Showing papers on "Metabolite published in 1979"

1 alpha, 25-Dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta.

Abstract: The biologically active metabolite of vitamin D, 1α, 25-dihydroxyvitamin D (1α, 25(OH)2D), is synthesised by successive hydroxylation of vitamin D in the liver and kidney1. The latter organ is the only known site of 1α-hydroxylation of 25-hydroxyvitamin D (25-OH-D)2 and is also a major site of production of 24,25-dihydroxyvitamin D (24,25(OH)2D), a metabolite involved in bone ossification3. However, 24-hydroxylation of 25-OH-D may also occur in rat intestinal homogenates and in cartilage cells4,5. Accumulating data show that, during pregnancy, 1α, 25(OH)2D increases in the maternal circulation6 but is absent or present only in small amounts in the fetus7,8. In comparison, 24,25(OH)2D accumulates in fetal tissues, particularly in the skeleton8. Little is known, however, about the independent metabolism of vitamin D in the fetoplacental unit. It has been reported9 that the nephrectomised pregnant rat can synthesise 1α, 25(OH)2D3 and 24,25(OH)2D3 and that the fetoplacental unit is the most likely site of production of such metabolites. Those data suggested that there may be independent vitamin D metabolism in the fetoplacental unit. We now report in vitro synthesis of 24,25(OH)2D3 by human placenta and of 1α,25(OH)2D3 and 24,25(OH)2D3 by human decidua. The human decidua is a development of the endometrium that is unique to pregnancy, originating from both epithelial and stromal cells and intimately attached to fetal structures, that is, the placenta and chorion.

Cocaine metabolism: cocaine and norcocaine hydrolysis by liver and serum esterases.

Abstract: The hydrolysis of cocaine and its N-demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.

Caffeine metabolism in the newborn

Abstract: The concentrations of caffeine and metabolites in urine have been examined as a function of age to explore the remarkably slow elimination of caffeine by human infants. Urine samples were obtained from 3 adults and 10 infants aged 8 days to 8 months during therapeutic treatment with caffeine. A high-performance liquid chromatographic (HPLC) procedure involving reversed-phase partition chromatography was developed to separate caffeine and 13 of its metabolites. During the first month of life, caffeine accounted for more than 85% of the identifiable products in urine. Caffeine remained the predominant component for the first 3 months, but its percentage decreased gradually to the adult value of less than 2% by the age of 7 to 9 months. This change reflected increasing metabolite production, not decreasing urinary caffeine concentration. The adult metabolite pattern of partially demethylated xanthines and urates was attained by 7 to 9 months. The data indicate that the 4-day plasma t1/2 of caffeine characteristic of the newborn depends in large part on slow urinary excretion of unchanged drug since there is little or no metabolism. Subsequent decrease in the t1/2 to about 4 hr by the age of 8 months correlates closely with the rise in metabolite production.

Mutagenic and alkylating metabolites of halo-ethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues. Evidence for oxirane formation by P450-linked microsomal mono-oxygenases.

Abstract: Mutagenicity, expressed as the number of his + revenants per μmole of test compound per hour of exposure, was estimated in two strains of S. typhimurium in the presence of a postmitochondrial mouse-liver supernatant, following exposure to vapours of one of a series of halo-olefins. Their activity was in the following descending order: 3,4-dichlorobutene-1 > 1-chlorobutadiene (technical grade) > 2-chlorobutadiene > vinyl bromide > vinylidene chloride > vinyl chloride; marginal mutagenicity was detected in the presence of 1,1,2-trichloroethylene and 1,1-difluoroethylene, and none with tetrachloroethylene and vinyl acetate. Liver fractions from humans converted vinyl chloride, vinyl bromide, vinylidene chloride and 2-chlorobutadiene into mutagens. In the plate incorporation assay, 1,4-dichlorobutene-2 was mutagenic per se, and addition of microsomal fractions from human or mouse liver enhanced the mutagenicity; a synthetic putative metabolite, 1,4-dichloro-2,3-epoxybutane was less mutagenic than the parent olefin in strain TA100. Treatment of rats with phenobarbital or 3-methylcholanthrene caused an up to 2-fold increase in the liver microsome-mediated mutagenicities of vinyl chloride and vinylidene chloride in S. typhimurium TA1530; while treatment with pregnenolone-16α-carbonitrile, aminoacetonitrile or disulfiram decreased the mutagenic effects. Vinyl chloride, and probably vinyl bromide, were shown to be epoxidized by mouse-liver microsomes; volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine and analysed spectrally. 2-Chlorobutadiene also yielded an alkylating intermediate, but 1,1-difluoroethylene, 1,1-dichloroethyleneand 1,1,2-trichloroethylene did not. 2-Chloro- and 1-chlorobutadiene, 3,4-dichlorobutene-1, 1,4-dichlorobutene-2 and its 2,3-epoxy derivative showed alkylating activity with 4-(4-nitrobenzyl)pyridine, which was not related quantitatively to mutagenic activity in S. typhimurium TA100 in the absence of a metabolic activation system. These data support the hypothesis that an oxidation of the double bond in certain halo-olefins, which is dependent on microsomal mono-oxygenases is a common pathway in the formation of biologically reactive intermediates. The relevance of the metabolites formed during such oxidative processes to the mutagenic, toxic and carcinogenic activities in vivo of some of the parent compounds is discussed.

Direct methods for measuring metabolite transport and distribution in mitochondria

Abstract: Publisher Summary This chapter describes the direct methods for studying mitochondrial transport, which are necessarily linked to the techniques for separating the mitochondria Since the rates of transport reactions are higher in mitochondria than in whole cells and bacteria, special emphasis will be devoted to the techniques developed for measuring kinetics It is possible to study (1) the efflux of endogenous substrates or of substrates from previously loaded mitochondria, (2) the uptake of added metabolites, and (3) the counterexchange by following the in and out movements of the exchanging metabolites The steady state distribution and the kinetic of transport between the two spaces are also examined The measurements of metabolite distribution are made after the separation of mitochondria from the incubation medium It is emphasized that the amount of metabolite within the mitochondria is relatively small, since the volume of the suspending medium is orders of magnitude larger than that of the intramitochondrial space

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. I. Isolation and identification of principal metabolites.

Abstract: 1. The anti-inflammatory agent diclofenac sodium (o-[(2,6-dichlorophenyl)amino]phenylacetic acid sodium salt) is extensively metabolized by rat, dog, baboon and man. The main metabolites were isolated from the urine of all species and from the bile of rat and dog and identified by spectroscopy. 2. Metabolism involves direct conjugation of the unchanged drug, or oxidation of the aromatic rings usually followed by conjugation. Sites of oxidation are either position 3' or 4' of the dichlorophenyl ring or, alternatively, position 5 of the phenyl ring attached to the acetic acid moiety. 3. In the urine of rat, baboon and man conjugates of the hydroxylated metabolites predominate, but the major metabolite in dog urine is the taurine conjugate of unchanged diclofenac. 4. In the bile of rat and dog, the main metabolite is the ester glucuroniade of unchanged diclofenac.

Candidiasis: detection by gas-liquid chromatography of D-arabinitol, a fungal metabolite, in human serum

Abstract: D-Arabinitol was identified as a major metabolite of Candida species in human subjects. Gas-liquid chromatography was used to measure the concentration of D-arabinitol in serum. The study included subjects who were healthy and cancer patients who had proven invasive candidiasis or were colonized with Candida. D-Arabinitol concentrations greater than 1.0 microgram per milliliter were found in serum from patients with invasive infection. This technique may prove valuable in the diagnosis of invasive candidiasis.

Mutagenic, cancerogenic and teratogenic effects of alcohol.

Abstract: Alcohol is mutagenic, cancerogenic and teratogenic in man. Ethanol is mutagenic via its first metabolite, acetaldehyde. This is substantiated by the findings that acetaldehyde induces chromosomal aberrations, sister-chromatid exchanges and cross-links between DNA strands. Methanol, a contaminant of many alcoholic beverages, is also mutagenic via its metabolite, formaldehyde. In addition, different indirect pathways may lead to mutations by alcohol. The cancerogenic activity of alcohol remains unverified by modern standard carcinogenicity tests. Ethanol and other alcohols, as well as aldehydes, inhibit RNA synthesis in cells and in cell-free transcriptional systems. A reduction of cellular RNA synthesis may play an important role in the mutagenic, carcinogenic and teratogenic activity of alcohol.

Assay of citric acid cycle intermediates and related compounds--update with tissue metabolite levels and intracellular distribution.

Abstract: Publisher Summary This chapter discusses new methods that have been developed in various laboratories for the assay of specific intermediates related to the citric acid cycle. Metabolite contents of different organs maintained in vitro under specified conditions are reviewed together with summaries of measured metabolite distributions between cytosol and mitochondria in liver. Considerable improvements have been made in commercial, relatively inexpensive, filter fluorometers and any one may be considered adequate for enzyme assays of metabolites in extracts of tissues and mitochondria at high sensitivity. The basic requirements are a stable light source, good mechanical placement of the cuvette in the machine, and an extended-range, and a zero-offset potentiometer. Several commercial fluorometers are available with automatic features for simultaneous assay of 20 or more samples. Commercial double-beam spectrophotometers are also capable of sensitivity similar to that of fluorometers for metabolite assay purposes, with an equivalent signal-to-noise ratio. Spectrophotometry also has advantages over fluorometry when the sample contains high concentrations of unwanted fluorochromes, as with perchloric acid extracts of tissues containing blood.

Plasma levels of chlorimipramine and its demethyl metabolite during treatment of depression.

Abstract: The biochemical and antidepressant effects of chlorimipramine (Cl) were studied in depressed patients after 3 wk of treatment. In vitro studies performed with rat brain slices incubated in human plasma showed that chlorimipramine and its demethyl metabolite (DMCI) are fairly specific blockers of serotonin and norepinephrine uptake. The uptake inhibition of serotonin and norepinephrine in plasma drawn from patients during treatment correlated with the plasma levels of parent drug and metabolite. Treatment with Cl caused a decrease of the main serotonin (5‐HIAA) and norepinephrine metabolites (HMPG) but had no significant mean effect on the major dopamine metabolite (HVA) or on tryptophan in cerebrospinal fluid (CSF). The 5‐HIAA decrease in CSF correlated with the plasma concentration of Cl within the range 80 to 360 nmole/l. The HMPG decrease in CSF correlated with plasma DMCI concentration. The patients were subdivided into two groups, those with “low” and those with “high” CSF 5‐HIAA. In patients with a “high” pretreatment CSF 5‐HIAA, there was a positive correlation between amelioration of depression and plasma level of DMCI. In this patient group there also was a correlation between HMPG alteration and amelioration of depression. This finding supports the hypothesis that patients with “high” CSF 5‐HIAA levels benefit from treatment with norepinephrine uptake blockers. In the patient group with “low” CSF 5‐HIAA, correlations between plasma levels of Cl and DMCI and amelioration were consistently negative, albeit not significant, supporting the idea that these patients are a biochemical subgroup within the depressive illness with a different reaction to antidepressant drugs than those with “high” CSF 5‐HIAA levels.

Studies on the different metabolic pathways of antipyrine in rats: influence of phenobarbital and 3-methylcholanthrene treatment.

Abstract: 1. The amounts of antipyrine and its metabolites excreted in 24 h urine after i.v. injection of 10 mg antipyrine into male Wistar rats were quantified after enzymic hydrolysis with beta-glucuronidase/aryl sulphatase. In 24 h 2.7% of the administered dose was excreted as unchanged antipyrine, 13.3% as 4-hydroxyantipyrine, 7.4% as norantipyrine, 28.9% as 3-hydroxymethylantipyrine and 1.1% as 3-carboxyantipyrine. 2. Treatment with phenobarbital decreased the antipyrine half-life from 65 to 30 min, but did not significantly change the urinary metabolite profile. Only the amount of 3-carboxyantipyrine was significantly different and increased from 1.1 to 2.6% dose. 3. 3-Methylcholanthrene treatment resulted in a decrease of antipyrine half-life from 72 to 37 min. After treatment 4-hydroxyantipyrine was increased from 13.4% to 25.6% dose, whereas 3-hydroxymethylantipyrine was decreased from 26.8% to 8.5% and 3-carboxyantipyrine from 1.3% to 0.2% of the dose respectively; norantipyrine was unchanged. 4. It is concluded that different types of hepatic cytochrome P-450 may be involved in the formation of 4-hydroxyantipyrine on one hand and the formation of 6-hydroxymethylantipyrine on the other. Another possibility is that in methylcholanthrene-treated animals another haemoprotein is formed that results in the formation of more 4-hydroxyantipyrine and less 3-hydroxymethylantipyrine. In any case, the urinary metabolite profile of antipyrine can be used to study changes in the activity of different cytochromes in drug metabolism studies.

Aldrin epoxidation, a highly sensitive indicator specific for cytochrome P-450-dependent mono-oxygenase activities.

Abstract: Aldrin epoxidation was studied in rat liver microsomes. The assay is very sensitive; amounts of less than 1 microgram of microsomal protein were sufficient for activity determination. The very low background, stability of the metabolite, and the complete separation of substrate and metabolite permit estimation of mono-oxygenase activities of less than 1 pmol per mg of protein per min by a simple procedure. Pretreatment of animals with the mono-oxygenase inducer phenobarbital (PB) increased the epoxidation rate 3-fold, whereas 3-methylcholanthrene (MC) treatment markedly depressed enzyme activity. Induction with MC did not change the apparent Km of the reaction, which was 18 muM. The Km in microsomes from PB-treated animals was 28 muM. These data suggest that the same or (a) similar form(s) of mono-oxygenase catalyze(s) the epoxidation in the three different microsomal preparations. SKF 525-A, metyrapone, and 7,8-benzoflavone were almost similarly active as inhibitors in microsomes from control and inducer-treated rats. Sensitivity to these inhibitors was low; 0.7 mM SKF 525-A and 0.4 mM 7,8-benzoflavone were necessary to reduce enzyme activity by 50%, whereas 0.5 mM metyrapone caused an inhibition of 10-45%. The activity of aldrin epoxidation in untreated rats increased almost parallel to the activity of ethylmorphine demethylation between 3 and 10 weeks of age. The rate of benzo[a]pyrene hydroxylation remained unchanged during this period. The results demonstrate that aldrin epoxidation offers a selective and sensitive assay for the activity of mono-oxygenases dependent on cytochrome P-450 forms.

Acetaminophen: potentially toxic metabolite formed by human fetal and adult liver microsomes and isolated fetal liver cells.

Abstract: A reactive metabolite of acetaminophen is hepatotoxic in humans when the drug is ingested in large overdoses. The ability of the human fetal and adult liver to oxidize acetaminophen by trapping the potentially toxic metabolite as a glutathione conjugate has been measured. Oxidation by fetal liver was approximately ten times slower than by adult liver. However, there was a definite increase in acetaminophen oxidation with fetal age. Isolated human fetal liver cells conjugated acetaminophen with sulfate but not with glucuronic acid. The results indicate that the human fetal liver is able to detoxify acetaminophen by conjugation. However, it also catalyzes the formation of an active metabolite of acetaminophen through oxidation. Hence the fetus remains at risk should a large dose of the drug cross into the fetal circulation.

Isolation and characterization of 1 alpha-hydroxy-23-carboxytetranorvitamin D: a major metabolite of 1,25-dihydroxyvitamin D3.

Abstract: The in vivo side-chain oxidation of 1 alpha,25-dihydroxyvitamin D3 was investigated by using a double-label radiotracer technique. Rats dosed with 1 alpha,25-dihydroxy-[3 alpha-3H]vitamin D3 and 1 alpha,25-dihydroxy[26,27-14C]vitamin D3 produced compounds with a high 3H/14C ratio. These compounds were found in sizable quantities in intestine and liver within 3 h after dosing. The major side-chain oxidized metabolite migrated as an acid on DEAE-Sephadex chromatography and contained no 14C. Methyl esterification of this compound with diazomethane proceeded in good yield and rendered the compound more amenable to chromatographic purification. The metabolite was isolated in several steps from rats dosed with 1 microgram of 1 alpha,25-dihydroxy[3 alpha-3H]vitamin D3. The metabolite was obtained in pure form as the methyl ester and was positively identified as 1 alpha,3 beta-dihydroxy-24-nor-9,10-seco-5,7,10(19)cholatrien-23-oic acid. The trivial name calcitroic acid is proposed for this major side-chain oxidized metabolite of 1,25-dihydroxyvitamin D3.

The production of the arachidonate metabolite HETE in vascular tissue.

Abstract: Blood platelets contain two enzyme systems which oxygenate arachidonic acid (AA). First, a cyclooxygenase1 which metabolises AA into the biologically active prostaglandin endoperoxides (PGG2 and PGH2) which are precursors for the platelet prostaglandins PGE2, PGF2α and thromboxane A2 (TxA2). And second, a lipoxygenase that metabolises AA into 12-L-hydroperoxy-5, 8, 10, 14-eicosatetraenoic acid (HPETE)1,2. HPETE is a labile intermediate which is nonenzymatically reduced in aqueous environments to a more stable metabolite, 12-L-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (HETE). HPETE is produced in significant quantities by aggregated platelets, but its exact biological role is not known. Turner et al.3 has suggested that HETE may be a mediator of neutrophil chemotaxis. Vane and coworkers demonstrated that 15-hydroperoxy-5, 8, 10, 14-eicosatetraenoic acid4,5 as well as HPETE were potent inhibitors of prostacyclin (PGI2), the major arachidonate metabolite in vascular tissue. These data suggest that HPETE, if present in vascular tissue, might act as endogenous regulator of PGI2 production. Using radiochemical techniques and mass sspectrometry we have now identified a lipoxygenase enzyme in vascular tissue (rabbit aorta) in which biosynthesis of HETE also occurred. The detection of HETE in this suggests a potential role of the lipoxygenated product as an endogenous regulator of PGI2 biosynthesis.

Acetaminophen kinetics in acutely poisoned patients

Abstract: A kinetic model of acetaminophen elimination over a wide dose range has been developed on the basis of (1) kinetic data from normal adults who received a usual dose (up to 2 gm) of the drug and (2) the composition of urinary metabolites of acetaminophen excreted within 24 hr by 29 patients who had ingested up to 26 gm of acetaminophen in suicide attempts (including 2 that were fatal and 5 with evidence of severe intoxication). The model consists of the following parallel pathways: conjugation with glucuronide by Michaelis-Menten kinetics, conjugation with sulfate by Michaelis-Menten kinetics, renal excretion of acetaminophen by apparent first-order kinetics, and formation of an oxidative metabolite (which is responsible for the hepatotoxicity of acetaminophen) by apparent first-order kinetics. There is good agreement between the model-predicted and actual urinary excretion of individual acetaminophen metabolites for doses of 0.8 to 26 gm and between model-predicted and actual plasma acetaminophen concentrations in both the low (normal subjects) and high (intoxicated subjects) concentration ranges. Computer simulations indicate that unsaturation of acetaminophen sulfate formation, previously shown to be feasible in vivo, should decrease the formation of the hepatotoxic metabolite. This prediction is consistent with experimental data obtained in preliminary studies on mice.

25-Hydroxyvitamin D3 26,23-lactone: a new in vivo metabolite of vitamin D

Abstract: A major vitamin D metabolite was isolated in pure form from the blood plasma of chicks either maintenance levels or large doses of vitamin D3. The isolation involved methanol-chloroform extraction and five column chromatographic procedures. The metabolite purification and elution position on these columns were followed by a competitive protein binding assay. The metabolite was identified, using high- and low-resolution mass spectrometry, 270-MHz proton nuclear magnetic resonance spectrometry, ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, and specific chemical reactions, as 3 beta,-25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-lactone. The trivial names 25-hydroxyvitamin D3 26,23-lactone or calcidiol 26,23-lactone are suggested for this compound.

Indole-3-lactic acid as a tryptophan metabolite produced by Bifidobacterium spp.

Abstract: Fifty-one strains of the genus Bifidobacterium have been found to accumulate indole-3-lactic acid in culture broth. The isolated metabolite was identified through mass and nuclear magnetic resonance spectroscopy. All the microorganisms tested, as resting cells, have been shown to be able to convert L-tryptophan into L-indole-3-lactic acid.

The anaerobic metabolism of metronidazole forms N-(2-hydroxyethyl)-oxamic acid.

Abstract: N-(2-hydroxyethyl)-oxamic acid is formed when metronidazole is reduced either chemically or by the action of the intestinal bacteria. When metronidazole, labeled with carbon-14 in the hydroxyethyl side chain, is administered by gavage to rats in doses of 200 mg/kg, an average of 1.4% of the administered radioactivity is recovered in the urine in the form of N-(2-hydroxyethyl)-oxamic acid. The presence of conjugated N-(2-hydroxyethyl)-oxamic acid in some samples was suggested by the detection of small additional amounts of the free acid after treatment of the urine with beta-glucuronidase. The metabolite is not found in the feces. In contrast N-(2-hydroxyethyl)-oxamic acid is not found in the urine or feces of germfree rats which receive metronidazole. Thus, the finding of N-(2-hydroxyethyl)-oxamic acid in the urine of rats which receive metronidazole appears to depend on the activity of the bacterial flora.