Vitamin B₁₂ status in health and disease: a critical review. Diagnosis of deficiency and insufficiency – clinical and laboratory pitfalls

Figures & data

Figure 1. The chemical structure of vitamin B12.

Figure 2. Vitamin B₁₂ absorption and intracellular processing via two enzymatic pathways. In the absence of vitamin B₁₂, 5-MTHF becomes metabolically trapped in this form producing a pseudo folate-deficient state (methyl-trap) and cannot be utilized for regeneration of THF. Cbl: cobalamin; CBS: cystathionine beta-synthase; dTMP: deoxythymidine monophosphate; dUMP: deoxyuridine monophosphate; DHFR: dihydrofolate reductase; HC: haptocorrin; holoTC: holotranscobalamin; HO-Cbl: hydroxocobalamin; IF: intrinsic factor; MS: methionine synthase; Me-Cbl: methylcobalamin; MTHFR: methylene tetrahydrofolate reductase; MMA: methylmalonic acid; MCM: methylmalonyl-CoA mutase; 5-MTHF: 5-methyltetrahydrofolate; SAH: S-Adenosyl homocysteine; SAM: S-Adenosyl methionine; THF: tetrahydrofolate; TS: thymidylate synthase; TC: transcobalamin.

Figure 3. Relationship between the intake and percentage of absorbed B₁₂ from a dose of [⁵⁸Co]-cyanocobalamin (Adams et al. [34]) and [¹³C]-cyanocobalamin (Devi et al. [35]) in n = 10–12 healthy volunteers.

Figure 4. The correlation between holoTC and serum B₁₂ in the low holoTC range, <25 pmol/L. The horizontal line on the y-axis represents the serum B₁₂ deficiency cutoff of 138 pmol/L used in the authors’ laboratory (AS-M and DJH).

Figure 5. The correlation between holoTC and serum B₁₂ in the holoTC range ≥25 pmol/L. The horizontal line on the y-axis represents the serum B₁₂ deficiency cutoff of 138 pmol/L and the vertical line represents the holoTC cutoff of 70 pmol/L for B₁₂ replete patients as used in authors’ laboratory (AS-M and DJH). Samples with holoTC results between 25–70 pmol/L are referred for additional testing.

Table 1. The commonly seen vitamin B₁₂ marker patterns in selected clinical scenarios.

Serum holoTC	Serum total B12	Plasma MMA	Plasma tHcy	Possible diagnosis
N	N	↑	↑	Suboptimal B12 status
↓	↓	N or ↑	↑	Mild B12 deficiency, on antibiotics
N	N	↑	N	Bacterial overgrowth, B12 replete
↓↓↓	↓↓↓	↑↑	↑↑	Pernicious anemia
N or ↓	↓	N or ↑	N	Pregnancy, B12 replete
↓	↓ ↓	↑	N or ↑	Pregnancy, B12 deficiency
N	N	N	↑ - ↑↑↑	Mild to severe folate deficiency, B12 replete
N or ↑	↓ ↓	N	N	Haptocorrin deficiency
↓↓↓	N or ↓ or ↑	↑↑	↑↑	Transcobalamin deficiency
N	N	↑↑↑	↑↑↑	CblC, D, F, J disorder
N	N	↑ ↑ ↑	N	CblA, B disorder
N	N	N	↑↑↑	CblE, G disorder
N or ↓	N or ↓	N or ↑	↑↑↑	Nitrous oxide abuse
↑↑	↑↑	N	N	On vitamin B12 injections
↑↑↑	↑↑↑	N	N	Chronic myeloid leukemia

Cbl: cobalamin; holoTC: holotranscobalamin; MMA: methylmalonic acid; tHcy: total plasma homocysteine. ↓↓↓: very low/undetectable concentration; ↓↓: low; ↓: decreased; N: within reference range; ↑: elevated; ↑↑: highly elevated; ↑↑↑: very highly elevated.

Table 2. Selected serum/plasma results from longitudinal studies of uncomplicated pregnancies: pre-conception, pregnancy, labor, post-delivery and cord samples.

Geo region	Variable (unit)	Methodology	Sampling time Pre-conception/weeks pregnancy/labor/cord/weeks postD (n)	Concentration (range)	Concentration/ range definition	Reference
Germany	Vitamin B12 (pmol/L)	FP IF Ligand assay	9–12 weeks (31)	257 (226–292)	Geometric Mean (95% CI)	Koebnick et al. 2002 [111]
			20–22 weeks (39)	239 (212–268)
			36–38 weeks (38)	178 (161–198)
	tHcy (µmol/L)	HPLC	9–12 weeks (31)	6.9 (6.2–7.6)	Mean (95^th CI)
			20–22 weeks (39)	5.9 (5.4–6.3)
			36–38 weeks (38)	6.6 (5.9–7.2)
Spain	Vitamin B12 (pmol/L)	Microbiological assay L. leichmannii	Pre-conception (89)	293 (155–535)	Geometric Mean (10^th-90^th percentiles)	Murphy et al. 2007 [109]
			8 weeks (88)	267 (144–449)
			20 weeks (90)	230 (123–432)
			32 weeks (90)	198 (107–339)
			Labor (84)	224 (117–444)
			Cord (82)	325 (146–641)
	holoTC (pmol/L)	MEIA	Pre-conception (56)	63 (38–98)
			8 weeks (58)	47 (31–74)
			20 weeks (61)	48 (34–78)
			32 weeks (60)	45 (26–82)
			Labor (49)	40 (23–79)
			Cord (23)	92 (45–214)
	MMA (nmol/L)	GC-MS	Pre-conception (88)	120 (90–170)
			8 weeks (87)	110 (90–170)
			20 weeks (89)	110 (80–150)
			32 weeks (90)	140 (90–200)
			Labor (83)	140 (90–210)
			Cord (72)	240 (130–400)
Denmark	Vitamin B12 (pmol/L)	MEIA	18 weeks (441)	216 (96–484)	Mean (±1.96SD)	Milman et al. 2007 [112]
			32 weeks (310)	169 (73–388)
			39 weeks (266)	154 (71–333)
			8 weeks PostD (170)	315 (148–672)
	MMA (nmol/L)	SID-MS	18 weeks (413)	110 (40–290)
			32 weeks (390)	130 (50–340)
			39 weeks (250)	150 (60–360)
			8 weeks PostD (160)	160 (80–350)
	Hcy (µmol/L)	GC-MS	18 weeks (416)	6.1 (3.4–11.0)
			32 weeks (291)	6.6 (3.9–11.1)
			39 weeks (252)	7.8 (4.7–12.8)
			8 weeks PostD (158)	11.0 (5.8–20.6)
Canada	Vitamin B12 (pmol/L)	Competitive-binding immunoenzymatic assay	12–16 weeks (188–342)	219 (210–229)	Mean (95^th CI)	Visentin et al. 2016 [113]
			Labor (188–342)	169 (162–176)
			Cord (188–342)	321 (300–344)
	MMA (nmol/L)	LC-MS/MS	12–16 weeks (188–342)	109 (105–114)
			Labor (188–342)	136 (129–142)
			Cord (188–342)	313 (297–331)
	tHcy (µmol/L)	Enzymatic assay	12–16 weeks (188–342)	5.0 (4.9–5.1)
			Labor (188–342)	6.0 (5.8–6.2)
			Cord (188–342)	4.9 (4.7–5.1)
USA	Vitamin B12 (pmol/L)	Electrochemiluminescent immunoassay	12–30 weeks (124)	343 (238–401)	Median (IQR)	Finkelstein et al. 2019 [114]
			Labor (75)	216 (173–312)
			Cord (58)	569 (479–844)
Canada	Vitamin B12 (pmol/L)	Binding Immuno Enzymatic Assay	8.3–13.9 weeks (596)	216 (89.9,519)	G-Mean (lower and upper limits [90% CI] of central 95% RI)	Schroder et al. 2019 [115]
			14.9–20.9 weeks (640)	198 (84.0, 473)
	holoTC (pmol/L)	MEIA	8.3–13.9 weeks (587)	83.6 (29.5, NA^a)
			14.9–20.9 weeks (567)	76.5 (26.0, NA^a)
	MMA (nmol/L)	LC-MS/MS	8.3–13.9 weeks (586)	130 (69.6, 371)
			14.9–20.9 weeks (601)	120 (51.0, 374)

CI: confidence interval; GC: gas chromatography; FP: fluorescence polarization; HPLC: high performance liquid chromatography; holoTC: holotranscobalamin; IQR: interquartile range; IF: intrinsic factor; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MS: mass spectrometry; MMA: methylmalonic acid; MEIA: microparticle enzyme immunoassay; NA: not applicable; postD: post-delivery; SID: stable isotope dilution; n: study size; tHcy: total plasma homocysteine.

^aThe upper limit for holoTC is undefined.

Figure 6. Empty nitrous oxide canisters in a London residential street, February 2020.

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