Alterations in hepatic one-carbon metabolism and related pathways following a high-fat dietary intervention.

Obesity frequently leads to insulin resistance and the development of hepatic steatosis. To characterize the molecular changes that promote hepatic steatosis, transcriptomics, proteomics, and metabolomics technologies were applied to liver samples from C57BL/6J mice obtained from two independent intervention trials. After 12 wk of high-fat feeding the animals became obese, hyperglycemic, and insulin resistant, had elevated levels of blood cholesterol and VLDL, and developed hepatic steatosis. Nutrigenomic analysis revealed alterations of key metabolites and enzyme transcript levels of hepatic one-carbon metabolism and related pathways. The hepatic oxidative capacity and the lipid milieu were significantly altered, which may play a key role in the development of insulin resistance. Additionally, high choline levels were observed after the high-fat diet. Previous studies have linked choline levels with insulin resistance and hepatic steatosis in conjunction with changes of certain metabolites and enzyme levels of one-carbon metabolism. The present results suggest that the coupling of high levels of choline and low levels of methionine plays an important role in the development of insulin resistance and liver steatosis. In conclusion, the complexities of the alterations induced by high-fat feeding are multifactorial, indicating that the interplay between several metabolic pathways is responsible for the pathological consequences.

Lowering and Stabilizing PSA Levels in Advanced-prostate Cancer Patients With Oral Methioninase.

BACKGROUND/AIM: Methionine addiction is a general and fundamental hallmark of cancer due to the excess use of methionine for transmethylation reactions, termed the "Hoffman Effect". Methionine addiction has been shown to be a highly-effective target for cancer therapy by methionine restriction with oral recombinant methioninase (o-rMETase) in preclinical studies, including patient- derived orthotopic xenograft (PDOX) mouse models of cancer. A clinical study of o-rMETase as a supplement showed a 70% reduction of PSA levels in a patient with bone-metastatic prostate cancer. MATERIALS AND METHODS: In the present study, two advanced prostate-cancer patients took o-rMETase as a supplement for approximately one month. RESULTS: One of the patients taking o-rMETase showed a 38% reduction of PSA levels and the second patient showed a 20% PSA reduction. CONCLUSION: o-rMETase shows promise for treating patients with advanced prostate cancer.

Chemical composition and anti-inflammatory activity of n-butanol extract of Piper sarmentosum Roxb. In the intestinal porcine epithelial cells (IPEC-J2).

ETHNOPHARMACOLOGICAL RELEVANCE: Piper sarmentosum Roxb. (PS) is a terrestrial herb primarily distributed in tropical and subtropical regions of Asia. It is widely used in folk medicine in certain countries of Southeast Asia for the treatment of fever, toothache, coughing and pleurisy, which showed the anti-inflammatory activity of PS. AIM OF THE STUDY: This study aimed to investigate the chemical constituents and the molecular mechanism and related metabolic pathway by which n-butanol extract of PS (PSE-NB) exerts its anti-inflammatory effects. MATERIALS AND METHODS: Chemical constituents of PSE-NB was analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique. Anti-inflammatory effects of PSE-NB were investigated in lipopolysaccharide (LPS)-induced IPEC-J2 cells. RESULTS: In total, 218 compounds, including 94 alkaloids and 26 phenolics were tentatively identified, which indicating alkaloids and phenolics were the main constituents of PSE-NB. In addition, the current cell experiment in vitro showed that PSE-NB (10-500 µg/mL) pre-treatment before LPS stimulation significantly decreased mRNA expression of IL-1ß, IL-6 and TNF-α in IPEC-J2 cells compared with LPS treatment (p < 0.05). PSE-NB improved mRNA expression of tight junction proteins (ZO-1 and Occludin) and NHE3, which were reduced by LPS stimulation (p < 0.05). Moreover, PSE-NB (10 µg/mL) alleviated LPS-induced protein expression of p65 and p-p65 (p < 0.05), and reduced p65 translocation into the nucleus induced by LPS. At the same time, metabolic pathway analysis indicated that PSE-NB exerts anti-inflammatory effects mainly via augmentation of methionine metabolism in IPEC-J2 cells. CONCLUSIONS: Taken together, the results suggested that alkaloids and phenolics were the main constituents in PSE-NB. PSE-NB might attenuate LPS-induced inflammatory responses in IPEC-J2 cells by regulating NF-κB signaling pathway and intracellular metabolic pattern.

Moderate or supranormal folic acid supplementation does not exert a protective effect for homocysteinemia and methylation markers in growing rats.

BACKGROUND/AIMS: Folic acid (FA) deficiency/supplementation effects seem to be dependent on age group and/or physiological status. The aim was to evaluate changes associated with rapid growth in relation to methionine metabolism in rats. METHODS: Four groups (n = 10 each) of male Sprague Dawley rats (5 weeks old) were on diets that varied in their FA content: 0 mg FA/kg diet (deficient), 2 mg FA/kg diet (control), 8 mg FA/kg diet (moderate supplementation), 40 mg FA/kg diet (supranormal supplementation). Animals were fed ad libitum for 30 days. Biomarkers of methionine metabolism and antioxidant status were evaluated. RESULTS: Serum total homocysteine concentration increased (p < 0.01) in FA deficient animals, with no differences between the supplemented groups. The hepatic 'methylation ratio' (S-adenosylmethionine/S-adenosylhomocysteine) of the FA content groups reached similar values, which were significantly higher compared to the deficient group. The brain 'methylation ratio', however, remained unmodified independently of FA content in the diet. FA deficiency induced hepatic DNA hypomethylation, and supranormal FA supplementation exerted the most protective effect (p < 0.01). Serum folate levels increased according to FA dietary level, whereas no differences were seen for vitamin B(12) and vitamin B(6). CONCLUSIONS: FA deficiency compromises methionine metabolism whereas supplementation does not show an additional positive effect compared to the control diet in growing animals.

The feasibility of 11C-methionine-PET in diagnosis of solitary lung nodules/masses when compared with 18F-FDG-PET.

OBJECTIVE: To differentiate between benign and malignant lesions of the lung, 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) has limitations such as a lower specificity in cases of non-specific inflammation. The positive predictive value is unsatisfactory in countries where inflammatory lung disorders are prevalent. We present the preliminary results of the usefulness of combining 11C-methionine-PET and 18F-FDG-PET in this context. METHODS: Fifteen patients with indeterminate solitary pulmonary nodules/masses (10 men, 5 women; average age 64.7 +/- 14.0 years, ranging from 25 to 87 years) were studied using 11C-methionine- and 18F-FDG-PET. Interpretations were primarily made on visual analysis with five-point scale and a consensus of two nuclear medicine physicians, using standardized uptake value as an accessory reference. Foci of abnormal radiotracer uptake were subsequently correlated with clinical follow-up, imaging modalities such as chest radiography, chest computed tomography (CT), serial PET studies, and pathology results from bronchoscopic biopsy and/or surgical specimen. RESULTS: Diagnoses were established in 14 patients. The 11C-methionine-PET and 18F-FDG-PET studies were both true positive in two cases of adenocarcinoma and true negative in two cases of clinical benign nodules. In one case of lymphoid hyperplasia both 11C-methionine-PET and 18F-FDG-PET showed false-positive findings. Discordant results were obtained in nine cases. In spite of the false-positive results of 18F-FDG-PET, 11C-methionine-PET was true negative in four cases with chronic inflammatory nodules and three cases of pulmonary tuberculosis. Furthermore, (11)C-methionine-PET was true positive in one case of lung metastasis of thyroid cancer, and in another with recurrence of gastric cancer, respectively, for which 18F-FDG-PET imaging was false negative. CONCLUSIONS: Our experience indicates that 11C-methionine-PET seems more specific and sensitive when compared with 18F-FDG-PET for the purpose of differentiating benign and malignant thoracic nodules/masses. The possibility of an FDG-avid lesion being malignant is decreased if it shows a negative result by 11C-methionine-PET.

Perinatal choline influences brain structure and function.

Choline is derived not only from the diet, but also from de novo synthesis. It is important for methyl-group metabolism, the formation of membranes, kidney function, and neurotransmission. When deprived of dietary choline, most adult men and postmenopausal women develop signs of organ dysfunction (fatty liver or muscle damage) and have a decreased capacity to convert homocysteine to methionine. Choline is critical during fetal development, when it influences stem cell proliferation and apoptosis, thereby altering brain structure and function (memory is permanently enhanced in rodents exposed to choline during the latter part of gestation).

Gene cloning and characterization of Pseudomonas putida L-methionine-alpha-deamino-gamma-mercaptomethane-lyase.

Methionine dependency has been reported in cancer cell lines and primary tumors. Thus, L-methionine deprivation might have potential value for the treatment of human cancers with a methionine requirement. L-Methionine-alpha-deamino-gamma-mercaptomethane-lyase has been reported to decrease plasma methionine levels and to inhibit tumor growth in experimental animals but has not been studied extensively because sufficient homogeneous enzyme was not available. In this study, we cloned the L-methioninase gene from Pseudomonas putida and isolated pure and abundant recombinant enzyme. Both L-methionine and L-cysteine in culture medium were completely degraded by 1 unit/ml purified enzyme. Two hundred and fifty units/kg L-methioninase administered i.v. to mice yielded 0.7 unit/ml of plasma concentration and lowered total plasma sulfur-containing amino acids by more than 75%. Although sensitivity to enzymatic methionine depletion differed among cell lines, leukemia cell lines were generally more sensitive than solid tumor cell lines. The availability of pure recombinant L-methioninase will allow in vivo studies on the antitumor activity and the potential toxicity of enzymatic methionine depletion.

Structural and activity characterization of human PHPT1 after oxidative modification.

Phosphohistidine phosphatase 1 (PHPT1), the only known phosphohistidine phosphatase in mammals, regulates phosphohistidine levels of several proteins including those involved in signaling, lipid metabolism, and potassium ion transport. While the high-resolution structure of human PHPT1 (hPHPT1) is available and residues important for substrate binding and catalytic activity have been reported, little is known about post-translational modifications that modulate hPHPT1 activity. Here we characterize the structural and functional impact of hPHPT1 oxidation upon exposure to a reactive oxygen species, hydrogen peroxide (H2O2). Specifically, liquid chromatography-tandem mass spectrometry was used to quantify site-specific oxidation of redox-sensitive residues of hPHPT1. Results from this study revealed that H2O2 exposure induces selective oxidation of hPHPT1 at Met95, a residue within the substrate binding region. Explicit solvent molecular dynamics simulations, however, predict only a minor effect of Met95 oxidation in the structure and dynamics of the apo-state of the hPHPT1 catalytic site, suggesting that if Met95 oxidation alters hPHPT1 activity, then it will do so by altering the stability of an intermediate state. Employing a novel mass spectrometry-based assay, we determined that H2O2-induced oxidation does not impact hPHPT1 function negatively; a result contrary to the common conception that protein oxidation is typically a loss-of-function modification.

Disruption of methyl group metabolism by ethanol.

Recent studies have focused on establishing a link between the pathogenesis of ethanol and the disruption of metabolic pathways in the liver. Ethanol alters hepatic methionine metabolism, leading to perturbation of S-adenosylmethionine-dependent transmethylation. Therefore, the supply of metabolically related nutrients such as folate may play a role in the hepatotoxic effects of ethanol.

Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis.

The reaction of radiolytically generated *OH with cytochrome c was investigated by mass spectrometry. Tryptic digestion and characterization of the oxidized peptides by MALDI-TOF and ESI tandem mass spectrometry identified eight different amino acid residues with oxidized side chains with no cleavage of the protein detected. Solvent-accessible aromatic and methionine residues are the most susceptible to oxidation by *OH. These results support the careful use of *OH in characterizing protein surfaces. Dose-response studies identified the residues most prone to oxidation to be Phe-36, Phe-46, and Met-80. Hydroxylation of Phe-36 and Phe-46 should serve as indicators of the presence of *OH in the mitochondrial intermembrane space. Using solutions containing 50 at.% (18)O, our study also provides a novel method of determining the source of oxygen during *OH-mediated oxidation of proteins and contributes to identification of the modified residue type, with Phe>Tyr>Met in (18)O incorporation. During aerobic radiolysis, UV-vis spectroscopy indicates that ferrocytochrome c reaches a steady state concomitant with reduction of the heme.

Effect of cystine on the methionine requirement of healthy Indian men determined by using the 24-h indicator amino acid balance approach.

BACKGROUND: The 1985 FAO/WHO/UNU requirement for methionine in healthy adults consuming a cystine-free diet is 13 mg.kg(-1).d(-1). It is unclear whether this daily requirement is influenced by dietary cystine. OBJECTIVE: We assessed the effect of 2 intakes of cystine (5 and 12 mg.kg(-1).d(-1)) on methionine requirements in well-nourished Indian men by using 7 test methionine intakes (3, 6, 9, 13, 18, 21 and 24 mg.kg(-1).d(-1)) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) methods. We combined these data with those from an experiment with zero cystine intake and in which the exact same method was used. DESIGN: Two studies were performed in which a diet containing either 5 or 12 mg cystine.kg(-1).d(-1) was fed to 21 well-nourished Indian men over three 7-d periods. The 24-h IAAO and 24-h IAAB values were measured on day 7 with the use of a 24-h intravenous [13C]leucine tracer infusion. The breakpoints in the relation between these values and methionine intake in each study were assessed by two-phase linear regression. RESULTS: Breakpoints in the response curve were obtained at methionine intakes of 20 (95% Fiellers CI: 17, 26) and 10 (95% Fiellers CI: 8, 16) mg.kg(-1).d(-1) with cystine intakes of 5 and 12 mg.kg(-1).d(-1) intakes, respectively, which suggested a sparing effect of cystine. Although the 5- and 12-mg cystine breakpoints differed from one another, they did not differ significantly from that estimated previously with 0 mg cystine. CONCLUSION: Cystine may spare the methionine requirement in healthy men, although the amount of sparing is difficult to quantify.

Dietary serine and cystine attenuate the homocysteine-raising effect of dietary methionine: a randomized crossover trial in humans.

BACKGROUND: A high plasma total homocysteine (tHcy) concentration is a risk factor for cardiovascular disease. The increase in tHcy induced by methionine, the sole dietary precursor of homocysteine, might be modulated by other amino acids present in dietary proteins. OBJECTIVES: Our objectives were to compare the postprandial effect of free and dietary methionine on plasma tHcy concentrations and to investigate whether serine and cystine modify the effect of free methionine on tHcy. DESIGN: We conducted a randomized crossover trial in 24 healthy men. Each subject ingested 4 meals on separate days, which were separated by 1 wk. tHcy concentrations were measured in the fasting state and at 2, 4, 6, 8, 10, and 24 h after meal ingestion. The meals were 1) a low-protein meal fortified with 30 mg methionine/kg body wt (reference, denoted by "Met"), 2) meal 1 additionally fortified with 60.6 mg serine/kg body wt (MetSer), 3) meal 1 additionally fortified with 12.3 mg cystine/kg body wt (MetCys), and 4) a protein-rich meal containing 30 mg methionine, 60.6 mg serine, and 12.3 mg cystine per kg body wt (Protein). RESULTS: The mean (+/-SD) fasting tHcy concentration was 9.1 +/- 2.7 micromol/L. Mean peak tHcy concentrations were 17.9 +/- 4.5, 14.3 +/- 3.3, 14.8 +/- 3.9, and 11.2 +/- 3.1 micromol/L after Met, MetSer, MetCys, and Protein, respectively. Compared with the mean 24-h area under the tHcy-by-time curve after Met, the mean curves after MetSer, MetCys, and Protein were 37%, 32%, and 77% smaller, respectively (all P < 0.0005). CONCLUSIONS: Dietary methionine increases tHcy much less than does free methionine. Serine and cystine attenuate the tHcy-raising effect of free methionine. Thus, dietary proteins with a high content of serine or cystine relative to methionine may lead to lower postprandial tHcy responses.

Alcohol, folate, methionine, and risk of incident breast cancer in the American Cancer Society Cancer Prevention Study II Nutrition Cohort.

Recent studies suggest that the increased risk of breast cancer associated with alcohol consumption may be reduced by adequate folate intake. We examined this question among 66,561 postmenopausal women in the American Cancer Society Cancer Prevention Study II Nutrition Cohort. A total of 1,303 incident cases had accrued during the first 5 years of follow-up. Cox proportional hazards models and stratified analysis were used to examine the relationship between alcohol, dietary and total folate intake, multivitamin use, dietary methionine, and breast cancer. We observed an increasing risk of breast cancer with increasing alcohol consumption (P for trend = 0.01). In the highest category of consumption (15 or more grams of ethanol/day), the risk of breast cancer was 1.26 (95% confidence interval, 1.04-1.53) compared with nonusers. We observed this association with higher alcohol consumption for in situ, localized, and regional disease. We found no association between risk of breast cancer and dietary folate, total folate, multivitamin use, or methionine intake. Furthermore, we found no evidence of an interaction between levels of dietary folate (P for interaction = 0.10) or total folate (P for interaction = 0.61) and alcohol. Nor did we find evidence of an interaction between alcohol consumption and recent or long-term multivitamin use (P for interaction = 0.27). Our results are consistent with a positive association with alcohol but do not support an association with folate or methionine intake or an interaction between folate and alcohol intake on risk of breast cancer.

Interaction between sodium 5,6-benzylidene-L-ascorbate and dopamine.

The interaction between sodium 5,6-benzylidene-L-ascorbate (SBA) and dopamine was investigated by three different parameters: radical intensity, prooxidant action and cytotoxic activity. Under alkaline conditions, SBA and dopamine produced doublet and quartet ESR signals, respectively. The addition of increasing concentrations of SBA completely scavenged the dopamine radical and replaced the latter with its own radical. On the other hand, dopamine accelerated the decay of SBA radical. These two compounds stimulated the methionine oxidation in culture medium, but in combination, their stimulation activities were weakened. Both of these two compounds dose-dependently reduced the viable cell number of human promyelocytic leukemics HL-60 cells. When these two compounds were mixed together before adding to HL-60 cells, both of their cytotoxic activities were diminished. The present study demonstrates the interaction between SBA and dopamine, which might modify their biological activities.

Growth hormone signaling is necessary for lifespan extension by dietary methionine.

Growth hormone significantly impacts lifespan in mammals. Mouse longevity is extended when growth hormone (GH) signaling is interrupted but markedly shortened with high-plasma hormone levels. Methionine metabolism is enhanced in growth hormone deficiency, for example, in the Ames dwarf, but suppressed in GH transgenic mice. Methionine intake affects also lifespan, and thus, GH mutant mice and respective wild-type littermates were fed 0.16%, 0.43%, or 1.3% methionine to evaluate the interaction between hormone status and methionine. All wild-type and GH transgenic mice lived longer when fed 0.16% methionine but not when fed higher levels. In contrast, animals without growth hormone signaling due to hormone deficiency or resistance did not respond to altered levels of methionine in terms of lifespan, body weight, or food consumption. Taken together, our results suggest that the presence of growth hormone is necessary to sense dietary methionine changes, thus strongly linking growth and lifespan to amino acid availability.

A meta-analysis on the diagnostic performance of (18)F-FDG and (11)C-methionine PET for differentiating brain tumors.

SUMMARY: (18)F-FDG-PET has been widely used in patients with brain tumors. However, the reported sensitivity and specificity of (18)F-FDG-PET for brain tumor differentiation varied greatly. We performed this meta-analysis to systematically assess the diagnostic performance of (18)F-FDG-PET in differentiating brain tumors. The diagnostic performance of (11)C-methionine PET was assessed for comparison. Relevant studies were searched in PubMed/MEDLINE, Scopus, and China National Knowledge Infrastructure (until February 2013). The methodologic quality of eligible studies was evaluated, and a meta-analysis was performed to obtain the combined diagnostic performance of (18)F-FDG and (11)C-methionine PET with a bivariate model. Thirty eligible studies, including 5 studies with both (18)F-FDG and (11)C-methionine PET data were enrolled. Pooled sensitivity, pooled specificity, and area under the receiver operating characteristic curve of (18)F-FDG-PET (n = 24) for differentiating brain tumors were 0.71 (95% CI, 0.63-0.78), 0.77 (95% CI, 0.67-0.85), and 0.80. Heterogeneity was found among (18)F-FDG studies. Subsequent subgroup analysis revealed that the disease status was a statistically significant source of the heterogeneity and that the sensitivity in the patients with recurrent brain tumor was markedly higher than those with suspected primary brain tumors. Pooled sensitivity, pooled specificity, and area under the receiver operating characteristic of (11)C-methionine PET (n = 11) were 0.91 (95% CI, 0.85-0.94), 0.86 (95% CI, 0.78-0.92), and 0.94. No significant statistical heterogeneity was found among (11)C-methionine studies. This meta-analysis suggested that (18)F-FDG-PET has limited diagnostic performance in brain tumor differentiation, though its performance may vary according to the status of brain tumor, whereas (11)C-methionine PET has excellent diagnostic accuracy in brain tumor differentiation.

14C-Methionine uptake as a potential marker of inflammatory processes after myocardial ischemia and reperfusion.

UNLABELLED: A relationship between l-[methyl-(11)C]methionine ((11)C-methionine) uptake and angiogenesis has been suggested in gliomas. However, methionine uptake in myocardial ischemia and reperfusion has received little attention. We investigated the serial changes and mechanisms of (14)C-methionine uptake in a rat model of myocardial ischemia and reperfusion. METHODS: The left coronary artery was occluded for 30 min, followed by reperfusion for 1-28 d. At the time of the study, (14)C-methionine (0.74 MBq) and (201)Tl (14.8 MBq) were injected intravenously at 20 and 10 min before sacrifice, respectively. One minute before sacrifice, the left coronary artery was reoccluded, and (99m)Tc-hexakis-2-methoxyisobutylisonitrile (150-180 MBq) was injected to verify the area at risk. Histologic sections of the heart were immunohistochemically analyzed using anti-CD68, anti-smooth-muscle α-actin (SMA), and antitroponin I and compared with the autoradiography findings. RESULTS: Both (14)C-methionine (uptake ratio, 0.71 ± 0.13) and (201)Tl uptake were reduced in the area at risk at 1 d after reperfusion. However, 3 d after reperfusion, an increased (14)C-methionine uptake (1.79 ± 0.23) was observed corresponding to the area of still-reduced (201)Tl uptake, and the (14)C-methionine uptake gradually declined until 28 d. The increased (14)C-methionine uptake area at 3 and 7 d corresponded well to the macrophage infiltrations demonstrated by positive CD68 staining. Anti-SMA staining appeared at 7 d, after which CD68 staining was gradually replaced by the SMA staining, suggesting that methionine uptake in the early phase after ischemia and reperfusion might reflect inflammatory activity. CONCLUSION: (14)C-methionine accumulated in the infarcted area, and its uptake corresponded closely to macrophage infiltration at 3-7 d after reperfusion. Methionine imaging may be useful for inflammatory imaging early after myocardial infarction.

Radical scavenging and anti-inflammatory properties of STW 5 (Iberogast) and its components.

A combination of ethanolic extracts from nine medicinal plants is successfully used in STW 5 (Iberogast((R))) for treatment of gastrointestinal disorders. To elucidate possible modes of action, the focus of this study is on antioxidant properties of the phytomedicine STW 5. In fact, functional gastrointestinal diseases, such as non-ulcer dyspepsia (NUD) and irritable bowel syndrome, are often initiated by or correlated to inflammatory processes, where oxidants such as reactive oxygen species (ROS) play a crucial role. Prominent in vivo sources of ROS generation are represented by the enzymes xanthine oxidase (XOD) or myeloperoxidase (MPO). Applying these enzymes in models in vitro, we show that STW 5 and its components possess strong antioxidant activities. Depending on the model investigated, even pro-oxidant activities of single components of STW 5 could be observed. Interestingly, these effects were absent in STW 5, indicating cooperation between the components. Moreover, if one of the component extracts of STW 5 is omitted, the antioxidant activity is reduced. Thus we conclude that all the single extracts combined in STW 5 are of importance for the therapeutic effect, working in concert. The component of STW 5 performing best in vitro differed with the model investigated, respectively, with ROS and ROS generators. In the XOD system, the extracts of lemon balm leaf and peppermint leaf showed the best antioxidant result, whereas concerning MPO driven chlorination reactions, bitter candy tuft extract was the most efficient antioxidant. Best protection against peroxynitrite induced oxidation of methionine like sulfur-compounds exhibited the STW 5 components lemon balm leaf, Matricaria flower and peppermint leaf.

Folic acid treatment increases homocysteine remethylation and methionine transmethylation in healthy subjects.

Folic acid treatment decreases plasma total homocysteine concentrations in healthy subjects, but the effects on homocysteine metabolism are unknown. In the present study, we investigated the effect of 3 weeks of oral treatment with 5 mg of folic acid on one-carbon flux rates in 12 healthy subjects, using in vivo stable isotope methods. In addition, we determined the effect of folic acid on blood concentrations of amino acids which may have regulatory roles in homocysteine metabolism, i.e. homocysteine, AdoMet (S-adenosylmethionine), AdoHcy (S-adenosylhomocysteine), serine and glycine. Primed, continuous infusions with [2H3-methyl-1-13C]methionine were used to determine flux rates of methionine transmethylation, homocysteine remethylation and homocysteine trans-sulphuration. Metabolic homocysteine clearance was defined as the ratio of trans-sulphuration and plasma homocysteine level. Folic acid treatment increased the homocysteine remethylation rate by 59% [95% CI (confidence interval), 13-97%; P = 0.02] and methionine transmethylation rate by 20% (95% CI, 3-41%; P=0.03). Plasma total homocysteine concentration (-18%; 95% CI, -28 to -9%; P<0.01) and the serine/glycine ratio (-20%; 95% CI, -63 to -6%; P<0.01) decreased significantly, and the AdoMet/AdoHcy ratio (11%; 95% CI, 1-20%; P = 0.02) increased significantly. Changes in one-carbon flux rates did not correlate significantly with changes in plasma concentration of these amino acids. In conclusion, folic acid treatment lowered plasma homocysteine concentration and increased whole-body remethylation and transmethylation flux in healthy subjects.

Effect of lamivudine therapy on the serum covalently closed-circular (ccc) DNA of chronic hepatitis B infection.

OBJECTIVE: To determine the effect of 1-yr lamivudine treatment on serum covalently closed-circular DNA (cccDNA) level. PATIENTS AND METHOD: Serum total HBV DNA and cccDNA levels at baseline, week 24, and week 52 were measured in 82 lamivudine-treated patients, 17 of whom received 1-yr placebo and acted as controls. RESULTS: There was a significant reduction in the cccDNA levels from baseline (median 3.0 x 10(6) copies/ml) to week 24 (33,476 copies/ml) and week 52 (48,694 copies/ml) (p < 0.001 for both). The median reduction in cccDNA level at week 24 and 52 were 2.21 and 2.12 logs, respectively, which were significantly greater than those of controls (0.31 log, p < 0.001; 0.2 log, p < 0.001, respectively). Fifteen patients (18.3%) developed YMDD mutations by week 52. Compared to patients without YMDD mutations, patients with YMDD mutations had significantly less median reduction of total HBV DNA level (4.44 vs 3.65 logs, respectively, p= 0.02) and cccDNA level (2.27 vs 1.65 logs, respectively, p= 0.016) at week 24 and significantly less median reduction of cccDNA at week 52 (2.35 vs 0.8 logs respectively, p < 0.001). CONCLUSIONS: One-year lamivudine treatment decreased serum cccDNA level by 2 logs. The chance of YMDD mutations at week 52 was related to the magnitude of viral suppression at week 24.