Novel Role of 5-Methyl-(6S)-Tetrahydrofolate in Mediating Endothelial Cell Tetrahydrobiopterin in Pregnancy and Implications for Gestational Hypertension.

BACKGROUND: Folate intake during pregnancy is essential for fetal development and maternal health. However, the specific effects of folic acid (FA) and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) on the prevention and treatment of hypertensive disorders of pregnancy remain unclear. We investigated whether FA and 5-MTHF have different effects on endothelial cell tetrahydrobiopterin (BH4) metabolism in pregnancy and the possible consequences for endothelial NO generation, maternal blood pressure, and fetal growth. METHODS: We analyzed the maternal blood pressure in pregnant wild-type (Gch1fl/fl) and Gch1fl/fl Tie2cre mice treated with either FA or 5-MTHF starting before pregnancy, mid-pregnancy or late pregnancy. BH4, superoxide, and NO bioavailability were determined in mouse and human models of endothelial cell BH4 deficiency by high-performance liquid chromatography. RESULTS: In vitro studies in mouse and human endothelial cells showed that treatment with 5-MTHF, but not FA, elevated BH4 levels, reduced superoxide production, and increased NO synthase activity. In primary endothelial cells isolated from women with hypertensive pregnancies, exposure to 5-MTHF, but not FA, restored the reduction in BH4 levels and NO synthase activity. In vivo studies in mice revealed that oral treatment with 5-MTHF, but not FA, prevented and treated hypertension in pregnancy when administered either before or during pregnancy, respectively, and normalized placental and fetal growth restriction if administered from mid-gestation onward. CONCLUSIONS: Collectively, these studies identify a critical role for 5-MTHF in endothelial cell function in pregnancy, related to endothelial cell BH4 availability and NO synthase activity. Thus, 5-MTHF represents a novel therapeutic agent that may potentially improve endothelial function in hypertensive disorders of pregnancy by targeting endothelial cell BH4.

An improved folate stable isotope dilution assay of unexploited food sources from Brazil.

Brazil has a diverse plant community, including underutilized non-conventional food crops (PANCs), which have the potential to be a rich source of food and contribute to food security. For assessing the folate content in a range of Brazilian PANCs, we extended the validation of an existing stable isotope dilution assay (SIDA) for the stably 13C-labelled 10-formyl-Pte[13C5]Glu (10-CHO-Pte[13C5]Glu). The SIDA method with an enzymatic treatment, purification step, and an LC-MS/MS measurement was validated regarding linearity, precision, LoD/LoQ, and recovery for 10-CHO-PteGlu. After successful validation, the study of some underutilized Brazilian non-conventional fruits and leaves from the São Paulo State University campus revealed them as an important source of folates. It provided the first insights into the folate content of unexploited food sources from Brazil. Pequi had the highest folate content among the fruits studied, with mean values of 333 µg/100 g based on fresh weight (FW). The analysis also shows that different cultivars of fruit or fruits from different growing locations have a high variability in folate content or other nutritional factors. In most fruits, the main vitamer was 5-CH3-H4folate, but jenipapo and taioba showed the highest content of 10-CHO-PteGlu with 28.22 µg/100 g (FW) in jenipapo peel and 75.64 µg/100 g (FW) in the taioba leaves. Thus, this study also provides results on the importance of the folate vitamer 10-CHO-PteGlu contributing to the total folate content.

Pharmacokinetics of Sodium and Calcium Salts of (6S)-5-Methyltetrahydrofolic Acid Compared to Folic Acid and Indirect Comparison of the Two Salts.

(6S)-5-Methyltetrahydrofolic acid ((6S)-5-Methyl-THF) salts and folic acid may differ in their abilities to raise plasma (6S)-5-Methyl-THF levels. We compared the area under the curve (AUC), Cmax, and Tmax of plasma (6S)-5-Methyl-THF after intakes of (6S)-5-Methyl-THF-Na salt (Arcofolin®) and folic acid. Moreover, we compared the AUCs after intakes of (6S)-5-Methyl-THF-Na and the calcium salt, (6S)-5-Methyl-THF-Ca, that were tested against folic acid in two independent studies. The study was randomized, double blind, and cross over. Twenty-four adults (12 men and 12 women) received a single oral dose of 436 µg (6S)-5-Methyl-THF-Na and an equimolar dose of folic acid (400 µg) on two kinetic days with two weeks washout period in between. The plasma concentrations of (6S)-5-Methyl-THF were measured at 9 time points between 0 and 8 h. We found that the AUC0-8 h of plasma (6S)-5-Methyl-THF (mean (SD) = 126.0 (33.6) vs. 56.0 (25.3) nmol/L*h) and Cmax (36.8 (10.8) vs. 11.1 (4.1) nmol/L) were higher after administration of (6S)-5-Methyl-THF-Na than after the administration of folic acid (p < 0.001 for both). These differences were present in men and women. Only administration of folic acid resulted in a transient increase in plasma unmetabolized folic acid (2.5 (2.0) nmol/L after 0.5 h and 4.7 (2.9) nmol/L after 1 h). Intake of (6S)-5-Methyl-THF-Na was safe. The ratios of the AUC0-8 h for (6S)-5-Methyl-THF-Na and (6S)-5-Methyl-THF-Ca to the corresponding folic acid reference group and the delta of these AUC0-8 h did not differ between the studies. In conclusion, a single oral dose of (6S)-5-Methyl-THF-Na caused higher AUC0-8 h and Cmax of plasma (6S)-5-Methyl-THF compared to folic acid. The Na- and Ca- salts of (6S)-5-Methyl-THF are not likely to differ in their pharmacokinetics. Further studies may investigate whether supplementation of the compounds for a longer time will lead to differences in circulating or intracellular/tissue folate concentrations.

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry.

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC-MS/MS system. The quantification limits were between 0.17nmol/L (5-formylTHF) and 1.79nmol/L (THF), and the assay was linear up to 100nmol/L (5-methylTHF) and 10nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r=0.939; p<0.001). The mean total folate from 32 apparently healthy subjects was 18.09nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.

Simultaneous quantification of S-adenosyl methionine and S-adenosyl homocysteine in human plasma by stable-isotope dilution ultra performance liquid chromatography tandem mass spectrometry.

S-adenosyl methionine (SAM) is an important methyl group donor that is formed from methionine. S-adenosyl homocysteine (SAH) is formed after demethylation of SAM and represents a potent inhibitor of many methyltransferases. We developed an improved stable-isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of SAM and SAH in biological samples. The method comprises a phenylboronic acid-containing solid-phase extraction procedure, serving for binding and clean-up of SAM and SAH. After extraction, samples were separated and detected using either a HPLC SymmetryShield RP(18) or an Acquity UPLC BEH C(18) column with a HPLC-MS/MS or an UPLC-MS/MS system. The best results were obtained by Acquity UPLC BEH C(18) column. In plasma samples, the estimated intraassay coefficients of variation (CVs) for SAM and SAH were 3.3% and 3.9%, respectively, the interassay CVs were 10.1% for SAM and 8.3% for SAH. Mean recovery of SAM and SAH at two different concentrations was 100.0% for SAM and 101.7% for SAH. The quantification limits were 0.5 and 0.7nmol/L for SAM and SAH, respectively. In 31 plasma samples, the mean concentrations (SD) were 85.5 (11.1)nmol/L for SAM and 13.3 (5.0)nmol/L for SAH with a SAM/SAH ratio of 7.0 (1.8). The new UPLC-MS/MS method showed very high sensitivity and selectivity for SAM and SAH, low CVs and fast sample preparation (40 samples in 60min) and analysis time (3min). This new assay can be used for large-scale clinical studies.

Decreased p66Shc promoter methylation in patients with end-stage renal disease.

BACKGROUND: p66Shc is a stress response protein and partially regulated by epigenetic modifications. Mice lacking p66Shc have reduced atherosclerosis, increased resistance to oxidative stress and a prolonged life time. The aim of the present study was to compare promoter methylation of the p66Shc gene between healthy controls and patients with end-stage renal disease (ESRD). There are two reasons for studying patients with ESRD. First, patients with ESRD have a disturbed homocysteine metabolism, and second an increased risk of morbidity and mortality from cardiovascular disease is a constant finding in these patients. METHODS: In our study, we measured fasting levels of homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 8-isoprostane in 22 patients and in 26 healthy, age- and sex-matched controls. The methylation of the p66Shc promoter and Line-1, as surrogate marker of whole genome methylation was quantified in peripheral blood mononuclear cells. RESULTS: In comparison to the control group, homocysteine, SAM, SAH, 8-isoprostane and whole genome methylation were significantly elevated in ESRD patients, while the p66Shc promoter methylation was significantly reduced. A significant correlation was found between SAH and p66Shc promoter methylation in the patient group. This observation underlines the role of SAH as a potent inhibitor of methyltransferases. Using backward regression analysis, we demonstrated that 8-isoprostane has a significant influence on p66Shc promoter methylation. In the control group and in patients with ESRD, increasing 8-isoprostane levels were linked to an elevated promoter methylation. CONCLUSIONS: Under physiological conditions, based on the results of the control group, the p66Shc expression is more silenced through epigenetic modifications. The atherosclerotic risk is dramatically increased in ESRD patients; therefore, our experimental results of methylation are in accordance with the clinical situation.

Folate and methylation status in relation to phosphorylated tau protein(181P) and beta-amyloid(1-42) in cerebrospinal fluid.

BACKGROUND: Increased plasma total homocysteine (tHcy) is a risk factor for neurological diseases, but the underlying pathophysiology has not been adequately explained. METHODS: We evaluated concentrations of tHcy, S-adenosyl homocysteine (SAH), S-adenosyl methionine (SAM), folate, and vitamin B(12) in cerebrospinal fluid (CSF) and plasma or serum from 182 patients with different neurological disorders. We measured concentrations of phosphorylated tau protein (P-tau)((181P)) and beta-amyloid(1-42) in the CSF. RESULTS: Aging was associated with higher concentrations of tHcy and SAH in the CSF, in addition to lower concentrations of CSF folate and lower SAM:SAH ratio. Concentrations of CSF SAH and CSF folate correlated significantly with those of P-tau (r = 0.46 and r = -0.28, respectively). Moreover, P-tau correlated negatively with SAM:SAH ratio (r = -0.40, P <0.001). The association between SAH and higher P-tau was observed in 3 age groups (<41, 41-60, and >60 years). CSF tHcy was predicted by concentrations of CSF cystathionine (beta = 0.478), folate (beta = -0.403), albumin (beta = 0.349), and age (beta = 0.298). CONCLUSIONS: tHcy concentration in the brain is related to age, B vitamins, and CSF albumin. Increase of CSF SAH is related to increased CSF P-tau; decreased degradation of P-tau might be a plausible explanation. Disturbed methyl group metabolism may be the link between hyperhomocysteinemia and neurodegeneration. Lowering tHcy and SAH might protect the brain by preventing P-tau accumulation.

Biomarkers of folate and vitamin B12 are related in blood and cerebrospinal fluid.

BACKGROUND: B-vitamins (folate, B(12)) are important micronutrients for brain function and essential cofactors for homocysteine (HCY) metabolism. Increased HCY has been related to neurological and psychiatric disorders. We studied the role of the B-vitamins in HCY metabolism in the brain. METHODS: We studied blood and cerebrospinal fluid (CSF) samples from 72 patients who underwent lumbar puncture. We measured HCY, methylmalonic acid (MMA), and cystathionine by gas chromatography-mass spectrometry; S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) by liquid chromatography-tandem mass spectrometry; and the B-vitamins by HPLC or immunoassays. RESULTS: Concentrations were lower in CSF than serum or plasma for HCY (0.09 vs 9.4 micromol/L), SAH (13.2 vs 16.8 nmol/L), cystathionine (54 vs 329 nmol/L), and holotranscobalamin (16 vs 63 pmol/L), whereas concentrations in CSF were higher for MMA (359 vs 186 nmol/L) and SAM (270 vs 113 nmol/L; all P <0.05). CSF concentrations of HCY correlated significantly with CSF folate (r = -0.46), CSF SAH (r = 0.48), CSF-albumin (r = 0.31), and age (r = 0.32). Aging was also associated with lower concentrations of CSF-folate and higher CSF-SAH. The relationship between serum and CSF folate depended on serum folate: the correlation (r) of serum and CSF-folate was 0.69 at serum folate <15.7 nmol/L. CSF concentrations of MMA and holotranscobalamin were not significantly correlated. CONCLUSIONS: CSF and serum/plasma concentrations of vitamin biomarkers are significantly correlated. Older age is associated with higher CSF-HCY and CSF-SAH and lower CSF-folate. These metabolic alterations may be important indicators of low folate status, hyperhomocysteinemia, and neurodegenerative diseases.

The vegetarian lifestyle and DNA methylation.

Vegetarians have a lower intake of vitamin B12 than omnivores do. Vitamin B12 deficiency (holotranscobalamin II <35 pmol/L or methylmalonic acid >271 nmol/L) was found in 58% of 71 vegetarians studied. Higher homocysteine levels (>12 micromol/L) found in 45% indicate disturbed remethylation of homocysteine to methionine. The methylation of DNA is strongly linked to homocysteine metabolism. Since DNA methylation is an important epigenetic factor in the regulation of gene expression, alteration of the methylation pattern has been associated with aging, cancer, atherosclerosis and other diseases. Three observations indicate that DNA methylation could be diminished by a vegetarian lifestyle. The vegetarian diet has a low content of methionine, remethylation of homocysteine is reduced by vitamin B12 deficiency and elevated homocysteine levels can induce the generation of S-adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases. In our study we observed a significant correlation between SAH and whole-genome methylation (r=-0.36, p<0.01). This observation underlines the role of SAH as a potent inhibitor of methyltransferases. The methylation status was not correlated with homocysteine or S-adenosylemethionine (SAM). These results indicate that the degree of methylation does not depend on the supply of methyl groups and that the reverse generation of SAH has no influence. In addition to whole-genome methylation, the specific promoter methylation of the p66Shc gene was studied. However, the latter did not correlate with SAH, SAM or homocysteine. Obviously, the promoter methylation of the p66Shc gene is controlled in a specific way, without following the general regulating influence of SAH. In conclusion, an inhibitory effect of SAH on whole-genome methylation was found, but from our data no interaction between vegetarian lifestyle and DNA methylation could be determined.

In vitro inhibition of fibrinolysis by apolipoprotein(a) and lipoprotein(a) is size- and concentration-dependent.

Lipoprotein(a) (Lp(a)) is considered an independent risk factor for atherosclerotic heart and circulatory diseases. The unique, polymorphic character of Lp(a) is based on its apolipoprotein(a) (apo(a)), which has remarkable structural analogies with plasminogen, an important protein for fibrinolysis. The formation of plasmin from plasminogen is a fundamental step in the dissolution of fibrin. Repression of this step may lead to a deceleration of fibrinolysis. It has been suggested that Lp(a) has antifibrinolytic properties through apo(a) and that the apo(a)-size polymorphism has a distinct influence on the prothrombotic properties of Lp(a). However, the results on this topic are controversial. Therefore we used a standardized in vitro fibrinolysis model to provide further information on the influence of Lp(a) on plasmin formation. Monitoring the time-course of plasmin formation, we investigated the inhibition of plasmin formation through dependence on Lp(a), respectively, free apo(a) concentration. Furthermore, we investigated the influence of three Lp(a)/apo(a) phenotypes ((22K)Lp(a), 22 kringle-4 repeats; (30K)Lp(a), 30 kringle-4 repeats; (35K)Lp(a), 35 kringle-4 repeats). Adding varying amounts of Lp(a) to our model, we observed that the rate of plasmin formation was inversely related to the Lp(a) concentration. At 0.1 micromol/l (30K)Lp(a), for example, the plasmin formation was reduced by 12.7% and decreased further by 40.7% at 0.25 micromol/l Lp(a). A similar but more distinct effect was observed when free (30K)apo(a) was added to the model (25.3% at 0.1 micromol/l vs. 59.3% at 0.25 micromol/l). Comparing the antifibrinolytic influence of different apo(a) phenotypes we found that the reduction of plasmin generation advanced with the size of apo(a). At 0.1 micromol/l Lp(a) the reduction of the plasmin formation increased in the order (22K)Lp(a), (30K)Lp(a) and (35K)Lp(a) from 3.7% to 10.7% and 22.3%, respectively. Experiments with different phenotypes of free apo(a) showed similar results (0.5 micromol/l: (22K)apo(a), 56.4% vs. (30K)Lp(a), 80.4%). Summarizing these results, our study indicates a distinct interrelation of Lp(a)/apo(a) phenotype and concentration with the formation of plasmin. From the antifibrinolytic Lp(a)/apo(a) effect in vitro it may be hypothesized that Lp(a)/apo(a) also has an inhibitory influence on in vivo fibrinolysis.

The role of genetic factors in the development of hyperhomocysteinemia.

Moderate hyperhomocysteinemia has been identified as a new independent risk factor for cardiovascular and neurodegenerative diseases. This fact has produced interest in the study of genetic variants involved in homocysteine metabolism and its relationship to pathogenesis. Recently, more than 15 different genes were studied for their relationship to plasma homocysteine levels. We determined the influence of genetic variants in five genes (5,10-methylenetetrahydrofolate reductase (MTHFR) 677C --> T, serine hydroxymethyltransferase (SHMT) 1420C --> T, thymidylate synthase (TS) 2R --> 3R, catechol-O-methyltransferase (COMT) 1947G --> A and transcobalamin (TC) 776C --> G) on plasma homocysteine, folic acid and parameters of vitamin B12 metabolism in 111 vegetarians (mean age: 46 +/- 15 years) and 118 healthy seniors (mean age: 82 +/- 6.5 years). Median homocysteine concentration in plasma was significantly influenced by the MTHFR genotypes in both populations. In the vegetarians the median homocysteine level was increased by 8 micromol/l in individuals homozygous for the mutation as compared to wild-type or heterozygous genotypes (20.4 micromol/l vs. 12.9 and 12.7 micromol/l, respectively). This unexpected increase was observed although the folate levels were in medium to elevated ranges. Our results suggest that vegetarians have a higher demand for folate to neutralize the genotype effect. Preclinical vitamin B12 deficiency in vegetarians may be the cause for disturbed remethylation and folate trap. Plasma homocysteine was not significantly influenced by the SHMT, TS, COMT and TC mutations. In addition, for the TC mutation a trend toward cellular vitamin B12 deficiency was observed. The methylmalonic acid (MMA) levels were slightly elevated and the holotranscobalamin-II (holoTC-II) levels decreased. In the vegetarian group a significant relationship between the COMT genotype and holoTC-II concentration in plasma was determined, whereas the high activity COMT genotype (G/G) resulted in increased levels (35 micromol/l vs. 21 micromol/l for heterozygous and low activity genotypes). The MMA levels were inversely correlated to holoTC-II concentrations. In conclusion, the study on vegetarians and seniors documents interesting lifestyle-genotype interactions. Although the TC and COMT mutations influence cellular vitamin B12 metabolism, this effect did not result in overt homocysteine elevation.

The impact of hyperhomocysteinemia as a cardiovascular risk factor in the prediction of coronary heart disease.

Coronary heart disease often occurs in the absence of traditional risk factors. Consequently, epidemiological studies exploring novel risk factors are necessary to improve the prediction of coronary heart disease. This study evaluated five promising markers of cardiovascular risk: homocysteine, C-reactive protein, fibrinogen, lipoprotein(a) (Lp(a)), free apolipoprotein(a) (apo(a)) and Lp(a) phenotypes. The study included 135 patients with angiographically confirmed atherosclerosis. The control group consisted of 93 sex- and age-matched individuals. The Mann-Whitney U-test was used for group comparison. New risk factors were evaluated by binary logistic regression. The odds ratios were calculated continuously for homocysteine in dependence on C-reactive protein. Low density lipoprotein (LDL)-cholesterol was nearly identical in controls and patients. Homocysteine, C-reactive protein, fibrinogen, high density lipoprotein (HDL)-cholesterol and Lp(a) discriminated highly significantly between both groups. The continuously calculated odds ratio for homocysteine demonstrated a distinct influence of C-reactive protein. In the group with high C-reactive protein levels, homocysteine levels above 9.6 micromol/l resulted in a markedly elevated risk (odds ratio 12), in the group with C-reactive protein levels below 5 mg/dl, a comparable risk increase was observed at a homocysteine level of 16.6 micromol/l. This data strongly suggests that plasma homocysteine helps identify individuals at risk, especially among those with elevated C-reactive protein levels.

Stimulatory effect of homocysteine on interleukin-8 expression in human endothelial cells.

Elevated plasma homocysteine is an independent risk factor for atherosclerosis. An important initial step of atherosclerosis is the adhesion and infiltration of monocytes to the lesion site. It has been shown that the pro-inflammatory cytokine interleukin-8 can rapidly cause rolling monocytes to adhere firmly onto monolayers expressing E-selectin. The objective of the present study was to investigate the effect of homocysteine on interleukin-8 production in human endothelial cells. Cells were incubated with various concentrations of homocysteine for 20 h. The gene expression was determined by real-time PCR and the interleukin-8 protein was measured by immunoassay analysis. Homocysteine enhanced the expression of interleukin-8 in a dose-dependent manner (181% of controls at 2.5 mmol/l homocysteine). Stimulation of gene expression was associated with a parallel increase in interleukin-8 protein synthesis (160% of controls at 5.0 mmol/l homocysteine). By co-incubation of endothelial cells with homocysteine and copper sulfate, a further elevation of interleukin-8 expression (251% of controls) was observed, whereas copper sulfate alone had no stimulatory effect. In conclusion, the present study demonstrated that homocysteine altered endothelial cell function by stimulating interleukin-8 expression, suggesting a contribution of homocysteine to the initiation and progression of atherosclerosis. The formation of homocysteine-induced oxidation products might serve as one of the underlying mechanisms of this effect.

Hyperhomocysteinemia: a new risk factor for degenerative diseases.

Hyperhomocysteinemia (HHCY) is a consequence of disturbed methionine metabolism. It results from enzyme and/or vitamin deficiency. Epidemiological and clinical studies have proven HHCY to be an independent risk factor for atherosclerotic cardiovascular diseases, stroke, peripheral arterial occlusive disease and venous thrombosis. Trials in progress may clarify the "causality" of high homocysteine (HCY) concentrations and will assess the value of HCY lowering therapy. HHCY is also seen as a risk factor for neurodegenerative diseases such as cognitive impairment, dementia, Alzheimer's disease, and also for depression. There is a high prevalence of HHCY as a syndrome of vitamin shortage in elderly subjects, which strongly increases with advancing age. Elderly people have a high frequency of vitamin B12 deficiency which is more reliably diagnosed by measurement of serum methylmalonic acid and holotranscobalamin II, the metabolically active B12 fraction, than by total serum vitamin B12. Subjects who follow a strict vegetarian diet also have a high prevalence of HHCY caused by vitamin B12 deficiency. For prevention of neurological damages an early diagnosis of vitamin B12 deficiency is important. Furthermore, HHCY is a factor in the pathogenesis of neural tube defects and preeclampsia. HCY should be measured in patients with a history of atherothrombotic vessel diseases, in patients with diabetes or hyperlipidemia, in renal patients, in adipose subjects, in elderly people, in vegetarians, in postmenopausal women, and in early pregnancy.