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Technical Briefs |
1 Department of Clinical Chemistry, University Hospital of Saarland, 66421 Homburg, Germany
2 Department of Biochemistry/College of Pharmacy, Damascus University, Syria
aaddress correspondence to this author at: Department of Clinical Chemistry/Central Laboratory, University Hospital of Saarland, Bldg. 40, Kirrberger Strasse, D-66421 Homburg/Saar, Germany; fax 49-6841-1623109, e-mail kchwher{at}uniklinik-saarland.de
Concern has emerged in America about subtle cobalamin (Cbl; vitamin B12) deficiency, especially in at-risk population groups such as the elderly and vegetarians (1). An optimal test to diagnose vitamin B12 deficiency is still not available (2)(3)(4). The determination of total serum vitamin B12 has a low diagnostic accuracy (2)(5). Measurements of homocysteine (HCY) and methylmalonic acid (MMA) have shown more specificity and sensitivity for subnormal Cbl status, but have disadvantages. HCY, for example, is also increased in folate and vitamin B6 deficiencies, as well as in renal insufficiency (4); in addition, MMA is expensive to measure, and it, too, increases in renal insufficiency (2)(4)(5). To improve specificity and sensitivity in diagnosis of vitamin B12 deficiency, holo-transcobalamin (holoTC) assays have been introduced (6). Because only transcobalamin II promotes the specific cellular uptake of Cbl, the Cbl subfraction attached to transcobalamin II represents the biologically active vitamin B12 fraction (6)(7)(8).
Our previous observations in Syrian individuals revealed a high prevalence of Cbl deficiency (
49%) when we used MMA as a metabolic marker for Cbl status (9). The present work was undertaken to further investigate the role of Cbl and folate status as determinants of hyperhomocysteinemia in Syrians.
We studied 222 patients [mean (SD) age, 52 (8) years; 192 males and 30 females] with angiographically defined stenosis 
50% in at least one major coronary artery. Exclusion criteria included recent myocardial infarction (3 months), acute diseases, and vitamin usage. Blood samples were collected 1 day before the angiography, and the angiography results were followed after that. The control group included 101 apparently healthy non-vitamin users and 10 individuals (7 males and 3 females) who had no stenosis [mean (SD) control age, 46 (9) years; total of 66 males and 45 females]. Only individual with creatinine concentrations within reference values were eligible for this study. Seventy-one percent of patients were hypertensive vs 25% of controls, 32% of patients had diabetes vs 11% of controls, and 23% of patients had never smoked vs 42% of controls. All participants gave informed consent.
We collected blood samples after a 12-h overnight fast, placed them directly on ice, and centrifuged them within 45 min (2000g for 20 min at 4 °C). Serum MMA and HCY were assayed by gas chromatography–mass spectrometry with deuterated MMA and HCY as internal standards (10)(11). The CVs for the MMA and HCY assays were 3.2% and 5.3%, respectively, at concentrations of 187 nmol/L and 7.8 µmol/L. Serum vitamin B12 and folate were measured by chemiluminescence immunoassay (Bayer). Serum holoTC was measured by RIA (Axis-Shield) (6). The CVs for holoTC assay were 8% and 5% at 38 and 98 pmol/L, respectively.
Patients and controls (males and females) displayed no differences in Cbl, HCY, MMA, and holoTC (data not shown). Therefore, all participants were collectively stratified according to MMA quartiles within the total group. The full metabolic profile and the B-vitamin concentrations according to MMA quartiles are presented in Table 1
. Serum folate did not differ significantly among MMA quartiles. Across MMA quartiles, holoTC and vitamin B12 showed a gradual decrease when MMA increased. Median HCY increased from 10.8 µmol/L in the lowest quartile to 15.4 µmol/L in the highest MMA quartile. Fig. 1
displays the interactions between serum folate, holoTC, and MMA as determinants of HCY concentrations. Median HCY increased within one folate range when MMA increased (or holoTC decreased). Conversely, HCY increased when serum folate decreased within one MMA or holoTC range.
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We found no significant differences in Cbl status biochemical indices between cardiovascular disease (CVD) patients and controls (data not shown). Similarly, a previous report failed to confirm a frank link between Cbl deficiency and increased CVD risk (2).
The holoTC and serum Cbl concentrations were highest in individuals within the lowest MMA quartile (Table 1
). In such a case, normal Cbl together with normal folate status may prevent HCY accumulation (median HCY, 10.8 µmol/L). However, in the highest MMA quartile, increased HCY (median, 15.4 µmol/L) may indicate impaired folate utilization despite normal serum folate. A role of Cbl in regulating folate metabolism has been suggested (12). Interestingly, given that HCY is an established CVD risk factor in the Syrian population (13), the median HCY concentrations in the first two MMA quartiles seem in accordance with the widely accepted cutoff value (12 µmol/L) above which HCY may become a risk factor (14). Also of note is that the median holoTC concentrations in the two highest MMA quartiles (34 and 24 pmol/L) were both below the cutoff (35 pmol/L) used in other population groups (2)(15).
Consistent with previous reports, both Cbl and folate status influenced HCY in the currently investigated group (Fig. 1
) (16)(17). The paradigm presented in Fig. 1
may justify concern about setting a recommended daily intake for folic acid or even a general reference range for folic acid in serum without considering Cbl status in different age ranges or socioeconomic groups (18). Our data confirm that the lowest HCY values may be achieved within the upper-normal range of serum folate when accompanied by better Cbl status (i.e., folic acid of 22–59 nmol/L and MMA of 84–181 nmol/L). The effect of moderately low Cbl status on HCY may be compensated for by higher folate status and vice versa (Fig. 1
).
Taken together, our current study emphasizes the importance of both Cbl and folate status as determinants of HCY. Furthermore, the present work has important implications for public health. It seems prudent that folate requirements be defined in the light of Cbl status.
References
The following articles in journals at HighWire Press have cited this article:
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  J. Brady, L. Wilson, L. McGregor, E. Valente, and L. Orning Active B12: A Rapid, Automated Assay for Holotranscobalamin on the Abbott AxSYM Analyzer Clin. Chem., March 1, 2008; 54(3): 567 - 573. [Abstract] [Full Text] [PDF]
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 K. M von Castel-Dunwoody, G. P. Kauwell, K. P Shelnutt, J. D Vaughn, E. R Griffin, D. R Maneval, D. W Theriaque, and L. B Bailey Transcobalamin 776C->G polymorphism negatively affects vitamin B-12 metabolism Am. J. Clinical Nutrition, June 1, 2005; 81(6): 1436 - 1441. [Abstract] [Full Text] [PDF]
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 R. Carmel, R. Green, D. S. Rosenblatt, and D. Watkins Update on Cobalamin, Folate, and Homocysteine Hematology, January 1, 2003; 2003(1): 62 - 81. [Abstract] [Full Text] [PDF]
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