holotranscobalamin.ppt

8/11/2019 holotranscobalamin.ppt

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Homocysteine

Homocysteine in Health and DiseaseMexico November 2010

The role ofHolotranscobalamin

in Vitamin B12metabolism anddiagnosis of VitaminB12 deficiency.


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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


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Cobalamin (Vitamin B12)

An essential nutrient(must be obtained fromthe diet).

Found in meat, fish and

dairy products.

Needed for producingred blood cells and for ahealthy nervous system.

Lack of B12 can cause

tiredness, dizziness andreduced sense of taste.

C63 H88 CoN 14 O 14 P
http://www.sciencephoto.com/image/7042/large/A6140085-Vitamin_B12_cyanocobalamin_molecule-SPL.jpg


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Vitamin B12 uptake

Adenosyl-B 12

Met

MMA

Methyl-B 12

Holotranscobalamin

(Biologically active)

Around 20% of circulating B12

Cobalamin

Holohaptocorrin(Biologically inert)

Around 80% of circulating B12

Specific receptor-mediated cellular uptake

Cobalaminbindingproteins

Hcy


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Development of B12 deficiency

Normal B 12 status

I II

Early serumdepletion

III IV V

Celldepletion

Damagedmetabolism

Clinicaldamage

B12 depletionNormal B12 deficiency

HoloTCMMA

HomocysteineSerum B12

Erythrocytes

MCVHb

NormalNormalNormalNormalNormal

NormalNormal

LowNormalNormalNormalNormal

NormalNormal

LowNormalNormalNormalNormal

NormalNormal

LowElevatedElevatedLowNormal

NormalNormal

LowElevatedElevatedLowMacrocytic

ElevatedLow

Adapted from Herbert V, Nut r i tion Sc ience a s a con t inua l ly un fo ld ing s to ry : the fo la t e and v i t amin B12 pa rad igmAmerican Journal of Clinical Nutrition 1987; 46: 387-402


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At risk groups

Patients at risk of B12 deficiency fall under 2 main categories:

1. Insufficient intakeo Vegetarians and vegans (lack of cobalamin in diet)

2. Reduced absorptiono Pernicious anaemiao 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)


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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 B12deficiency (Total B12 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


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Diagnosing Vitamin B12 deficiency

There is no gold standard test for determining B12 deficiencyand 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


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Assessment of B12 deficiency symptomsSymptoms 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


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Laboratory Tests

Four biochemical tests are routinely available for assessment of VitaminB12 status:

o Total Serum B12

o Methylmalonic Acid (MMA)

o Homocysteine

o Holotranscobalamin (HoloTC or Active-B12)


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Assessment of B12 deficiency laboratory tests

Total Serum B12

o Measures total, not bio-active B12.

o Levels


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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 elderlyeven among supposedly healthy persons.

o Can be falsely elevated in renal insufficiency particularly problematic inthe elderly.

o Highly sensitive but poor specificity

(1) Chanarin I and Metz J Brit J Haematol 1997;97:695-697


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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 inthe elderly.

o Sensitive but poor specificity - elevated levels may indicate folatedeficiency

(1) Chanarin I and Metz J Brit J Haematol 1997;97:695-697


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Holotranscobalamin

o Many recent studies show improved sensitivity and specificity overTotal 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.


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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 laboratoryvariation)

Must be an early marker of deficiency


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Correlation of Total B12 with HoloTC

0

50

100

150

200

250

300

350

0 100 200 300 400 500 600 700 800 900

Total Serum B12 pmol/L

H o

l o T C p m o

l / L

n = 468

Spearman correlation r = 0.74


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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 SerumB12 (in this case 151-300

pmol/L) contains a number ofsamples which are actually

deficient, as shown by a HoloTClevel


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Low Total B12 but normal HoloTC

Mild Haptocorrin deficiency features low Haptocorrin and low Total B12levels and may account for up to 15% of all low Total B 12 levels .Haptocorrin deficiency should be added to the list of common causes of lowTotal B12 results.

(Note: the definition of low Total B12 varied but generally


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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


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HoloTC distribution in population groups

Omnivores (median age 41 yrs)

Vegans

LV/LOV

Elderly

Serum concentration of holoTC, pmol/L

256115816243281

100

80

60

40

20

0

C u m u

l a t i v e

d i s t r i b u t i o n o

f h o

l o T C %

Herrmann et al CurrentDrug Metabolism, 2005,

6, 47-53


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B12 in clinical conditions - Pregnancy

o Total B12 is known todecline in pregnancy.

o Blood samples weretaken at 18, 32 and 39weeks gestation and 8weeks post-partum fromhealthy pregnant Danishwomen >18 years of age.

o There was a statisticallysignificant drop in TotalB12 levels but nostatistically significantchange in HoloTC levels .

0

50

100

150

200

250

300

350

18th 32nd 39th 8pp

Gestation

p m o

l / L

Total B12HoloTC

HoloTC but NOT Total B12 can be used to assess B12status during pregnancy


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B12 in clinical conditions Cognitive decline Cognitive function was

assessed in n=2741elderly subjects (meanaged 75.7 years) andreassessed in survivors(n=472) 10 years later.

Rate of cognitivedecline was significantlypredicted by HoloTClevel. A doubling ofHoloTC levels wasassociated with a 30%slower rate of decline .

Cognitive decline wasNOT predicted by TotalB12 status.

Clarke et al American Journal of Clinical Nutrition, 2007 Nov;86(5):1384-91.

Total B12 (pmol/l)

0 200 400 600 800

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140 160 180

0

0.2

0.4

0.6

0.8

1

HoloTC (pmol/l)

ControlsAlzheimer


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Performance of Total B12 and HoloTC versus MMA

from Obeid R Herrmann W Clin Chem Lab Med 2007;45:1746-50


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Performance of Total B12 and HoloTC

Number of subjects(number of subjects withB12 deficiency)

MMA cut-offused to define

deficiency(mol/L)

AUCHoloTC

AUCTotal 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

(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

Across >3000 patients, HoloTC consistently outperforms Total B12,regardless of MMA cut-off


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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 trueB12 status in the tissues.


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Proportion of samples in grey-zone

MMA HoloTC Total B12

Grey-zone 0.310 to 1.402 mol/L 19.6 to 29.9 pmol/L 79 to 238 pmol/L

Samples ingrey-zone (n)

349/700 96/699 313/700

Samples ingrey-zone

(%)

50% 14% 45%

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).

A high proportion of samples in the grey-zone restricts clinicalutility


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Summary can HoloTC replace Total B12?

o Must be a better marker of Vitamin B12 statusHoloTC measures only the biologically-available form of B12.HoloTC appears to be a better measure of B12 status then Total B12 whenusing either MMA or RBC-B12 as the true definition.

o Must offer improved sensitivity and specificityHoloTC appears to be more sensitive and more specific than Total B12.

o Pre-analytical steps (main source of laboratory variation) should beremoved.No pre-analytical steps.

o Must be an early marker of deficiency.Changes in HoloTC levels occur earlier than Total B12 inVitamin B12 depletion.


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Using Active-B12 in the routine laboratory

1. Resolution of grey-zone Total B12 results

2. Replacement for Total B12 assay.


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Resolution of grey-zone Total B12 results

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

Subjects at risk of B 12 deficiency

Total B 12 250 pmol/L

Measure HoloTC

Likely deficient Unlikely deficient

Resolve B 12 indeterminate

samples


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Replacement for Total B12 assay

Subjects at risk of B 12 deficiency

HoloTC 35 pmol/L

Likely deficient Unlikely deficient*

Note that all suggested cut-offs will be dependent on the population served by the laboratory

*Renal patients should be further investigatedwith creatinine and/or MMA


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HoloTC in practice Melbourne Pathology, Australia

Protocol offers the test to every patient with Total B12


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HoloTC in practice GSTS Pathology, UK

Primary interest is the development and application of novelmarkers of vitamins to improve patient care.

The majority (up to 80%) of serum vitamin B12 is not bio -available. Currentassays measure total vitamin B12 which leads to a grey area where deficientpatients can be missed - there is a poor correlation between circulatory totalB12 and B12 status at the tissue level. Conversely patients caninappropriately be classified to a deficient state with the inconvenience andexpense of long term supplementation regimes.

The Nutristasis Unit will be providing this new nutritional marker fromSeptember and GSTS will be the first to offer it outside of researchenvironment in the UK.


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Quotes

Accumulating evidence indicates that this assay may enhance the predictive power for identifyingvitamin 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 ofvitamin B-12 deficiency....On the basis of the data we present in this review, we predict that holoTC willalso 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 withserum 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 ofB12 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


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Conclusions

o Vitamin B12 deficiency is a significant public health concern,especially for the elderly.

o Early detection is vital to avoid progression to irreversibleneurological damage but lack of consensus and lack of definition ofdeficiency hampers diagnosis.

o Symptoms, haematological signs and laboratory tests can beconflicting.

o HoloTC appears to be an earlier, more sensitive and specific markerthan the current Total B12 test.


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