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PubMed2000

Styrene-induced changes in amacrine retinal cells: an experimental study in the rat.

Dopamine (DA) is synthesized in amacrine cells and released upon membrane depolarization in a calcium-dependent way. Thus, it is recognized to function as a major neurotransmitter or modulator in vertebrate retina. Owing to DA modulating activity on cone-horizontal cells transmission, depletion or dysfunction of amacrine cells could interfere with chromatic processing, accounting for the acquired dyschromatopsia described among styrene-exposed workers. The present study has been designed to test the hypothesis that amacrine cells represent a vulnerable target of styrene in subchronically exposed rats. Ten female Sprague-Dawley rats were exposed to 300 ppm styrene 6 h/day, 5 days/week, for 12 weeks; ten rats exposed to fresh air served as a control group. Whole mounted retinas were used for the morphometry of tyrosine hydroxylase (TH) immunoreactive cells (IR). DA content and TH activity were measured by HPLC and electrochemical detection and glutathione (GSH) was measured by HPLC tandem mass spectrometry (LC-MS/MS). In treated rats, morphometric analysis showed a loss of TH-IR amacrine cells (6.2/mm2 vs. 8.7/mm2 recorded in controls, p = 0.002), without any peripheral-central variation in cell loss. DA content was also lower in exposed, as compared to control animals (208.64 vs. 267.98 microg/g w.w., p = 0.004). The activity of TH in the whole retina was similar in styrene-exposed and control rats when expressed as a function of the wet weight, whereas it was much higher in styrene-exposed rats (+64%) when expressed as a function of the number of TH-IR amacrine cells (p < 0.001). Finally, retinal GSH was reduced by 30% in exposed as compared to control rats (p = 0.01). In summary, retinal TH-IR cells were sensitive to styrene exposure, which seems to cause both structural and functional changes, represented by cell loss and DA depletion, respectively. These findings confirm the vulnerability of dopaminergic systems to styrene toxicity, providing some insights on the possible mechanism of loss in chromatic discrimination recorded among workers occupationally-exposed to styrene.

Animals↗

PubMed2001

Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency.

We identified a new peroxisomal disorder caused by a deficiency of the enzyme alpha-methylacyl-coenzyme A (CoA) racemase. Patients with this disorder show elevated plasma levels of pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid (DHCA and THCA), which are all substrates for the peroxisomal beta-oxidation system. alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (2S)-isomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. In this study we used liquid chromatography/tandem mass spectrometry (LC-MS/MS) to analyze the bile acid intermediates that accumulate in plasma from patients with a deficiency of alpha-methylacyl-CoA racemase and, for comparison, in plasma from patients with Zellweger syndrome and patients with cholestatic liver disease.We found that racemase-deficient patients accumulate exclusively the (R)-isomer of free and taurine-conjugated DHCA and THCA, whereas in plasma of patients with Zellweger syndrome and patients with cholestatic liver disease both isomers were present. On the basis of these results we describe an easy and reliable method for the diagnosis of alpha-methylacyl-CoA racemase-deficient patients by plasma analysis. Our results also show that alpha-methylacyl-CoA racemase plays a unique role in bile acid formation. - Ferdinandusse, S., H. Overmars, S. Denis, H. R. Waterham, R. J. A. Wanders, and P. Vreken. Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency. J. Lipid Res. 2001. 42: 137;-141.

Bile Acids and Salts↗

PubMed2001

Verapamil metabolism in distinct regions of the heart and in cultures of cardiomyocytes of adult rats.

A substantial number of drugs act either directly or indirectly on the heart, but surprisingly, little is known about drug oxidation in the heart. We therefore investigated the metabolism of the calcium antagonist verapamil in microsomal fractions isolated from the left and right ventricle of heart muscle and in primary cultures of cardiomyocytes of adult rats. Metabolism of verapamil proceeded predominantly with microsomal fractions isolated from the right ventricle of rat heart, and in liquid chromatographic-tandem mass spectrometry (LC-MS/MS) and LC-MS(3) experiments four metabolites (M1-M4) could be identified. Furthermore, the intermediate biotransformation products M5 to M8 could additionally be identified in cultures of primary cardiomyocytes, thus providing new insight into the mechanisms of the N-dealkylation and O-demethylation pathway of verapamil. We show metabolism of verapamil to be predominant in the right ventricle of the heart, and the data reported herein may explain metabolic inactivation and/or adverse drug reactions of certain cardiovascular drugs on the basis of tissue specific metabolism.

Animals↗

PubMed2001

Analysis of dicarboxylic acids by tandem mass spectrometry. High-throughput quantitative measurement of methylmalonic acid in serum, plasma, and urine.

BACKGROUND: Methylmalonic acid (MMA) is a dicarboxylic acid whose concentration can be increased in blood and urine in patients with an inborn error of metabolism or vitamin B(12) deficiency. We developed a method for the selective analysis of dicarboxylic acids that exploits the high specificity of tandem mass spectrometry (MS/MS) and the substantial difference in fragmentation patterns of the isomers methylmalonic (MMA) and succinic acid (SA). METHODS: Dicarboxylic acids were extracted from samples with methyl-tert-butyl ether and derivatized with butanolic HCl to form dibutyl esters. The derivative was injected into the liquid chromatography (LC)-MS/MS system using TurboIonSpray (nebulizer-assisted electrospray) ionization and quantified by the multiple reaction monitoring mode of MS/MS. RESULTS: The assay for MMA was linear up to 150 micromol/L. The total imprecision was < or =7.5% at both low and high concentrations. The limits of quantification and detection were 0.1 and 0.05 micromol/L, respectively. The degree of interference from SA could be predicted from the branching ratios of the major product ions. CONCLUSIONS: The method is specific for dicarboxylic acids. The LC-MS/MS analysis for MMA requires minimal chromatographic separation and takes <60 s per sample. The entire analysis, including sample preparation, for a batch of 100 specimens can be performed in <4 h.

Chromatography, Liquid↗

PubMedDate not supplied

Analysis of agricultural commodities and foods for Alternaria mycotoxins.

Fungi of the genus Alternaria are parasitic on plants and other organic materials. A. alternata is a frequently occurring species of particular interest because it produces a number of mycotoxins, including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxins I, II, and III (ATX-I, -II, and -III), and L-tenuazonic acid (TeA). Cleanup procedures of analytical methods for foods and foodstuffs include solvent partition, generally used for TeA, and solid-phase extraction columns for AOH, AME, and ATX-I. These Alternaria mycotoxins have been determined by TLC, GC, and more usually LC, mainly with ultraviolet detection, although fluorescence and electrochemical detection have also been used for Alternaria toxins other than TeA. A Zn2+ salt is usually added to the LC mobile phase for TeA. Recently, atmospheric pressure chemical ionization and electrospray LC/MS and LC-MS/MS have been applied to the determination and confirmation of AOH and AME in apple juice and other fruit beverages at sub ng/mL levels. Natural occurrences of AOH, AME, and in some cases other Alternaria toxins have been reported in various fruits, including tomatoes, olives, mandarins, melons, peppers, apples, and raspberries. They have been found also in processed fruit products such as apple juice, other fruit beverages and tomato products, wheat and other grains, sunflower seeds, oilseed rape meal, and pecans.

Alternaria↗

PubMed2002

Interlaboratory variation of plasma total homocysteine measurements: results of three successive homocysteine proficiency testing surveys.

BACKGROUND: Numerous studies have demonstrated that increased plasma total homocysteine (tHcy), whether measured after fasting or after a methionine load, is associated with increased risk for cardiovascular and thromboembolic diseases. However, little information is available regarding interlaboratory variation of tHcy measurements, especially at the increased tHcy concentrations observed after loading. METHODS: We conducted three Homocysteine Proficiency Testing Surveys at 6-month intervals. Sets of five plasma pools with endogenous tHcy concentrations ranging from 5 to 48 micromol/L were sent to participants. We received 11, 23, and 17 responses in the first, second, and third surveys, respectively. The following methods were used by participating laboratories: fluorescence polarization immunoassay (FPIA); HPLC with fluorescent detection (HPLC-FD), further subdivided by type of reduction/derivatization; HPLC with electrochemical detection (HPLC-ED); amino acid analyzer with ninhydrin detection; and liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). RESULTS: In surveys 1 and 2, no notable differences among the mean tHcy values obtained by the different methods performed were observed. In survey 3, tHcy values obtained by the FPIA method were significantly lower (P <0.05) at increased tHcy concentrations (34 micromol/L) compared with values obtained by HPLC-FD regardless of reduction/derivatization agents used. Our laboratory confirmed the observation that tHcy values obtained by the FPIA method differed from those obtained by HPLC-FD at increased tHcy concentrations by reanalyzing each pool 10 times by FPIA and HPLC-FD using tributylphosphine-ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (P <0.001 for tHcy >19 micro mol/L). The mean among-method variations in surveys 1, 2, and 3 were 19%, 12%, and 9.6%, respectively. When results of the three surveys were combined, the mean among-method variation on 170 samples was 13%. Within-method variation was lowest for the FPIA method (4.4%), and ranged from 11-20% for HPLC methods. CONCLUSIONS: Various degrees of imprecision and lack of correlation among tHcy methods indicate that there is a need to improve analytical precision, decrease analytical difference, and standardize tHcy measurements among laboratories.

Chromatography, High Pressure Liquid↗

PubMed2002

Contents of four active components in different commercial crude drugs and preparations of danshen (Salvia miltiorrhiza).

AIM: To detect the contents of four active components of Salvia miltiorrhiza in various commercially available danshen crude drugs and preparations. METHODS: Commercially available danshen crude drugs from different sources, as well as danshen pills and intravenous injection preparations containing danshen alone or in combination with other herbs were collected. The composition of these danshen samples was analyzed using HPLC. Specifically, the amounts of magnesium tanshinoate B (MTB), danshensu, isotanshinone HA, and cryptotanshinone were determined. In some of these samples, the content of MTB was further confirmed by liquid chromatography-tandem mass spectrometer (LC-MS)/MS method. RESULTS: There were great variations in the amount of the four active ingredients in the commercially available danshen crude drugs and drug preparations in this study. The amount of MTB was the highest among the four components measured in the crude drugs. However, the amounts of MTB in all danshen preparations were much lower than those in crude drugs. The 2 lipophilic components, isotanshinone HA and cryptotanshinone, were very low or not detectable in both injection and oral preparations. CONCLUSION: MTB can be used to standardize the various forms of danshen crude drugs and drug preparations from different sources. In view of the variation in the amounts of MTB and other components, improvement in the production methods of danshen preparations is essential to ensure consistent amount of its active ingredients and reproducible pharmacological actions.

Chromatography, High Pressure Liquid↗

PubMed2003

Novel monitoring concepts to acquire new water quality knowledge.

Two novel water quality monitoring concepts were developed: the HPLC-fingerprint for the monitoring of yet unidentified pollutants and the HPLC-Toxprint for the recognition of (unknown) toxic or genotoxic compounds. The paper describes applications of both concepts. The HPLC-fingerprint is used for the evaluation of the overall water quality in addition to the monitoring of individual pollutants. Based on their occurrence (frequency, concentration, location) a listing of unknown priority pollutants is set up. Participating waterworks monitor for these compounds using a dedicated HPLC-DAD library that contains the required compound data (UV-absorbance spectrum, retention time index). In five years of experience with this concept, the HPLC-fingerprint was also found very suitable for the retrieval of new priority pollutants in existing HPLC-fingerprint databases, providing historic data on these new compounds. The HPLC-fingerprint concept was also used as an Early Warning System for accidental spills or sabotage. The HPLC-Toxprint was successfully applied in identifying genotoxicity (in the umu-test) in various waste water samples. By the application of LC-MS/MS genotoxicity could be assigned to acridine-derivatives in one of these wastewaters. To enable the evaluation of drinking water resources, the sensitivity of the HPLC-Toxprint was improved, now allowing the detection of pollutants with a 10% genotoxic potential as compared to 2-aminoanthracene (the positive control compound) at concentrations as low as 0.1 microg/l.

Chromatography, High Pressure Liquid↗

PubMed2003

The behavior of estrogenic substances in the biological treatment process of sewage.

A study was conducted for about one year on the fate and behavior of estrogens, namely 17beta-estradiol (E2), estrone (E1), and estriol (E3) in an activated sludge process of a pilot scale plant supplied with domestic sewage. A simultaneous analytical method for these three substances using LC-MS/MS was developed and applied to sewage samples. The average removal of E2 was 94.7%, while that of E3 was 96.9%. In contrast, the average removal of E1 was relatively low at 69.2% with a maximum concentration of 55.4 ng/L detected in the treated water. The theoretical values of estrogenic activity calculated from the concentrations of each natural estrogen in treated water were found to correlate with the values of estrogenic activity measured by a yeast estrogen screening assay. The effect of E2 on estrogenic activity in influent was found to be high, while that of E1 in treated water was considerably higher. In batch treatment tests on E2, E2 turned into E1 immediately after being charged. After three hours of aeration, the values of both E1 and E2 were around threshold limits. It was determined from this that E1 and E2 were substances that could be degraded by biological treatment. As the removal of E2 was found to be sufficiently high at times, optimization of operational conditions based on E1 removal should be important for reducing estrogenic activity in treated water.

Biodegradation, Environmental↗

PubMedDate not supplied

Determination of ephedra alkaloids by liquid chromatography/tandem mass spectrometry.

In conjunction with an AOAC Task Group on dietary supplements, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was validated for measurement of 6 major alkaloids in raw ephedra sinica herb, ephedra extracts, ephedra tablets, complex dietary supplements containing ephedra, and a high-protein drink mix containing ephedra. The amount of ephedrine-type alkaloids present was determined by LC with mass selective detection. Six replicates of each matrix were analyzed on 3 separate occasions. The presence of 6 ephedrine-type alkaloids was detected at a level > 0.5 microg/g based on a 0.5 g sample. The standard curve range for this assay is from 0.02 to 1.0 microg/mL. Appropriate dilutions covered a wide range of specific alkaloid concentrations. The calibration curves for all 6 analytes had correlation coefficients > 0.995.

Alkaloids↗

PubMed1996

Disposition of indinavir, a potent HIV-1 protease inhibitor, after an oral dose in humans.

Indinavir, N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-tertiary- butylaminocarbonyl-4-(3-pyridylmethyl)piperazino]-4(S)- hydroxy-2(R)-phenylmethylpentanamide (L-735,524,MK-639, ayl-4- Crixivan), is a potent and specific inhibitor of the HIV-1(3 protease for the treatment of AIDS. Disposition of [14C]indinavir was investigated in six healthy subjects after single oral administration of 400 mg. AUC, Cmax, and Tmax values for indinavir were 492 microM x min, 4.7 microM, and 50 min, respectively. The AUC value for the total radioactivity in plasma was 1.9 times higher than that of indinavir, indicating the presence of metabolites. The major excretory route was through feces, and the minor through urine. Mean recovery of radioactivity in the feces was 83.4%. In the urine, mean recoveries of the total radioactivity and unchanged indinavir were 18.7% and 11.0% of the dose, respectively. HPLC radioactivity and LC-MS/MS analyses of urine showed the presence of indinavir and low levels of quaternary pyridine N-glucuronide (M1), 2',3'-trans-dihydroxyindanylpyridine N-oxide (M2), 2',3'-trans-dihydroxyindan (M3) and pyridine N-oxide (M4a) analogs, and despyridylmethyl analogs of M3 (M5) and indinavir (M6). M5 and M6 were the major metabolites in urine. The metabolic profile in plasma was similar to that in urine. Quantitatively, the metabolites in feces accounted for >47% of the dose, which along with the urinary excretion of approximately 19%, suggested that the absorption of the drug was appreciable. In the feces, radioactivity was predominantly due to M3, M5, M6, and the parent compound. Thus, in urine and feces, the prominent metabolic pathways were oxidations and oxidative N-dealkylations. Excretion of the quaternary N-glucuronide metabolite in the urine, which is a minor metabolite in human, was specific to primates.

Adult↗

PubMed1997

Studies on the metabolic activation of disulfiram in rat. Evidence for electrophilic S-oxygenated metabolites as inhibitors of aldehyde dehydrogenase and precursors of urinary N-acetylcysteine conjugates.

Recent studies on the mechanism by which disulfiram inhibits aldehyde dehydrogenase have provided evidence for the formation of reactive intermediates that are thought to carbamoylate, and thereby inactivate the enzyme. In our study, rats were dosed with either disulfiram (0.25 mmol kg-1 i.p.) or its reduced metabolite diethyldithiocarbamate (DDTC; 0.5 mmol kg-1 i.p.) and urine was collected for the analysis of metabolites derived from putative reactive intermediates. By means of ionspray LC-MS/MS, two novel N-acetylcysteine (NAC) conjugates, i.e., N-acetyl-S-(N, N-diethylcarbamoyl)cysteine and N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine, were identified in urine specimens. Quantitative analyses indicated that, over the 0- to 24-hr period after drug administration, urinary excretion of N-acetyl-S-(N, N-diethylcarbamoyl)cysteine accounted for 7.5 +/- 4.0 and 6.2 +/- 1.0%, respectively, of the dose of disulfiram and diethyldithiocarbamate, while the corresponding thiocarbamoyl conjugate, N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine, accounted for a further 0.5 +/- 0.3 and 0.3 +/- 0.1%, respectively, of the dose. These conjugates are believed to derive from reactive sulfoxide and sulfone metabolites of disulfiram, namely S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO), S-methyl-N, N-diethylthiocarbamate sulfone (DETC-MeSO2), S-methyl-N, N-diethyldithiocarbamate sulfoxide (DDTC-MeSO) and S-methyl-N, N-diethyldithiocarbamate sulfone (DDTC-MeSO2), which were found to carbamoylate N-acetylcysteine in vitro with the following rank order of reactivity: DDTC-MeSO2 > DETC-MeSO2 > DDTC-MeSO > DETC-MeSO. In vitro experiments with aldehyde dehydrogenase showed that all four S-oxygenated metabolites inhibited the enzyme effectively. Furthermore, inclusion of NAC in incubation media attenuated significantly the inhibition by DDTC-MeSO2, DETC-MeSO2 and DDTC-MeSO, but had little effect on that by DETC-MeSO. Our results are consistent with the hypothesis that disulfiram and diethyldithiocarbamate undergo activation by a sequence of metabolic reactions leading to the formation of electrophilic S-methyl sulfoxides and sulfones that carbamoylate, and thereby inhibit, aldehyde dehydrogenase and possibly other enzymes.

Acetylcysteine↗

PubMed1997

Oral and topical absorption, disposition kinetics, and the metabolic fate of trans-methyl styryl ketone in the male Fischer 344 rat.

trans-Methyl styryl ketone (MSK; trans-4-phenyl-3-buten-2-one) is a beta-unsaturated ketone that has a wide range of uses in industry and is present in numerous consumer products. Although MSK has been shown to be positive in several in vitro mutagenic assays, it does not seem to be overtly toxic in animal models. This lack of toxicity may relate to its poor absorption and/or rapid elimination. However, little is known about the fate of MSK in the body. Studies were conducted to characterize the absorption, and disposition kinetics of MSK after intravenous, oral, and topical administration to male Fischer 344 rats. After intravenous administration of [14C]MSK (20 mg/kg, 120 microCi/kg), blood concentration-time data could be characterized with a biexponential equation and apparent first-order elimination kinetics. The following pharmacokinetic parameter values were obtained (mean +/- SD): terminal disposition half-life, 17.7 +/- 0.08 min; apparent steady-state volume of distribution, 0.89 +/- 0.14 liters/kg; systemic body clearance, 68.9 +/- 10.0 ml/kg *min; and mean residence time, 13.1 +/- 2.2 min. Within 48 hr, 95.5% of the dose was excreted in the urine and 2.7% in the feces. The major blood metabolite after intravenous administration was identified by GC/MS as the 4-phenyl-3-buten-2-ol (methyl styryl carbinol). After oral administration of [14C]MSK (200 mg/kg, 100 microCI/kg), approximately 96.6% of the dosed radioactivity was recovered in the urine and 4.8% in the faces within 48 hr. Major urinary metabolites identified by LC-MS/MS and quantified by HPLC radioassay were N-phenylacetyl-L-glycine (64.9% of dose) and N-benzyl-L-glycine (9.9% of dose). Parent compound could not be detected in the blood after oral administration, and 14C-equivalents in the blood never exceeded 1.3% of the dose. Results suggest near-total presystemic elimination of the oral dose. After topical application of [14C]MSK (250 mg/kg, 50 microCi/kg), > 60% of the dose was absorbed, and the majority of the dose was excreted into the urine (55% of dose) in the form of metabolites. Urinary metabolites were similar to those described after oral administration. 14C-equivalents were not detected in the blood at any time after topical administration. These results indicate that MSK is almost totally metabolized before systemic distribution after oral or topical administration. The systemic exposure dose of MSK seems to be exceedingly low at the doses studied herein.

Administration, Oral↗

PubMed1997

Orally active inhibitors of human leukocyte elastase. III. Identification and characterization of metabolites of L-694,458 by liquid chromatography-tandem mass spectrometry.

The in vitro and in vivo metabolism of N-[1(R)-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2(S)-[4-[(4-methy l-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (L-694,458) was studied in male Sprague-Dawley rats and rhesus monkeys. Analysis by LC-MS/MS and NMR revealed that the major metabolite generated in incubations with rat liver microsomes resulted from N-oxidation of the piperazine group, while the major metabolite generated in monkey liver microsomes was the catechol that resulted from O-dealkylation of the methylenedioxyphenyl group. Other metabolites observed in these incubations include the piperazine N-desmethyl, several monohydroxylated derivatives of the parent compound, and three products that resulted from cleavage of the beta-lactam ring. Incubations of parent compound with rat hepatocytes in culture generated two major metabolites that resulted from cleavage of the piperazine ring with the loss of an ethylene group from one side of the ring; one of these metabolites retained the piperazine N-methyl group, while the other did not. The metabolite profiles in vivo were similar to those observed in vitro, but they were much more complex owing to secondary and, in some cases, tertiary biotransformations of many of the primary metabolites. Bile obtained from orally dosed rats contained more than 40 parent-related components, and many of these metabolites had arisen from piperazine ring cleavage.

Administration, Oral↗

PubMed1997

Absorption, disposition, and metabolism of trans-methyl styryl ketone in female B6C3F1 mice.

trans-Methyl styryl ketone (MSK; trans-4-phenyl-3-buten-2-one) is a beta-unsaturated ketone that has a wide range of uses in industry, as well as consumer products. MSK does not appear to be overtly toxic in animal models, however, it has been shown to be mutagenic in several in vitro assays after S-9 activation. In this study experiments were conducted to characterize MSK absorption, distribution, metabolism, and elimination after iv, oral, and topical administration to female B6C3F1 mice. After iv administration, [14C]MSK (20 mg/kg; 120 microCi/kg) was rapidly cleared from the blood as evidenced by the following pharmacokinetic values (mean +/- SD): terminal disposition half-life (t1/2), 7.98 +/- 1.72 min; mean residence time, 5.6 +/- 1.7 min; steady-state apparent volume of distribution (Vss), 3.33 +/- 0.75 liters/kg; and systemic body clearance (CLs), 0.53 +/- 0.05 liters/min/kg. Within 48 hr, 92.4% of the dose was excreted in the urine and 3.5% in the feces. The major blood metabolites after iv administration were identified by GC-MS as the 4-phenyl-3-buten-2-ol (methyl styryl carbinol), 4-hydroxy-4-phenyl-2-butanone, and benzyl alcohol. After oral administration of [14C]MSK (200 mg/kg; 100 microCi/kg), 95% of the dosed radioactivity was recovered in the urine and 1.2% in the feces within 48 hr. Major urinary metabolites were identified by LC-MS/MS as N-phenylacetyl-l-glycine (35.1% of dose) and N-benzyl-L-glycine (19.1% of dose). Only a small amount of MSK was detected in the blood after oral administration ( approximately 0.73 microg/ml at 10 min), and [14C]-equivalents in the blood never exceeded 2.8% of the dose. Ater topical application of [14C]MSK (250 mg/kg; 50 microCi/kg), approximately 40% of the dose was absorbed and 84.5% of the absorbed dose was excreted into the urine (36% of the total dose). Urinary metabolites were similar to those described for oral administration. Importantly, [14C]-equivalents were not detected in the blood at any time after dermal administration. These results indicate that the rate of MSK clearance is equivalent to its rate of absorption, and tissue exposure to intact MSK is expected to be limited.

Administration, Cutaneous↗

PubMed2026

Unveiling crosstalk regulations within the polyamine pathway and between polyamine and purine pathways in Aedes aegypti females.

We previously demonstrated that ornithine decarboxylase (ODC) deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti. To further examine the role of the polyamine pathway in Ae. aegypti nitrogen metabolism, we evaluated the expression of three additional genes encoding proteins involved in the biosynthetic pathway: S-adenosylmethionine decarboxylase, spermidine synthase (SdS), spermine synthase (SmS), and seven genes encoding proteins involved in the catabolic pathway in fat body, midgut and Malpighian tubules by qPCR. Distinct transcriptional profiles were observed in mosquito tissues during the first gonotrophic cycle. SdS and SmS showed a differential protein expression pattern in fat body of sugar- and blood-fed mosquitoes. Genetic silencing of SdS, SmS or SdS and SmS by RNA interference (RNAi) decreased female survival. Mosquitoes with SdS or SmS deficiency exhibited a reduction of 5G1 trypsin level in the midgut at 24 h post-blood meal (PBM) , a delay in blood digestion, and a decrease in uric acid concentration in the excreta at 48 h PBM. RNAi-mediated SdS knockdown also caused a decrease in SmS protein level and vice-versa, RNAi-driven SmS deficiency resulted in a decrease in SdS protein abundance. Notably, ODC knockdown reduced SdS, SmS, xanthine dehydrogenase-1 protein levels, and decreased specific metabolite concentrations in fat body at 24 h PBM. In addition, RNAi-mediated ODC, SdS and SmS knockdown impacted transcript levels of genes involved in polyamine and purine pathways in fat body at 24 h PBM. Our findings uncover unique crosstalk regulations within the polyamine pathway and between polyamine and purine pathways.

Ammonia metabolism↗

PubMed2026

Proteomic characterization of acidic aqueous extracts from Vicia faba L. pod valves identifies chitinase as a major co-extracted protein macromolecule.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Vicia faba↗

PubMed2026

Improved comprehensive profiling of fecal bile acids through chemical derivatization combined with HPLC-MS/MS analysis.

Bile acids (BAs) facilitate the digestion and absorption of fats and influence lipid and glucose homeostasis, making them potential therapeutic targets for obesity and related metabolic disorders. The liver and intestinal microbiota modify BAs structurally, generating diverse chemical forms and isomers. Comprehensive profiling of the BA pool is critical for understanding their key biological functions and as a therapeutic approach for related diseases. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is usually chosen as the preferred method for BA detection due to the complex chemical structures, the wide range of actual concentrations and the complexity of fecal sample matrices. However, free BAs are difficult to ionize, resulting in low detection signals and a lack of characteristic structural fragments to assist in structural identification. In this method, the labeling reagent (2-aminoethyl) trimethylammonium (AETMA) is employed to label the carboxyl group of BAs. Compared with underivatized BAs, the detection sensitivity of unconjugated BAs was enhanced by 25-180 fold, while that of conjugated BAs increased by 6-160 fold. It also generates unique fragment ions and enhances MS response, facilitating the discovery of potential BAs. Methodological parameters were validated using 38 BAs as representatives. Through methodological validation, it was verified that the precision, recovery, matrix effect and stability parameters of the method met acceptable criteria. We also identified 61 confirmed BAs and 55 additional candidate BAs in human pooled fecal samples. It has been successfully applied to fecal BA analysis in obese populations, providing valuable insights into potential therapeutic strategies for obesity.

Tandem Mass Spectrometry↗