Ether Glucuronide Research Articles

The formation of β-glucuronidase resistant glucuronides by the intramolecular rearrangement of glucuronic acid conjugates at mild alkaline pH

It is well known to carbohydrate chemists that substituted sugars may undergo facile rearrangement involving the migration of the aglycone from —OH to adjacent —OH. Despite the importance of glycoside conjugates, notably involving glucuronic acid, in the metabolism of xenobiotics, drug metabolism workers have neglected this phenomenon. The potential rearrangement of glucuronides from the biosynthetic C-1 isomers to other positional and stereo-isomers is important, since only 1- O-substituted β- d-glucosiduronates are substrates for β-glucuronidase, which is commonly used to identify such conjugates. The intramolecular rearrangement of clofibryl glucuronide has been studied over the pH range 5.2–8.6, by enzymic hydrolysis with β-glucuronidase, and by HPLC. The amount of clofibric acid released from the conjugate by β-glucuronidase falls with increasing pH of preincubation above pH 7.4, and this is accompanied by the appearance of three new peaks, each containing both clofibric and glucuronic acids, in the HPLC traces of the incubation mixtures. Similar experiments with three ether glucuronides, those of p-nitrophenol, phenolphthalein and 7-hydroxycoumarin, did not show any conversion to β-glucuronidase resistant forms. The phenomenon of intramolecular rearrangement of ester glucuronides must be considered whenever β-glucuronidase is used in the analysis of conjugates of carboxylic acids.

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

Metabolic conjugation of some carboxylic acids in the horse.

1. 14C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques. 2. [14C]Benzoic acid (5 mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24 h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D5]benzoic acid together with [14C]benzoic acid to a pony permitted the mass spectral determination of metabolites of the exogenous benzoic acid metabolites in the presence of the same endogenous compounds. 3. [14C]Salicylic acid (35 mg/kg) was eliminated rapidly in the urine, 98% of the 14C dose being excreted in 24 h. The major excretion product was unchanged salicylate (94% of dose). Gentisic acid, salicyluric acid and the ester and ether glucuronides of salicylic acid were very minor metabolites. 4. 2-Naphthyl[14C]acetic acid (2 mg/kg) was excreted very slowly in the urine, with 53 and 77% of the 14C dose being recovered in six days. 2-Naphthylacetylglycine was the major metabolite (26 and 38% dose) and in addition, the glucuronic acid and taurine conjugates were excreted together with unchanged 2-naphthylacetic acid. 5. The study has shown that the horse can utilize glycine, taurine and glucuronic acid for conjugation of xenobiotic carboxylic acids, and that the relative extents of these pathways are governed by the structure of the carboxylic acid.

Analysis of bile acid glucuronides in urine. Identification of 3 alpha, 6 alpha, 12 alpha-trihydroxy-5 beta-cholanoic acid.

A method is described for the analysis of bile acid glucuronides in urine. Following extraction on Amberlite XAD-2 and separation of bile acid conjugates on DEAP-LH-20, the fractions containing bile acid glucuronides are methyl esterified and filtered through DEAP-LH-20, yielding a fraction of purified bile acid glucuronide methyl esters. Individual glucuronides are separated in a reversed phase system on Lipidex 1000 and are analyzed as TMS ethers by GC/MS. Oxidation with periodate, which transforms the glucuronyl moiety into a formate ester, followed by oxidation with chromic acid and conversion of keto groups to O-methyloximes, permits determination of the site of conjugation with glucuronic acid. The daily excretion of bile acid glucuronides in urine of healthy subjects was 0.8–16.2 μmol (12–36% of the total bile acid excretion in urine). Only ether glucuronides were observed, most of which were otherwise unconjugated. A major part of the bile acids were hydroxylated at C-6, and 3α,6α,12α-trihydroxy-5β-cholanoic acid was identified as a quantitatively important new bile acid. The glucuronyl moiety was attached at C-6 of hyodeoxycholic acid and at C-3 of cholic and chenodeoxycholic acids. The results may indicate a possible coupling of glucuronic acid conjugation with 6α-hydroxylation of secondary bile acids.

Gas chromatography of ether glucuronides as methyl-trifluoroacetyl derivatives

Gas chromatography of ether glucuronides as methyl-trifluoroacetyl derivatives

Permethylation and mass spectrometry of intact ether glucuronides at the low nanomolar level

A group of steroid glucuronides and one drug glucuronide were permethylated with the dimethylsulfinylmethide anion and methyl iodide. All permethylated products were easily recognized as glucuronides from their mass spectra. Overmethylation was observed in those steroid samples which contained isolated or α , β-unsaturated ketones, but this did not hinder the interpretation of the spectra. The technique has further application for the study of drug metabolism.

Mass spectral identification of probenecid metabolites in rat bile

The metabolism of probenecid (p-(di-n-propylsulfamyl)benzoic acid) by the liver has been studied in biliary cannulated rats with ligated renal pedicles. The unchanged drug, N-depropylated probenecid, and three glucuronides of metabolites of this drug readily appeared in the bile. When rats were given a single 50 mg/kg dose of [ 14C] probenecid, 96% of the administered radioactivity was excreted in the bile of renal-ligated rats in 8 hr. Approximately 64% of the drug in this system appeared as metabolites. Four major metabolites were identified by their gas-liquid chromatograms and mass spectra. They were the N-dealkylated drug, an ester glucuronide of a side chain oxidized metabolite (metabolite B) and two ether glucuronides of side-chain hydroxylated drug (metabolites A and C). None of the previously reported acyl glucuronide of probenecid was found in rat bile and none of the metabolites included in this paper have been reported before. In the homozygotous Gunn rat, reported to be deficient in glucuronidating enzymes, the same biliary metabolites as appeared in Sprague-Dawley rats, were found. It is proposed that one may be able to very rapidly assess the importance of drug metabolism for a new agent if it is administered to renal-ligated animals, collected in bile, and then subjected to GLC-MS procedures. The overall technique appears to have several advantages over the usual methods of collecting and assaying urine for drugs and their metabolites.

The metabolism of cis- and trans-decalin.

1. The metabolism of cis- and trans-decalin in the rabbit has been investigated. 2. Both hydrocarbons were oxidized to racemic secondary alcohols and excreted as ether glucuronides in amounts equal to about 60% of the dose administered. The principal glucuronides were isolated as triacetyl methyl esters and as sodium salts. 3. cis-Decalin gave rise mainly to (+/-)-cis-cis-2-decalol, together with a little cis-trans-2-decalol, and trans-decalin mainly to (+/-)-trans-cis-2-decalol and a small amount of trans-trans-2-decalol. 4. These results suggest that biological oxidation of the decalins does not occur via a free-radical mechanism. An attempt is made to explain why racemic alcohols are obtained, rather than the more typical optically active products of enzymic reaction, and a mechanism is proposed. It is suggested that enzymes similar to steroid hydroxylases are involved.

Method for the Direct Measurement of Acetylsalicylic Acid in Human Blood

A procedure was developed for directly determining acetylsalicylic acid in human blood specimens. The method instantly stops enzymatic hydrolysis, removes salicylic acid and conjugates of salicylic acid by reaction with ceric ammonium nitrate, automatically hydrolyzes acetylsalicylic acid, and determines the resulting salicylic acid fluorometrically. It sensitively measures acetylsalicylic acid in the presence of salicylic acid, salicylamide, salicyluric acid and salicylic acid ether glucuronide (O-carboxyphenyl glucuronide). A procedure was developed for directly determining acetylsalicylic acid in human blood specimens. The method instantly stops enzymatic hydrolysis, removes salicylic acid and conjugates of salicylic acid by reaction with ceric ammonium nitrate, automatically hydrolyzes acetylsalicylic acid, and determines the resulting salicylic acid fluorometrically. It sensitively measures acetylsalicylic acid in the presence of salicylic acid, salicylamide, salicyluric acid and salicylic acid ether glucuronide (O-carboxyphenyl glucuronide).

The metabolism of metronidazole (1 -2′-hydroxyethyl-2-methyl-5-nitroimidazole)

The metabolism of metronidazole has been studied in man and dog, and was similar in both species. The urine contained unchanged metronidazole (I), the corresponding acid, 2-methyl-5-nitroimidazole-1-ylacetic acid (II), and a second metabolite which has been tentatively identified as the ether glucuronide of metronidazole (III).

(GLUCURONIC ACID EXCRETION IN URINE OF RABBITS BY EXPOSURE TO TOLUENE, XYLENE AND CARBON DISULFIDE.)

Free-glucuronic acid and glucuronides excretion seem to be necessary to discuss toxicity and detoxition mechanism of various chemical substances in animal body. Present report was made with rabbits exposed to toluene, xylene and carbon disulfide in relatively low concentratons. Namely, the concentrations of these organic solvents were 211±19.3ppm in toluene, 209±28.5ppm in xylene and 108±17.5ppm in carbon disulfide respectively. Exposure chambers were designed for dynamic exposure. The time of exposure was 6 hours in all cases of these experiments.Analyses of urinary free-glucuronic acid and ether glucuronide were made by the naphtoresorcin picrate methods. Both wrs corrected by the concentration of urinary creatinine. Therefore, the value of glucuronic acid excretion in urine was expressed as G/C and OG/C ratio. The following results were obtained.1) The immediate decrease of G/C ratio in urine was observed after exposure of toluene. The reelevation to pre-experimental level after a cessation of the exposure would be comparatively slow. These responses mentioned above were shown more markedly in summer than that in winter. Also the response of OG/C ratio in urine resembled the change of G/C ratio exclusively.2) In the case of xylene exposure, the range of increase as to glucuronic acid excretion was from 117% to 129% in G/C ratio and from 121% to 142% in OG/C ratio. Two days after the exposure no increase of glucuronic acid in urine was observed. Metabolites of xylene in animal body would be apparently conjugated with glucronic acid and then eliminated into the urine shortly.3) Transient decreases of G/C and OG/C ratio were shown after exposure of carbon disulfide as well as that in the case of toluene inhalation. However, they were slight as compared with the decrease of G/C ratio by exposure to carbon disulfide in higher concentration.

A glucuronide metabolite of sulfamethomidine in human urine

Human urine collected after the oral administration of sulfamethomidine contains a glucuronide metabolite in addition to the N 4-acetyl conjugate. Urinary sulfamethomidine glucuronide is an ether glucuronide having the glucuronic acid moiety linked to the pyrimidine nucleus. The Bratton-Marshall test procedure was employed to form azo derivatives of sulfanilamide, sulfanilic acid, hydroxysulfanilic acids, sulfamethomidine, and of sulfamethomidine glucuronide and its aglycon. These pigmented products were shown to be separable by paper chromatography and are considered to have general application to the metabolic study of sulfonamides and other aromatic amines. The azo product of sulfamethomidine glucuronide was partially purified by column chromatography with Florisil as the adsorbent.

Metabolisme d'un conservateur alimentaire: L'acide parahydroxybenzoique et ses esters

We made a study of the form in which p-hydroxybenzoic acid and its methyl,ethyl and propyl esters are eliminated by the rat, and of the rate at which the phenolic acid and its metabolites appear in the urine. We were able, by means of paper chromatography and the use of Millon's reagent, to show the presence in the urine of p-hydroxybenzoic acid, p-hydroxyhippuric acid, an ester and ether glucuronides, an ethereal sulphate, and an unidentified compound. Study of the kinetic process of the appearance of the phenolic acid and its metabolites in the urine showed that free p-hydroxybenzoic acid appeared first, followed by the glucuronide and p-hydroxyhippuric acid, the concentration of which increased as that of the free p-hydroxybenzoic acid fell off. During the experiment the concentration of the phenolic acid in the blood remained extremely low.