salicylates and salicylacyl-glucuronide

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C18 column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3-4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2-20 micrograms/ml and linearity was observed from 0.1-200 micrograms/ml and 5-2000 micrograms/ml for SA in plasma and urine, respectively. The method was validated to 0.2 microgram/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.

Topics: Chromatography, High Pressure Liquid; Gentisates; Glucuronates; Hippurates; Humans; Hydroxybenzoates; Reproducibility of Results; Salicylates; Salicylic Acid; Sensitivity and Specificity

Many acyl glucuronides are labile, reactive and form covalent adducts with proteins. In the present experiment, the stability of salicyl acyl glucuronide (SAG), its reactivity with serum albumin in vitro and the influence of renal failure in rats on the disposition of SAG and covalent binding of salicylate (SA) to rat plasma proteins were investigated. In vitro studies showed that SAG was hydrolyzed to SA or undergoes isomerization to positional isomers. The half-life of SAG was 1.3 hr in 0.15 M phosphate buffer at pH 7.4 and 37 degrees C, but the stability of its isomers was much greater with an apparent half-life of 19 hr. Incubation of SAG in solutions of human serum albumin revealed the formation of covalent adducts with the protein, with maximal binding of 2.8% of total SA equivalents added to the solution. For in vivo studies, one group of rats was administered 5 mg/kg of uranyl nitrate i.p. to induce renal failure. After administration of 100 mg/kg of SA i.v., the AUC 0-26 hr of SAG in rats with renal failure was 9 times higher than that observed in control rats. The apparent clearance of SA decreased from 64 +/- 21 ml/hr in control rats 28 +/- 8.7 ml/hr in rats with renal failure. The level of SA covalent adducts with plasma proteins reached about 0.5 ng/mg of protein in control rats, whereas in rats with renal failure the binding was increased significantly and achieved an average peak concentration of 18 ng/mg of protein when measured 26 hr after dosing. The data indicate that reactive SAG can accumulate in renal failure which then increases covalent addition of SA to proteins. Such binding may have a role in enhancing the potential for toxicity of acidic drugs such as SA in renal disease.

Topics: Animals; Binding, Competitive; Blood Proteins; Glucuronates; Rats; Renal Insufficiency; Salicylates

Salicyl acyl glucuronide (SAG) is a significant metabolite of salicylic acid (SA) and aspirin. We have shown that, under physiological conditions in vitro, SAG undergoes rearrangement in a manner consistent with acyl migration to its 2-, 3- and 4-O-acyl positional isomers as the predominant pathway (T1/2 values were 1.4-1.7 hr in buffer at pH 7.4 and 37 degrees). Incubation of SAG or a mixture of its rearrangement isomers (iso-SAG) (each at approximately 50 micrograms SA equivalents/mL) with human serum albumin (HSA, at approximately 40 mg/mL) revealed the formation of covalent adducts with the protein, with peak concentrations of 1-2 micrograms SA equivalents/mL. The data support a role for the rearrangement/glycation mechanism of adduct formation. Covalent adducts of SA were also detected in the plasma of humans taking aspirin (at > or = 1200 mg/day), but the concentrations were low (<< 100 ng SA equivalents/mL). Reactivity of SAG thus provides a mechanism (though of uncertain quantitative importance) of covalent attachment of the salicyl moiety of aspirin to tissue macromolecules, which is in addition to its well-known acetylating capacity.

Topics: Animals; Aspirin; Buffers; Glucuronates; Half-Life; Humans; Hydrogen-Ion Concentration; Rats; Salicylates; Salicylic Acid; Serum Albumin

A novel direct high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of three salicylate glucuronide conjugates and other salicylate metabolites in human urine has been developed. Salicylate glucuronide conjugates were purified by HPLC from the urine of a volunteer after oral administration of aspirin and identified by selective hydrolysis with beta-glucuronidase and with sodium hydroxide. This method gave high reproducibility with coefficients of variation less than 10%. The total urinary recovery of salicylic acid after a single 1.2-g dose of soluble aspirin was greater than 90%. This assay has been successfully used to re-evaluate the capacity-limited pharmacokinetics of salicylic acid in humans.

Topics: Administration, Oral; Aspirin; Chromatography, High Pressure Liquid; Glucuronates; Hippurates; Humans; Salicylates

1. Metabolism of aspirin was studied in 10 human volunteers who took a therapeutic dose (600 mg) by mouth and in nine patients who took aspirin in overdose. 2. Salicyluric acid was the major urinary metabolite in volunteers (63.1 +/- 8.4% of dose in 0-8 h). In overdose patients, salicyluric acid in urine was decreased (30.0 +/- 8.2%, 0-24 h, P less than 0.001) and there was increased elimination of salicyclic acid (34.1%, P less than 0.005), salicyl acyl glucuronide (14.4%, P less than 0.05) and gentisuric acid (5.3%). 3. Metabolism of orally administered 14C-aspirin in rats over a 10-fold dose range (10-100 mg/kg) resulted in excretion of 81-91% dose in urine in the first 24 h. Salicyclic acid was the major urinary metabolite (43-51% dose). Excretion of salicyluric acid decreased with increasing dose, whereas gentisic acid and salicyl phenolic and acyl glucuronides increased. 4. The profile of aspirin metabolites was qualitatively similar in man and rat but there were quantitative differences. Limited capacity to form salicyluric acid was observed in both species. Dependence on this pathway in rat was low and was compensated by increased utilization of other routes; dependence on salicyluric acid formation in man was high and in overdose, compensation by other routes was incomplete.

Topics: Adolescent; Adult; Animals; Aspirin; Female; Gentisates; Glucuronates; Hippurates; Humans; Kinetics; Male; Middle Aged; Rats; Rats, Inbred Strains; Salicylates; Salicylic Acid