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Synthesis of benzyl 2, 4, 5, 6-tetra-O-benzyl-L-gulonate (IX), a prospective starting material leading to β-keto-L-gulonic acid (II), was described. Oxidation of IX with the Pfitzner-Moffatt reagent gave a mixture of β-keto esters (X, XI) epimeric at C-2 and their enol-form, which was debenzylated by catalytic hydrogenation to afford L-xylulose (III), decarboxylation product of II, as the principal product and probably 2, 3-diketo-L-gulonic acid (XII).

A quantitative chemical method for the determination of urinary d -glucaric acid is described. Following the separation of d -glucaric acid by ion-exchange column chromatography using Dowex 1 X-8 borate, a portion of the sample is treated under neutral conditions (pH ca. 6.8) with periodic acid; the glyoxylic acid obtained is condensed with phenylhydrazine. The glyoxylic acid phenylhydrazone thus formed undergoes mild oxidation with potassium ferricyanide in the presence of excess phenylhydrazine, resulting in the intensely colored 1,5-diphenylformazan, the absorbancy of which is determined at 520 mμ. This method has been applied to determine d -glucaric acid excretion in human, rat, and guinea-pig urine; the results are compared with those obtained by the enzymic method of Marsh.

The nuclear magnetic resonance spectra of anomeric pairs of acetylated aryl D-glycopyranosides were measured in deuteriochloroform at 60 MHz. The shift reagent Eu (FOD)3 proved to be useful to distinguish the H-1 proton of the β-anomer and permitted the assignment of all the ring protons in some cases. The virtual long-range coupling of the H-5 proton with the H-3 proton of aryl β-D-glucopyranosiduronates was confirmed with an aid of the shift reagent. All of the acetoxy signals were well resolved by the addition of the shift reagent.

Serum D-glucaric acid (V) level was determined by the colorimetric method, especially after oral administration of D-glucuronolactone (I) or D-glucarolactones (II, III) to man. The highest level was observed at one hour after administration simultaneously with the lowest level of serum β-glucuronidase activity as well as the maximal β-glucuronidase inhibition of serum. In vivo conversion of I into β-glucuronidase inhibitor was thus demonstrated.

Uridine diphosphate (UDP)-glucuronosyltransferase (GT) active on androsterone (AD) and 4-nitrophenol (NP) was solubilized from male rat liver microsomes of the Wistar strain. The precipitate obtained in the 60%-satd. ammonium sulfate was purified by diethylaminoethyl (DEAE)-cellulose chromatography and affinity chromatography on UDP-hexanolamine Sepharose 4B. DEAE-cellulose chromatography showed the existence of two peaks of GT active on AD and NP. Peak I was found in rats with the high-activity and low-activity phenotypes in terms of AD glucuronidation and had high NP-GT and low AD-GT activities. In contrast, peak II was found only in rats with the high-activity phenotype, corresponded to high AD-GT activity and had comparatively low NP-GT activities. The corresponding peak in rats with the low-activity phenotype had only NP-GT activity. Comparison of Km values for AD obtained from microsomes and purified enzymes provides evidence that AD-GT isoenzyme should be deficient in Wistar rats with the low-activity phenotype and that AD glucuronidation should be catalyzed poorly by other GT isoenzyme in these rats.

A jaundiced rat strain was derived from a cross between Gunn and Wistar-Imamichi rats, and inbreeding was continued by forced heterozygosis with jaundice locus. These Gunn rats have black pigment on heads and a black stripe on their backs similar to Long-Evans rats. Wistar rats with low activity in androsterone (AD) glucuronidation were selected and inbred (LA Wistar rats). The levels of hepatic uridine diphosphate-glucuronosyltransferase (GT) and sulfotransferase (ST) activities as well as cytochrome P-450 contents were compared in these mutant strains. Gunn rats were devoid of bilirubin (BL) GT activity but had high AD GT activity. LA Wistar rats had very low AD GT activity but showed high BL GT activity. Native and Triton X-100-stimulated GT activities toward 2-aminophenol and 4-nitrophenol (NP) were much lower in Gunn rats than in LA Wistar rats. When N-nitrosodiethylamine was added to the incubation media, these GT activities were stimulated equally to high levels in both mutants. N-Nitrodiethylamine provided a similar stimulatory effect on NP GT activity. There were no significant differences in ST activities toward cortisol, AD and NP and cytochrome P-450 contents in the two mutant strains. These results indicate that Gunn and LA Wistar rats have a different deficiency in GT isoenzymes.

Examination of twelve nitrosamines and seven nitramines revealed that nitramines modify UDP-glucuronosyltransferase activity in a manner similar to that of nitrosamines. Only N,N-diethyl-substituted nitrosamine and nitramine significantly stimulated transferase activity toward 2-aminophenol and 4-nitrophenol but not toward phenolphthalein and androsterone. Elongation of the alkyl chains or introduction of carboxy, hydroxy, or oxo groups into the alkyl chains did not result in stimulatory ability, and some of these compounds inhibited the transferase activity.

A new and simple method for the determination of rat liver microsomal uridine diphosphate glucuronosyltransferase activity toward 4-nitrophenol, phenolphthalein, and testosterone has been developed with the use of high-pressure liquid chromatography (hplc). After incubation of the substrate with microsomal fractions in the presence of uridine diphosphate glucuronic acid, the reaction is stopped by heating, mixed with methanol, and centrifuged to give the supernatant, which is analyzed directly by hplc. The unreacted substrate as well as its glucuronide can be quantitated from the peak-height method. When the pure glucuronide is not available, the standard curve for the glucuronide is obtained from the enzymatically produced glucuronide.

A mixture of testosterone-4-14C and testosterone-1,2-3H-17-glucosiduronate was intraperitoneally administered into male and female rats with bile fistulas. Biliary metabolites were separated and purififd by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the blue. 5β-Androstane-3α, 17β-diol was found principally in monoglucosiduronate fraction and was produced preferentially from the injected conjugate in both sexes. Very marked sex differences from the injected conjugate in both sexes. Very marked sex differences were observed in the following metabolites: Androsterone was present only in the female as monoglucosidironate, which was preferentially derived from testosterone. 5α-Androstane-3α,17β-diol was identified in both monoglucosiduronate and diconjugate fractions of the female, which was formed significanrly more from the conjugate than testosterone. These findings provide evidence that testosterone glucosiduronate could be converted directly into 5α-steroids as well as 5β-ones in vivo . In marked contrast, the major portion of testosterone was metabolized to polar steroids in the male.

Gunn rats, which have defects in bilirubin and 4-nitrophenol UDP-glucuronyltransferases (GT), were crossed with LA Wistar rats with a defect in androsterone GT. The F1 hybrids showed normal GT activities towards androsterone, bilirubin and 4-nitrophenol, demonstrating that Gunn and LA (‘low activity’) Wistar rats inherit a homozygous dominant trait for androsterone GT and bilirubin GT respectively. The F2 progeny showed four different combinations of bilirubin and androsterone GT activities: defects in both GT activities, a single defect in bilirubin GT activity, a single defect in androsterone GT activity and two normal GT activities. They were segregated in the approximate ratio of 1:3:3:9, which is compatible with Mendel's Principle of Independent Assortment. These results provide evidence that androsterone GT and bilirubin GT are located on different chromosomes. In the F2 generation, defective bilirubin and 4-nitrophenol GT activities were not segregated, indicating that these two mutant genes are closely linked on the same chromosome.

Estradiol benzoate (EB), testosterone propionate (TP), progesterone (PG), corticosterone (CS), 3-methylcholanthrene (MC) and phenobarbital (PB) were administered to Wistar rats and their effects on hepatic sulfotransferase (ST) activities toward androsterone (AD) and 4-nitrophenol (NP) were determined. ST activity toward AD was increased by pretreatment with EB and PG in male rats. ST activity toward NP was increased by administration of TP and PG in females, whereas the enzyme activity was suppressed by pretreatment with EB in males. Administration of CS, MC and PB did not significantly affect ST activities toward AD and NP. These results indicate that sex difference in rat liver ST activities appears to be primarily regulated by androgenic, estrogenic and progestational hormones.

The fate of 4- 14 C-testosterone 17-N-acetyl-glucosaminide has been studied in two subjects. Following intravenous administration urinary excretion of radioactivity was rapid. Testosterone N-acetylglucosaminide was the principal metabolites; etiocholanolone and androsterone conjugated with glucuronic acid were present in a ratio of 7.3. Following oral administration only traces of metabolites conjugated with the aminosugar were present. Etiocholanolone and androsterone were obtained in ratio of 7.3. In both studies etiocholane-3α,17β-diol was the predominant non-ketonic metabolite. These results were compared with the metabolites obtained following labelled testosterone and testosterone 17-glucosiduronate. The results demonstrate that 1) the cleavage of the glycosidic linkage is almost complete by the oral route, 2) ring A is reduced prior to hydrolysis of the aminosugar and 3) there is a directive influence of the aminosugar toward 5β-reduction similar to that of a glucosiduronate group at C-17.

The structure of the principal colored product formed in the determination of glyoxylic acid which involves the oxidation of glyoxylic acid phenylhydrazone in the presence of excess phenylhydrazine is unequivocally identified with 1,5-diphenylformazan, as opposed to the structure, 1,5-diphenylformazan-3-carboxylic acid, previously proposed.

D-Mannaric acid (II) was isolated and identified from human urine after oral administration of D-mannuronolactone (I). It was also isolated and identified from the incubation mixture containing I, nicotinamide adenine dinucleotide (NAD) and D-glucuronolactone dehydrogenase preparation purified from guinea pig liver, thus demonstrating the conversion of I into II in mammalian systems likewise the conversion of D-glucuronolactone (III) into D-glucaric acid (IV).