Steinar Hustad

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.

Betaine : a key modulator of one-carbon metabolism and homocysteine status

Abstract Betaine serves as a methyl donor in a reaction converting homocysteine to methionine, catalysed by the enzyme betaine-homocysteine methyltransferase. It has been used for years to lower the concentration of plasma total homocysteine (tHcy) in patients with homocystinuria, and has recently been shown to reduce fasting and in particular post-methionine load (PML) tHcy in healthy subjects. Betaine exists in plasma at concentrations of about 30μmol/L; it varies 10-fold (from 9 to 90μmol/L) between individuals, but the intra-individual variability is small. Major determinants are choline, dimethylglycine and folate in plasma, folic acid intake and gender. Recent studies have demonstrated that plasma betaine is a stronger determinant of PML tHcy than are vitamin B6 and folate. The betaine-PML tHcy relationship is attenuated after supplementation with B-vitamins, and is most pronounced in subjects with low folate. Betaine shows a weaker association with fasting tHcy (than with PML tHcy), and also this association is most pronounced in subjects with low folate. In pregnancy, plasma betaine declines until gestational week 20, and thereafter remains constant. From gestational week 20 onwards, fasting tHcy shows a strong inverse association with plasma betaine, and betaine becomes a stronger predictor than folate of fasting tHcy. To conclude, betaine status is a component of an individuals biochemical make-up with ramifications to one-carbon metabolism. Betaine status should be investigated in pathologies related to altered metabolism of homocysteine and folate, including cardiovascular disease, cancer and neural tube defects.

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.

Quantitative profiling of folate and one-carbon metabolism in large-scale epidemiological studies by mass spectrometry

Abstract Background: Derangements of one-carbon metabolism have been related to the development of chronic diseases. Metabolic profiling as part of epidemiological studies in this area should include intermediates involved in the transfer of one-carbon units, cofactors for the relevant enzymes and markers of inflammation, kidney function and smoking. Methods: We established five platforms that measured 6–16 analytes each. Platforms A (gas chromatography-mass spectrometry; GC-MS) and B (gas chromatography-tandem mass spectrometry; GC-MS/MS) involved methylchloroformate derivatization of primary amines, thiols and carboxylic acids. Platform C determined basic compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using an ether-linked phenyl reversed-phase column. Platforms D and E (LC-MS/MS) exploited the efficient ionization and high sensitivity obtained for a wide range of analytes, using a mobile phase containing a high concentration of acetic acid. The chromatographic run times ranged from 3 to 8 min. Results: The analyte concentrations ranged from 0.2 nmol/L to 400 μmol/L. Platforms A and B both measured methylmalonic acid, total homocysteine and related amino acids. Platform B also included sarcosine, cystathionine, tryptophan and kynurenine. Platform C was optimized for the measurement of choline and betaine, but also included arginine, asymmetric and symmetric dimethylarginine and creatinine. A diversity of low abundance compounds mainly occurring in the nanomolar range were measured on platform D. These were vitamin B2 and B6 species, neopterin, cotinine and tryptophan metabolites. Platform E measured folates and folate catabolites. Conclusions: Approximately 40 analytes related to one-carbon metabolism were determined in less than 1 mL of plasma/serum using five complementary analytical platforms. As a method control, several metabolites were measured on two or more platforms. Logistics and data handling were carried out by specially designed software. This strategy allows profiling of one-carbon metabolism in large-scale epidemiological studies. Clin Chem Lab Med 2007;45:1737–45.

Kinetic Modeling of Storage Effects on Biomarkers Related to B Vitamin Status and One-Carbon Metabolism

BACKGROUND Biomarkers and metabolites related to B vitamin function and one-carbon metabolism have been studied as predictors of chronic diseases in studies based on samples stored in biobanks. For most biomarkers, stability data are lacking or fragmentary. METHODS Degradation and accumulation kinetics of 32 biomarkers were determined at 23 °C in serum and plasma (EDTA, heparin, and citrate) collected from 16 individuals and stored for up to 8 days. In frozen serum (-25 °C), stability was studied cross-sectionally in 650 archival samples stored for up to 29 years. Concentration vs time curves were fitted to monoexponential, biexponential, linear, and nonlinear models. RESULTS For many biomarkers, stability was highest in EDTA plasma. Storage effects were similar at room temperature and at -25 °C; notable exceptions were methionine, which could be recovered as methionine sulfoxide, and cystathionine, which decreased in frozen samples. Cobalamin, betaine, dimethylglycine, sarcosine, total homocysteine, total cysteine, tryptophan, asymetric and symmetric dimethyl argenine, creatinine, and methylmalonic acid were essentially stable under all conditions. Most B vitamins (folate and vitamins B2 and B6) were unstable; choline increased markedly, and some amino acids also increased, particularly in serum. The kynurenines showed variable stability. For many biomarkers, degradation (folate and flavin mononucleotide) or accumulation (pyridoxal, riboflavin, choline, amino acids) kinetics at room temperature were non-first order. CONCLUSIONS Data on stability and deterioration kinetics for individual biomarkers are required to optimize procedures for handling serum and plasma, and for addressing preanalytical bias in epidemiological and clinical studies.

Plasma Vitamins B2, B6, and B12, and Related Genetic Variants as Predictors of Colorectal Cancer Risk

Background: B-vitamins are essential for one-carbon metabolism and have been linked to colorectal cancer. Although associations with folate have frequently been studied, studies on other plasma vitamins B2, B6, and B12 and colorectal cancer are scarce or inconclusive. Methods: We carried out a nested case-control study within the European Prospective Investigation into Cancer and Nutrition, including 1,365 incident colorectal cancer cases and 2,319 controls matched for study center, age, and sex. We measured the sum of B2 species riboflavin and flavin mononucleotide, and the sum of B6 species pyridoxal 5′-phosphate, pyridoxal, and 4-pyridoxic acid as indicators for vitamin B2 and B6 status, as well as vitamin B12 in plasma samples collected at baseline. In addition, we determined eight polymorphisms related to one-carbon metabolism. Relative risks for colorectal cancer were estimated using conditional logistic regression, adjusted for smoking, education, physical activity, body mass index, alcohol consumption, and intakes of fiber and red and processed meat. Results: The relative risks comparing highest to lowest quintile were 0.71 [95% confidence interval (95% CI), 0.56-0.91; Ptrend = 0.02] for vitamin B2, 0.68 (95% CI, 0.53-0.87; Ptrend <0.001) for vitamin B6, and 1.02 (95% CI, 0.80-1.29; Ptrend = 0.19) for vitamin B12. The associations for vitamin B6 were stronger in males who consumed ≥30 g alcohol/day. The polymorphisms were not associated with colorectal cancer. Conclusions: Higher plasma concentrations of vitamins B2 and B6 are associated with a lower colorectal cancer risk. Impact: This European population-based study is the first to indicate that vitamin B2 is inversely associated with colorectal cancer, and is in agreement with previously suggested inverse associations of vitamin B6 with colorectal cancer. Cancer Epidemiol Biomarkers Prev; 19(10); 2549–61. ©2010 AACR.

Seasonal and age-related differences in serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway.

Abstract Objective. The aim of this study was to investigate the seasonal and age-related variation of vitamin D and PTH serum concentrations in a large general patient population in Western Norway. Design. A retrospective study was conducted at the Hormone laboratory, Haukeland University Hospital, Bergen, Norway. All analyses of 25-hydroxyvitamin D (25(OH)D) (n = 8325), 1,25-dihydroxyvitamin D (1,25(OH)2D) (n = 4509) and PTH (n = 4203) requested from private practitioners from 2005 to 2008 were included. All three analytes were available in 1551 subjects. Subjects. Mean age of the study population was 49.8 years and 70.9% of the samples were from women. Results. The highest concentrations of 25(OH)D and 1,25(OH)2D were observed in July–September. In April 43% of the studied population had 25(OH)D concentrations below 50 nmol/L. There was a positive correlation between 25(OH)D and 1,25(OH)2D (p < 0.001). The levels of 25(OH)D and PTH were negatively correlated (p < 0.001) while 1,25(OH)2D and PTH showed a weak positive correlation (p = 0.015). We observed higher concentrations of 25(OH)D (p = 0.003) and lower 1,25(OH)2D levels (p < 0.001) in the older age groups. PTH increased throughout the whole age span (p < 0.001). Conclusion. We observed a seasonal variation in 25(OH)D and 1,25(OH)2D with low serum concentrations during winter/early spring while PTH showed an inverse pattern. Higher levels of PTH in winter and the elderly may reflect an impaired vitamin D status that may affect calcium homeostasis and bone health.

Multisteroid LC–MS/MS assay for glucocorticoids and androgens and its application in Addison's disease

Objective Liquid chromatography–tandem mass spectrometry (LC–MS/MS) offers superior analytical specificity compared with immunoassays, but it is not available in many regions and hospitals due to expensive instrumentation and tedious sample preparation. Thus, we developed an automated, high-throughput LC–MS/MS assay for simultaneous quantification of ten endogenous and synthetic steroids targeting diseases of the hypothalamic–pituitary–adrenal axis and gonads. Methods Deuterated internal standards were added to 85 μl serum and processed by liquid–liquid extraction. Cortisol, cortisone, prednisolone, prednisone, 11-deoxycortisol, dexamethasone, testosterone, androstenedione and progesterone were resolved by ultra-high-pressure chromatography on a reversed-phase column in 6.1 min and detected by triple-quadrupole mass spectrometry. The method was used to assess steroid profiles in women with Addisons disease (AD, n=156) and blood donors (BDs, n=102). Results Precisions ranged from 4.5 to 10.1% relative standard deviations (RSD), accuracies from 95 to 108% and extraction recoveries from 60 to 84%. The method was practically free of matrix effects and robust to individual differences in serum composition. Most postmenopausal AD women had extremely low androstenedione concentrations, below 0.14 nmol/l, and median testosterone concentrations of 0.15 nmol/l (interquartile range 0.00–0.41), considerably lower than those of postmenopausal BDs (1.28 nmol/l (0.96–1.64) and 0.65 nmol/l (0.56–1.10) respectively). AD women in fertile years had androstenedione concentrations of 1.18 nmol/l (0.71–1.76) and testosterone concentrations of 0.44 nmol/l (0.22–0.63), approximately half of those found in BDs of corresponding age. Conclusion This LC–MS/MS assay provides highly sensitive and specific assessments of glucocorticoids and androgens with low sample volumes and is suitable for endocrine laboratories and research. Its utility has been demonstrated in a large cohort of women with AD, and the data suggest that women with AD are particularly androgen deficient after menopause.

Analytical Recovery of Folate Degradation Products Formed in Human Serum and Plasma at Room Temperature

Folate is not stable in serum and plasma. This may impair laboratory diagnostics and distort the outcome of epidemiological studies on folate and chronic diseases. The present study was designed to determine the kinetics of folate loss in human serum and plasma (collected into tubes containing EDTA, heparin, or citrate) at room temperature and the recovery of folate as 4-alpha-hydroxy-5-methyltetrahydrofolate (hmTHF) or p-aminobenzoylglutamate (pABG) equivalents. Different folate species and pABG were determined by liquid chromatography-tandem MS and microbiologically active folate was measured by a Lactobacillus rhamnosus assay. Concentrations of 5mTHF and microbiologically active folate had a parallel and rapid decrease in EDTA plasma to approximately 60% of the initial concentration after 24 h. In serum, heparin plasma, and citrate plasma, folate decreased more slowly to approximately 50% after 192 h. The loss of 5mTHF that occurred within 48 h was totally recovered as hmTHF. Folate measured as pABG equivalents decreased slowly to approximately 80% in 192 h and the decline was essentially matrix independent. In conclusion, the degradation of 5mTHF and microbiologically active folate in serum and plasma at room temperature can largely be corrected for by determining hmTHF or measuring folate as pABG equivalents. Moreover, results obtained using conventional folate assays may be biased by improper sample handling or if samples contained high concentrations of hmTHF.

Vitamins B2 and B6 and Genetic Polymorphisms Related to One-Carbon Metabolism as Risk Factors for Gastric Adenocarcinoma in the European Prospective Investigation into Cancer and Nutrition

B vitamins and polymorphisms in genes coding for enzymes involved in one-carbon metabolism may affect DNA synthesis and methylation and thereby be implicated in carcinogenesis. Previous data on vitamins B2 and B6 and genetic polymorphisms other than those involving MTHFR as risk factors for gastric cancer (GC) are sparse and inconsistent. In this case-control study nested within the European Prospective Investigation into Cancer and Nutrition cohort, cases (n = 235) and controls (n = 601) were matched for study center, age, sex, and time of blood sampling. B2 and B6 species were measured in plasma, and the sum of riboflavin and flavin mononucleotide was used as the main exposure variable for vitamin B2 status, whereas the sum of pyridoxal 5′-phosphate, pyridoxal, and 4-pyridoxic acid was used to define vitamin B6 status. In addition, we determined eight polymorphisms related to one-carbon metabolism. Relative risks for GC risk were calculated with conditional logistic regression, adjusted for Helicobacter pylori infection status and smoking status. Adjusted relative risks per quartile (95% confidence interval, Ptrend) were 0.85 (0.72-1.01, 0.06) for vitamin B2 and 0.78 (0.65-0.93, <0.01) for vitamin B6. Both relations were stronger in individuals with severe chronic atrophic gastritis. The polymorphisms were not associated with GC risk and did not modify the observed vitamin-cancer associations. In summary, results from this large European cohort study showed an inverse association between vitamin B2 and GC risk, which is borderline significant, and a significant inverse association between vitamin B6 and GC risk. Cancer Epidemiol Biomarkers Prev; 19(1); 28–38