Acid Metabolite In Plasma Research Articles

Effect of moxibustion on plasma amino acid metabolism in rats with knee osteoarthritis.

To observe the effect of moxibustion at "Zusanli "(ST36) on the plasma amino acid metabolism in rats with knee osteoarthritis (KOA), and to explore the amino acid metabolism mechanism of moxibustion in repairing cartilage injury in KOA. A total of 30 SD rats were randomly divided into normal, model and moxibustion groups, with 10 rats in each group. Rats in the model and moxibustion groups were injected with the mixture of L-cysteine and papain into bilateral knee joint cavity to make the KOA model. The moxibustion group received moxibustion at bilateral ST36 for 30 min, once daily for 30 days. At the end of the experiment, the swelling degree of knee joint was calculated, the mechanical pain threshold was measured by the Von Frey filament, the cartilage tissue injury was observed by HE staining, the matrix metalloproteinase-13 (MMP-13) content in the synovial tissue was detected by enzyme-linked immunosorbent assay (ELISA), and the differential amino acid metabolites in plasma were detected and screened by liquid chromatography-mass spectrometry (LC-MS). Compared with the normal group, the model group showed irregular cartilage surface, decreased number of chondrocytes, uneven distribution, and local clusters of chondrocytes；the contour of the tide line was blurred. The degree of joint swelling in the model group was higher than that in the normal group (P<0.01), the mechanical pain threshold was lower (P<0.01), and the content of MMP-13 in synovial tissue was higher (P<0.01). The contents of proline and tryptophan in the model group were down-regulated (P<0.01, P<0.05). Compared with the model group, the cartilage tissue damage and knee joint swelling were decreased(P<0.05), mechanical pain threshold was increased(P<0.05), MMP-13 content in synovial tissue and levels of glutamate and histidine expression were decreased (P<0.01, P<0.05). Moxibustion at ST36 significantly alleviated arthritis-related swelling and pain in KOA model rats, attenuated cartilage damage, and regulated levels of certain plasma amino acid metabolites. Moxibustion may regulate KOA cartilage synthesis and degradation through amino acid metabolic pathways such as proline, tryptophan, glutamate and histidine, exerting anti-inflammatory, analgesic, and protection of cartilage injury effects.

Systemic and Ocular Anti-Inflammatory Mechanisms of Green Tea Extract on Endotoxin-Induced Ocular Inflammation.

IntroductionGreen tea extract (GTE) alleviated ocular inflammations in endotoxin-induced uveitis (EIU) rat model induced by lipopolysaccharide (LPS) but the underlying mechanism is unclear.ObjectivesTo investigate the systematic and local mechanisms of the alleviation by untargeted metabolomics using liquid chromatography-tandem mass spectrometryMethodsSprague-Dawley rats were divided into control group, LPS treatment group, and LPS treatment group treated with GTE two hours after LPS injection. The eyes were monitored by slip lamp and electroretinography examination after 24 hours. The plasma and retina were collected for metabolomics analysisResultsIn LPS treated rats, the iris showed hyperemia. Plasma prostaglandins, arachidonic acids, corticosteroid metabolites, and bile acid metabolites increased. In the retina, histamine antagonists, corticosteroids, membrane phospholipids, free antioxidants, and sugars also increased but fatty acid metabolites, N-acetylglucosamine-6-sulphate, pyrocatechol, and adipic acid decreased. After GTE treatment, the a- and b- waves of electroretinography increased by 13%. Plasma phosphorylcholine lipids increased but plasma prostaglandin E1, cholanic metabolites, and glutarylglycine decreased. In the retina, tetranor-PGAM, pantothenic derivatives, 2-ethylacylcarinitine, and kynuramine levels decreased but anti-oxidative seleno-peptide level increased. Only phospholipids, fatty acids, and arachidonic acid metabolites in plasma and in the retina had significant correlation (p < 0.05, r > 0.4 or r < -0.4).ConclusionsThe results showed GTE indirectly induced systemic phosphorylcholine lipids to suppress inflammatory responses, hepatic damage, and respiratory mitochondrial stress in EIU rats induced by LPS. Phospholipids may be a therapeutic target of GTE for anterior chamber inflammation

Simultaneous quantification of anthraquinone glycosides, aglycones, and glucuronic acid metabolites in rat plasma and tissues after oral administration of raw and steamed rhubarb in blood stasis rats by UHPLC-MS/MS.

Rhubarb is a widely used herbal medicine. To achieve different effects, rhubarb is usually steamed with rice wine (steamed rhubarb). This steaming treatment increases the blood-activating and stasis-removing effects of rhubarb. A specific and accurate ultra high performance liquid chromatography with tandem mass spectrometry method was established for simultaneous determination of anthraquinone glycosides, aglycones, and glucuronic acid metabolites in plasma and tissues after administration of raw rhubarb and steamed rhubarb in blood stasis rats. Chromatographic separation was performed on ACQUITY UPLC BEH Shield RP 18 column using the mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Satisfactory linearity, precision, accuracy, extraction recovery, and matrix effect have been achieved. From pharmacokinetic study, it showed that glucuronic acid metabolites were found abundantly in plasma as bioactive components. The lower area under concentration-time curve, maximum concentration, and higher apparent volume of distribution (P<0.01), body clearance (P<0.01) values in steamed rhubarb showed that most components of steamed rhubarb have lower bioavailability in plasma compared with raw rhubarb. But it found these components were mainly distributed in spleen and liver with large blood flow and perfusion rates. The pharmacokinetics and tissue distribution studies of anthraquinone components will provide helpful information for clinical application of steamed rhubarb and raw rhubarb.

The Folate Concentration and/or Folic Acid Metabolites in Plasma as Factor for COVID-19 Infection.

Pregnant women appear to be more susceptible to infectious diseases than women in reproductive age. According to the California Department of Public Health pregnant women were 9.6-folds more likely to be hospitalized during the 2009 influenza outbreak when compared to non-pregnant women in reproductive age. In contrast, it was reported that of 16,749 COVID-19 patients that were hospitalized in the UK, the probability for pregnant women to require in-patient care due to infection by SARS-CoV-2 was 0.95 versus non-pregnant women. Therefore 9.6/0.95 = 10.10, which brings us to the conclusion that pregnant women are 10.10-folds less likely to be hospitalized for a SARS-CoV-2 infection than for the 2009 H1N1 pandemic. Folic acid supplementation during pregnancy could be the factor that is protecting these patients against SARS-CoV-2 infection. Two independent papers that used informatic simulation proved that folic acid reduced the replication of this virus. One of them showed that folic acid inhibits the furin protease which the virus needs in order to enter its host cell, while the other one explained that folic acid inactivates protease 3CLpro, a protein that the virus needs to replicate. Nonetheless the probability that folic acid blocks two different proteins is very low, therefore the mechanism by which folic acid has apparently protected pregnant women during the COVID-19 pandemic has not been determined.

The dose-response effect on polyphenol bioavailability after intake of white and red wine pomace products by Wistar rats.

Wine pomace by-products are an important source of phenolic acids with significant health benefits. However, phenolic acid bioavailability in vivo has been little studied and there are few comparative studies on bioavailability between red and white wine pomace and the effect of intake of different doses. The qualitative and quantitative profile of phenolic acid metabolites in plasma and urine samples from Wistar rats was obtained by gas chromatography/mass detection, after oral administration of four doses (50, 100, 150, and 300 mg) of both the red and the white wine pomace products (rWPP and wWPP, respectively). The antioxidant capacity of the plasma samples assessed by both the ABTS and the FRAP levels was also evaluated. The results showed that neither the bioavailability nor the antioxidant capacity in vivo of the rWPP increased at high doses. However, both parameters were dependent on the intake of the wWPP.

High Altitude Hypoxia Impacts Omega‐3 Fatty Acid Metabolites in Plasma of Fetal and Newborn Sheep

Perinatal hypoxia has profound effect on an infant's development, with physiological impairment of multiple organ systems. The present study explored the effect of hypoxia due to long term high altitude exposure during gestation and after birth on lipid mediators by measuring the metabolites that foreshadow oxidative stress and inflammation, which are primary drivers of dysfunction. We tested the hypothesis that long term hypoxia reduces the amount of oxylipin and endocannabinoid production, which are important mediators of oxidative stress and inflammation. To test this hypothesis pregnant sheep and newborn lambs were housed at a low altitude (700 m) or exposed to an altitude of 3,800 meters starting gestation day 30. UPLC‐MS/MS analysis was used to investigate the lipid mediator composition in plasma collected from veins of near‐term fetal and newborn animals. The results show that hypoxia causes an overwhelming effect on omega‐3 fatty acids and their derivatives, which are crucial in late‐stage fetal development. Several of the omega‐3 fatty acids reduced with hypoxia have well known effects on inflammation, vascular tone, and oxidative stress. Furthermore, we tracked the origin of the identified oxylipins and endocannabinoids by tracing their pathways. The cytochrome P450 (CYP) pathway enzymes and the subsequent activity of soluble epoxide hydrolase (sEH) are prominent synthesizers of the oxylipins such as epoxyeicosatrienoic acids (EETs) and epoxyoctadecenoic acids (EpOMEs). Previous studies have suggested the role of EETs and EpOMEs in vasodilation, a feature in inflammation. Based on these findings we demonstrated that a majority of the affected oxylipins were produced from CYP and sEH enzymes, which are important to vascular function. These findings provide novel insight into our understanding of lipid metabolites to hypoxia‐induced dysfunction in the developing fetus and newborn. In the long term this may help us develop novel therapies that target inflammatory pathways induced by pre and post‐natal hypoxia.Support or Funding InformationThis material is based upon work supported by NIH grants P01HD083132 and 1U24DK097154 through a pilot project grant to Sean Wilson. VL is a recipient of the American Physiological Society's Short‐Term Research Education Program to Increase Diversity in Health‐Related Research (STRIDE) Fellowship funded by the APS and a grant from the National Heart, Lung and Blood Institute R25HL115473.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Host Nrf2 –glutathione Redox Dysregulation Precedes TNF-α Elevation and Predicts Severity of Graft Versus Host Disease in Experimental Transplantation

AbstractAbstract 4107Graft-versus-host disease (GVHD) is characterized by cytokine and chemokine dysregulation that predicts and also contributes to disease severity. Intermediates in sulfur amino acid (SAA) metabolism are known to modulate cytokines implicated in GVHD, such as TNF-α and IL-2. In this report, a new redox metabolomics assay was used to simultaneously profile 40 SAAs and other amino acid metabolites in plasma and liver 0, 4, and 10 days following experimental allogeneic (Balb/C→ B6, Allo) and syngeneic ((B6Thy1.1→ B6, Syn) bone marrow transplantation (BMT). At day 4 post transplantation (Day +4), prior to increase in plasma TNF-α and subsequent GVHD histopathology, there was a significant decline in both plasma and hepatic glutathione (GSH), resulting in a more oxidized redox potential in Allo relative to Syn mice. Specifically, at Day +4, plasma free GSH concentrations in Allo mice were ∼65% lower than Syn controls (p<0.005), and were accompanied by a ∼3-fold increase in plasma GSSG (p=0.012). The elevated GSSG/GSH ratio indicates a significant shift toward a more oxidized state. Loss of GSH was not due substrate limitation because plasma concentrations of total cysteine, the rate-limiting substrate for GSH synthesis, were significantly higher in Allo vs Syn mice at Day +10, indicating an allo-specific block in GSH synthesis. The plasma GSH redox potential on both Day +4 and Day +10 was significantly lower in Allo (−116.7 ± 5.8 mV, −129.0 ± 11.3 mV, respectively) compared to Syn (−157.9 ± 6.7 mV, −158 ± 2 mV) mice, in which the steady-state GSH redox potential remained similar to values in wild-type controls. Early GSH depletion appears to be a metabolic event associated solely with GVHD pathology, and not a consequence of effects of the conditioning regimen, as shown by the striking difference in hepatic GSH redox potential in an unconditioned paternal to F1 hybrid transplantation model (B6 → B6D2F1) in which some mice develop GVHD (Allo/GVHD+) and some do not (Allo/GVHD-). Specifically, the GSH/GSSG ratio was significantly higher in the Allo/GVHD-compared to Allo/GVHD+ group (17.0 ± 4.91 vs 0.315 ± 0.0601, p=0.015). At day+10, Allo/GVHD+ and Allo/GVHD-mice could be segregated based on hepatic total GSH, free GSH and total cystinylglycine concentrations (Figure 1). Increase in plasma TNF-α lagged behind the redox changes, with no significant differences detected between Syn and Allo mice at Day +4. Also observed at Day +4 in Allo mice was a significant decline in the hepatic nuclear concentration of NF E2-related factor 2 (Nrf2), the transcription factor regulating multiple antioxidant genes that contain the antioxidant response element (ARE) DNA sequence. This decline was accompanied by a decrease in protein and mRNA abundance of phase 2 antioxidant enzymes. For example, hepatic γ-glutamyl cysteine ligase catalytic unit (GCLC) mRNA abundance was decreased by ∼50% (P<0.0001) at Day +4 and further declined to ∼80% by Day +10 (P<0.0004) in Allo compared to Syn mice. Consistent with expectations, hepatic nuclear Nrf2 and TNF-α concentrations at Day +10 were inversely correlated (r2=0.8, p=0.01) in Allo mice (Figure 2 and 3). Together, these observations suggest that down-regulation of the Nrf2-GSH axis precedes stable elevation of TNF-α, and is an early predictor of the severity of GVHD in experimental transplantation. Future experiments will explore potential therapeutic implications of modulating the Nrf2 system in GVHD. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Determination of Clopidogrel Main Metabolite in Plasma: A Useful Tool for Monitoring Therapy?

This study was performed to determine whether analysis of clopidogrel and its main carboxylic acid metabolite in plasma provides additional information about the wide variability of platelet aggregation inhibition in clopidogrel-treated patients with peripheral arterial occlusive disease. Consecutive outpatients (n = 56) with stable peripheral arterial occlusive disease treated with 75 mg clopidogrel daily, without co-administration of aspirin, were investigated. With use of a standardized questionnaire, the time of drug intake was documented. Blood sampling was performed within 24 hours after the most recent drug intake. Platelet function was measured by optical aggregometry using adenosine diphosphate (ADP) (2 mumol/L) as the agonist. Plasma concentrations of clopidogrel and its main metabolite, clopidogrel carboxylic acid, were quantitated using high-performance liquid chromatography analysis coupled to mass spectrometry. In 95% (53/56) of patients, clopidogrel carboxylic acid was detected. In 40% (22/56) of patients, the ADP-induced aggregation response was within the normal range despite clopidogrel treatment. In 14% (3/22) of these patients, neither clopidogrel nor its main metabolite could be detected. Two of these patients agreed to ingest 75 mg/d clopidogrel under observation and to undergo blood sampling after 2, 12, and 24 hours. Clopidogrel carboxylic acid and a significant inhibition of platelet aggregation were detected even after 24 hours in both patients, confirming noncompliance as the reason for the lack of inhibition of ADP-induced platelet aggregation observed in the initial measurements. In the subgroup of patients who had taken clopidogrel within 4 hours before blood sampling, a large range of carboxylic acid concentrations was detected, indicating a high variability of drug metabolism among patients. In conclusion, determining clopidogrel metabolite plasma concentrations could be a useful tool for identifying poor compliance and variable metabolism in clopidogrel-treated patients. Nevertheless, in the majority of clopidogrel-treated patients, the variability of platelet response is not caused by noncompliance.

Effect of dietary fat on oral bioavailability of tepoxalin in dogs

A pharmacokinetic study was conducted to compare the oral bioavailability of tepoxalin and its pharmacologically active acid metabolite in fasted dogs and dogs fed either a low-fat or high-fat commercial diet. Using a cross-over design, six beagles were administered tepoxalin (10 mg/kg) intravenously (i.v.) and orally (p.o.) after being fed one of three diets (fasted, low-fat, or high-fat). Thereafter, blood samples were collected at frequent intervals, concentrations of tepoxalin and acid metabolite in plasma were determined by high performance liquid chromatography, and pharmacokinetic parameters were estimated. After i.v. dosing, the mean (+/-SD) half-life of elimination (t(1/2(beta))) was 2.45 +/- 1.47 h. After p.o. administration, plasma concentrations of acid metabolite were consistently higher than corresponding concentrations of the parent tepoxalin, indicating that tepoxalin is subject to a substantial first-pass effect. Mean (+/-SD) peak concentrations of tepoxalin were significantly higher after feeding of low-fat (1.08 +/- 0.37 microg/mL) and high-fat (1.19 +/- 0.29 microg/mL) diets than in fasted dogs (0.53 +/- 0.20 microg/mL), suggesting that feeding improves oral bioavailability.

Retinoic Acid Metabolites in Plasma Are Higher after Intake of Liver Paste Compared with a Vitamin A Supplement in Women

The objectives of the present study were to compare the bioavailability of vitamin A from liver paste and from a vitamin A supplement at three nutritionally relevant levels of intake, and to estimate levels of "safe" intake based on concentrations of retinoic acid and its metabolites in plasma after a single dose of vitamin A from liver paste. Women (n = 35; 19-47 y of age) consumed 3.0, 7.5 or 15 mg vitamin A as liver paste or as a vitamin A supplement with a test meal in a randomized design, with a combined crossover (two sources) and parallel approach (three dosages). Retinyl esters and retinoic acid (RA) metabolites were quantified in blood samples at 2-24 h after dosing. The areas under the time-response curves (AUC) were calculated to evaluate responses in plasma vitamin A after intake of liver paste and the vitamin A supplements. For retinyl esters, the AUC was significantly affected by the dosage, but not by the source. The formation of 13-cis-RA, 13-cis-4-oxo-RA, and to a lesser extent all-trans-RA was significantly higher after consumption of liver paste compared with the supplement, especially at higher dosages. Long-term baseline concentrations of retinol were not affected by a single intake of vitamin A. In conclusion, the bioavailability of vitamin A from single doses of liver paste and a vitamin A supplement does not differ, but the plasma concentrations of RA metabolites are higher after intake of liver paste. Thus, pregnant women should indeed limit the intake of vitamin A from liver products.

Simultaneous determination of modafinil and its acid metabolite by high-performance liquid chromatography in human plasma

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of modafinil and its acid metabolite in human plasma has been developed. The method is based on a liquid—liquid extraction followed by isocratic reversed-phase HPLC with ultraviolet absorbance detection at 236 nm. The eluent used was acetonitrile—water—acetic acid (150:420:12, v/v/v). The run time was 45 min. The method provided a detection limit of 0.04 mg/l for modafinil and the acid metabolite, a quantitation limit of 0.13 mg/l for modafinil and 0.14 mg/l for the acid metabolite. A good linear relationship was obtained in the concentration range studied (0.1–20 mg/l) for both compounds and the method was sufficiently accurate and precise to support clinical pharmacokinetic studies. To our knowledge this is the first described method for determination of modafinil and its acid metabolite in plasma.

Reverse-Phase High-Performance Liquid Chromatography Analysis of Arachidonic Acid Metabolites in Plasma after Stimulation of Whole Blood ex Vivo

Following the stimulation of heparinized blood ex vivo, aliquots of plasma were denatured with organic solvents containing the internal standards prostaglandin (PG) B2 and 19-hydroxy-PGB2. Precipitated material was removed by centrifugation and the supernatants were directly analyzed by reverse-phase HPLC with on-line extraction and uv detection. Stimulation of blood with A23187 lead to the formation of both leukocyte and platelet arachidonic acid metabolites as the 5-lipoxygenase products leukotriene (LT) B4, 5-hydroxy-eicosatetraenoic acid (5-HETE), 20-hydroxy-LTB4 and 20-carboxy-LTB4, the 12-lipoxygenase product 12-HETE, and the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHTrE) were detected in plasma; in some plasma samples LTC4 and/or LTE4 were also detected. Stimulation of blood with zymosan lead to the synthesis of LTB4 and 5-HETE as major products and of 12-HETE. Recoveries of 20-hydroxy-LTB4, LTB4, 5-HETE, 12-HETE, and HHTrE added to plasma were high (≥90%) while those of LTC4 and LTE4 were lower (50-70%); however, washing of the precipitated protein pellet resulted in >90% recoveries for all metabolites including the cysteinyl-leukotrienes. Levels of arachidonic acid metabolites in native plasma samples stored at −20°C were stable for at least 28 days, while significant loss of material was observed over the same period of time in denatured plasma samples. Finally, we made the critical observation that the capacity for A23187- (but not zymosan-, ionomycin-, or LPS and FMLP-) induced arachidonic acid metabolite synthesis in blood decreased by 80% within 1 h of blood collection. The profiling of 5-lipoxygenase products in synovial fluid from an arthritic patient illustrates the applicability of the method to protein-rich matrices other than plasma. An application of the method is shown through the demonstration of the inhibition of LTB4 formation ex vivo following the in vivo administration of a LT synthesis inhibitor to rabbits.

Determination of four carboxylic acid metabolites of felodipine in plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method with ultraviolet detection at 220 nm was developed to determine four carboxylic acid metabolites in plasma following therapeutic doses of the calcium antagonist felodipine. After the addition of an internal standard the analytes were isolated by liquid-liquid and solid-phase extraction. The metabolites were applied to a C2 cartridge in their free acid form, but they were transformed and retained as ion pairs with tetrabutylammonium during a wash with phosphate buffer (pH 7), prior to automated elution and injection by the Varian AASP system onto the analytical C18 column. Using a sample volume of 1 ml of plasma, the lower limit of determination for the metabolites was about 20 nmol/l. The influence of the pH of the mobile phase on the retention time of the metabolites and the structural requirements for the internal standard were studied. The method was applied to plasma samples from four dogs collected after an oral dose of felodipine. The plasma concentration-time profiles of the metabolites gave useful information about the mechanisms by which they were formed and eliminated.

Determination of terfenadine and terfenadine acid metabolite in plasma using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

This work describes the methodology for the analysis of terfenadine and the acid metabolite of terfenadine in plasma using high-performance liquid chromatography. The use of solid-phase extraction allows the use of robotic or manual sample preparation for the efficient clean-up of terfenadine and terfenadine acid metabolite from plasma. Additional selectivity is obtained through the use of fluorescence detection. For terfenadine, the validated quantitation range of this method is 10.0–84.2 ng/ml with coefficients of variation of 5.7–30%. For terfenadine acid metabolite, the validated quantitation range of this method is 8.2–500 ng/ml with coefficients of variation of 4.1–24%.

High-performance liquid chromatographic assay for the simultaneous monitoring of mefloquine and its acid metabolite in biological samples using protein precipitation and ion-pair extraction

A high-performance liquid chromatographic (HPLC) method is presented for the simultaneous determination of mefloquine and its acid metabolite in plasma and whole blood. Plasma and whole blood are deproteinized with a combination of zinc and acetonitrile before extraction. Mefloquine and its acid metabolite are extracted simultaneously at pH 4 by methyl tert.-butyl ether, where mefloquine is extracted as an ion pair with heptanesulphonate. After evaporation of the organic phase, the residue is dissolved in mobile phase and injected on to the chromatographic column. A reversed-phase column (Spherisorb ODS-1) is used with acetonitrile—phosphate buffer (0.1 mol/l, pH 2.5) (42:58) containing 40 mmol/l perchlorate as the mobile phase. N,N-Dioctylamine was added to the mobile phase to give a concentration of 0.1% and the pH was adjusted to 2.3–2.7 with concentrated phosphoric acid. The method permits the determination of 0.10 μmol/l (30 ng/ml) mefloquine and its acid metabolite in plasma. The coefficient of variation was 5–6% at the therapeutic level (mefloquine 1–4 μmol/1, its carboxylic metabolite 2–6 μmol/1) in 0.5-ml samples. An alternative method is also described with a similar clean-up procedure that uses protein precipitation with zinc—acetonitrile as a sample pretreatment for therapeutic monitoring of mefloquine and metabolite in plasma and whole blood. Using this method, 0.25 μmol/l mefloquine and its metabolite can be determined. The results from the two methods correlate well.

Leukotrienes LTB4 and LTC4 in thermally injured patients' plasma and burn blister fluid.

Radioimmunoassays for 5(S), 12(R)-dihydroxyeicosa-6, 14-cis, 8,10-trans-tetraenoic acid (leukotriene B4, LTB4) and 5(S)-hydroxy-6(R)-L-gamma-glutamyl-L-cysteinyl-glycinyleicosa-7,9- trans, 11,14-cis-tetraenoic acid (leukotriene C4, LTC4) have been used to quantify the concentrations of these arachidonic acid metabolites in plasma (12 patients) and blister fluid (6 patients) of burned patients. The results of this study demonstrate that, in general, burn plasma LTB4, and LTC4 were not elevated; however, in individual cases, transient high levels of leukotrienes were observed. Correlation between leukotriene "peaks" and the clinical course could not be established. High levels of LTB4 and LTC4 in burn blister fluid suggest their participation in local inflammatory reactions.

Determination of the agonist-antagonist anxiolytic agent 6-(2-chlorophenyl)-4-hydroxy-4 H-imidazo[1,5- a]-[1,4]benzodiazepine-3-carboxamide and its benzophenone carboxylic acid metabolite in plasma by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of 6-(2-chlorophenyl)-4-hydroxy-4 H-imidazo[1,5- a]-[1,4]benzodiazepine-3-carboxamide (I) and its benzophenone carboxylic acid metabolite, 1-[2-(2-chlorobenzoyl)phenyl]-4-(aminocarbonyl)-1 H-imidazole-5-carboxylic acid (II) in plasma. The assay involves the extraction of both compounds into benzene from buffered plasma (pH 5.4) and subsequent analysis by reversed-phase HPLC. The overall recovery of I and II is 98.3 ± 9.4 and 59.7 ± 15.7% for dog plasma, 86.0 ± 14.7 and 52.8 ± 15.1% for rat plasma and 98.1 ± 9.3 and 66.9 ± 18.0% for human plasma, respectively. The sensitivity limit of the assay for I and II is 20.0 and 40.0 ng/ml of plasma using ultraviolet detection at 254 nm. The assay was used in studies in dog and rat.

Influence of steroidal and nonsteroidal anti-inflammatory agents on the accumulation of arachidonic acid metabolites in plasma and lung lymph after endotoxemia in awake sheep. Measurements of prostacyclin and thromboxane metabolites and 12-HETE.

The influence of methylprednisolone and meclofenamate on endotoxin-induced release of 3 arachidonic acid metabolites was studied in unanesthetized sheep. Concentrations in plasma and lung lymph of prostacyclin and thromboxane (Tx) A2 metabolites (6-keto-PGF1 alpha and TxB2, respectively) were measured by radioimmunoassay. Concentrations of 12-HETE in lung lymph were measured by stable isotope dilution assay employing gas chromatography-mass spectroscopy. Thromboxane B2 concentrations increased quickly to peak values during the first hour after endotoxin infusion, then decreased to baseline by 1.5 hr. 6-keto-PGF1 alpha concentrations increased more gradually to peak values between 1 and 2 h after endotoxin infusion and remained elevated at 2.5 h. Lymph concentrations of both cyclooxygenase metabolites exceeded those in blood plasma. Methylprednisolone significantly inhibited accumulation of 6-keto-PGF1 alpha in lymph and plasma, but did not significantly inhibit accumulation of TxB2 in lymph or plasma. The combination of meclofenamate and methylprednisolone completely inhibited accumulation of TxB2 and 6-keto-PGF1 alpha in lymph and plasma. The concentration of 12-HETE in lung lymph increased significantly to peak values by 2.5 h after endotoxemia, and methylprednisolone, with or without meclofenamate, inhibited accumulation of 12-HETE in lung lymph. These data support participation of TxA2 in acute pulmonary hypertension after endotoxemia. That methylprednisolone treatment inhibited accumulation of 6-keto-PGF1 alpha and prevented the increase in lung vascular permeability suggests that prostacyclin production is a consequence of lung vascular injury. Increased lung lymph concentrations of the lipoxygenation product, 12-HETE, were coincident with physiologic evidence of increased lung vascular permeability, but whether release of lipoxygenase products after endotoxemia contributes to or results from lung vascular injury remains to be established.

Infantile Refsum's disease: Biochemical findings suggesting multiple peroxisomal dysfunction

Infantile Refsum's disease was diagnosed in three male patients, presenting with facial dysmorphia, retinitis pigmentosa, neurosensory hearing loss, hepatomegaly, osteopenia and delayed growth and psychomotor development. An elevated plasma phytanic acid concentration and a deficient phytanic acid oxidase activity in fibroblasts were found with an accumulation of very long chain fatty acids in plasma and fibroblasts. There were elevated pipecolic acid levels in plasma, urine and CSF, and abnormal bile acid metabolites in plasma. Deficient activity of acylCoA: dihydroxyacetone phosphate acyl transferase was found in thrombocytes and fibroblasts of these patients as well as an impaired de novo plasmalogen biosynthesis in fibroblasts. These biochemical abnormalities, previously described in the Zellweger syndrome, suggest multiple peroxisomal dysfunction in our patients.

Determination of 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester and its acid metabolite in plasma and urine by high-performance liquid chromatography

Two separate, rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assays were developed for the determination of 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester (I) and its acid metabolite, 4-amino-5-ethyl-3-thiophenecarboxylic acid (II), in plasma and urine. The analysis of I is performed directly on a hexane extract of plasma or urine (buffered to pH 11) by normal-phase HPLC analysis using a 10-μm silica gel column with an eluting solvent of hexane—ethanol (95:5) and UV detection of the effluent at 254 nm. A methyl analogue, 4-amino-5-methyl-3-thiophenecarboxylic acid methyl ester, was used as the internal standard. The analysis of II is performed on the residue of either a diethyl-ether-washed protein-free filtrate of plasma or a methylene chloride—isopropanol (95:5) extract of urine (buffered to pH 5.3) using a 10-μm alkyl phenyl (reversed-phase) column with an eluting solvent of water—methanol—1 M phosphoric acid, pH 2.5 (70:30:0.05) with UV detection of the effluent at 254 nm. An isopropyl analogue, 4-amino-5-isopropylthiophene-3-carboxylic acid (IV), was used as the internal standard. The assay of compounds I and II were applied to the determination of plasma and urine concentrations of I and II in the dog and in man following oral administration of I ṡ HCl. The data obtained demonstrated the extremely rapid and virtually complete deesterification of I (ester) to II (acid) in both species.