Abstract
The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation. Glucuronidation of 20-HETE by UDP-glucuronosyltransferases (UGTs) is thought to be a primary pathway of 20-HETE elimination in humans. The present study identified major UGT enzymes responsible for 20-HETE glucuronidation and investigated their genetic influence on the glucuronidation reaction using human livers (n = 44). Twelve recombinant UGTs were screened to identify major contributors to 20-HETE glucuronidation. Based on these results, UGT2B7, UGT1A9, and UGT1A3 exhibited as major contributors to 20-HETE glucuronidation. The Km values of 20-HETE glucuronidation by UGT1A3, UGT1A9, and UGT2B7 were 78.4, 22.2, and 14.8 μM, respectively, while Vmax values were 1.33, 1.78, and 1.62 nmol/min/mg protein, respectively. Protein expression levels and genetic variants of UGT1A3, UGT1A9, and UGT2B7 were analyzed in human livers using Western blotting and genotyping, respectively. Glucuronidation of 20-HETE was significantly correlated with the protein levels of UGT2B7 (r(2) = 0.33, P < 0.001) and UGT1A9 (r(2) = 0.31, P < 0.001), but not UGT1A3 (r(2) = 0.02, P > 0.05). A correlation between genotype and 20-HETE glucuronidation revealed that UGT2B7 802C>T, UGT1A9 -118T9>T10, and UGT1A9 1399T>C significantly altered 20-HETE glucuronide formation (P < 0.05-0.001). Increased levels of 20-HETE comprise a risk factor for cardiovascular diseases, and the present data may increase our understanding of 20-HETE metabolism and cardiovascular complications.
Highlights
The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation
Assays for 20-HETE glucuronidation were performed in a reaction containing 20 M 20-HETE, 15 g of pooled human liver microsome (HLM), and 1.5 mg/ml of alamethicin in 0.5 M Tris-HCl buffer, and 20-HETE glucuronide was detected using LC-MS/MS
The 20-HETE glucuronide was detected at a retention time of 2.91 min in samples incubated with uridine diphosphate glucuronic acid (UDPGA)
Summary
The compound 20-HETE is involved in numerous physiological functions, including blood pressure and platelet aggregation. The present study identified major UGT enzymes responsible for 20HETE glucuronidation and investigated their genetic influence on the glucuronidation reaction using human livers (n = 44). In a study conducted on mice to identify biomarkers for rofecoxib-induced cardiotoxicity, 20-HETE increased by 120-fold after rofecoxib treatment compared with the control groups [6]. This increase in 20-HETE was correlated with increased platelet aggregation and reduced bleeding time, suggesting that rofecoxib increases the levels of 20-HETE. The excretion of 20-HETE glucuronide in urine differs widely among individuals by approximately 10-fold [14], and such variations in 20-HETE levels may affect blood hemostasis or other physiological functions. Urinary excretion of 20-HETE glucoronide provides little information on the regulation of 20-HETE production and its levels in blood, which suggests that the metabolism of 20-HETE in the liver coupled with filtration by the kidney could be the most important determinant of the urinary excretion and circulating levels [17]
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