Jörn Schneede

Biological and clinical implications of the MTHFR C677T polymorphism

The enzyme methylenetetrahydrofolate reductase (MTHFR) directs folate species either to DNA synthesis or to homocysteine (Hcy) remethylation. The common MTHFR C677T polymorphism affects the activity of the enzyme and hence folate distribution. Under conditions of impaired folate status, the homozygous TT genotype has been regarded as harmful because it is associated with a high concentration of plasma total Hcy, increased risk of neural tube defects and colorectal neoplasias, and can also predispose individuals to adverse effects from drugs with antifolate effects. The MTHFR C677T polymorphism shows no consistent correlation with cardiovascular risk and longevity but, in combination with positive folate balance, the TT genotype is associated with decreased risk of colorectal neoplasias. Because of the high prevalence of this polymorphism in most populations, the TT variant might represent an ancestral genetic adaptation to living constraints (tissue injury or unbalanced vitamin intake) that has become a determinant of disease profiles in modern times.

Effect of Flexible Sigmoidoscopy Screening on Colorectal Cancer Incidence and Mortality A Randomized Clinical Trial

IMPORTANCE Colorectal cancer is a major health burden. Screening is recommended in many countries. OBJECTIVE To estimate the effectiveness of flexible sigmoidoscopy screening on colorectal cancer incidence and mortality in a population-based trial. DESIGN, SETTING, AND PARTICIPANTS Randomized clinical trial of 100,210 individuals aged 50 to 64 years, identified from the population of Oslo city and Telemark County, Norway. Screening was performed in 1999-2000 (55-64-year age group) and in 2001 (50-54-year age group), with follow-up ending December 31, 2011. Of those selected, 1415 were excluded due to prior colorectal cancer, emigration, or death, and 3 could not be traced in the population registry. INTERVENTIONS Participants randomized to the screening group were invited to undergo screening. Within the screening group, participants were randomized 1:1 to receive once-only flexible sigmoidoscopy or combination of once-only flexible sigmoidoscopy and fecal occult blood testing (FOBT). Participants with positive screening test results (cancer, adenoma, polyp ≥10 mm, or positive FOBT) were offered colonoscopy. The control group received no intervention. MAIN OUTCOMES AND MEASURES Colorectal cancer incidence and mortality. RESULTS A total of 98,792 participants were included in the intention-to-screen analyses, of whom 78,220 comprised the control group and 20,572 comprised the screening group (10,283 randomized to receive a flexible sigmoidoscopy and 10,289 to receive flexible sigmoidoscopy and FOBT). Adherence with screening was 63%. After a median of 10.9 years, 71 participants died of colorectal cancer in the screening group vs 330 in the control group (31.4 vs 43.1 deaths per 100,000 person-years; absolute rate difference, 11.7 [95% CI, 3.0-20.4]; hazard ratio [HR], 0.73 [95% CI, 0.56-0.94]). Colorectal cancer was diagnosed in 253 participants in the screening group vs 1086 in the control group (112.6 vs 141.0 cases per 100,000 person-years; absolute rate difference, 28.4 [95% CI, 12.1-44.7]; HR, 0.80 [95% CI, 0.70-0.92]). Colorectal cancer incidence was reduced in both the 50- to 54-year age group (HR, 0.68; 95% CI, 0.49-0.94) and the 55- to 64-year age group (HR, 0.83; 95% CI, 0.71-0.96). There was no difference between the flexible sigmoidoscopy only vs the flexible sigmoidoscopy and FOBT screening groups. CONCLUSIONS AND RELEVANCE In Norway, once-only flexible sigmoidoscopy screening or flexible sigmoidoscopy and FOBT reduced colorectal cancer incidence and mortality on a population level compared with no screening. Screening was effective both in the 50- to 54-year and the 55- to 64-year age groups. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00119912.

The Methylenetetrahydrofolate Reductase 677C→T Polymorphism as a Modulator of a B Vitamin Network with Major Effects on Homocysteine Metabolism

Folates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use riboflavin, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C-->T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 micromol/liter, 10.9 micromol/liter, and 13.3 micromol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C-->T polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P<or=.006). Conversely, the MTHFR polymorphism influenced B vitamin effects, which were strongest in the TT group, in which the estimated tHcy difference between subjects with vitamin concentrations in the lowest compared with the highest quartile was 5.4 micromol/liter for folate, 4.1 micromol/liter for riboflavin, 3.2 micromol/liter for cobalamin, and 2.1 micromol/liter for vitamin B6. Furthermore, interactions between B vitamins were observed, and B vitamins were more strongly related to plasma tHcy when concentrations of other B vitamins were low. The study provides comprehensive data on the MTHFR-B vitamin network, which has major effects on the transfer of one-carbon units. Individuals with the TT genotype were particularly sensitive to the status of several B vitamins and might be candidates for personalized nutritional recommendations.

Methylmalonic Acid and Homocysteine in Plasma as Indicators of Functional Cobalamin Deficiency in Infants on Macrobiotic Diets

ABSTRACT: Methylmalonic acid and total honnxyslcine in plasma and scrum have previously been used as indicators of intracellular cobalamin function in adults. To assess the usefulness of quantitation of these metabolites in the diagnosis of dietary cobalamin deficiency in infants, they were determined in plasma from 41 infants (aged l0–20 mo) on a macrobiotic diet and in 50 healthy group-matched omnivorous controls. In the macrobiotic infants, both methylmalonic acid and total homocysteine were markedly increased compared with controls (8-fold and 2-fold, respectively). Both metabolites showed an inverse relation to the plasma cobalamin level. The very low cobalamin content of the macrobiotic diet and low plasma cobalamin in macrobiotic infants makes an impaired cobalamin function likely in these infants. We therefore used dietary group as an independent indicator of cobalamin status. Different test parameters for cobalamin status were evaluated by comparing their ability to discriminate between the two dietary groups. Logistic regression analysis showed that methylmalonic acid followed by total homocysteine and cobalamin, in that order, were the strongest predictors of dietary group. Mean corpuscular volume and Mb had low discriminative power. We conclude that the determination of methylmalonic acid and total homocysteine represents a sensitive and specific test for the diagnosis and follow-up of nutritional cobalamin deficiency in infants. Furthermore, the finding of high methylmalonic acid and total homocysteine in plasma of most macrobiotic infants demonstrates a functional cobalamin deficiency in these subjects.

Quantification of methylmalonic acid in human plasma with hydrophilic interaction liquid chromatography separation and mass spectrometric detection

BACKGROUND Measurement of methylmalonic acid (MMA) in serum or plasma is useful for diagnosing cobalamin deficiency. We developed a method for quantifying MMA in plasma based on hydrophilic interaction liquid chromatography (HILIC) and single-stage negative electrospray ionization (ESI) mass spectrometry. METHODS We deproteinized plasma samples (200 microL) with 800 microL acidified acetonitrile containing 0.17 micromol/L deuterated MMA (D(3)-MMA) internal standard, centrifuged the samples, and injected 4 microL of the supernatant into the LC-MS instrument. Separation was achieved within 3 min on a Merck SeQuant ZIC-HILIC column with a mobile phase consisting of 4 volumes acetonitrile plus 1 volume 100 mmol/L ammonium acetate buffer, pH 4.5, at a flow rate of 400 microL/min. Subsequent column washing and reconditioning contributed to a total run time of 10 min. MMA and D(3)-MMA were quantified by single-ion monitoring (m/z 117.2 and 120.2, respectively) in negative ESI mode at a drying-gas flow rate of 10 L/min, 300 degrees C, and a capillary voltage of 3.0 kV. RESULTS The estimated limits of MMA quantification and detection were 0.09 micromol/L and 0.03 micromol/L, respectively, in plasma. The assay was linear to 200 micromol/L. Interassay and intraassay CVs were < or = 5% at all tested concentrations. Recoveries were 90%-93%. CONCLUSIONS This robust assay allows analysis of MMA in human plasma without derivatization. Sample preparation is simple and suitable for automation.

Biomarkers Related to One-Carbon Metabolism as Potential Risk Factors for Distal Colorectal Adenomas

Background: Efficient one-carbon metabolism, which requires adequate supply of methyl group donors and B-vitamins, may protect against colorectal carcinogenesis. However, plasma folate and vitamins B2 and B12 have inconsistently been associated with colorectal cancer risk, and there have been no previous studies relating plasma concentrations of methionine, choline, and betaine to this outcome. Methods: This study comprised 10,601 individuals, 50 to 64 years of age, participating in the Norwegian Colorectal Cancer Prevention (NORCCAP) screening study. Using logistic regression analyses, we crosssectionally investigated associations between distal colorectal adenoma occurrence–potential precursor lesions of colorectal carcinomas–and plasma concentrations of methyl group donors and B-vitamins, and polymorphisms of genes related to one-carbon metabolism. Results: Screening revealed 1,809 subjects (17.1%) with at least one adenoma. The occurrence of high-risk adenomas (observed in 421 subjects) was inversely associated with plasma concentrations of methionine (highest versus lowest quartile: odds ratio (OR) = 0.61; 95% confidence interval (CI) = 0.45–0.83), betaine: OR = 0.74; 95% CI = 0.54–1.02, the vitamin B2 form flavin-mononucleotide (FMN): OR = 0.65; 95% CI = 0.49–0.88, and the vitamin B6 form pyridoxal 5′-phosphate (PLP): OR = 0.69; 95% CI = 0.51–0.95, but not with folate, choline, vitamin B12 concentrations, or with the studied polymorphisms. High methionine concentration in combination with high vitamin B2 or B6 concentrations was associated with lower occurrence of high-risk adenomas compared with these factors individually. Conclusions: High plasma concentrations of methionine and betaine, and vitamins B2 and B6 may reduce risk of developing colorectal adenomas. Impact: In addition to B-vitamins, methyl group donors such as methionine and betaine may play a role in colorectal carcinogenesis. Cancer Epidemiol Biomarkers Prev; 20(8); 1726–35. ©2011 AACR.

Transcobalamin Polymorphism 67A->G, but Not 776C->G, Affects Serum Holotranscobalamin in a Cohort of Healthy Middle-Aged Men and Women

Two polymorphic variants in the gene coding for transcobalamin II (TCN2), TCN2 776C- > G and TCN2 67A- > G, may alter serum holotranscobalamin (holoTC), which in turn may affect cellular uptake of cobalamin (Cbl) and thereby Cbl status indicators. We studied the effects of TCN2 776C- > G and TCN2 67A- > G on blood concentrations of holoTC, Cbl, methylmalonic acid (MMA), and total homocysteine (tHcy) in 2411 individuals (50-64 y) that had been selected on the basis of these TCN2 genotypes from 10601 Norwegian inhabitants. The serum holoTC concentration was lower in TCN2 67AG (55 ± 0.75 pmol/L) and 67GG (48 ± 2.14 pmol/L) than in 67AA (62 ± 0.67 pmol/L) (P < 0.001) but did not differ among TCN2 776C- > G genotypes. The polymorphisms interacted as serum holoTC determinants (P = 0.001) and the presence of TCN2 67AG and GG in strata of 776CC and CG, but not 776GG, increased the risk of having serum holoTC < 45.6 pmol/L [tertile 1 vs. tertiles 2 and 3: OR = 2.5 (95% CI 1.8-3.5) for 67AG; OR = 5.7 (95% CI 3.5-9.1) for 67GG in 776CC; OR = 2.1 (95% CI 1.6-2.9) for 67AG; and OR = 4.5 (95% CI 2.4-8.2) for 67GG in 776CG; all P < 0.001]. Plasma MMA, tHcy, and Cbl were not affected by either polymorphism. In summary, serum holoTC, but not plasma Cbl, MMA, or tHcy, varied according to TCN2 67A- > G genotypes. It remains to be determined whether this polymorphic effect on serum holoTC alters its diagnostic utility as Cbl status indicator.

Determinants of Plasma Homocysteine

The concentration of total homocysteine in plasma is influenced by a diversity of genetic and acquired factors, and by interactions between such factors. The most prevalent genetic cause of hyperhomocysteinemia is the C677T polymorphism of the methylenetetrahydrofolate reductase gene, which predisposes to hyperhomocysteinemia under conditions of impaired folate status. Among the physiological and life-style determinants, increasing age, male sex, poor nutrition with low vitamin intake, smoking, heavy coffee consumption cause high homocysteine, whereas young age, premenopausal state, pregnancy, vitamins like folate and cobalamin, and exercise are associated with low homocysteine. Several drugs may influence the homocysteine level by acting as vitamin antagonists, and among these, methotrexate and nitrous oxide cause a rapid increase in homocysteine by interfering with folate and cobalamin functions, respectively. Some sulfhydrylcontaining drugs reduce homocysteine, probably via disulphide exchange reactions, whereas the effect of steroid hormones on homocysteine is complex and their mechanisms are conjectural. Cyclosporin A increases homocysteine, possibly by a mechanism independent of interference with renal function. The diseases which most often and profoundly increase homocysteine are folate and cobalamin deficiencies and renal failure. Some proliferative (psoriasis) and malignant (leukemia) diseases may increase homocysteine, probably by directing folates towards DNA synthesis. Hyperhomocysteinemia has been associated with diabetes, but this is most likely secondary to impaired renal function, since factors like insulin itself and glomerular hyperfiltration seem to reduce homocysteine.

Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiazomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-319]. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiazomethane derivatives were detected with a laser-induced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH > 4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the pKa value (5.2–5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operational parameters (voltage, temperature and capillary length) were tested, and were largely found to comply with validated theoretical models for capillary zone electrophoresis. In an optimal system, as judged by high number of theoretical plates, high resolution and short run times, the MMA derivative was separated from related 1-pyrenylmethyl monoesters. The laser-induced fluorescence detection afforded a limit of detection of about 40 nmol/1 (signal-to-noise ratio of 5) for the MMA derivative. Under optimal conditions, we were able to detect <1 μmol/1 endogenous MMA in human serum.

Dietary Folate Intake and Breast Cancer Risk: European Prospective Investigation Into Cancer and Nutrition

BACKGROUND There is limited evidence on the association between dietary folate intake and the risk of breast cancer (BC) by hormone receptor expression in the tumors. We investigated the relationship between dietary folate and BC risk using data from the European Prospective Investigation into Cancer and Nutrition (EPIC). METHODS A total of 367993 women age 35 to 70 years were recruited in 10 European countries. During a median follow-up of 11.5 years, 11575 women with BC were identified. Dietary folate intake was estimated from country-specific dietary questionnaires. Cox proportional hazards regression models were used to quantify the association between dietary variables and BC risk. BC tumors were classified by receptor status. Subgroup analyses were performed by menopausal status and alcohol intake. Intake of other B vitamins was considered. All statistical tests were two-sided. RESULTS A borderline inverse association was observed between dietary folate and BC risk (hazard ratio comparing top vs bottom quintile [HRQ5-Q1] = 0.92, 95% CI = 0.83 to 1.01, P trend = .037). In premenopausal women, we observed a statistically significant trend towards lower risk in estrogen receptor-negative BC (HRQ5-Q1 = 0.66, 95% CI = 0.45 to 0.96, P trend = .042) and progesterone receptor-negative BC (HRQ5-Q1 = 0.70, 95% CI = 0.51 to 0.97, P trend = .021). No associations were found in postmenopausal women. A 14% reduction in BC risk was observed when comparing the highest with the lowest dietary folate tertiles in women having a high (>12 alcoholic drinks/week) alcohol intake (HRT3-T1 = 0.86, 95% CI = 0.75 to 0.98, P interaction = .035). CONCLUSIONS Higher dietary folate intake may be associated with a lower risk of sex hormone receptor-negative BC in premenopausal women.