Macro-B12 and Unexpectedly High Levels of Plasma B12: A Critical Review.

A low total plasma vitamin B12 supports a clinical suspicion of B12 deficiency, while the interpretation of an unexpectedly normal/high level is marred by controversies. Here, we critically review current knowledge on B12 in blood plasma, including the presence of the so-called "macro-B12". The latter form is most often defined as the fraction of B12 that can be removed by precipitation with polyethylene glycol (PEG), a nonspecific procedure that also removes protein polymers and antibody-bound analytes. Plasma B12 includes B12 attached to transcobalamin and haptocorrin, and an increased concentration of one or both proteins almost always causes an elevation of B12. The total plasma B12 is measured by automated competitive binding assays, often incorrectly referred to as immunoassays, since the binding protein is intrinsic factor and not an antibody. An unexpectedly high level of B12 may be further explored using immunological measurements of haptocorrin and transcobalamin (optionally combined with e.g., size-exclusion chromatography). Nonspecific methods, such as PEG precipitation, are likely to give misleading results and cannot be recommended. Currently, the need for evaluation of a high B12 of unknown etiology is limited since other tests (such as measurements of methylmalonic acid) may better guide the diagnosis of B12 deficiency.

Reference intervals and stability of haptocorrin and holotranscobalamin in Danish children and elderly.

BACKGROUND: Haptocorrin (HC) and holotranscobalamin (holoTC) carry vitamin B12 (B12) in the circulation and can be useful biomarkers for evaluating B12 status. The concentration of both proteins depends on age, but data on reference intervals for children and the elderly are sparse. Similarly, not much is known about the effect of preanalytical factors. METHODS: HC plasma samples from healthy elderly > 65 years (n = 124) were analysed, and both HC and holoTC were analysed in paediatric serum samples ≤ 18 years (n = 400). Furthermore, we investigated assay precision and stability. RESULTS: HC and holoTC were effected by age. We established reference intervals for HC: 2-10 years, 369-1237 pmol/L; 11-18 years, 314-1128 pmol/L; 65-82 years, 242-680 pmol/L and for holoTC: 2-10 years, 46-206 pmol/L; 11-18 years, 30-178 pmol/L. Analytical coefficients of variations of 6.0-6.8% and 7.9-15.7% were found for HC and holoTC, respectively. HC were affected when stored at room temperature and by freeze/thaw. HoloTC was stable at room temperature and after delayed centrifugation. CONCLUSION: We present novel 95% age-related reference limits for HC and HoloTC in children, and for HC both in children and elderly. Moreover, we found HoloTC to be fairly stable when stored, whereas HC was more vulnerable to preanalytical factors.

"Macro transcobalamin causing raised vitamin B12: Case-based laboratory investigation".

Determination of plasma vitamin B12 (B12) is a frequently requested laboratory analysis, mainly employed to establish B12 deficiency. However, an increased level of B12 is a common unexpected finding that may be related to an increased concentration of one of the B12 binding proteins, haptocorrin or transcobalamin. This paper describes the extensive laboratory evaluation of a patient with an elevated level of plasma B12 with various well-established assays. Initial studies suggested the presence of a macromolecule consisting of haptocorrin bound B12. Specific determinations of the B12-binding proteins revealed normal amounts of haptocorrin but a markedly increase in both total and B12 saturated transcobalamin (holo-TC). The results are in accord with the presence of macro-transcobalamin. These experiments reveal that determination of the nature of the B12-macromolecules is troublesome due to differences in assays applied to measure these proteins. In addition, this publication creates awareness of macro-holo-TC as a cause of an unexplained increased B12 level.

Clinical Utility of Serum Holotranscobalamin Measurements in Patients with First-Ever Ischemic Stroke.

BACKGROUND: Whether holotranscobalamin (holoTC) indicates B12 deficiency more sensitively than total vitamin B12 (B12) is unclear. This study is aimed at determining the impact of serum holoTC level as a risk factor for ischemic stroke and investigating its association with disease severity and short-term outcomes. METHODS: Serum holoTC, total B12, and homocysteine levels were compared between 130 stroke patients and 138 healthy controls. Biomarker level correlations with disease severity and stroke functional outcomes were investigated. RESULTS: holoTC levels were lower and homocysteine levels were higher in stroke patients than in healthy controls (P < 0.05). The holoTC/total B12 ratio and homocysteine level significantly predicted ischemic stroke in the multivariable regression analysis (P < 0.05). Along with hyperhomocysteinemia, patients more often had holoTC than total B12 deficiency (6.2% vs. 3.1%). holoTC levels negatively correlated with homocysteine levels (partial R -0.165, P < 0.05) in stroke patients in multiple linear regression analyses, but not total B12 levels. The holoTC level and holoTC/total B12 ratio, but not homocysteine and total B12 levels, negatively correlated with the National Institute of Health Stroke Scale (partial R, -0.405 and -0.207, respectively, P < 0.01). CONCLUSIONS: Measurements of serum holoTC levels combined with total B12 and homocysteine levels may provide valuable information for predicting ischemic stroke and its severity and short-term outcomes of ischemic stroke patients.

Relationship between cobalt transporter II gene rs9606756 site mutant and serum homocysteine level and recurrent cerebral infarction in young and middle-aged people.

BACKGROUND: This study aimed to investigate the effect of cobalt transporter II gene (rs1801198, rs2301957, rs9606756) polymorphism on serum homocysteine level and its correlation with young and middle recurrent cerebral infarction. METHODS: A total of 348 young and middle-aged patients with cerebral infarction admitted to The Third Affiliated Hospital of Qiqihar Medical University from January 2017 to March 2018 were enrolled. The patients were divided into recurrent and non-recurrent groups according to follow-up. DNA was extracted from the peripheral blood of patients, and the DNA samples were genotyped by IlluminaBeadArray technology to detect the gene polymorphisms of cobalt transporter II (TCN2) sites (rs1801198, rs2301957, rs9606756), and the homocysteine (hcy) level was determined by cyclic enzymatic method. VitB12 and folate levels were measured by chemiluminescence immunoassay, and holo transcobalamin (holoTC) expression levels were detected by enzyme-linked immunosorbent assay. RESULTS: The frequency of alleles of rs9606756 mutation in the recurrent group was higher than that in the non-recurrent group (P<0.05), and the Hcy level in rs9606756 locus genotype AG+GG was significantly higher than the AA genotype in the recurrent group (P=0.031). Pearson correlation analysis showed that Hcy levels were associated with different genotypes of rs9606756 in the recurrent group (r=0.483, P=0.0003). The rs9606756 allele AA in SH-SY5Y cells was replaced with GG by point mutation experiment. The Hcy metabolism levels of wild and mutant cells were detected. The accumulation level of Hcy in the mutant group was significantly increased (P=0.007). The holoTC in the supernatant was significantly reduced in the mutant (P=0.032). CONCLUSIONS: The TCN2 gene rs9606756 mutation is closely related to the level of Hcy metabolism in young and middle-aged patients, which may affect the recurrence of cerebral infarction. It is of great significance to further understand the pathogenesis, prevention and treatment of recurrent cerebral infarction in young and middle-aged patients.

Effects of Prolonged Whey Protein Supplementation and Resistance Training on Biomarkers of Vitamin B12 Status: A 1-Year Randomized Intervention in Healthy Older Adults (the CALM Study).

We investigated the effect of long-term whey supplementation on biomarkers of B12 status in healthy older adults subjected to different schemes of supplements and exercise. The total study population examined at baseline consisted of 167 healthy older adults (age ≥ 65 year) who were randomized to 1-y intervention with two daily supplements of (1) whey protein (3.1 µg B12/day) (WHEY-ALL), (2) collagen (1.3 µg B12/day) (COLL), or (3) maltodextrin (0.3 µg B12/day) (CARB). WHEY-ALL was comprised of three groups, who performed heavy resistance training (HRTW), light resistance training (LITW), or no training (WHEY). Dietary intake was assessed through 3-d dietary records. For the longitudinal part of the study, we included only the participants (n = 110), who met the criteria of ≥ 50% compliance to the nutritional intervention and ≥ 66% and ≥ 75% compliance to the heavy and light training, respectively. Fasting blood samples collected at baseline and 12 months and non-fasting samples collected at 6 and 18 months were examined for methylmalonic acid, B12 and holotranscobalamin. At baseline, the study population (n = 167) had an overall adequate dietary B12 intake of median (range) 5.3 (0.7-65) µg/day and median B12 biomarker values within reference intervals. The whey intervention (WHEY-ALL) caused an increase in B12 (P < 0.0001) and holotranscobalamin (P < 0.0001). In addition, methylmalonic acid decreased in the LITW group (P = 0.04). No change in B12 biomarkers was observed during the intervention with collagen or carbohydrate, and the training schedules induced no changes. In conclusion, longer-term daily whey intake increased plasma B12 and holotranscobalamin in older individuals. No effect of intervention with collagen or carbohydrate or different training regimes was observed. Interestingly, the biomarkers of B12 status appeared to be affected by fasting vs. non-fasting conditions during sample collection.

Vitamin B12 and its binding proteins in patients with non-small cell lung cancer referred to fast-track diagnostic work-up for lung cancer.

High cobalamin levels have previously been associated with short-term cancer risk, including lung cancer. We explored whether levels of cobalamin and/or its binding proteins are useful as a diagnostic tool in patients suspected of non-small cell lung cancer. We included 889 patients referred for fast-track diagnosis of lung cancer to Aarhus University Hospital, Denmark. We analyzed plasma concentrations of cobalamin, transcobalamin, holotranscobalamin and haptocorrin. Information on lung cancer diagnosis was retrieved from a national database. The study cohort showed levels above reference intervals for cobalamin 12%, holotranscobalamin 25%, transcobalamin 9% and haptocorrin 36% (all p-values <.05). We observed no difference in cobalamin or holotranscobalamin levels when comparing patients diagnosed with non-small cell lung cancer (n = 161, 18%) to patients without lung cancer (n = 742, 80%), while transcobalamin showed minor differences. Haptocorrin was significantly higher in those with cancer, mainly among patients with adenocarcinoma (n = 94). A comparison of patients with the highest vs. lowest quartile levels of haptocorrin yielded an adjusted odds ratio for adenocarcinoma of 2.39 (95% confidence interval: 1.26-4.55). However, ROC curve analyzes showed haptocorrin (AUC = 0.55) and total transcobalamin (AUC = 0.56) to be poor diagnostic markers for lung cancer. A high proportion of patients suspected for non-small cell lung cancer showed increased levels of cobalamin-binding proteins. We thereby confirm the association between non-small cell lung cancer and high cobalamin levels and found that haptocorrin was the major underlying factor causing high cobalamin levels. However, none of these biomarkers were of diagnostic use among patients referred for suspected lung cancer.

Seminal but not Serum Levels of Holotranscobalamin are Altered in Morbid Obesity and Correlate with Semen Quality: A Pilot Single Centre Study.

Vitamin B12 (cobalamin) is an essential cofactor in the one-carbon metabolism. One-carbon metabolism is a set of complex biochemical reactions, through which methyl groups are utilised or generated, and thus plays a vital role to many cellular functions in humans. Low levels of cobalamin have been associated to metabolic/reproductive pathologies. However, cobalamin status has never been investigated in morbid obesity in relation with the reduced semen quality. We analysed the cross-sectional data of 47-morbidly-obese and 21 lean men at Careggi University Hospital and evaluated total cobalamin (CBL) and holotranscobalamin (the active form of B12; holoTC) levels in serum and semen. Both seminal and serum concentrations of holoTC and CBL were lower in morbidly obese compared to lean men, although the difference did not reach any statistical significance for serum holoTC. Seminal CBL and holoTC were significantly higher than serum levels in both groups. Significant positive correlations were observed between seminal holoTC and total sperm motility (r = 0.394, p = 0.012), sperm concentration (r = 0.401, p = 0.009), total sperm number (r = 0.343, p = 0.028), and negative correlation with semen pH (r = -0.535, p = 0.0001). ROC analysis supported seminal holoTC as the best predictor of sperm number (AUC = 0.769 ± 0.08, p = 0.006). Our findings suggest that seminal rather than serum levels of holoTC may represent a good marker of semen quality in morbidly obese subjects.

Early changes in vitamin B12 uptake and biomarker status following Roux-en-Y gastric bypass and sleeve gastrectomy.

BACKGROUND & AIMS: Bariatric surgery increases the risk of micronutrient deficiencies, including vitamin B12 (B12) deficiency. We analysed early changes in biomarkers of B12 status following bariatric surgery. METHODS: We prospectively included adult patients (n = 27) referred for either Roux-en-Y Gastric Bypass (RYGB) (n = 19) or Sleeve Gastrectomy (SG) (n = 8). Blood samples were drawn before surgery and 2 and 6 months following surgery for measurement of B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA). The B12 absorption capacity was estimated from the increase in plasma holoTC two days after a standardised oral B12 challenge. RESULTS: B12 status decreased following both RYBG and SG. While a decrease in plasma B12 was not evident until 6 months postoperatively, we observed a statistically significant decrease in plasma holoTC and increase in MMA already 2 months postoperatively. These changes were more pronounced at 6 months post surgery. Correspondingly, the B12 absorption capacity was decreased following surgery. CONCLUSIONS: HoloTC and MMA were superior to B12 to detect early changes in B12 status following bariatric surgery. Our data challenge the current concept that liver B12 stores secure long-term maintenance of B12 status. They indicate that B12 treatment in pharmacological doses may be warranted immediately after surgery.

Proton Pump Inhibitors, H2-Receptor Antagonists, Metformin, and Vitamin B-12 Deficiency: Clinical Implications.

There is clear evidence that proton-pump inhibitors (PPIs), H2-receptor antagonists (H2RAs), and metformin can reduce serum vitamin B-12 concentrations by inhibiting the absorption of the vitamin. However, it is unclear if the effects of these drugs on serum vitamin B-12 are associated with increased risk of biochemical or functional deficiency (as is indicated by elevated blood concentrations of homocysteine and methylmalonic acid) or clinical deficiency (including megaloblastic anemia and neurologic disorders such as peripheral neuropathy and cognitive dysfunction). This review provides an overview of vitamin B-12 absorption and biochemistry and the mechanisms by which PPIs, H2RAs, and metformin affect these functions. It also summarizes the literature relating the use of these drugs to the risk of vitamin B-12 deficiency. Also discussed is that strategies for assessing vitamin B-12 status and diagnosing vitamin B-12 deficiency have evolved in recent years beyond solely measuring serum total vitamin B-12. Multiple analyte testing, a strategy in which ≥2 of 4 biomarkers of vitamin B-12 status-serum total vitamin B-12, holotranscobalamin, homocysteine, and methylmalonic acid-are measured, increases sensitivity and specificity for diagnosing vitamin B-12 deficiency. It is concluded that randomized controlled trials are now needed that use the strategy of multiple analyte testing to determine if PPIs, H2RAs, and metformin do indeed increase the risk of vitamin B-12 deficiency. Until these studies are conducted, a reasonable recommendation for physicians and their patients who are taking these drugs is to monitor vitamin B-12 status and to provide vitamin B-12 supplements if altered blood biomarkers or clinical signs consistent with low or deficient vitamin B-12 status develop.

Foods and Supplements Associated with Vitamin B12 Biomarkers among Vegetarian and Non-Vegetarian Participants of the Adventist Health Study-2 (AHS-2) Calibration Study.

To investigate the association between plasma concentration of vitamin B12 and B12 intake from supplements, fortified foods, and animal source foods among vegetarians and non-vegetarians, we conducted a cross-sectional analysis among 728 participants of the Adventist Health Study 2 (AHS-2) calibration study. The median age of participants was 58 years, 65.4% were female, and 50.3% were White. We used six 24 h dietary recalls to measure B12 intake, serum vitamin B12, and holotranscobalamin (holoTC) concentration. B12 supplements had a significantly positive association with plasma B12 among all subjects (p trend < 0.001), especially among vegans and lacto-ovo vegetarians (p trend < 0.001). Among non-users of B12 supplements, B12 intake from milk substitutes was significantly positively associated with holoTC (p trend < 0.004) and serum B12 (p trend < 0.030). In non-vegetarians, holoTC was significantly positively associated with B12 intake from eggs, while serum B12 was significantly positively associated with B12 intake from milk in the upper tertile compared to the lower, and B12 intake from meat in the middle compared to the lower tertile intake (p < 0.011). Supplements containing B12 followed by B12 intake from milk substitutes were significant contributors of plasma vitamin B12 concentration.

Measurement of total Transcobalamin employing a commercially available assay for Active B12.

INTRODUCTION: Vitamin B12 deficiency is mostly caused by insufficient gastro-intestinal absorption and in rare conditions by Transcobalamin (TC) deficiency. Unsaturated Transcobalamin (apoTC) can be measured by a binding assay using radiolabeled cobalamin. The Active B12 test analyzes saturated Transcobalamin (holoTC) and we hypothesize that this test can be used to measure total TC by additional in vitro saturation with cobalamin. METHODS: Serum was saturated in vitro (16 times dilution) with a cyanocobalamin solution and total TC was selectively measured with the Abbott Active B12 test. ApoTC was calculated by subtracting endogenous holoTC from total TC after correction for dilution. Linearity was determined with a pool serum dilution series. Precision was investigated according to the CLSI EP15 protocol. Method comparison was performed against a binding assay using radiolabeled cobalamin. Reference values were determined in 100 healthy controls. RESULTS: The method was linear in the range of 240 to 1933pmol/L (R2=0.997, lack of fit F=1.61). Precision of low- and high-pool total TC in serum were; 5.2% and 4.3% respectively. Method comparison against a radiolabeled cobalamin binding assay showed a proportional bias of 30% (y=0.70x+126). Total TC reference values were determined at 500-1276pmol/L. CONCLUSION: We describe a rapid method to quantify total TC, which can be implemented on routine platforms using commercial Active B12 tests. In addition, apoTC can be assessed by subtracting endogenous holoTC concentration which can be measured in the same run, securing the same calibration level for all three parameters (holoTC, apoTC and total TC). This method is applicable in clinical diagnostics and in larger epidemiological studies.

Biological variation of holotranscobalamin and cobalamin in healthy individuals.

Data on biological variation for cobalamin and holotranscobalamin (holoTC) are limited. The aim of this study was to determine within-subject (CVI) and between-subject (CVG) biological variations for these analytes in a healthy population. We collected blood samples from 15 healthy volunteers (12 women and three men, 22-66 years) on the same weekday for 10 consecutive weeks. Serum samples were stored at -80 °C until analysis in duplicate in a single analytical run. The CVI and CVG were estimated by nested ANOVA. The CVI (95% CI) for cobalamin and holoTC was 6.7% (5.7-7.7) and 13.0% (11.5-15.0), respectively. The corresponding CVG was 24.1% (16.4-36.0) and 24.6% (16.3-37.7). The analytical variation (CVA) (95% CI) was 3.5% (3.2-4.0) for cobalamin and 2.4% (2.1-2.6) for holoTC. The index of individuality (II) was low (<0.6) for both cobalamin and holoTC and the reference change value (RCV) was 20.1% for cobalamin and 36.6% for holoTC. Our study describes the components of biological variation of cobalamin and holoTC in a healthy population, contributing to a better clinical interpretation of these biomarkers.

Vitamin B12 deficiency after esophagectomy with gastric tube reconstruction for esophageal cancer.

The aim of this study is to determine the prevalence and incidence of vitamin B12 deficiency after esophagectomy for cancer. It is unknown if patients after esophagectomy with gastric tube reconstruction are at an increased risk for vitamin B12 deficiency. A cross-sectional cohort (group A) and a prospective cohort (group B) of patients who underwent esophagectomy for cancer in two tertiary referral centers in the Netherlands were included. Serum levels of holo-transcobalamin (Holo-TC) and methyl malonic acid (MMA) were determined. Vitamin B12 deficiency was defined as Holo-TC < 21 pmol/L and/or MMA > 0.45 µmol/L. Vitamin B12 status was assessed in group A at a single time point between one and three years postoperatively and before and every three months after resection in group B. Ninety-nine patients were analyzed in group A. The median time between surgery and analysis of vitamin B12 deficiency was 19.3 months. In 11 of 99 (11%) patients, vitamin B12 deficiency was detected. In group B, 5 of 88 (5.6%) patients had vitamin B12 deficiency preoperatively, and another 9 (10.2%) patients developed vitamin B12 deficiency after the operation at a median time of 6 months postoperatively. The estimated one-year incidence of vitamin B12 deficiency was 18.2%. None of the patients with vitamin B12 deficiency had a megaloblastic anemia. Vitamin B12 deficiency can be anticipated in 18% of patients after esophagectomy with gastric tube reconstruction for cancer. During follow-up, Holo-TC and MMA levels should be measured to detect vitamin B12 deficiency and commence treatment timely.

Folate and Vitamin B12-Related Biomarkers in Relation to Brain Volumes.

AIM: We investigated cross-sectional associations between circulating homocysteine, folate, biomarkers of vitamin B12 status and brain volumes. We furthermore compared brain volumes of participants who received daily folic acid and vitamin B12 supplementation with participants who did not. METHODS: Participants of the B-PROOF study (n = 2919) were assigned to 400 µg folic acid and 500 µg vitamin B12, or a placebo. After two years of intervention, T1-weighted magnetic resonance imaging (MRI) scans were made in a random subsample (n = 218) to obtain grey and white matter volume, and total brain volume (TBV). Plasma homocysteine, serum folate, vitamin B12, holotranscobalamin, and methylmalonic acid concentrations were measured. RESULTS: Multiple linear regression analyses showed inverse associations between plasma homocysteine with TBV (ß = -0.91, 95% CI -1.85-0.03; p = 0.06) and between serum folate and TBV (ß = -0.20, 95% CI -0.38, -0.02; p = 0.03). No significant associations were observed for serum vitamin B12 and holotranscobalamin. Fully adjusted ANCOVA models showed that the group that received B-vitamins had a lower TBV (adjusted mean 1064, 95% CI 1058-1069 mL) than the non-supplemented group (1072, 95% CI 1067-1078 mL, p = 0.03). CONCLUSIONS: Results were contradictory, with higher Hcy levels associated with lower TBV, but also with higher folate levels associated with lower TBV. In addition, the lack of a baseline measurement withholds us from giving recommendations on whether folic acid and vitamin B12 supplementation will be beneficial above and beyond normal dietary intake for brain health.

Low holo-transcobalamin levels are prevalent in vegetarians and is associated with coronary artery disease in Indian population.

Coronary artery disease (CAD) has been increasing alarmingly in India. We had earlier shown that vitamin B12 deficiency is associated with CAD in Indian population. However, only about a quarter of the total vitamin B12 is internalised in the cells by the proteins transcobalamin II. Vitamin B12-bound transcobalamin II (holotranscobalamin, holoTC) is thus referred to as biologically active B12. In this study, we ascertained the levels of holoTC in 501 CAD cases and 1253 healthy controls and for the first time show that holoTC levels are significantly lower (p = 2.57E-4) in CAD (26.81 pmol/l) cases as compared to controls (29.97 pmol/l).

An International Standard for holotranscobalamin (holoTC): international collaborative study to assign a holoTC value to the International Standard for vitamin B12 and serum folate.

BACKGROUND: Investigation of possible B12 and folate deficiencies requires measurement of these vitamins in serum. There is evidence that holotranscobalamin (holoTC), the active portion of B12 available to cells, is a more specific marker of early B12 deficiency than total B12. The availability of immunoassays for holoTC prompted an international collaborative study to assign a holoTC value to the World Health Organization (WHO) 1st International Standard (IS) for vitamin B12 and serum folate, 03/178. METHODS: The IS, 03/178, and three serum samples with different holoTC levels were assayed by 12 laboratories in eight countries using manual and automated immunoassays for holoTC; one laboratory additionally performed an in-house assay. Fourteen sets of data were analysed. RESULTS: Overall, the IS, 03/178, and the three serum samples demonstrated assay linearity and parallelism. An overall geometric mean (GM) holoTC value of 106.8 pmol/L was obtained for 03/178, with an inter-laboratory geometric coefficient of variation (GCV) of 10.5%. There was a reduction in inter-laboratory variability when the holoTC levels in the serum samples were determined relative to the IS with an assigned holoTC value rather than to the assays' calibration. Accelerated degradation studies showed that 03/178 was sufficiently stable to serve as an IS for holoTC. CONCLUSIONS: The WHO Expert Committee on Biological Standardization endorsed the proposal to assign a holoTC value of 107 pmol/L to 03/178, corresponding to 0.107 pmol per ampoule, for use as the 1st IS for vitamin B12, serum folate, and holoTC.

Biochemical Identification of Vitamin B12 Deficiency in a Medical Office.

BACKGROUND: A vitamin B12 deficiency can be an underlying cause or a deteriorating factor in several diseases. Nevertheless, early identification of such a deficiency remains a problem. Holotranscobalamin (HTC) is presently considered to be the gold standard. We tested the predictive power of other B12 parameters by comparing them with HTC. METHODS: The blood of 77 patients from a medical office was tested for HTC, total B12 (CLIA [chemiluminescent immunoassay] and MTP [microbiological test with microtitre plates]), MMA (methylmalonic acid), HCY (homocysteine), and MCV (mean cell volume). The parameters were correlated and sensitivity, specificity, PPV (positive predictive value), NPV (negative predictive value), LR+ (positive likelihood ratio), and LR- (negative likelihood ratio) in comparison to HTC were determined. A ROC analysis was also performed. RESULTS: At a cutoff value of 35 pmol/L for HTC, the total B12 CLIA (cutoff 211 ng/L) qualified 53% of individuals as having a B12 deficiency. The total B12 MTP (cutoff 288 ng/L) classified 71% as having a B12 deficiency. Specificity was similar in both cases (CLIA, 93%; MTP, 95%). With a cutoff value of 10 µmol/L for homocysteine, the best negative prediction was achieved. MVA has a low sensitivity (41%) and a high specificity (90%). Based on the ROC analysis, which indicated superiority of the B12-MTP, the reference levels of B12-CLIA and B12-MTP were raised to 304 and 368 ng/L, respectively. Thus, a probable B12 deficiency was identified in 94% of cases with either method and with a comparable specificity. CONCLUSIONS: If total B12 is applied to identify B12 deficiency, the cutoff values should be elevated to 304 (B12-CLIA) and 368 ng/L (B12-MTP) to improve the predictive power. The negative-predictive power of HCY can be useful in daily routine. HTC has a broad grey area of uncertainty and MMA should only be applied as a confirmatory test.