Serum holoTC Research Articles

Vitamin B12 and the Risk of Neural Tube Defects in a Folic-Acid-Fortified Population

Low maternal vitamin B(12) status may be a risk factor for neural tube defects (NTDs). Prior studies used relatively insensitive measures of B(12), did not adjust for folate levels, and were conducted in countries without folic acid food fortification. In Canada, flour has been fortified with folic acid since mid-1997. We completed a population-based case-control study in Ontario. We measured serum holotranscobalamin (holoTC), a sensitive indicator of B(12) status, at 15 to 20 weeks' gestation. There were 89 women with an NTD and 422 unaffected pregnant controls. A low serum holoTC was defined as less than 55.3 pmol/L, the bottom quartile value in the controls. The geometric mean serum holoTC levels were 67.8 pmol/L in cases and 81.2 pmol/L in controls. There was a trend of increasing risk with lower levels of holoTC, reaching an adjusted odds ratio of 2.9 (95% confidence interval = 1.2-6.9) when comparing the lowest versus highest quartile. There was almost a tripling in the risk for NTD in the presence of low maternal B(12) status, measured by holoTC. The benefits of adding synthetic B(12) to current recommendations for periconceptional folic acid tablet supplements or folic-acid-fortified foods need to be considered. It remains to be determined what fraction of NTD cases in a universally folate-fortified environment might be prevented by higher periconceptional intake of B(12).

Detection of Vitamin B12 Deficiency in Older People by Measuring Vitamin B12 or the Active Fraction of Vitamin B12, Holotranscobalamin

Impaired vitamin B(12) function and decreased vitamin B(12) status have been associated with neurological and cognitive impairment. Current assays analyze total vitamin B(12) concentration, only a small percentage of which is metabolically active. Concentrations of this active component, carried on holotranscobalamin (holoTC), may be of greater relevance than total vitamin B(12). We compared the utility of serum holoTC with conventional vitamin B(12) for detection of vitamin B(12) deficiency in a population-based study of older people, using increased methylmalonic acid (MMA) concentrations as a marker of metabolic vitamin B(12) deficiency in the overall population (n = 2403) and in subsets with normal (n = 1651) and abnormal (n = 752) renal function. Among all participants, 6% had definite (MMA >0.75 micromol/L) and 16% had probable (MMA >0.45 micromol/L) metabolic vitamin B(12) deficiency. In receiver operating characteristic curves for detection of definite vitamin B(12) deficiency, holoTC had a greater area under the curve (AUC) compared with vitamin B(12) in all participants (0.85 vs 0.76; P <0.001) and in subsets with normal (AUC: 0.87 vs 0.79; P <0.001) and abnormal (AUC: 0.85 vs 0.74; P = 0.002) renal function. Similar findings were observed for detection of moderate vitamin B(12) deficiency. Whereas the positive predictive value for both holoTC and vitamin B(12) was greater for detection of probable than definite vitamin B(12) deficiency, both tests were associated with more false-positive than true-positive test results. HoloTC has a modestly superior diagnostic accuracy compared with conventional vitamin B(12) for the detection of vitamin B(12) deficiency, but neither test can be recommended to screen asymptomatic populations.

Holotranscobalamin in laboratory diagnosis of cobalamin deficiency compared to total cobalamin and methylmalonic acid

Cobalamin-saturated transcobalamin, also called holotranscobalamin (holoTC), constitutes only between 6% and 20% of total plasma B(12). Serum concentration of holoTC is a new marker in laboratory diagnosis of cobalamin deficiency. We tested the utility of holoTC in assessing vitamin B(12) status. We measured concentrations of holoTC and methylmalonic acid (MMA) in 1018 serum samples that were referred to our laboratory for total cobalamin testing. Concentrations of MMA were lower in females compared to males and this difference was no more significant after adjusting for serum creatinine. Moreover, age was associated with higher concentrations of serum MMA, higher holoTC and slightly higher concentrations of total cobalamin. Higher concentrations of serum creatinine were associated with higher concentrations of MMA and holoTC. However, no association between serum creatinine and total cobalamin was observed. Only subjects with normal serum creatinine showed a negative correlation between serum holoTC and MMA (r= -0.36, p<0.001). In subjects with MMA > or =300 nmol/L and holoTC < or =35 pmol/L, concentrations of total cobalamin were well within the normal range (median; 25th/75th percentiles=212; 171/272 pmol/L). Receiver operating characteristic (ROC) curve analysis displayed a higher sensitivity and specificity for holoTC compared with vitamin B(12) for detecting concentrations of MMA > or =300 nmol/L in individuals with normal renal function. Compared to total cobalamin, we observed a better performance of holoTC assay in detecting elevated concentrations of MMA in subjects with normal renal function. The majority of subjects with combined low holoTC and elevated MMA had normal concentrations of total cobalamin. HoloTC can be used as a first line parameter in detecting cobalamin deficiency.

Transcobalamin 776C→G polymorphism negatively affects vitamin B-12 metabolism

A common genetic polymorphism [transcobalamin (TC) 776C-->G] may affect the function of transcobalamin, the protein required for vitamin B-12 cellular uptake and metabolism. Remethylation of homocysteine is dependent on the production of 5-methyltetrahydrofolate and adequate vitamin B-12 for the methionine synthase reaction. The objectives were to assess the influence of the TC 776C--> G polymorphism on concentrations of the transcobalamin-vitamin B-12 complex (holo-TC) and to determine the combined effects of the TC 776C-->G and methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphisms and vitamin B-12 status on homocysteine concentrations. Healthy, nonpregnant women (n = 359; aged 20-30 y) were screened to determine plasma vitamin B-12, serum holo-TC, and plasma homocysteine concentrations and TC 776C-->G and MTHFR 677C-->T genotypes. The serum holo-TC concentration for women with the variant TC 776 GG genotype was significantly different (P = 0.0213) from that for subjects with the CC genotype (74 +/- 37 and 87 +/- 33 pmol/L, respectively). An inverse relation was observed between plasma homocysteine concentrations and both serum holo-TC (P </= 0.0001) and plasma vitamin B-12 (P </= 0.0001) concentrations, regardless of genotype. These data suggest that the TC 776C-->G polymorphism negatively affects the serum holo-TC concentration and provide additional evidence that vitamin B-12 status modulates the homocysteine concentration in this population.

Circadian variation of holo-transcobalamin (holo-TC) and related markers

We examined the circadian variation of holo-transcobalamin (holo-TC), total transcobalamin (total TC) and plasma cobalamins, and the association between food intake and fluctuations in the biochemical markers. A total of 17 healthy women (mean age 33 years; range 24-40) participated. The subjects were admitted at 08:00 h after an overnight fast, and blood samples were obtained every 20 min for 24 h from 12:00 h. Holo-TC and total TC were measured by an ELISA, and plasma cobalamins were measured by a routine method (Centaur, Bayer). All subjects, except one, had 12:00 h levels within the reference interval for all variables studied, and all subjects had normal renal function as judged from plasma creatinine. We found a small intra-individual variation (12%, 10%, 10%) and a considerable inter-individual variation (56%, 26%, 43%) in the level of holo-TC, total TC and plasma cobalamins, respectively. During the night, the absolute values of all components decreased, as did plasma albumin. There was no systematic association between intake of food and fluctuation of the biochemical markers. In conclusion, we found no significant circadian variation in serum holo-TC in healthy vitamin-replete subjects on a standard diet. This supports the view that holo-TC is a marker of long-term vitamin B(12) status, and that samples may also be obtained from non-fasting subjects.

Influence of cobalamin deficiency compared with that of cobalamin absorption on serum holo-transcobalamin II

Cobalamin attached to transcobalamin II (TC II), known as holo-TC II, is the active cobalamin fraction taken up by tissues. Holo-TC II is also the form in which absorbed cobalamin enters the circulation from the ileum. Therefore, holo-TC II has been proposed variously as a marker of cobalamin adequacy, cobalamin absorption, or both, including even its advocacy as a surrogate Schilling test. Such claims carry conflicting diagnostic implications because metabolic adequacy and absorption are not identical. The objective was to examine metabolic and absorptive influences on holo-TC II. Treated patients with pernicious anemia (PA), who have abnormal absorption but a normal metabolic status, were chosen as the model to differentiate between the effects of the 2 cobalamin-related characteristics. Serum holo-TC II and indexes of cobalamin metabolism in 23 treated patients were compared with those of 6 untreated PA patients (abnormal absorption and metabolic status) and 33 control subjects (normal absorption and metabolic status). Holo-TC II, which correlated directly with cobalamin and inversely with homocysteine, was significantly higher in treated PA patients in metabolic remission than in untreated PA patients (74 +/- 59 compared with 9 +/- 6 pmol/L) and was significantly lower than in control subjects (105 +/- 58 pmol/L), although the latter difference was small and the values overlapped greatly. Metabolic cobalamin status is a major determinant of serum holo-TC II. Absorption status may have mild influence as well, although other explanations remain possible. Serum holo-TC II cannot be used clinically to diagnose cobalamin malabsorption because of overlap with normal values. The influences on holo-TC II are complex and require careful analysis.

Biochemical signs of impaired cobalamin status during and after radiotherapy for rectal cancer

Purpose The aim of the study was to investigate whether pelvic radiotherapy for rectal cancer had a negative impact on cobalamin status. Methods and materials Consecutive patients receiving pelvic radiotherapy (50 Gy) for rectal cancer were evaluated prospectively ( n = 54). Serum cobalamin, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) were measured at start and end of radiotherapy, at follow-up 4–6 weeks and 1 year ( n = 23) after radiotherapy. Results Mean serum cobalamin decreased from 306 pmol/L before treatment to 267 pmol/L at the end of radiotherapy ( p < 0.001), 247 pmol/L 4–6 weeks after radiotherapy ( p < 0.001), and 249 pmol/L 1 year after radiotherapy ( p = 0.02). Mean serum MMA was 0.16 μmol/L pretreatment, 0.17 μmol/L at the end of radiotherapy (n.s.), and increased to 0.19 μmol/L after 4–6 weeks ( p = 0.007), and to 0.21 μmol/L after 1 year ( p < 0.001). There was no change in serum tHcy. Mean serum holoTC was reduced from 111 pmol/L pretreatment to 93 pmol/L 4–6 weeks after radiotherapy ( p = 0.002). Conclusions The data suggest rapid and persistent decrease in cobalamin status after radiotherapy for rectal cancer, as reflected by reduced serum cobalamin combined with increased serum MMA. This observation, though modest, may motivate routine monitoring of cobalamin status at follow-up after radiotherapy.

Transcobalamin Polymorphism and Serum Holo-Transcobalamin in Relation to Alzheimer’s Disease

Isoforms of the vitamin B₁₂ carrier protein transcobalamin (TC) might influence its cellular availability and contribute to the association between disrupted single-carbon metabolism and Alzheimer’s disease (AD). We therefore investigated the relationships between the TC 776C>G (Pro259Arg) genetic polymorphism, total serum cobalamin and holo-TC levels, and disease onset in 70 patients with clinically diagnosed AD and 74 healthy elderly controls. TC 776C>G polymorphism was also determined for 94 histopathologically confirmed AD patients and 107 controls. Serum holo-TC levels were significantly higher in TC 776C homozygotes (p = 0.04). Kaplan-Meier survival functions differed between homozygous genotypes (Cox’s F-Test F(42, 46) = 2.1; p = 0.008) and between 776C homozygotes and heterozygotes (Cox’s F test F(46, 108) = 1.7; p = 0.02). Proportionately fewer TC 776C homozygotes appear to develop AD at any given age, but this will require confirmation in a longitudinal study.

Quantification of Holo-Transcobalamin, a Marker of Vitamin B12 Deficiency

We report a new method for measurement of holo-transcobalamin (holoTC), in which magnetic beads coated with vitamin B12 (cobalamins) precipitate apo-transcobalamin (apoTC) and the holoTC present in the supernatant is measured by ELISA. Serum holoTC denotes the part of vitamin B12 accessible for the cells of the body and is considered to be a sensitive marker of vitamin B12 deficiency (1)(2)(3). Serum holoTC is not easily measured because it accounts for only approximately one-third of the circulating vitamin B12 and because the major part of TC circulates unsaturated with vitamin B12 (apoTC) (1)(2)(3). In the few published attempts to measure holoTC, TC is separated from the other vitamin B12-binding protein, haptocorrin, before quantification of vitamin B12. This allows a direct measurement of the cobalamins attached to TC (4)(5)(6) or an indirect calculation of holoTC from measurement of total plasma cobalamins and the plasma cobalamins not attached to TC (7)(8)(9). We have chosen another path. We produced B12 beads and removed apoTC before ELISA quantification (10) of the protein moiety of holoTC. Vitamin B12 (400 mg; Fluka) was modified by acid treatment (11) to produce derivatives with a free COOH group. Magnetic beads (DB M-270 amine; DYNAL) from 10 mL of the original suspension were incubated overnight at room temperature with 10 mL of 2 mmol/L derivatives in 0.1 mol/L 2-morpholinoethane sulfonic acid, pH 5.5, in the presence of 10 mmol/L 1-ethyl-3-(dimethylaminopropyl)carbodiimide (Sigma). The beads were then washed twice with 50 mL of 1 mol/L Tris, pH 7.5, twice with 50 mL of 1 mol/L ammonium acetate, pH 4.6, and 10 times with 50 mL of 0.1 mol/L Tris–0.5 mol/L NaCl, …