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o-Hydroxymandelic acid and m-hydroxymandelic acid have been identified in human urine by gas chromatography mass spectrometry selected ion monitoring. After solvent extraction the urinary acids were converted to their O-trifluoroacetoxy methyl ester derivatives which were identified by comparison of the retention times and relative intensities of the characteristic m/z 374 and m/z 315 ions with those from authentic samples. 4,6-[2H3]-o-hydroxymandelic acid and 2,4,6-[2H3]-m-hydroxymandelic acid were synthesized for use as internal standards in the quantitative estimation of the isomeric hydroxymandelic acids excreted in urine. In ten normal adults the following results were obtained: o-hydroxymandelic acid 4--16 ng mg-1 creatinine and m-hydroxymandelic acid 11--71 ng mg-1 creatinine. Acid hydrolysis of the urine or ingestion of a diet of known composition did not affect these results, indicating that these compounds are excreted as the free acids and probably arise by metabolism of the corresponding phenylethanolamine(s).
Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.
We evaluted measurement of urinary 4-hydroxyphenyl acetic acid as a potential screening method for small-bowel disease and bacterial overgrowth syndromes in 360 unselected acutely ill infants and children. Control data were obtained on 120 healthy children, ages 1.5 to 15 years, from a general medical practice, 48 healthy infants, ages one to five years, from local day nurseries, and 150 healthy babies, ages less than one to eight days. Comparative data were from 300 acutely ill hospitalized babies and children, ranging in age from less than one day to 15 years and without clinical evidence for small-bowel disease and bacterial overgrowth syndrome. No false-negative results and only 2% false-positive results were observed. Among the 10 patients whose urinary excretion of the analyte was considered to be abnormal were patients with Giardia lamblia infestation, ileal resection with blind loop, and other diseases of the small intestine associated with bacterial overgrowth. We conclude that measurement of 4-hydroxyphenylacetic acid excretion is useful in screening for such diseases.
1. Aromatic acids were extracted from brain and liver of rats with phenylketonuria-like characteristics produced by administration of phenylalanine, either alone or in combination with p-chlorophenylalanine. The metabolism of the aromatic acids in these tissues was measured by gas chromatography. 2. At 1h after an intraperitoneal injection of l-phenylalanine (1g/kg) in 23-day-old rats, the phenyl-lactate concentration was 2.2mug/g in the liver and 0.43mug/g in the brain, and the concentration of o-hydroxyphenylacetate was 0.26mug/g in the liver. 3. Phenylacetate concentrations in brain and liver were 0.26 and 0.14mug/g respectively. 4. Suckling rats produced phenyl-lactate less rapidly than weanling rats, but accumulated higher concentrations in longer-term experiments. 5. Intraperitoneal injections of phenyl-lactic acid showed that this compound could directly penetrate the blood-brain barrier, and could produce similar brain/liver ratios of phenyllactate to those found after phenylalanine injection. 6. Qualitative and quantitative similarities in urinary excretion of aromatic acids between the rats used in this study and human patients with uncontrolled phenylketonuria indicate that a patient with a circulating phenylalanine concentration of the order of those achieved in the experimental animal may have aromatic acid concentrations in brain and liver comparable with those found in the rats used in the present study.
Rabbit aortic strips (nerve-free, reserpine-pretreated or normal) whose noradrenaline-metabolizing enzymes were inhibited (by in vitro treatment with 0.5 mM pargyline for 30 min and by the presence of 0.1mM U-0521) were exposed to 1.18 muM labelled (-)- or (+)noradrenaline for 30 min. At the end of the incubation period some strips were used for analysis of radioactive (i.e., of noradrenaline and its metabolites), while for others the efflux of radioactivity was determined during 250 min of wash out with amine-free solution. An estimate of the original distribution of the amine into the various extraneuronal and neuronal compartments of the tissue was obtained by compartmental analysis of the efflux curves. 1. The mechanisms responsible for the accumulation of radioactivity in extraneuronal and axoplasmic compartments lack stereoselectivity; the rate constants for the efflux of radioactivity from these compartments are the same for (-)- and (+)noradrenaline. 2. The accumulation of radioactivity in storage vesicles is stereospecific with preference for the (-)isomer. 3. Despite the use of enzyme inhibitors, the "late neuronal efflux" of radioactivity (i.e., the efflux collected between the 200th and 250th min of wash out) contained a considerable proportion of metabolites of noradrenaline. The metabolism of noradrenaline was stereoselective: while dihydroxyphenylglycol (DOPEG) was the predominant metabolite in the efflux from strips incubated with (-)noradrenaline, a considerable part of the efflux from strips incubated with the (+) isomer consisted of dihydroxymandelic acid and "O-methylated and deaminated" metabolites (in addition to DOPEG).
Benzoylformate decarboxylase forms a covalent intermediate from thiamin diphosphate (TDP) and benzoylformate, alpha-mandelylTDP. This loses carbon dioxide to form a carbanion (enamine). Protonation of the carbanion and elimination of benzaldehyde regenerate enzyme-bound TDP. We synthesized alpha-mandelylthiamin and found that the rate of the loss of carbon dioxide is one-millionth that of the enzymic reaction. Thus, the enzyme provides an environment that facilitates the unimolecular decarboxylation process. However, the resulting nonenzymic carbanion reacts very rapidly to give products that lead to the irreversible destruction of the cofactor. This contrasts with the normal process on the enzyme. Brønsted acids on the enzyme may divert the reaction to the benzaldehyde precursor, or the enzyme may block access to the pathway that leads to destruction of the cofactor.
The paper describes spontaneous decomposition of D,L-2,3-diphenyltartaric acid in propane-2-ol, which takes place via radicals at ambient temperature. The primary product of the decomposition is short-living ketyl radicals of phenylglyoxalic acid, which are captured and stabilized by means of the spin trap of N-terc-butyl-alpha-phenylnitrone. The spontaneous decomposition of D,L-2,3-diphenyltartaric acid in propane-2-ol served as the source of radicals for the determination of the antioxidative effect of known antioxidants: C-vitamin, E-vitamin, and selenomethionine. It was found that the above-mentioned antioxidants are effective scavengers of radicals of phenylglyoxalic acid. On the basis of these data, the method can be recommended for testing the antioxidative properties of other substances as potential antioxidants.
To examine the disposition of [3H]norepinephrine ([3H]NE) in adrenergically innervated veins, helical strips of canine saphenous veins were incubated in Krebs-Ringer solution containing D,L[3H]NE (2 X 10(-7) M) for 2 h. [3H]NE and its metabolites were measured in extracts of veins and in superfusate (Krebs-Ringer) collected during basal conditions and during release of [3H]NE evoked by electrical stimulation (1-8 Hz), tyramine (5 X 10(-6) to 5 X 10(-4) M), or high concentrations of potassium (35-100 meq/liter). During basal conditions, the efflux from veins comprised mainly metabolits of [3H]NE, especially 3,4-dihydroxphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG); this pattern was unchanged by cocaine treatment, and monoamine oxidase inhibition reduced the formation of DOPEG. During evoked release of NE, the major metabolites in the perfusate were DOPEG, MOPEG, and normetanephrine, and their proportions differed with the stimulus used: O-methylated metabolites in the perfusate always increased more than did the deaminated catechol compounds; DOPEG and MOPEG were released in greater amounts than the corresponding acids; and cocaine treatment caused a higher content of all metabolites except DOPEG. 3-Methoxy-4-hydroxymandelic acid was also formed by the vein but was retained in the tissue.
Beta-lactam antibiotics restrict bacterial growth by inhibiting DD-peptidases. These enzymes catalyze the final transpeptidation step in bacterial cell wall biosynthesis. Although much structural information is now available for these enzymes, the mechanism of the actual transpeptidation reaction has not been studied in detail. The reaction is known to involve a double-displacement mechanism with an acyl-enzyme intermediate, which can be attacked by water, specific amino acids, peptides, and other acyl acceptors. We describe in this paper an investigation of acyl acceptor specificity and assess the need for general base catalysis in the deacylation transition state of the Streptomyces R61 DD-peptidase. We show, by the criterion of solvent deuterium kinetic isotope effect measurements and proton inventories, that the transition states of specific and nonspecific substrates are very similar, at least with respect to proton motion. The transition states for attack (tetrahedral intermediate formation) by d-amino acids and Gly-l-Xaa dipeptides do not include a general base catalyst, while such catalysis is essential for reaction with water and d-alpha-hydroxy acids. D-Alpha-hydroxy acids act as acyl acceptors for glycyl substrates but not for more specific d-alanyl substrates; hydroxy acids actually behave, more generally, as mixed inhibitors of the DD-peptidase. The structural and mechanistic bases of these observations are discussed; they should inform transition state analogue design.
Heterozygotes for phenylketonuria and controls were given oral loads of 100 mg and 200 mg L-phenylalanine per kilogram body weight. The concentrations of urinary aromatic acids were determined by gas-chromatography after isolation by ion-exchange chromatography and ethylacetate extraction. On an intake of 100 mg L-phenylalanine per kilogram, controls and carriers of classical phenylketonuria excreted nearly the same amounts of aromatic acids (P greater than 0.05). However on an intake of 200 mg per kilogram L-phenylalanine they could be distinguished from one another (P less than 0.001).
1. A single oral dose of [(14)C]Chlorfenvinphos to rats is quantitatively eliminated in 4 days. Rats do not show a sex difference in the elimination pattern and show only a small degree of biological variation in the total excretion data. Of the label 87.2% is excreted in the urine (67.5% in the first day after dosage), 11.2% in the faeces and 1.4% in the expired gases; less than 0.9% of (14)C is present in the gut and contents after 4 days. 2. After oral administration of [(14)C]Chlorfenvinphos to dogs, 94.0% (91.8-97.6%) of the (14)C is excreted in the urine and faeces during 4 days. Dogs do not show a sex difference in the pattern of elimination, and excretion of radioactivity in the urine is very rapid: 86.0% of (14)C during 0-24hr. 3. Chlorfenvinphos is completely metabolized in rats and dogs: unchanged Chlorfenvinphos is absent from the urine and from the carcass, when elimination is complete. In rats, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 32.3% of a dose of Chlorfenvinphos, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid for 41.0%, 2,4-dichloromandelic acid for 7.0%, 2,4-dichlorophenylethanediol glucuronide for 2.6% and 2,4-dichlorohippuric acid for 4.3%; in dogs, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 69.6%, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid] uronic acid for 3.6%, 2,4-dichloromandelic acid for 13.4% and 2,4-dichlorophenylethanediol glucuronide for 2.7%. 4. Dogs and rats show a species difference in the rate of excretion of (14)C in the urine, and in the proportions of the metabolites, with the exception of 2,4-dichlorophenylethanediol glucuronide, that are excreted in the urine. Alternative explanations for the latter species difference are suggested. 5. 2-Chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate and 2,4-dichlorophenacyl chloride probably lie on the main metabolic pathway of Chlorfenvinphos, since, in common with that insecticide, they give rise to [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid and 2,4-dichloromandelic acid as major metabolites in the urine. 6. The proposed scheme for the metabolism of Chlorfenvinphos represents a detoxication mechanism.
We designed a rapid, simple and sensitive method for the determination of norepinephrine (NE) and its metabolites by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. NE, 3,4-dihydroxymandelic acid (DOMA), and 3,4-dihydroxyphenylglycol (DOPEG) were adsorbed on alumina and eluted with 0.2 N HCl. From the remaining solution, normetanephrine and 3-methoxy-4-hydroxyphenylglycol (MOPEG) were extracted with ethyl acetate in the presence of both borate buffer and K2HPO4. Vanillylmandelic acid was extracted with ethyl acetate after acidification of the solution with concentrated HCl. The combined ethyl acetate phase was evaporated and the residue was dissolved in 0.1 N HCl. A 50 mu1 aliquot of each eluate or solution was injected onto the HPLC. Detection limits ranged from 300 pg to 1 ng per initial sample. We used this method to determine substances in the medium following incubation of the rat vas deferens. Approximately 110 and 80 ng/g/10 min of DOPEG and MOPEG, respectively, were present under normal conditions. The electrical stimulation of tissues from the rat vas deferens led to increases in the levels of NE, DOPEG, DOMA and MOPEG. Normetanephrine and vanillylmandelic acid were not detected in the medium. This is probably the first documentation of the endogenous levels of NE and all its metabolites in medium containing tissue of the sympathetic nervous system.
Ethyl glucuronide (EtG), a metabolite of ethanol, is a marker of recent alcohol consumption. In the past few years, its analysis in body fluids has attracted considerable attention because it closes a gap between short time and long time alcohol markers such as ethanol and carbohydrate-deficient transferrin, respectively. The capillary zone electrophoresis (CZE) analysis of EtG in model mixtures and human serum is reported using uncoated and coated fused-silica capillaries together with acidic buffers in the pH range between 3.2 and 4.4 and indirect detection. In these approaches, separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) is based upon transient isotachophoretic stacking referred to as sample self-stacking. The selection of a favorable bufferco-ion and pH is shown to be crucial for optimized sensitivity. Abuffercomposed of 10 mM nicotinic acid and epsilon-aminocaproic acid (pH 4.3) is demonstrated to provide a detection limit for EtG in serum of 0.1 microg/ml, a value that is relevant for clinical and forensic purposes.
Our earlier work demonstrated that the sweetener sucralose, C12 H19 CI3 O8, mixed with water had no effect on intraoral plaque pH. The current study compared the effect on resting plaque pH of sucralose to sucrose when these sweeteners were used in hot coffee at equivalent sweetness levels. Twelve subjects with an identified acidogenic plaque were tested at dicrete sessions, using coffee as vehicle with: (1) sucrose; (2) sucralose; (3) sucralose plus maltodextrin (SM); (4) sucralose plus dextrose and maltodextrin (SMD), and (5) no additional sweetener. Each subject rinsed for 1 min with the test rinse, expectorated, and plaque pH was measured at six dental sites for 60 min using an antimony touch electrode method. Data were summarized for baseline pH, delta pH (baseline pH minus lowest pH attained), minimum pH, and area under the pH curve (AUC). Baseline pH was not different throughout all tests. Quantification of AUC in the various groups showed that sucralose with coffee had no statistically significant impact on plaque acidogenesis. AUC, minimum pH and delta pH were least changed by coffee and sucralose, while the SM and SMD combinations generally led to intermediate changes as compared with coffee sweetened with sucrose or sucralose. Because of its acidic nature, unsweetened coffee led to a modest pH depression, the effect of which appears to be blunted by sucralose. This study confirms that sucralose is non-acidogenic and indicates that sucralose may reduce the acidogenic potential of coffee.
Noradrenaline synthesis and metabolism of dopamine was evaluated in three patients with familial dysautonomia and compared with that of six normal subjects. Each patient and subject was infused with 104.8 muCi of dopamine-2-(14)C dissolved in 1000 ml of physiological saline. The urine was collected during the infusion period and at intervals thereafter. Using a specially designed flow monitor system, the various biosynthetic and metabolic products of dopamine were separated, identified, and their radioactivity measured. The results indicate that in familial dysautonomia the synthesis of noradrenaline is significantly decreased; this is reflected by a decrease in recovery of radioactive noradrenaline as well as various metabolic products of noradrenaline, i.e. 3-methoxy-4-hydroxymandelic acid (MOMA), normetadrenaline, and normetadrenaline conjugate. Concomitant with this decrease in noradrenaline synthesis, there was a shift towards dopamine metabolism as reflected by an increase in the recovery of primary and secondary dopamine metabolites; 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylacetic acid (HVA), 3-methoxytyrosine, and respective conjugates, etc. Whereas all dysautonomic patients showed the same general metabolic pattern as was expected, they varied in degree.
In groups of women taking oral contraceptives and in control groups of women, the serum levels of cortisol, protein-bound iodine, and total thyroxine were measured together with the T(3) binding index. The daily excretion in the urine of free cortisol, 17-hydroxycorticosteroids, 17-ketosteroids, pregnanediol, pregnanetriol, total oestrogens, total catecholamines, and 4-hydroxy-3-methoxymandelic acid was also assayed. The frequency distribution of the values obtained indicates that oral contraceptives have a marked influence on the endocrine environment. The smallest deviations were observed in urinary excretion of total catecholamines and of 4-hydroxy-3-methoxymandelic acid. In some individuals the hormone assays were continued throughout the menstrual cycle. The morning and afternoon levels of serum cortisol tended to increase during the period when the oral contraceptive was being taken.