Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.

Development of an assay using 4-trifluoromethylumbelliferyl as a marker substrate for assessment of drug-drug interactions to multiple isoforms of UDP-glucuronosyltransferases.

Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs) and is one of the most important pathways for elimination of xenobiotics. The aim of the present study was to develop an in vitro assay for assessment of drug-drug interactions related to UGTs applicable to early drug discovery. 4-Trifluoromethylumbelliferyl was tested as a marker substrate for six human recombinant expressed UGT isoforms: 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7. It was shown that 4-trifluoromethylumbelliferyl was glucuronidated by all UGTs tested, except UGT1A4. By using a short HPLC gradient (7 min) and fluorescence detection, the enzyme kinetic parameters for these reactions were obtained. All reactions were found to follow classical Michaelis-Menten kinetics, with K(m) values between 29 microM (UGT1A9) and 80 microM (UGT1A3). The method was validated by using several known competitive inhibitors of UGTs. The most potent inhibition was observed for the reaction between 17alpha-ethynylestradiol and UGT1A1 (K(i) = 10.5 microM), and the weakest interaction was detected for acetaminophen and UGT1A9 (IC(50) > 1 mM). Taken together, we report the development of an assay using 4-trifluoromethylumbelliferyl as a marker substrate for five different human UGT isoforms suitable for the assessment of drug-drug interactions related to UGTs during early drug discovery.

Expression of glutathione transferase isoenzymes in the human H295R adrenal cell line and the effect of forskolin.

In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111-118; L. Staffas, L. Mankowitz, M. Söderström, A. Blanck, I. Porsch-Hällström, C. Sundberg, B. Mannervik, B. Olin, J. Rydström, and J.W. DePierre, Biochem J, 1992, 286, 65-72) isoenzymes of GST, primarily of the mu class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45-50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells.