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December 1, 2020

A Deeper Dive Into B12 - Part 3

Welcome to part 3 of the ODX B12 Deficiency Series. In this post, the ODX Research Team reviews how we can use FBCA biomarkers and other assessment techniques to uncover vitamin B12 deficiency.vitamin B12 deficiency and what happens when get there.

Biomarkers and how we can assess for B12 deficiency

Dicken Weatherby, N.D. and Beth Ellen DiLuglio, MS, RDN, LDN

The ODX B12 Series

  1. Vitamin B12 Part 1 - Biochemistry and Physiology
  2. Vitamin B12 Part 2 - The Road to B12 Deficiency
  3. Vitamin B12 Part 3 - Biomarkers & Assessing B12 Deficiency
  4. Vitamin B12 Part 4 - How to Correct B12 Deficiency

A full clinical assessment of B12 status includes a review of signs and symptoms as well as extended blood chemistry analysis.

Assessment of B12 status can be complex because: [1]

 

  • Most patients presenting with B12 deficiency do not have anemia.
  • If clinicians wait until serum B12 drops below the standard range, it is likely that synthesis of DNA and cell maturation have already become impaired.
  • Assessment of related biomarkers can help identify those in early stages of B12 insufficiency who may be asymptomatic
  • Related biomarkers may help identify those who present with symptoms but with basic B12 labs, including serum B12, within the standard range.
  • If MCV is significantly elevated (e.g. up to 130 fL) but serum B12 is optimal, folate deficiency may be a compounding factor.
  • Concurrent thalassemia or iron deficiency may mask macrocytic changes in the cell.
  • Patients with spinal cord degeneration or peripheral neuropathy due to B12 deficiency may not have overtly abnormal hematological parameters as telltale red flags
  • The severity of neurological impairment may not correlate with the severity of megaloblastic anemia

The US Institute of Medicine deficiency cut-off for B12 deficiency is set at 203 pg/mL (150 pmol/L). However, researchers recognize an inverse relationship between adverse outcomes and serum B12 (even at “normal” levels).

Adverse outcomes include an increase in homocysteine and methylmalonic acid as serum B12 drops below 542 pg/mL (400 pmol/L).[2]

Although elevated homocysteine may not be exclusive to B12 insufficiency, levels above 9 umol/L should be investigated further,[3] especially considering that atherosclerosis increases progressively with a homocysteine level above 11 umol/L.[4]

Insufficiency of B12, reflected by a serum level of less than 407 pg/mL (300 pmol/L) occurs in up to 60% of the population and should be addressed before progressing to overt deficiency.[5]

Assessment of at least 2 biomarkers is recommended to identify a subclinical cobalamin deficiency (SCCD), as serum B12 alone does not reveal intracellular adequacy or function..[6]

Biomarker

Optimal

Serum B12 

450-800 pg/mL (investigate further if below 542 pg/mL (400 pmol/L) [7] or 881 pg/mL (650 pmol/L) or more [8]

Holotranscobalamin

54-188 pmol/L (investigate further if holotranscobalamin 25-70 pmol/L[9])

Homocysteine

5-7 umol/L

MCV

82-89 fL

Methylmalonic Acid (MMA)

0-260 nmol/L [10]

RDW

11.7-13%  

Fedosov’s Wellness Score

A variety of approaches for identifying B12 insufficiency/deficiency have been proposed, including a ”combined indicator” cB12 formula known as the Fedosov’s Wellness Score accounts for both very high and very low B12 status.[11]

cB12 = log10[ (holo-TC x serum B12) / (MMA x tHcy) ]−(age factor)

The Fedosov’s score can broken down into diagnostic levels:[12]

Ranges of combined vitamin B12 status, their equivalence to single cut-off values, and clinical interpretation.

Combined vitamin B12 status

Equivalence to single cut-points

Interpretation

Elevated B12 >1.5

B12 > 650
Holo-TC > 190
tHcy < 8.0
MMA < 0.11

The biological effects of high vitamin B12 are not fully understood

Adequate B12 −0.5 to 1.5

186 < B12 < 650
37 < holo-TC < 190
13.6 > tHcy > 8.0
0.35 < MMA < 0.11

Expected to support normal B12-dependent functions

Low B12 −1.5 to −0.5

119 < B12 < 186
20 < holo-TC < 37
19.2 > tHcy > 13.6
0.84 < MMA < 0.35

Subclinical deficiency. No hematological changes, subtle neurological impairment

Possible B12 deficiency −2.5 to −1.5

116 < B12 < 119
8.4 < holo-TC < 20
51 > tHcy > 19.2
1.7 < MMA < 0.84

Potential manifestations of vitamin B12 deficiency

Probable B12 deficiency

< −2.5

B12 < 116
holo-TC < 8.4
tHcy > 51
MMA > 1.7

Clinical manifestations of vitamin B12 deficiency

Units: B12 and holo-TC are expressed in pmol/L, and tHcy and MMA in μmol/L. Table adapted from Fedosov et al. (2015).[13]

Source: Hannibal, Luciana et al. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.” Frontiers in molecular biosciences vol. 3 27. 27 Jun. 2016.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

One retrospective cohort study of a mixed population utilized the 4 biomarker Fedosov formula and provided “marker cutoffs for detecting subclinical B12 deficiency (4cB12 ≤ -0.5 and >-1.5) with a sensitivity or specificity of 99% at a given specificity and sensitivity, as well as the optimum decision point” with the following results: [14]

Marker

Cutoff 99% sensitivity

Corresponding specificity (%)

Cutoff 99% specificity

Corresponding sensitivity (%)

Optimum decision point

Corresponding sensitivity/specificity (%)

HoloTC (pmol/L)

<73

44.1

<25

27.5

<45

85.7/81.2

B12 (pmol/L)

<351

36.7

<142

28.2

<229

86.1/77.7

MMA (nmol/L)

>152

37.8

>480

29.3

>245

81.8/83.4

Hcy (μmol/L)

>8

12.6

>29

11.5

>15

67.7/76.7

Markers of oxidative stress may be elevated in B12 deficiency as demonstrated in one clinical study of 51 individuals with serum B12 below 211 pg/mL (156 pmol/L), and 53 controls. Those with B12 deficiency had significantly lower glutathione and total antioxidant levels and significantly elevated malondialdehyde levels.[15]

Neuropsychological consequences are of B12 deficiency may be especially troubling in young vegetarians. With mean levels of serum B12 below 238 pg/mL (175 pmol/L), subjects had significantly higher levels of methylmalonic acid (mean 285 nmol/L) and significantly lower serum folate (mean 16.1 nmol/L). Vegetarians also had significantly greater incidence of depression, paresthesias, peripheral neuropathy, and psychosis compared to omnivores.[16]

Unfortunately, vegetarians often resist recommendations of B12 supplementation.[17]

Elevated B12 should also be assessed further

A serum B12 level above 1355 pg/mL (1000 pmol/L) may not necessarily be uncommon and may be related to changes in B12 binding proteins. However, a rise in haptocorrin may be associated with malignancy. Evaluation of unsaturated vitamin B12 binding capacity (UBBC) may be performed for further evaluation of elevated serum B12.[18]

Standard ranges include:

UBBC                            670-1200 ng/L

Haptocorrin                   49-143 ng/L

Transcobalamin             402-930 ng/L

Autoantibodies to        Elevated
transcobalamin            

Elevated serum B12 in a deficiency state

Serum B12 levels reflect both free and bound B12 and do not assess intracellular B12. Therefore, serum B12 may be elevated despite insufficiency or functional deficiency.[19]  

  • Serum levels may be above normal even if insufficiency is present.
  • Increased levels could be due to excess supplementation, increased transport proteins, or a hematological or autoimmune process.
  • Chronic disorders such as renal failure, cancer, or hepatic disease may promote elevated serum B12.
  • B12 may also be sequestered by immune cells and be unavailable to cells.
  • B12 may be elevated in critical illness and, when combined with an elevated CRP, may be associated with poorer outcomes and mortality.
  • Assess for other markers of B12 if a functional deficiency is suspected.
    • Increased MCV, homocysteine, and methylmalonic acid
    • Low holotranscobalamin
  • A functional B12 deficiency reflects a decrease in cellular uptake, processing, transport, or utilization.
  • Oxidative stress can also cause a local functional B12 deficiency, which can be seen in diabetes and Alzheimer’s. In such cases glutathione or vitamin C may be of therapeutic value.

An elevated serum methylmalonic acid level above 260 nmol/L along with an elevated serum B12 may be seen in functional B12 deficiency. However, this scenario may reflect kidney dysfunction and reduced renal clearance of MMA instead of B12 insufficiency. In such cases, a second biomarker such as holotranscobalamin should be evaluated. [20]

A significantly elevated B12 should be always be investigated further as it could be a sign of disease including leukemia, polycythemia vera, and hypereosinophilic syndrome.[21]

Unexplained elevated B12 may also be associated with other hematological cancers, solid neoplasms, kidney disease, and liver disease.[22]

Be sure to follow any clues to B12 insufficiency that may be hiding in basic and specialized blood chemistry panels. Identify any insufficiency or metabolic dysfunction early, before any debilitating symptoms set in.

Next Up: Vitamin B12 Part 4 - How to Correct B12 Deficiency

References

[1] Harrington, Dominic J. “Laboratory assessment of vitamin B12 status.” Journal of clinical pathology vol. 70,2 (2017): 168-173. 

[2] Smith, A David et al. “Vitamin B12.” Advances in food and nutrition research vol. 83 (2018): 215-279. 

[3] Hannibal, Luciana et al. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.” Frontiers in molecular biosciences vol. 3 27. 27 Jun. 2016, 

[4] University of Michigan. Pathology Handbook. Accessed October 25, 2020 from https://www.pathology.med.umich.edu/handbook/#/details/519

[5] Smith, A David et al. “Vitamin B12.” Advances in food and nutrition research vol. 83 (2018): 215-279. 

[6] Allen, Lindsay H et al. “Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review.” The Journal of nutrition vol. 148,suppl_4 (2018): 1995S-2027S. 

[7] Smith, A David et al. “Vitamin B12.” Advances in food and nutrition research vol. 83 (2018): 215-279. 

[8] Hannibal, Luciana et al. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.” Frontiers in molecular biosciences vol. 3 27. 27 Jun. 2016, 

[9] Harrington, Dominic J. “Laboratory assessment of vitamin B12 status.” Journal of clinical pathology vol. 70,2 (2017): 168-173. 

[10] Hannibal, Luciana et al. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.” Frontiers in molecular biosciences vol. 3 27. 27 Jun. 2016, 

[11] Harrington, Dominic J. “Laboratory assessment of vitamin B12 status.” Journal of clinical pathology vol. 70,2 (2017): 168-173. 

[12] Hannibal, Luciana et al. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.” Frontiers in molecular biosciences vol. 3 27. 27 Jun. 2016, 

[13] Fedosov, S. N., et al. "Combined indicator of vitamin B 12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points." Clinical Chemistry and Laboratory Medicine 10 (2015). 

[14] Jarquin Campos, Araceli et al. “Diagnostic Accuracy of Holotranscobalamin, Vitamin B12, Methylmalonic Acid, and Homocysteine in Detecting B12 Deficiency in a Large, Mixed Patient Population.” Disease markers vol. 2020 7468506. 7 Feb. 2020, 

[15] Misra, Usha Kant et al. “Oxidative Stress Markers in Vitamin B12 Deficiency.” Molecular neurobiology vol. 54,2 (2017): 1278-1284. 

[16] Kapoor, Aneel et al. “Neuropsychiatric and neurological problems among Vitamin B12 deficient young vegetarians.” Neurosciences (Riyadh, Saudi Arabia) vol. 22,3 (2017): 228-232.

[17] Rizzo, Gianluca et al. “Vitamin B12 among Vegetarians: Status, Assessment and Supplementation.” Nutrients vol. 8,12 767. 29 Nov. 2016.

[18] Harrington, Dominic J. “Laboratory assessment of vitamin B12 status.” Journal of clinical pathology vol. 70,2 (2017): 168-173. 

[19] Vollbracht, Claudia et al. “Supraphysiological vitamin B12 serum concentrations without supplementation - the pitfalls of interpretation.” QJM : monthly journal of the Association of Physicians, hcz164. 28 Jun. 2019.

[20] Hannibal, Luciana et al. “Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.” Frontiers in molecular biosciences vol. 3 27. 27 Jun. 2016, 

[21] Ermens, A A M et al. “Significance of elevated cobalamin (vitamin B12) levels in blood.” Clinical biochemistry vol. 36,8 (2003): 585-90. 

[22] Andrès, E et al. “The pathophysiology of elevated vitamin B12 in clinical practice.” QJM : monthly journal of the Association of Physicians vol. 106,6 (2013): 505-15.

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