Characterization of chemical constituents and metabolites in rat plasma after oral administration of San Miao Wan by ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap mass spectrometry.

San Miao Wan (SMW), composed of Phellodendri Chinensis Cortex, Atractylodis Lanceae Rhizoma and Achyranthis Bidentatae Radix, is widely used for the treatment of gout, hyperuricemia and other diseases. In the present study, an overall identification strategy based on ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) method was established to characterize the multiple chemical constituents of SMW and its metabolites in rat plasma after oral administration of SMW. A total of 76 constituents including alkaloids, organic acids, lactones, terpenes, saponins, sterones and others types of components were identified in the extract of SMW. After the oral administration of SMW, 47 prototype constituents and 66 metabolites were identified in rat plasma samples. The related metabolic pathways mainly involved reduction, demethylation, hydroxylation, methylation and glucuronide conjunction. The proposed method could be a useful approach to identify the chemical constituents of SMW and its metabolic components. Our study provide a universal strategy for the analysis of the components and metabolites of the traditional Chinese medicine prescription (TCP) extracts and plasma after administration using UPLC-Q-Exactive Orbitrap/MS method. It will assist with clarifying the substance basis of effective components in SMW. It also provides a rapid method for overall analysis of chemical constituents and metabolites of SMW.

Pharmacokinetics of Single and Multiple Doses of Omega-3 Carboxylic Acids in Healthy Chinese Subjects: A Phase I, Open-Label Study.

In patients with coronary heart disease undergoing primary prevention, hypertriglyceridemia is a residual risk for cardiovascular events. Omega-3 carboxylic acid (OM3-CA), a mixture of the free fatty acid forms of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may be beneficial in reducing triglyceride levels. As part of the clinical development program of OM3-CA in China, this phase I study evaluated the pharmacokinetics, safety, and tolerability profile of OM3-CA in healthy subjects. The pharmacokinetic results of this study were also compared with those of available data for Western populations. Fourteen healthy Chinese subjects (aged 18-45 years) received once-daily oral OM3-CA 4 g for 14 consecutive days. Pharmacokinetic parameters were assessed from both baseline-uncorrected and baseline-corrected plasma concentrations vs time profile of EPA, DHA, and EPA plus DHA. Following single and multiple oral doses of OM3-CA, the absorption of EPA, DHA, and EPA plus DHA was steady with median tmax occurring at 5.5-6 hours after both single and multiple dosing. Close to steady-state concentrations in plasma were reached after 14 days of continuous once-daily dosing, and accumulation was confirmed for EPA, DHA, and EPA plus DHA. Of the 14 subjects treated with OM3-CA, 6 (42.9%) reported at least 1 adverse event (diarrhea) during the study, which was determined as mild and treatment emergent. No serious adverse events were reported. In summary, the pharmacokinetic profile of oral OM3-CA 4 g after single and multiple dosing in healthy Chinese subjects is consistent with that observed in other ethnic populations.

Metabonomic analysis of serum reveals antifatigue effects of Yi Guan Jian on fatigue mice using gas chromatography coupled to mass spectrometry.

Yi Guan Jian (YGJ), one of the most commonly used traditional Chinese medicines, has been reported to possess significant antifatigue effects. However, the mechanisms underlying its antifatigue effects remain largely unresolved. In this study, a metabonomics approach, involving gas chromatography coupled to mass spectrometry and a multivariate statistical technique, was developed to estimate the extent to which YGJ alleviated the exhausting swimming-induced fatigue of mice. High-dose treatment with YGJ significantly extended the swimming time of fatigued mice. Significant alterations of metabolites involving amino acids, organic acids and carbohydrates were observed in the serum of fatigued mice, which were reversed by YGJ treatment while biochemical indexes returned to normal. These metabolic changes suggest that the antifatigue effect of YGJ is associated with the impairement of amino acid, organic acids and carbohydrates. It also appears that YGJ can induce significant metabolic alterations independent of the exhausting swimming-induced metabolic changes. The significantly altered metabolites induced by YGJ intervention include l-2-amino-acetoacetate, taurine, fumaric acid, malic acid, oxoadipic acid and l-aspartate, all of which are associated with antifatigue properties. This suggests that YGJ exerts chemopreventive effects via antifatigue mechanisms.

Effects of Food on the Pharmacokinetics of Omega-3-Carboxylic Acids in Healthy Japanese Male Subjects: A Phase I, Randomized, Open-label, Three-period, Crossover Trial.

AIMS: Omega-3-carboxylic acids (OM3-CA) contain omega-3 free fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as carboxylic acids. Food intake is known to affect the bioavailability of ethyl ester fatty acid formulations. We conducted a phase I study to investigate the effects of the timing of OM3-CA administration relative to food intake on the pharmacokinetics of EPA and DHA. METHODS: In this randomized, open-label, three-period crossover study, Japanese healthy male subjects were administered 4×1 g OM3-CA capsules with continued fasting, before a meal, or after a meal. All subjects fasted for ≥10 h prior to drug/meal administration. The primary objective was to examine the effect of meal timing on the pharmacokinetics of EPA and DHA after OM3-CA administration. The secondary objectives were to examine the safety and tolerability of OM3-CA. RESULTS: A total of 42 Japanese subjects was enrolled in the study. The baseline-adjusted maximum concentration and area under the concentration-time curve from 0 to 72 h for EPA, DHA, and EPA +DHA were lower in the fasting and before meal conditions than in the after meal condition. The maximum total EPA, total DHA, and total EPA+DHA concentrations were reached later when administered in fasting conditions than in fed conditions, indicating slower absorption in fasting conditions. Diarrhea was reported by five, six, and no subjects in the fasting, before meal, and after meal conditions, respectively. CONCLUSIONS: The timing of OM3-CA administration relative to food intake influences the systemic bioavailability of EPA and DHA in healthy Japanese male subjects. TRIAL REGISTRATION: NCT02372344.

[Metabonomic analysis of the serum from rat model with abnormal balgam syndrome of Uyghur medicine].

OBJECTIVE: To investigate correlation between the change in metabolic components of serum and the abnormal balgam syndrome by using a rat model of abnormal balgam syndrome. METHODS: Male Wistar rats were randomly divided into a control group and a test group. According to Uyghur medicine theory, the test group of rats were given wet cold diet (seeds of spinach and parsley, 24 hours) in a cold (6 °C) and humid (85%-95%, 10 hours) environment for 40 days to establish the rat model of abnormal balgam syndrome. 1H MR based metabonomic analysis of serum was performed. Data was analyzed using Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) software. RESULTS: Compared with the control group, the serum components including glutamate, phenylalanine, tyrosine, citric acid, ß-hydrocxy butyrate, acetoacetate, pyruvic acid and creatine were decreased, while the glucose, lactic acid, low density lipoprotein and very low density lipoprotein were increased in the test group (P<0.05). CONCLUSION: The low energy production and consumption in the rat model of abnormal balgam syndrome suggests that the dysfunctional metabolisms of three major nutrients might be the molecular basis for the abnormal balgam syndrome.

[Serum metabolome by gas chromatography-mass spectrometry (GC-MS) in patients with ulcerative colitis and celiac disease].

Metabolomics is the emerging science of measurement and analysis of metabolome--the complete set of low molecular weight compounds in a cell, tissue, organ or whole organism. One of the aims of metabolomics is to research the response of an organism to a pathophysiological insult by measuring the concentrations of small molecule metabolites in biofluids and tissues and its dynamics. Intestinal microbiota is most probably involved in the development and maintenance of autoimmune inflammation in ulcerative colitis and celiac disease. Gas chromatography-mass spectrometry (GC - MS) of serum generates comprehensive metabolic profiles, reflecting integrated human (systemic) and gut microbial metabolism which may be altered in disease states. The aim of this study was to investigate GC - MS-based serum metabolomic profiles in UC and CD patients. Serum metabolic profiles were collected from 75 individuals: 20 patients with mild-moderate active UC, 35 CD patients, and 20 healthy controls (HC). We characterized 84 serum metabolites by use GC-MS. 18 metabolites at least have a combined (human + microbial) origin. In serum of UC patients, phenylacetic acid (PAA), 4-hydroxyphenylacetic acid (4-HPAA), 3-indolylacetic acid (IAA), succinic acid (SA) and fumaric acid (FA) were the metabolites most prominently increased, whereas 3-phenylpropionic acid (PPA) was significantly decreased. Serum of CD patients showed significant increases in IAA, 3-indolepropionic acid (IPA), SA and FA. Increased serum levels of succinic acid suggest its possible damaging effect on intestinal mucosa especially in ulcerative colitis. Orally administered butyrate + inulin as supplement to mesalazine in UC or gluten free diet in CD was effective in reducing disease activity with a marked improvement of serum metabolomic profiles (including SA reduction) and gut microbiota in both diseases. There were no any adverse events.

The effect of menaquinone-7 supplementation on circulating species of matrix Gla protein.

OBJECTIVE: To investigate whether menaquinone-7 (MK-7) supplementation increases carboxylation of MGP. DESIGN: A randomized, double-blind, placebo-controlled trial was performed. Sixty participants (40-65 y) were randomly allocated to supplementation of 180 µg/d, 360 µg/d of MK-7 or placebo during 12 weeks. At baseline, after 4 and 12 weeks, desphospho-uncarboxylated MGP (dp-ucMGP), desphospho-carboxylated MGP (dp-cMGP) and total uncarboxylated MGP (t-ucMGP) were measured by ELISA techniques. Furthermore, the ratio of uncarboxylated osteocalcin (ucOC) to carboxylated osteocalcin (cOC) was used as proxy of vitamin K status and various cardiovascular risk factors were measured. RESULTS: Dp-ucMGP decreased significantly and dose-dependently in the 180 µg and 360 µg MK-7 supplementation groups (P time*treatment < 0.001) after 12 weeks, by 31% and 46% respectively, while dp-ucMGP levels remained unchanged after placebo treatment. The osteocalcin ratio also decreased significantly after 12-week supplementation with 180 µg (60%) and 360 µg (74%) MK-7 (P time*treatment < 0.001), while levels remained unchanged after placebo treatment. These results indicate improved vitamin K status and good compliance to the study treatment. Changes over time of dp-cMGP (p = 0.42) and t-ucMGP (p = 0.23) levels did not differ between treatment arms. Other cardiovascular risk factors did not differ between treatments arms. CONCLUSIONS: Menaquinone supplementation dose-dependently decreases dp-ucMGP concentrations, but does not affect other MGP species. Dp-ucMGP may serve as a non-invasive marker of vitamin K status.

The effect of menaquinone-7 (vitamin K2) supplementation on osteocalcin carboxylation in healthy prepubertal children.

Vitamin K contributes to bone health, probably through its role as cofactor in the carboxylation of osteocalcin. Intervention studies in adults have demonstrated that markedly higher osteocalcin carboxylation is obtained by intakes of vitamin K well above the current recommended dietary intake. However, the relationship between increased vitamin K2 intake and enhanced osteocalcin carboxylation has never been shown in healthy children. The objective was to study the effect of 45 microg menaquinone-7 (MK-7; one of the vitamin K2 species) on the circulating levels of undercarboxylated osteocalcin (ucOC) and carboxylated osteocalcin (cOC) in healthy prepubertal children. We hypothesised that MK-7 supplementation will reduce the ucOC:cOC ratio (UCR), indicating an improved vitamin K status. The present study is a double-blind randomised placebo-controlled trial examining the effect of 8 weeks MK-7 supplementation on the carboxylation of osteocalcin in healthy children (n 55). Serum levels of ucOC, cOC and MK-7 were measured at baseline and after 8 weeks, together with bone markers and coagulation parameters. The UCR was used as an indicator of vitamin K status. In the MK-7-supplemented group (n 28), the circulating concentration of inactive ucOC reduced and the UCR improved whereas the concentration of MK-7 increased. Within the placebo group, ucOC, cOC, UCR and MK-7 did not significantly change over time. In both groups, bone markers and coagulation parameters remained constant over time. These findings demonstrate that in healthy, prepubertal children, modest supplementation with MK-7 increases circulating concentrations of MK-7 and increases osteocalcin carboxylation.

Response of serum carboxylated and undercarboxylated osteocalcin to alendronate monotherapy and combined therapy with vitamin K2 in postmenopausal women.

Alendronate decreases the risk of femoral neck fracture by suppressing bone turnover, and also decreases the serum total osteocalcin level. A low serum carboxylated osteocalcin level or high undercarboxylated osteocalcin level could be risk factors for femoral neck fracture. Vitamin K mediates the carboxylation of osteocalcin, but the effect of alendronate therapy with or without vitamin K(2) supplementation remains unknown. Forty-eight postmenopausal women were enrolled in a 1-year prospective randomized trial and assigned to alendronate monotherapy (5 mg/day) (group A, n = 26) or vitamin K(2) (45 mg/day) plus alendronate (5 mg/day) (group AK, n = 22). Bone mineral density was measured by dual-energy X-ray absorptiometry at 0 and 12 months; bone turnover parameters were measured at 0, 3, and 12 months. Four patients discontinued alendronate therapy, and we analyzed the remaining 44 patients (23 in group A and 21 in group AK) who completed 1 year of treatment. Alendronate decreased undercarboxylated osteocalcin; carboxylated osteocalcin was not affected. Addition of vitamin K(2) enhanced the decrease of undercarboxylated osteocalcin levels and led to a greater increase of femoral neck bone mineral density. Alendronate monotherapy does not decrease carboxylation of osteocalcin, and combination of vitamin K(2) and alendronate brings further benefits on both osteocalcin carboxylation and BMD of femoral neck in postmenopausal women with osteoporosis.

Diet- or warfarin-induced vitamin K insufficiency elevates circulating undercarboxylated osteocalcin without altering skeletal status in growing female rats.

To further characterize the skeletal role of vitamin K (K), markers of bone turnover, density, and strength were evaluated in rats with diet- or warfarin (W)-induced K insufficiency. One hundred two, 7-week-old, female rats were randomly assigned to low K (phylloquinone [K1], 20 microg/kg diet), control K (K1, 1300 microg/kg diet), low-dose W (W, 1.5 mg/kg control diet), or high-dose W plus K (W/K1, 10/100 mg/kg diet). Femur bone mineral content (BMC) and bone mineral density (BMD), plasma prothrombin time (PT) and prothrombin concentration (PC), and serum total alkaline phosphatase (ALP) and skeletal alkaline phosphatase (sALP) were measured at baseline and days 20, 40, 60, and 80. Serum total osteocalcin (OC) and undercarboxylated osteocalcin (ucOC) and femur length (FL) were measured at baseline and day 80. Left femur OC was measured and biomechanical testing of the right femur and third lumbar vertebral body was performed at day 80. Low dietary K elevated circulating ucOC (17% higher than control; p < 0.0001) at day 80. Furthermore, in both W groups, essentially all circulating OC was undercarboxylated and femur OC was lower than control (p < 0.0001). However, there was no change in femur percent ucOC, suggesting deposition of less newly synthesized OC. No between group differences were observed in PT, ALP, sALP, FL, BMC, BMD, or bone strength. In conclusion, skeletal K insufficiency can be induced by W or diet manipulation. This does not hinder peak bone mass attainment in female rats; however, W causes less newly synthesized OC to be deposited in bone.

The response to L-carnitine and glycine therapy in isovaleric acidaemia.

The profound metabolic disturbances which occur in isovaleric acidaemia are due to the intramitochondrial accumulation of isovaleryl coenzyme A (CoA) with a consequent reduction in the availability of free CoA. Secondary carnitine insufficiency is also a feature of this and other disorders of organic acid metabolism. A patient who presented at 2.5 years of age was diagnosed using capillary GC-MS as having isovaleric acidaemia. She showed the full spectrum of abnormal organic acids previously associated with the 'neonatal' form of the disease despite her late presentation, indicating that it is inappropriate to refer to acute early and late onset forms of isovaleric acidaemia. Instead, a spectrum of disease exists, determined by environmental factors, residual enzyme activities and modifying effects of different phenotypes in different individuals. She also showed evidence of carnitine insufficiency. An oral challenge with L-carnitine resulted in the excretion of large amounts of urinary acylcarnitines which were shown by use of fast atom bombardment mass spectrometry to be primarily isovalerylcarnitine. Regular glycine supplementation caused no significant increase in urinary isovalerylglycine and had to be stopped because of side-effects after 5 days. An oral L-carnitine challenge during glycine supplementation resulted in a marked increase in isovalerylglycine excretion, again associated with the excretion of large amounts of isovalerylcarnitine. Carnitine acts by removing (detoxifying) intramitochondrial isovaleryl groups and, in the presence of glycine, it promotes the formation of isovalerylglycine. We believe L-carnitine supplementation is of value in the treatment of isovaleric acidaemia and that, in the present case, L-carnitine together with a moderate dietary restriction has proved to be the optimum form of therapy.