalamin and HoloTC levels
(1)
o Levels increase with age and elevated levels are common in the elderly
even among supposedly healthy persons.
o Can be falsely elevated in renal insufficiency – particularly problematic in
the elderly.
o Highly sensitive but poor specificity
(1) Chanarin I and Metz J Brit J Haematol 1997;97:695-697
Assessment of B12 deficiency – laboratory tests
Homocysteine
o Also a functional marker of intracellular cobalamin metabolism.
o Can be elevated despite normal cobalamin and HoloTC levels
(1)
o Levels increase with age
o Can be falsely elevated in renal insufficiency – particularly problematic in
the elderly.
o Sensitive but poor specificity - elevated levels may indicate folate
deficiency
(1) Chanarin I and Metz J Brit J Haematol 1997;97:695-697
Assessment of B12 deficiency – laboratory tests
Holotranscobalamin
o Many recent studies show improved sensitivity and specificity over
Total B12.
o Changes in HoloTC levels precede changes in other markers.
o Widely regarded to be a better indicator than Total B12 of Vitamin B12
status.
Current State – Unmet Needs
1.
•
•
•
Accurate diagnosis of vitamin B12 status
Diffused and varied signs and symptoms.
Potentially misleading haematological parameters.
Limitations of current front-line Total B12 test and other reflex
tests.
2. Early diagnosis of B12 deficiency
Needed in order to initiate treatment before disease
symptoms manifest and/or become irreversible
(neurological).
Can HoloTC meet these needs?
o Should HoloTC replace Total B12 as the front line test?
• Must be a better marker of Vitamin B12 status
• Must offer improved sensitivity and specificity
• Should have no Pre-analytical steps (main source of laboratory
variation)
• Must be an early marker of deficiency
Correlation of Total B12 with HoloTC
350
300
HoloTC pmol/L
250
200
150
100
50
0
0
100
200
300
400
500
600
Total Serum B12 pmol/L
n = 468
Spearman correlation r = 0.74
700
800
900
Correlation of Total B12 with HoloTC
n=468 patients
350
300
HoloTC pmol/L
250
n=13
n=344
Low
B12,
high
HoloTC
High B12,
high HoloTC
85 results (18%)
show a
discrepancy
between Total
B12 and HoloTC.
200
More importantly,
72 patients
(15%) have Total
B12 levels
>150pmol/L but
are deficient by
HoloTC.
150
100
50
0
0
100
n=39
Low B12,
low HoloTC
200
300
400
500
600
Total Serum B12 pmol/L
700
n=72
High B12,
low HoloTC
800
900
Resolution of grey-zone B12 results
A = deficient by Total B12 test
B = indeterminate by Total B12 test
C = replete by Total B12 test
The grey-zone for Total Serum
B12 (in this case 151-300
pmol/L) contains a number of
samples which are actually
deficient, as shown by a HoloTC
level <20pmol/L.
A percentage of patients with Total B12
levels <150pmol/L are actually replete
Raw Data provided by Prof. W Herrmann, Universitätskliniken des Saarlandes, Homburg, Germany
Low Total B12 but normal HoloTC
Mild Haptocorrin deficiency features low Haptocorrin and low Total B12
levels and may account for up to 15% of all low Total B12 levels.
Haptocorrin deficiency should be added to the list of common causes of low
Total B12 results.
(Note: the definition of “low Total B12” varied but generally <140-180pmol/L).
Expected Values of HoloTC
Study
n
Median [Mean]
pmol/L
90% [95%]
Reference Interval (pmol/L)
1
93
54
17 – 114
2
79
54
16 – 122
3
105
[61]
[24 – 157]
4
281
[50]
[19 – 134]
(1) Herrmann W et al Clin Chem Lab Med 2003;41: 1478-88
(2) Herrmann W et al Am J Clin Nutr 2003;78:131-67
(3) Ulleland M et al. Clin Chem 2002; 48(3): 526 - 532.
(4) Abbott AxSYM Package Insert
Cumulative distribution of holoTC %
HoloTC distribution in population groups
LV/LOV
100
Vegans
80
60
Omnivores
(median age 41 yrs)
Elderly
40
Herrmann et al Current
Drug Metabolism, 2005,
6, 47-53
20
0
1
28
43
62
81
115
256
Serum concentration of holoTC, pmol/L
B12 in clinical conditions - Pregnancy
350
o
o
Total B12 is known to
decline in pregnancy.
Blood samples were
taken at 18, 32 and 39
weeks gestation and 8
weeks post-partum from
healthy pregnant Danish
women >18 years of age.
There was a statistically
significant drop in Total
B12 levels but no
statistically significant
change in HoloTC levels.
300
250
pmol/L
o
200
Total B12
HoloTC
150
100
50
0
18th
32nd
39th
Gestation
HoloTC but NOT Total B12 can be used to assess B12
status during pregnancy
8pp
B12 in clinical conditions – Cognitive decline
• Cognitive function was
assessed in n=2741
elderly subjects (mean
aged 75.7 years) and
reassessed in survivors
(n=472) 10 years later.
• Rate of cognitive
decline was significantly
predicted by HoloTC
level. A doubling of
HoloTC levels was
associated with a 30%
slower rate of decline.
• Cognitive decline was
NOT predicted by Total
B12 status.
1
1
Alzheimer
Controls
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
0 20 40 60 80 100 120 140 160 180
HoloTC (pmol/l)
Clarke et al American Journal of Clinical Nutrition, 2007 Nov;86(5):1384-91.
0
200
400
600
Total B12 (pmol/l)
800
Sensitivity and Specificity – Total B12 v. HoloTC
o In order to assess Sensitivity and Specificity of a test, there must be
a ‘gold standard’ or true definition against which both tests can be
compared.
o Early studies used MMA as the true definition of B12 deficiency and
compared Total B12 to HoloTC.
Performance of Total B12 and HoloTC versus MMA
from Obeid R Herrmann W Clin Chem Lab Med 2007;45:1746-50
Performance of Total B12 and HoloTC
Number of subjects
(number of subjects
with
B12 deficiency)
MMA cut-off
used to define
deficiency
(µmol/L)
AUC
HoloTC
AUC
Total B12
Reference
806 (24)
>0.75
0.90
0.85
1
1651 (70)
>0.75
0.87
0.79
2
1651 (129)
>0.45
0.80
0.73
2
759 (174)
>0.30
0.71
0.60
3
204 (68)
>0.27
0.88
0.84
4
Across >3000 patients, HoloTC consistently outperforms Total B12,
regardless of MMA cut-off
(1) Hvas A Nexo E J Intern Med 2005;257:289-98
(2) Clarke et al Clin Chem 2007;53:963-70
(3) Obeid R Herrmann W Clin Chem Lab Med 2007;45:1746-50
(4) Herrmann et al Clin Chem Lab Med 2003;41:1478-88
New definition of B12 deficiency
o Use of MMA as the gold standard is flawed due to poor specificity.
o A recent study used Red Blood Cell B12 as a measure of the true
B12 status in the tissues.
Performance of markers against RBC-B12
1
0.9
Sensitivity (true positives)
0.8
AUC
0.7
No discrimination
0.6
Active B12 pmol/L
0.5
S.B12 pmol/L
0.4
MMA umol/l
HoloTC
0.899
Total B12 0.801
0.3
MMA
0.2
0.776
0.1
0
0
0.2
0.4
0.6
0.8
1
1 - Specificity (false positives)
Marker
Cut-off
Sensitivity
Specificity
HoloTC
<20pmol/L
55%
96%
Total B12
<150pmol/L
46%
88%
MMA
>0.36µmol/L
81%
63%
Using this novel approach, HoloTC is still superior to
Total B12 and can now be shown to be superior to MMA
Proportion of samples in grey-zone
Potential clinical use was assessed by use of a grey-zone with limits of 60%
for ruling-in deficiency (PPV) and 98% for ruling-out deficiency (NPV).
MMA
HoloTC
Total B12
Grey-zone
0.310 to 1.402
µmol/L
19.6 to 29.9 pmol/L
79 to 238 pmol/L
Samples in
grey-zone (n)
349/700
96/699
313/700
Samples in
grey-zone
(%)
50%
14%
45%
A high proportion of samples in the grey-zone restricts clinical
utility
Summary – can HoloTC replace Total B12?
o
Must be a better marker of Vitamin B12 status
HoloTC measures only the biologically-available form of B12.
HoloTC appears to be a better measure of B12 status then Total B12 when
using either MMA or RBC-B12 as the true definition.
o
Must offer improved sensitivity and specificity
HoloTC appears to be more sensitive and more specific than Total B12.
o
Pre-analytical steps (main source of laboratory variation) should be
removed.
No pre-analytical steps.
o
Must be an early marker of deficiency.
Changes in HoloTC levels occur earlier than Total B12 in
Vitamin B12 depletion.
Using Active-B12 in the routine laboratory
1. Resolution of grey-zone Total B12 results
2. Replacement for Total B12 assay.
Resolution of grey-zone Total B12 results
Subjects at risk of B12 deficiency
Total B12 <150 pmol/L
Total B12 150-250 pmol/L
Total B12 >250 pmol/L
Measure HoloTC
Likely deficient
Unlikely deficient
Resolve B12
indeterminate
samples
Adapted from Schneede J., Scan J Clin Lab Invest 2003; 63: 369 – 376
Note that all suggested cut-offs will be dependent on the population served by the laboratory
Replacement for Total B12 assay
Subjects at risk of B12 deficiency
HoloTC <35 pmol/L
Likely deficient
HoloTC >35 pmol/L
Unlikely deficient*
*Renal patients should be further investigated
with creatinine and/or MMA
Note that all suggested cut-offs will be dependent on the population served by the laboratory
Axis-Shield EIA
o Axis-Shield part number FMABT100.
o Ready-to-use reagents, 96-well break-apart micro-titre plate.
o Includes six assay calibrators and two kit controls (Low and High).
o Alkaline phosphatase conjugate with pNPP substrate, read at 405nm.
o Serum or serum-separator collection tubes.
HoloTC in practice – Melbourne Pathology, Australia
Protocol offers the test to every patient with Total B12 <200pmol/L
“….From our experience reviewing these cases, Active B12 has advantage over total
B12 in assessing B12 status of an individual. Active B12 was helpful in excluding
B12 deficiency falsely ‘identified’ by low total B12 such as in pregnancy. It is also
helpful in confirming true B12 deficiency missed by a normal total B12 in patients
such as those with iron deficiency and hypothyroidism.”
“Active B12 has fulfilled our expectation in being a robust assay that improves the
assessment of vitamin B12 status. It provides reassurance to us that we have
contributed to improved patient care. The routine availability of this assay
differentiates our laboratory as a progressive provider of quality pathology.”
HoloTC in practice – Erasmus Medical Centre, Netherlands
HoloTC is offered routinely with a cut-off of 32pmol/L
“Active-B12 appears to be a better predictor of disturbed B12-dependent
metabolism than Total B12.”
“Accepting MMA >0.45µmol/L as a reference standard for Vitamin B12 deficiency
the Active-B12 assay demonstrates a better sensitivity and specificity in
detecting Vitamin B12 deficiency than the Total B12 assay in a mixed collection of
diagnostic samples.”
“For the detection of B12-deficiency Active-B12 can replace Total B12 as a firstline diagnostic aid; no reason for combination with Total B12.”
HoloTC in practice – GSTS Pathology, UK
“Primary interest is the development and application of novel
markers of vitamins to improve patient care.”
“The majority (up to 80%) of serum vitamin B12 is not bio-available. Current
assays measure total vitamin B12 which leads to a grey area where deficient
patients can be missed - there is a poor correlation between circulatory total
B12 and B12 status at the tissue level. Conversely patients can
inappropriately be classified to a deficient state with the inconvenience and
expense of long term supplementation regimes.”
“The Nutristasis Unit will be providing this new nutritional marker from
September and GSTS will be the first to offer it outside of research
environment in the UK.”
Quotes
“Accumulating evidence indicates that this assay may enhance the predictive power for identifying
vitamin B12 deficiency, either alone or when used in conjunction with other available tests” (1)
“Today, we can conclude that holoTC seems more suitable than total vitamin B-12 for diagnosis of
vitamin B-12 deficiency....On the basis of the data we present in this review, we predict that holoTC will
also be an excellent marker for monitoring a population’s vitamin B-12 status” (2)
“This study supports the use of holoTC as the first-line diagnostic procedure for vitamin B(12) status” (3)
“HoloTC levels were more sensitive than the serum vitamin B12 levels for indicating vitamin B12 status.
If the serum vitamin B12 level is 151-300 pmol/L, the levels of holoTC alone or in combination with
serum vitamin B12 levels are likely to be more useful markers than serum vitamin B12 levels alone” (4)
“From these results it can be concluded that serum concentration of holoTC is a much better predictor of
B12 status than total B12” (5)
(1) Green AJCN Jul 2011;94(suppl):666S-72S
(2) Nexo E, Hoffmann-Lucke E. AJCN Jul 2011;94(1):359S-65S
(3) Valente et al Clin Chem. 2011;57:6 856-863
(4) Woo et al Korean J Lab Med. 2010 Apr;30(2):185-9
(5) Herrmann Current Drug Metabolism, 2005, 6, 47-53
Conclusions
o Vitamin B12 deficiency is a significant public health concern,
especially for the elderly.
o Early detection is vital to avoid progression to irreversible
neurological damage but lack of consensus and lack of definition of
deficiency hampers diagnosis.
o Symptoms, haematological signs and laboratory tests can be
conflicting.
o HoloTC appears to be an earlier, more sensitive and specific marker
than the current Total B12 test.
www.active-b12.com