Infant Rhesus Macaque Brain α-Tocopherol Stereoisomer Profile Is Differentially Impacted by the Source of α-Tocopherol in Infant Formula.

BACKGROUND: α-Tocopherol (αT) in its natural form [2'R, 4'R, 8'R αT (RRR-αT)] is more bioactive than synthetic α-tocopherol (all rac-αT). All rac-αT is widely used in infant formulas, but its accretion in formula-fed infant brain is unknown. OBJECTIVE: We sought to compare αT and stereoisomer status in infant rhesus macaques (Macaca mulatta) fed infant formula (RRR-αT or all rac-αT) with a reference group fed a mixed diet of breast milk and maternal diet. METHODS: From 1 d after birth until 6 mo of age, infants (n = 23) were either nursery reared and exclusively fed 1 of 2 formulas by staff personnel or were community housed with their mothers and consumed a mixed reference diet of breast milk (69 mL/d at 6 mo) transitioning to monkey diet at ∼2 mo (MF; n = 8). Formulas contained either 21 µmol RRR-αT/L (NAT-F; n = 8) or 30 µmol all rac-αT/L (SYN-F; n = 7). Total αT and αT stereoisomers were analyzed in breast milk at 2, 4, and 6 mo and in monkey plasma and liver and 6 brain regions at 6 mo of age. α-Tocopherol transfer protein (α-TTP), lipoprotein αT, and urinary α-carboxyethyl-hydroxychroman (α-CEHC) were measured. One-way ANOVA with Tukey's post-hoc test was used for analysis. RESULTS: At study termination, plasma, liver, lipoprotein, and brain total αT did not differ between groups. However, the NAT-F-fed group had higher RRR-αT than the SYN-F-fed group (P < 0.01) and the MF group (P < 0.0001) in plasma (1.7- and 2.7-fold) and brain (1.5- and 2.5-fold). Synthetic αT 2R stereoisomers (SYNTH-2R) were generally 3- and 7-fold lower in brain regions of the NAT-F group compared with those of the SYN-F and MF groups (P < 0.05). SYNTH-2R stereoisomers were 2-fold higher in MF than SYN-F (P < 0.0001). The plasma percentage of SYNTH-2R was negatively correlated with the brain percentage of RRR-αT (r = -0.99, P < 0.0001). Brain αT profiles were not explained by α-TTP mRNA or protein expression. Urine α-CEHC was 3 times higher in the NAT-F than in the MF group (P < 0.01). CONCLUSIONS: Consumption of infant formulas with natural (NAT-F) compared with synthetic (SYN-F) αT differentially impacted brain αT stereoisomer profiles in infant rhesus macaques. Future studies should assess the functional implications of αT stereoisomer profiles on brain health.

Simultaneous determination of 4-hydroxyphenyl lactic acid, 4-hydroxyphenyl acetic acid, and 3,4-hydroxyphenyl propionic acid in human urine by ultra-high performance liquid chromatography with fluorescence detection.

A simple and reliable method was established for simultaneous determination of 4-hydroxyphenyl acetic acid, 4-hydroxyphenyl lactic acid, and 3,4-hydroxyphenyl propionic acid in human urine by high-performance liquid chromatography with fluorescence detection. Solid-phase extraction was used to eliminate the interferences in urine. The separation of three analytes was achieved using a C18 column and a mobile phase formed by a 95:5 v/v mixture of 50 mmol/L ammonium acetate buffer at pH 6.8 that contained 5 mmol/L tetrabutyl ammonium bromide and acetonitrile. Under the optimized conditions, the detection limits of 4-hydroxyphenyl acetic acid, 4-hydroxyphenyl lactic acid, and 3,4-hydroxyphenyl propionic acid were 4.8 × 10-3 , 8.80 × 10-3 , and 9.00 × 10-3 mg/L, respectively, and the recoveries were in the range of 85.0-120.0% with relative standard deviations of 1.5-3.1%. This method was used to analyze urine samples from breast cancer patients, healthy people and post-surgery breast cancer patients. Significant differences in urinary levels of 4-hydroxyphenyl acetic acid and 4-hydroxyphenyl lactic acid could be found between the breast cancer patients group and other two groups. No effect of age and sex was observed on the urinary levels of 4-hydroxyphenyl acetic acid and 4-hydroxyphenyl lactic acid. This method might be helpful for cancer biomarkers discovery in urine.

Flavanol-Enriched Cocoa Powder Alters the Intestinal Microbiota, Tissue and Fluid Metabolite Profiles, and Intestinal Gene Expression in Pigs.

BACKGROUND: Consumption of cocoa-derived polyphenols has been associated with several health benefits; however, their effects on the intestinal microbiome and related features of host intestinal health are not adequately understood. OBJECTIVE: The objective of this study was to determine the effects of eating flavanol-enriched cocoa powder on the composition of the gut microbiota, tissue metabolite profiles, and intestinal immune status. METHODS: Male pigs (5 mo old, 28 kg mean body weight) were supplemented with 0, 2.5, 10, or 20 g flavanol-enriched cocoa powder/d for 27 d. Metabolites in serum, urine, the proximal colon contents, liver, and adipose tissue; bacterial abundance in the intestinal contents and feces; and intestinal tissue gene expression of inflammatory markers and Toll-like receptors (TLRs) were then determined. RESULTS: O-methyl-epicatechin-glucuronide conjugates dose-dependently increased (P< 0.01) in the urine (35- to 204-fold), serum (6- to 186-fold), and adipose tissue (34- to 1144-fold) of pigs fed cocoa powder. The concentration of 3-hydroxyphenylpropionic acid isomers in urine decreased as the dose of cocoa powder fed to pigs increased (75-85%,P< 0.05). Compared with the unsupplemented pigs, the abundance ofLactobacillusspecies was greater in the feces (7-fold,P= 0.005) and that ofBifidobacteriumspecies was greater in the proximal colon contents (9-fold,P= 0.01) in pigs fed only 20 or 10 g cocoa powder/d, respectively. Moreover, consumption of cocoa powder reducedTLR9gene expression in ileal Peyer's patches (67-80%,P< 0.05) and mesenteric lymph nodes (43-71%,P< 0.05) of pigs fed 2.5-20 g cocoa powder/d compared with pigs not supplemented with cocoa powder. CONCLUSION: This study demonstrates that consumption of cocoa powder by pigs can contribute to gut health by enhancing the abundance ofLactobacillusandBifidobacteriumspecies and modulating markers of localized intestinal immunity.

Divergent Urinary Metabolic Phenotypes between Major Depressive Disorder and Bipolar Disorder Identified by a Combined GC-MS and NMR Spectroscopic Metabonomic Approach.

Bipolar disorder (BD) is a complex debilitating mental disorder that is often misdiagnosed as major depressive disorder (MDD). Therefore, a large percentage of BD subjects are incorrectly treated with antidepressants in clinical practice. To address this challenge, objective laboratory-based tests are needed to discriminate BD from MDD patients. Here, a combined gas chromatography-mass spectrometry (GC-MS)-based and nuclear magnetic resonance (NMR) spectroscopic-based metabonomic approach was performed to profile urine samples from 76 MDD and 43 BD subjects (training set) to identify the differential metabolites. Samples from 126 healthy controls were included as metabolic controls. A candidate biomarker panel was identified by further analyzing these differential metabolites. A testing set of, 50 MDD and 28 BD subjects was then used to independently validate the diagnostic efficacy of the identified panel using an area under the receiver operating characteristic curve (AUC). A total of 20 differential metabolites responsible for the discrimination between MDD and BD subjects were identified. A panel consisting of six candidate urinary metabolite biomarkers (propionate, formate, (R*,S*)2,3-dihydroxybutanoic acid, 2,4-dihydroxypyrimidine, phenylalanine, and ß-alanine) was identified. This panel could distinguish BD from MDD subjects with an AUC of 0.913 and 0.896 in the training and testing sets, respectively. These results reveal divergent urinary metabolic phenotypes between MDD and BD. The identified urinary biomarkers can aid in the future development of an objective laboratory-based diagnostic test for distinguishing BD from MDD patients.

Alkylresorcinol metabolites in urine correlate with the intake of whole grains and cereal fibre in free-living Swedish adults.

Alkylresorcinols (AR) have been established as short/medium-term biomarkers for whole grain (WG) wheat and rye intake; and AR metabolites, 3,5-dihydroxybenzoic acid and 3-(3,5-dihydroxyphenyl)-propanoic acid, have been suggested as complementary biomarkers to AR. The present study examined the medium-term reproducibility and relative validity of urinary AR metabolites as biomarkers for WG and cereal fibre intake. A total of sixty-six free-living Swedes completed 3 d weighed food records and provided single 24 h urine collections and morning urine spot samples on two occasions, 2-3 months apart. The medium-term reproducibility of urinary AR metabolites was moderate when assessed in 24 h collections and lower in creatinine (CR)-adjusted morning urine. Mean AR metabolite 24 h excretions correlated well with total WG (r(s) 0·31-0·52, P < 0·05) and cereal fibre (r(s) 0·46-0·58, P < 0·001) intake on both occasions. As expected, correlations with WG (r(s) 0·28-0·38, P < 0·05) and cereal fibre (r(s) 0·35-0·42, P < 0·01) were weaker for mean CR-adjusted AR metabolite concentrations in spot samples of morning urine, although the adjusted concentrations correlated well with 24 h urinary excretion (r(s) 0·69-0·73, P < 0·001). Adjustment for intra-individual variations substantially improved the correlations between intake and excretion. These findings suggest that urinary AR metabolites can successfully reflect the medium-term intake of WG and cereal fibre when adjusted for intra-individual variation in this population, where rye was the major contributor to high WG intake. The performance of urinary AR metabolites as medium-term biomarkers appears to be comparable to that of fasting plasma AR concentration in this population.

Oxidative stress parameters in urine from patients with disorders of propionate metabolism: a beneficial effect of L:-carnitine supplementation.

Propionic (PA) and methylmalonic (MMA) acidurias are inherited disorders caused by deficiency of propionyl-CoA carboxylase and methylmalonyl-CoA mutase, respectively. Affected patients present acute metabolic crises in the neonatal period and long-term neurological deficits. Treatments of these diseases include a protein restricted diet and L: -carnitine supplementation. L: -Carnitine is widely used in the therapy of these diseases to prevent secondary L: -carnitine deficiency and promote detoxification, and several recent in vitro and in vivo studies have reported antioxidant and antiperoxidative effects of this compound. In this study, we evaluated the oxidative stress parameters, isoprostane and di-tyrosine levels, and the antioxidant capacity, in urine from patients with PA and MMA at the diagnosis, and during treatment with L: -carnitine and protein-restricted diet. We verified a significant increase of isoprostanes and di-tyrosine, as well as a significant reduction of the antioxidant capacity in urine from these patients at diagnosis, as compared to controls. Furthermore, treated patients presented a marked reduction of isoprostanes and di-tyrosine levels in relation to untreated patients. In addition, patients with higher levels of protein and lipid oxidative damage, determined by di-tyrosine and isoprostanes levels, also presented lower urinary concentrations of total and free L: -carnitine. In conclusion, the present results indicate that treatment with low protein diet and L: -carnitine significantly reduces urinary biomarkers of protein and lipid oxidative damage in patients with disorders of propionate metabolism and that L: -carnitine supplementation may be specially involved in this protection.

Pharmacokinetics of alkylresorcinol metabolites in human urine.

Wholegrain cereals are reported to promote beneficial health effects. Wholegrain wheat and rye are almost exclusive sources of alkylresorcinols, and intact alkylresorcinols together with their plasma and urinary metabolites, 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA), have been proposed as biomarkers of the intake of these foods in humans. The pharmacokinetics of alkylresorcinols and their metabolites in plasma have been determined but not that of the urinary metabolites. We aimed to characterise the urinary pharmacokinetics of alkylresorcinol metabolites in humans to evaluate their potential as biomarkers of wholegrain wheat and rye. A group of fifteen volunteers followed a low-alkylresorcinol diet for 2 d before ingesting a single dose of rye bread, containing 100 mg alkylresorcinols. Urine was collected between baseline (0 h) and 25 h after administration. Thereafter alkylresorcinol metabolites were quantified by HPLC with coulometric electrode array detection. Maximum excretion rates were observed at 5-6 h for both metabolites, DHPPA being predominant over DHBA and also possessing a greater area under the curve0-25 h. Total urinary recovery between 0 and 25 h yielded 43 % of ingested alkylresorcinols, and at 25 h significant amounts of metabolites were still retained in the body, suggesting that even a spot urine sample may be sufficient to indicate whether or not wholegrain wheat or rye is a daily dietary component. These results support the use of urinary DHPPA and DHBA as biomarkers of wholegrain wheat and rye and enable new potential for studying the association between wholegrain intake and diseases, even in the absence of dietary data.

Effects of clofibric acid on the biliary excretion of benoxaprofen glucuronide and taurine conjugate in rats.

Benoxaprofen (BOP) is a 2-methyl propionic acid derivative with anti-inflammatory activity. BOP has an asymmetric carbon, and receives chiral inversion from R to S in vivo. BOP is metabolized to glucuronide (BOP-G) and taurine conjugate (BOP-T). The configuration of BOP-G is mainly S, and that of BOP-T is R. Chiral inversion of R to S of the propionic acid moiety and amino acid conjugation of carboxyl compounds proceed via an acyl CoA intermediate. It is known that fibrates, used in hyperlipidemia, induce acyl CoA synthetase and increase CoA concentration. We administered racemic BOP (10 mg/kg body weight) to rats (CFA+) pre-administered clofibric acid (CFA, 280 mg/kg/day), and studied BOP, BOP-G, and BOP-T enantiomer concentrations in plasma and bile up to 12 h after administration. The findings were compared with those in rats (CFA-) that had not received CFA. Furthermore, we studied the amounts of BOP-G enantiomer produced by glucuronidation in vitro using microsomes pretreated with CFA. The amounts of (S)-BOP-G in CFA+ rats were 2.7-fold larger than that in CFA- rats. Although (R)-BOP-T was excreted in CFA- rats, BOP-T could not be detected in CFA+ rats. Plasma clearance values of racemic BOP and (S)-BOP in CFA+ rats were 5-fold and 6-fold larger than those in CFA- rats, respectively. (S)-BOP-G formation activities were higher than (R)-BOP-G formation activities in both CFA+and CFA- microsomes. These findings suggest that CFA increases biliary excretion of (S)-BOP-G and facilitates plasma elimination of BOP, and further suggests that CFA predominantly induces chiral inversion to S rather than metabolic reaction to (R)-BOP-T, resulting in an increase of (S)-BOP-G.

Urinary oxidized, but not enzymatic vitamin E metabolites are inversely associated with measures of glucose homeostasis in middle-aged healthy individuals.

BACKGROUND & AIMS: Damage induced by lipid peroxidation has been associated with impaired glucose homeostasis. Vitamin E (α-tocopherol, α-TOH) competitively reacts with lipid peroxyl radicals to mitigate oxidative damage, and forms oxidized vitamin E metabolites. Accordingly, we aimed to investigate the associations between α-TOH metabolites (oxidized and enzymatic) in both circulation and urine and measures of glucose homeostasis in the general middle-aged population. METHODS: This cross-sectional study was embedded in the population-based Netherlands Epidemiology of Obesity (NEO) Study. α-TOH metabolites in blood (α-TOH and α-CEHC-SO3) and urine [sulfate (SO3) and glucuronide (GLU) of both α-TLHQ (oxidized) and α-CEHC (enzymatic)] were quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS-MS). Measures of glucose homeostasis (HOMA-B, HOMA-IR, Insulinogenic index and Matsuda index) were obtained from fasting and postprandial blood samples. Multivariable linear regression analyses were performed to assess the associations of α-TOH metabolites and measures of glucose homeostasis. RESULTS: We included 498 participants (45% men) with mean (SD) age of 55.8 (6.1) years who did not use glucose-lowering medication. While blood α-TOH was not associated with measures of glucose homeostasis, urinary oxidized metabolites (α-TLHQ-SO3/GLU) were associated with HOMA-IR and Matsuda index. For example, a one-SD higher α-TLHQ-SO3 was associated with 0.92 (95% CI: 0.87, 0.97) fold lower HOMA-IR and 1.06 (1.01, 1.11) fold higher Matsuda index, respectively. Similar results were obtained for the urinary α-TLHQ to α-CEHC ratio as a measure of oxidized-over-enzymatic conversion of α-TOH. CONCLUSION: Higher urinary levels of oxidized α-TOH metabolites as well as higher oxidized-to-enzymatic α-TOH metabolite ratio, but not circulating α-TOH or enzymatic metabolites, were associated with lower insulin resistance. Rather than circulating α-TOH, estimates of the conversion of α-TOH might be informative in relation to health and disease.

Plasma alkylresorcinol metabolites as potential biomarkers of whole-grain wheat and rye cereal fibre intakes in women.

It has been demonstrated that intact plasma alkylresorcinols (AR) and urinary AR metabolites could be used as biomarkers of whole-grain intake. Thereafter, we developed the method for the plasma AR metabolites, which is more convenient and requires less sample pretreatment than the analysis of intact plasma AR. The aim of the present study is to evaluate whether AR metabolites measured in plasma, in the same population, could also be considered as useful biomarkers of cereal fibre. Fifty-six women were recruited in a cross-sectional and observational study. Dietary intake (5-d record) and plasma AR metabolites (3,5-dihydroxybenzoic acid, DHBA; 3-(3,5-dihydroxyphenyl)-1-propanoic acid, DHPPA) were measured. The relationship between plasma AR metabolites and cereal fibre intake was examined using partial correlation and stepwise regression. Cereal fibre intake correlated significantly with plasma DHBA (r 0.411; P = 0.002) and DHPPA (r 0.463; P = 0.000) even after adjustment for BMI and age. Thus, plasma AR metabolites correlate with cereal fibre intake as noted with plasma intact AR and urinary AR metabolites. We observed that plasma DHPPA was the independent predictor of cereal fibre intake, explaining 18 % of the variance (adjusted r(2) 0.176; P = 0.002). In epidemiological screening, it might be easier to obtain and to collect plasma than urine samples. In addition, the plasma AR metabolites half-life seems longer than those of intact plasma AR, and their measurements are more convenient, and faster. Thus, sum of plasma AR metabolites and more specifically plasma DHPPA seems to be good and specific biomarkers of cereal fibre intake.

Metabolomics of the diabetic nephropathy: behind the fingerprint of development and progression indicators. / Metabolómica de la nefropatía diabética: tras la huella de indicadores de desarrollo y progresión.

Current diagnostic methods are not very sensitive to detect the initial stages diabetic nephropathy of type 2. In this work, a review of metabolomic approximation studies for the identification of biomarkers of this disease with potential to differentiate between early stages, evaluate and direct treatment and help slow kidney damage. Using public (Pubmed and Google Scholar) and private (Scopus and Web of Knowledge) databases, a systematic search of the information published related to metabolomics of diabetic nephropathy in different biospecimens (urine, serum, plasma and blood) was made. Later, the MetaboAnalyst 4.0 software was used to identify the metabolic pathways associated with these metabolites. Groups of potential metabolites were identified for monitoring diabetic nephropathy with the available literature data. In the urine, oxide-3-hydroxyisovalerate, TMAO, aconite and citrate and hydroxypropionate derivatives are highlighted; meanwhile, in the serum: citrate, creatinine, arginine and its derivatives; and in the plasma: amino acids such as histidine, methionine and arginine has a potential contribution. Using MetaboAnalyst 4.0 the metabolic pathways related to these metabolites were related. The search for biomarkers to measure the progression of diabetic nephropathy, together with analytical strategies for their detection and quantification, are the starting point for designing new methods of clinical chemistry analysis. The association between the metabolic pathway dysfunction could be useful for the overall assessment of the treatment and clinical follow-up of this disease.

Daily consumption of an aqueous green tea extract supplement does not impair liver function or alter cardiovascular disease risk biomarkers in healthy men.

Regular consumption of green tea polyphenols (GTP) is thought to reduce the risk of cardiovascular disease (CVD) but has also been associated with liver toxicity. The present trial aimed to assess the safety and potential CVD health beneficial effects of daily GTP consumption. We conducted a placebo-controlled parallel study to evaluate the chronic effects of GTP on liver function and CVD risk biomarkers in healthy men. Volunteers (treatment: n = 17, BMI 26.7 +/- 3.3 kg/m(2), age 41 +/- 9 y; placebo, n = 16, BMI 25.4 +/- 3.3 kg/m(2), age 40 +/- 10 y) consumed for 3 wk 6 capsules per day (2 before each principal meal) containing green tea extracts (equivalent to 714 mg/d GTP) or placebo. At the beginning and end of the intervention period, we collected blood samples from fasting subjects and measured vascular tone using Laser Doppler Iontophoresis. Biomarkers of liver function and CVD risk (including blood pressure, plasma lipids, and asymmetric dimethylarginine) were unaffected by GTP consumption. After treatment, the ratio of total:HDL cholesterol was significantly reduced in participants taking GTP capsules compared with baseline. Endothelial-dependent and -independent vascular reactivity did not significantly differ between treatments. In conclusion, the present data suggests that the daily consumption of high doses of GTP by healthy men for 3 wk is safe but without effects on CVD risk biomarkers other than the total:HDL cholesterol ratio.

Occurrence of S-methyl thioesters in urines of humans after they have eaten asparagus.

Gas chromatography-mass spectrometry was used to determine the odor-causing agent (or agents) present in the urines of humans after they have eaten asparagus. S-Methyl thioacrylate and S-methyl 3-(methylthio)thiopropionate were identified from methylene chloride extracts of such urines and appear to be the odor-causing compounds. Methanethiol, the previously reported odor-causing agent, was not detected in these methylene chloride extracts.

Vitamin E supplementation and hepatic drug metabolism in humans.

Meta-analyses studies suggest that high-dose vitamin E may be associated with increased mortality in some populations. Vitamin E may increase the production of CYP3A4 in the liver, and this could lead to an increase in drug metabolism, potentially lowering the efficacy of therapeutic drugs. We hypothesized that upregulation of CYP3A4 by alpha-tocopherol (alpha-TOH) would decrease the plasma concentration of the CYP3A4 substrate midazolam. Baseline metabolism of midazolam (1 mg intravenously) was determined in 12 healthy subjects before randomization into 2 groups of 6 to receive either RRR-alpha-TOH (750 IU/d) or placebo for 3 weeks. At completion, subjects were given an additional 1 mg intravenous bolus of midazolam. Plasma midazolam, 1-hydroxy-midazolam, and urinary alpha-TOH metabolite excretion were measured using gas chromatography mass spectrometry. Serum alpha-TOH was measured using high performance liquid chromatography with electrochemical detection. Serum alpha-TOH increased by 100% (P = 0.002) and urinary alpha-TOH metabolite excretion increased 20-fold in the treatment group versus placebo (P = 0.001). There was no effect on the area under time curve of midazolam in subjects taking alpha-TOH compared with placebo. These findings do not support the hypothesis that alpha-TOH supplementation interferes with hepatic CYP3A4-mediated drug metabolism.

Urinary 3-(3,5-dihydroxyphenyl)-1-propanoic acid, an alkylresorcinol metabolite, is a potential biomarker of whole-grain intake in a U.S. population.

5-n-Alkylresorcinols (AR) are a major group of phenolic compounds in whole-grain wheat, rye, and barley. As such, they may serve as potential biomarkers of whole-grain intake, because they are quantifiable intact in plasma and as metabolites in urine. We examined relationships between 12-h urinary excretion of AR metabolite 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA) and self-reported habitual intake of whole-grain foods measured by 3-d food record (3DFR) and FFQ. Urine samples from 100 men and women were analyzed for DHPPA using HPLC with coularray detection. DHPPA excretion ranged from 1.3 to 99.4 (mean +/- SE, 14.0 +/- 1.5) mumol/12 h. Whole-grain food intake, as determined by 3DFR and FFQ and adjusted for BMI and energy and fiber intake, was significantly associated with 12-h urinary DHPPA excretion. Based on 3DFR, whole-grain wheat + rye consumers had a 44% higher DHPPA excretion than nonconsumers [ratio of excretion (95% CI) = 1.44 (1.04, 1.97); P = 0.029]. Using whole-grain intake estimated by FFQ, a serving increase in whole-grain wheat + rye intake increased DHPPA excretion by 94% [ratio of excretion (95% CI) = 1.94 (1.35, 2.78); P = 0.001] and a serving increase in whole grains as defined more broadly in epidemiologic studies of whole-grain intake and disease risk (whole-grain wheat, rye, oats, and corn) increased DHPPA by 67% [ratio of excretion (95% CI) = 1.67 (1.28, 2.17); P < 0.0001]. This study supports the potential utility of urinary DHPPA as a biomarker of whole-grain intake in a U.S. population.

Accumulation of methylguanidine and changes in guanidino compound levels in plasma, urine, and kidneys of furosemide-treated rats.

Antidiuresis and renal diseases alter the levels of guanidino compounds (GCs) in various tissues. Therefore, we hypothesized that diuresis could also disturb GC metabolism, storage, and elimination. In this study, rats were made diuretic to analyze GC levels in plasma, urine, and kidneys. Furosemide was chosen because of its wide use in various human pathologies. Rats were injected intraperitoneally 5 or 10 mg furosemide spread over a 24-hour cycle. Urine was collected over a period of 24 hours before and during furosemide treatment. Plasma was obtained from arterial blood. Renal zones were dissected. The GCs were determined by liquid chromatography. Five milligrams of furosemide provoked a significant increase in plasma and urine levels of GCs compared with those of the controls. The renal distribution and content of GCs were weakly modified by furosemide except for methylguanidine (MG). The level of MG was enhanced by 10 to 16 times in all renal zones. The MG level was 60% higher in renal zones of rats treated with 10 rather than 5 mg furosemide. The fractional excretion of MG was decreased by furosemide. Our data suggest that MG accumulation in kidney and plasma was caused by furosemide, which might induce MG synthesis, and that MG washout from tissue cells into urine by furosemide through the kidney may cause an increase in MG in the kidney.

A male case with CDKL5-associated encephalopathy manifesting transient methylmalonic acidemia.

Mutations in the X-linked gene CDKL5 cause early-onset epileptic encephalopathy and severe developmental delay. Because this disorder predominantly affects females, the full clinical spectrum of male patients remains elusive. We herein report a 16-year-old boy, who suffered from intractable seizures 20 days after birth. Serial electroencephalograms detected recurrent focal epileptiform discharges from age 4 months, which evolved to hypsarrhythmia later in infancy. Mass-spectrometric analyses revealed increase in urinary excretion of methylmalonic acid without perturbed concentrations of propionic acid, homocystein and methionine. Whole-exome sequencing identified a de novo, truncating mutation in CDKL5 (NM_003159.2:c.419dupA, p.Asn140Lysfs*8). Targeted sequencing excluded concomitant mutations in methylmalonic academia-associated genes. No methylmalonic acidemia has been reported in children with CDKL5 disorder. Extensive analyses on organic acid metabolism for males with CDKL5 mutations will gain more insight into their biochemical profiles in infancy.

[Determination of deltamethrin and its toxicity biomarkers in rabbit urine by high performance liquid chromatography-tandem mass spectrometry].

A method was developed for the determination of biomarkers related to toxicity of deltamethrin in rabbit urine by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The target analytes in this method are as follows:deltamethrin and its two metabolites (1R-cis)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropane carboxylic acid (dibromochrysanthemic acid) and 3-phenoxybenzoic acid (3-PBA), and five toxic biomarkers, viz. serotonin hydrochloride (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), 3-nitropropionic acid (3-NPA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and 6-methoxyguanine. Urine samples were cleaned by matrix solid-phase dispersion extraction (MSPD) with diatomite; and protein was precipitated with trichloroacetic acid; and then the sample solutions were purified with hydrophilic-lipophilic balance (HLB) solid-phase extraction cartridges. The biomarkers were analyzed with electrospray ionization (ESI) in a positive and negative switching scan mode, in which the positive scan mode was used for deltamethrin, 5-HT, 5-HIAA, 8-OHdG, and 6-methoxyguanine, and the negative scan mode was used for (1R-cis)-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropane, 3-PBA, and 3-NPA. The target compounds were quantified with the external standard using matrix calibration curves. The linear regression curves of the eight target compounds were linear with correlation coefficients no less than 0.9914. The LOD and LOQ of 5-HIAA were 20 µg/L and 50 µg/L, respectively, and the LODs and LOQs of the other analytes were 0.2-5.0 µg/L and 0.5-10 µg/L, respectively. The average recoveries of the analytes spiked in rabbit urine ranged from 74.2% to 98.7% at three levels, with relative standard deviations (RSDs) no more than 12%. The method was simple, fast, accurate, sensitive, and suitable for the detection for the exposure evaluation of deltamethrin.

Increased guanidino species in murine and human succinate semialdehyde dehydrogenase (SSADH) deficiency.

Mice with targeted deletion of the GABA-degradative enzyme succinate semialdehyde dehydrogenase (SSADH; Aldh5a1; OMIM 271,980) manifest globally elevated GABA and regionally decreased arginine in brain extracts. We examined the hypothesis that arginine-glycine amidinotransferase catalyzed the formation of guanidinobutyrate (GB) from increased GABA by quantifying guanidinoacetate (GA), guanidinopropionate (GP) and GB in brain extracts employing stable isotope dilution gas chromatographic-mass spectrometry. GA and GB were up to 4- and 22-fold elevated, respectively, in total and regional (cerebellum, hippocampus, cortex) brain extracts derived from SSADH(-/-) mice. Corresponding analyses of urine and cerebrospinal fluid derived from SSADH-deficient patients revealed significant (P<0.05) elevations of GA and GB in urine, as well as GB levels in CSF. These data suggest that GB may be an additional marker of SSADH deficiency, implicate additional pathways of pathophysiology, and identify the second instance of elevated GB in a human inborn error of metabolism.

Biotin-response organicaciduria. Multiple carboxylase defects and complementation studies with propionicacidemia in cultured fibroblasts.

Fibroblast cultures from two individuals with biotin-responsive organicacidemia were found to have a pleiotropic deficiency of propionyl-CoA carboxylase, beta-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase activities after growth in biotin limited culture medium, conditions which do not affect the carboxylase activities of normal cells. All three enzyme activities were restored to normal levels after transferring the mutant strains to biotin-rich medium. Both patients excreted abnormal levels of an array of metabolic intermediates, including beta-methylcrotonate, beta-hydroxyisovalerate, beta-hydroxypropionate, and lactate, which reflect metabolic blocks at all three carboxylase sites.14 mutants deficient in only propionyl-CoA carboxylase activity from patients with propionicacidemia and the two biotin-responsive strains were examined for complementation with seven previously mapped pcc mutants. No new pcc complementation groups were identified. Nine of the mutants were mapped to group pccA. The remaining 12 mutants mapped to pccBC or its B or C subgroups, confirming the complex nature of this group. The biotin-responsive mutants failed to complement each other but did complement mutants from all the pcc groups. Thus biotin-responsive organicacidemia is defined by a new complementation group, bio. The results obtained in this study suggest that the bio mutants have a defect of either biotin transport or a common holocarboxylase synthetase required for the biotin activation of all three mitochondrial carboxylases.