RESUMO
Long acting inhaled muscarinic receptor antagonists, such as tiotropium, are widely used as bronchodilator therapy for chronic obstructive pulmonary disease (COPD). Although this class of compounds is generally considered to be safe and well tolerated in COPD patients the cardiovascular safety of tiotropium has recently been questioned. We describe a rat in vivo model that allows the concurrent assessment of muscarinic antagonist potency, bronchodilator efficacy and a potential for side effects, and we use this model to compare tiotropium with NVA237 (glycopyrronium bromide), a recently approved inhaled muscarinic antagonist for COPD. Anaesthetized Brown Norway rats were dosed intratracheally at 1 or 6h prior to receiving increasing doses of intravenous methacholine. Changes in airway resistance and cardiovascular function were recorded and therapeutic indices were calculated against the ED50 values for the inhibition of methacholine-induced bronchoconstriction. At both time points studied, greater therapeutic indices for hypotension and bradycardia were observed with glycopyrronium (19.5 and 28.5 fold at 1h; >200 fold at 6h) than with tiotropium (1.5 and 4.2 fold at 1h; 4.6 and 5.5 fold at 6h). Pharmacokinetic, protein plasma binding and rat muscarinic receptor binding properties for both compounds were determined and used to generate an integrated model of systemic M2 muscarinic receptor occupancy, which predicted significantly higher M2 receptor blockade at ED50 doses with tiotropium than with glycopyrronium. In our preclinical model there was an improved safety profile for glycopyrronium when compared with tiotropium.
Assuntos
Broncoconstrição/efeitos dos fármacos , Broncodilatadores/farmacocinética , Sistema Cardiovascular/efeitos dos fármacos , Glicopirrolato/farmacocinética , Antagonistas Muscarínicos/farmacocinética , Derivados da Escopolamina/farmacocinética , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Bradicardia/induzido quimicamente , Bradicardia/fisiopatologia , Testes de Provocação Brônquica , Broncodilatadores/administração & dosagem , Broncodilatadores/sangue , Broncodilatadores/toxicidade , Sistema Cardiovascular/fisiopatologia , Glicopirrolato/administração & dosagem , Glicopirrolato/sangue , Glicopirrolato/toxicidade , Frequência Cardíaca/efeitos dos fármacos , Hipotensão/induzido quimicamente , Hipotensão/fisiopatologia , Masculino , Modelos Biológicos , Antagonistas Muscarínicos/administração & dosagem , Antagonistas Muscarínicos/sangue , Antagonistas Muscarínicos/toxicidade , Ligação Proteica , Ensaio Radioligante , Ratos Endogâmicos BN , Receptor Muscarínico M2/efeitos dos fármacos , Receptor Muscarínico M2/metabolismo , Medição de Risco , Derivados da Escopolamina/administração & dosagem , Derivados da Escopolamina/sangue , Derivados da Escopolamina/toxicidade , Brometo de TiotrópioRESUMO
The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (P450s) is described. cDNAs from seven canine P450s were subcloned into inducible expression plasmids and, for the first time, cotransformed and expressed with a canine P450 oxidoreductase in Escherichia coli to produce active, full-length, native sequence P450s. Enzyme expression levels, although variable, were generally sufficient to enable short incubation times and to limit the total protein present in enzyme incubations. Steady-state kinetics of CYP1A1, 2C21, and 2D15 Bactosomes demonstrated similarities with dog liver microsomes or Baculosomes. However, 3A12 lacked substrate inhibition in the formation of 1'-OH midazolam, and 2B11 displayed non-Michaelis-Menten kinetics, suggesting possible differences in protein interaction effects. In monitoring the metabolites of common P450 substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1'-hydroxylation were shown to be relatively selective reactions catalyzed by CYP1A1, 2B11, and 2D15, respectively. 1'-OH midazolam was formed in higher quantities by CYP2B11 and 2C21 than by 3A12, raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant P450s was produced to make up for the lack of commercially available canine P450 isoforms. The Bactosomes are expected to facilitate reaction phenotyping and metabolic drug-drug interaction assessment in canine drug development and to enable the study of interspecies differences in P450-mediated drug metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Proteínas de Membrana/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Animais , Sequência de Bases , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA , DNA Complementar , Cães , Proteínas de Membrana/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Glucuronidation represents a novel mechanism of intrinsic drug resistance in colon cancer cells. To safely reverse this mechanism in vivo, it is essential to identify which isoforms of UDP-glucuronosyltransferases are responsible for catalysing this drug metabolism in tumour tissue. MATERIALS AND METHODS: LC-MS was applied to measure rates of glucuronidation of two anticancer compounds (SN-38 and NU/ICRF 505) by patient colon cancer biopsies and paired normal colon. RESULTS: Three independent lines of enquiry indicated that, in the tumour specimens, SN-38 was glucuronidated primarily by UGT1A1, the isozyme generally recognised as being responsible for hepatic detoxification of this compound, while with NU/ICRF 505 two candidate isoforms emerged - UGT1A8 and/or UGT1A10 - both of which are not normally expressed in the liver. CONCLUSION: These data suggest that tumour selective modulation of this drug resistance mechanism in patients may be feasible with NU/ICRF 505 but more difficult to realise with SN-38. De novo drug resistance is recognised as contributing significantly to the poor response rates of colorectal cancer (CRC) to chemotherapy (1). Nonetheless, the underlying mechanisms responsible for drug insensitivity remain
Assuntos
Adenocarcinoma/metabolismo , Antraquinonas/farmacocinética , Antineoplásicos Fitogênicos/farmacocinética , Camptotecina/análogos & derivados , Neoplasias do Colo/metabolismo , Tirosina/análogos & derivados , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Animais , Antraquinonas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacocinética , Camptotecina/farmacologia , Colo/enzimologia , Colo/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Cricetinae , Resistencia a Medicamentos Antineoplásicos , Glucuronídeos/biossíntese , Glucuronosiltransferase/metabolismo , Células HT29 , Humanos , Irinotecano , Tirosina/farmacocinética , Tirosina/farmacologiaRESUMO
Colon cancer exhibits inherent insensitivity to chemotherapy by mechanisms that are poorly characterized. We have shown that human colon cancer cells are efficient in drug conjugation catalyzed by UDP-glucuronosyltransferases (UGTs) and now report on the role of glucuronidation in de novo resistance to two topoisomerase I inhibitors. Identification of the UGT responsible for glucuronidation of SN-38 and the anthraquinone NU/ICRF 505 was achieved by first using a panel of human cDNA-expressed isozymes to measure conjugating activity. HT29 colon cancer cells were then probed by reverse transcriptase-PCR, Western Blot analysis, and liquid chromatography with mass spectrometry for their profile and activity of UGT isozymes and screened for effective inhibitors of glucuronidation. Expression analysis was also conducted in colon cancer biopsies and paired adjacent normal colon specimens. UGT1A9 was identified as the isozyme catalyzing biotransformation of the two compounds in HT29 cells and propofol as an effective competitive inhibitor of this metabolism. Inhibition of glucuronidation resulted in up to a 5-fold enhancement in drug activity. The majority of colon cancer biopsies studies expressed UGT protein at levels greater than in HT29 cells but with marked interpatient variations and proficiently glucuronidated the two anticancer drugs. A range of UGT aglycones were capable of modulating glucuronidation in the biopies with octylgallate being 10-fold more potent (ID(50) 24 microM) than propofol. In a subset of tumors (33%), UGT protein levels and activity exceeded that of paired normal colon. Glucuronidation may represent a mechanism of intrinsic drug resistance in colon cancer open to modulation by a range of food additives and proprietary medicines.
Assuntos
Antraquinonas/metabolismo , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Aditivos Alimentares/farmacologia , Glucuronídeos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Biópsia , Catálise , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Irinotecano , Isoenzimas/metabolismo , Propofol/metabolismo , Propofol/farmacologia , Inibidores da Topoisomerase IRESUMO
Valproic acid glucuronidation kinetics were carried our with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high K(m) values with microsomes and expressed UGTs (2.3-5.2mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively (K(i)=1.6+/-0.06mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/metabolismo , Esteroides/metabolismo , Ácido Valproico/farmacologia , Animais , Ligação Competitiva , Células Cultivadas , Cricetinae , Glucuronosiltransferase/efeitos dos fármacos , Glucuronosiltransferase/genética , Humanos , Cinética , Microssomos Hepáticos/metabolismoRESUMO
Glucuronidation is a major route of clearance for a diverse set of both drug and endogenous substrates. The present study was undertaken to redress the lack of molecular information currently available on drug glucuronidation by the dog, a species widely used in metabolism studies by the pharmaceutical industry. A novel dog uridine diphosphate glucuronosyltransferase (UGT), designated UGT2B31 (GenBank Accession Number: AY135176), has been isolated from a dog cDNA library, expressed in V79 cells and characterised using various methods: (i) UGT2B31 sequence has been compared with mammalian UGT sequences using both sequence alignments and phylogenetic analysis; and (ii) the substrate specificity of UGT2B31 has been determined using functional analysis and compared with that obtained using UGT2B7 and dog liver microsomes. The following results were obtained: (i) sequence alignments between UGT2B31 and UGT2B15 gave the greatest degree of identity (76%); however, human UGT2B4, human UGT2B7, monkey UGT2B9 (all 75%), and rat UGT2B1 (73%) also gave a high degree of identity; (ii) phylogenetic analysis determined UGT2B31 to be most closely related to rat UGT2B1; (iii) UGT2B31 displayed a substrate specificity similar to human UGT2B7 and rat UGT2B1, catalysing the glucuronidation of phenols, opioids, and carboxylic acid-containing drugs; and (iv) UGT2B31 only formed morphine-3-glucuronide; however, kinetic analysis determined the K(m) of this reaction to be similar to that observed with UGT2B7 (both approximately 1300 microM). The results suggest that UGT2B31 plays a crucial role in drug detoxification by the dog and may be the canine equivalent of human UGT2B7.
Assuntos
Glucuronosiltransferase/genética , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Biotransformação , Linhagem Celular , Clonagem Molecular , Cricetinae , Cães , Biblioteca Gênica , Glucuronosiltransferase/classificação , Glucuronosiltransferase/metabolismo , Humanos , Pulmão , Mamíferos , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TransfecçãoRESUMO
As part of a program to identify novel mechanisms of resistance to topoisomerase I (topo I) inhibitors, the cellular pharmacology of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of clinically used irinotecan (CPT-11) and NU/ICRF 505, an anthraquinone-tyrosine conjugate, has been investigated in two human colorectal cancer (CRC) cell lines. Two novel metabolites of NU/ICRF 505 (M1 and M2) and a single metabolite of SN-38 (M1) were detected by high performance liquid chromatography in the culture medium of HT29 cells but were absent in HCT116 cells. Identities of all three metabolites were established by a combination of biochemical and physicochemical techniques. M1 of SN-38 was the C10-(beta)-glucuronide of the parent lactone while M1 of NU/ICRF 505 was the C4-O-glucuronide and M2 the tyrosine-O-glucuronide, both of the parent compound. Drug transport studies revealed that by 24hr HT29 cells had effectively cleared 82.5% of NU/ICRF 505 (10 microM) into the culture medium as the two glucuronides. In contrast, intracellular concentrations of NU/ICRF 505 were maintained in HCT116 cells in the absence of glucuronidation at a level 550 times greater than in HT29 cells. HT29 cells cleared 40.9% of SN-38 (1 microM) as the glucuronide to the culture medium, while the parent drug was maintained at a level 2-fold greater in HCT116 cells. Enhanced drug clearance due to glucuronidation may contribute to intrinsic drug resistance of human CRC.
Assuntos
Antraquinonas/farmacocinética , Camptotecina/farmacocinética , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacocinética , Glucuronídeos/farmacocinética , Inibidores da Topoisomerase I , Tirosina/análogos & derivados , Tirosina/farmacocinética , Antraquinonas/metabolismo , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Neoplasias do Colo/patologia , Glucuronatos/metabolismo , Células HT29 , Humanos , Irinotecano , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Taxa de Depuração Metabólica , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation.
Assuntos
Canais de Cátion TRPM/antagonistas & inibidores , Anilidas/farmacologia , Animais , Regulação da Temperatura Corporal , Carbamatos/farmacologia , Feminino , Gânglios Espinais/metabolismo , Cobaias , Células HEK293 , Humanos , Masculino , Mentol/análogos & derivados , Mentol/farmacologia , Músculo Liso/metabolismo , Neurônios/metabolismo , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/fisiologia , Uretra/metabolismo , Bexiga Urinária/metabolismoRESUMO
UGT1A6 and UGT1A9 have both been demonstrated to rapidly glucuronidate simple phenolic compounds. A series of simple phenols were selected and screened with both isoforms and then used as model substrates for the generation of V(max) and K(m) values. UGT1A6 showed a more restricted acceptance of phenolic substrates compared with UGT1A9. However, the affinity of UGT1A6 for these compounds exhibited higher K(m) values than UGT1A9, although rates of turnover were similar. Molecular surface-weighted holistic invariant molecular descriptors were generated for each substrate and used to produce the first quantitative structure activity relationship models generated for expressed human UGTs. Models relating log of the K(m) value to the generated descriptors correlated well with the experimental data r(2) value of 0.996 for UGT1A6 and r(2) value of 0.83 for UGT1A9. Cross validation by a leave-one-out method also showed good predictive capability within the subset with a q(2) value of 0.98 for UGT1A6 and q(2) value of 0.73 for UGT1A9. Empirically, UGT1A6 V(max) decreased as the 4-substituent increased in size, and a trend was observed when UGT1A6 V(max) was plotted against molecular volume. The larger UGT1A6 substrates were typified by low activity and lower K(m) values than their smaller counterparts. Extrapolating from this, it was demonstrated that phenols with large 4-substituents, which were not UGT1A6 substrates, could inhibit 4-ethylphenol glucuronidation. The K(m) values for UGT1A9 showed a similar relationship to UGT1A6 but with much lower K(m) values and greater variability in range of this value.
Assuntos
Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Fenóis/farmacocinética , Linhagem Celular , Humanos , Isoenzimas/metabolismo , Cinética , Estrutura Molecular , Fenóis/química , Fenóis/metabolismo , Relação Quantitativa Estrutura-AtividadeRESUMO
HMR1098, a novel KATP-blocking agent, is metabolized to form an S-glucuronide in rat and dog bile. Synthesis of the S-glucuronide metabolite was studied in human liver and kidney microsomes. Recombinant UPD-glucuronosyltransferases (UGTs) were screened for activity, and kinetic analysis was performed to identify the isoform or isoforms responsible for the formation of this novel S-glucuronide in humans. S-Glucuronidation is relatively rare, but from this study it appears that S-glucuronides are not generated exclusively by a single UGT isoform. From the panel of recombinant isoforms used, both UGT1A1 and UGT1A9 catalyzed the glucuronidation of HMR1098. The Vmax values in both instances were similar, but the Km for UGT1A1 was substantially lower than that measured for UGT1A9, 82 microM compared with 233 microM, respectively. Liver and kidney microsomes displayed similar Km values, but the Vmax in kidney was more than 20-fold less than in liver microsomes, which is suggestive of a significant role for the bilirubin UGT in catalysis of HMR1098, although other UGTs may play a secondary role.
Assuntos
Benzamidas/metabolismo , Glucuronídeos/biossíntese , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Linhagem Celular , Células Clonais , Cricetinae , Cricetulus , Glucuronídeos/síntese química , Glucuronídeos/isolamento & purificação , Glucuronosiltransferase/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Rim/enzimologia , Cinética , Propofol/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sulfonamidas/síntese química , Sulfonamidas/isolamento & purificação , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
Conjugation of a structurally diverse set of 53 catechol compounds was studied in vitro using six recombinant human sulfotransferases (SULTs), five UDP-glucuronosyltransferases (UGT) and the soluble form of catechol O-methyltransferase (S-COMT) as catalyst. The catechol set comprised endogenous compounds, such as catecholamines and catecholestrogens, drugs, natural plant constituents, and other catechols with diverse substituent properties and substitution patterns. Most of the catechols studied were substrates of S-COMT and four SULT isoforms (1A1, 1A2, 1A3, and 1B1), but the rates of conjugation varied considerably, depending on the substrate structure and the enzyme form. SULT1E1 sulfated fewer catechols. Only low activities were observed for SULT1C2. UGT1A9 glucuronidated catechols representing various structural classes, and almost half of the studied compounds were glucuronidated at a high rate. The other UGT enzymes (1A1, 1A6, 2B7, and 2B15) showed narrower substrate specificity for catechols, but each glucuronidated some catechols at a high rate. Dependence of specificity and rate of conjugation on the molecular structure of the substrate was characterized by structure-activity relationship analysis and quantitative structure-activity relationship modeling. Twelve structural descriptors were used to characterize lipophilicity/polar interaction properties, steric properties, and electronic effects of the substituents modifying the catechol structure. PLS models explaining more than 80% and predicting more than 70% of the variance in conjugation activity were derived for the representative enzyme forms SULT1A3, UGT1A9, and S-COMT. Several structural factors governing the conjugation of catechol hormones, metabolites, and drugs were identified. The results have significant implications for predicting the metabolic fate of catechols.
Assuntos
Catecol O-Metiltransferase/química , Catecóis/química , Glucuronosiltransferase/química , Sulfotransferases/química , Catecol O-Metiltransferase/metabolismo , Catecóis/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Sulfotransferases/metabolismoRESUMO
The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v., and metabolites were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human liver microsomes were incubated with [12-(3)H]DHA and cofactors for either glucuronidation or cytochrome P450-catalyzed oxidation. Human liver cytosol was incubated with cofactor for sulfation. Metabolites were detected by HPLC-MS and/or HPLC with radiochemical detection. Metabolism of DHA by recombinant human UDP-glucuronosyltransferases (UGTs) was studied. HPLC-MS analysis of urine identified alpha-DHA-beta-glucuronide (alpha-DHA-G) and a product characterized as the tetrahydrofuran isomer of alpha-DHA-G. DHA was present only in very small amounts. The ratio of the tetrahydrofuran isomer, alpha-DHA-G, was highly variable (median 0.75; range 0.09-64). Nevertheless, alpha-DHA-G was generally the major urinary product of DHA glucuronidation in patients. The tetrahydrofuran isomer appeared to be at least partly a product of nonenzymic reactions occurring in urine and was readily formed from alpha-DHA-G by iron-mediated isomerization. In human liver microsomal incubations, DHA-G (diastereomer unspecified) was the only metabolite found (V(max) 177 +/- 47 pmol min(-1) mg(-1), K(m) 90 +/- 16 microM). Alpha-DHA-G was formed in incubations of DHA with expressed UGT1A9 (K(m) 32 microM, V(max) 8.9 pmol min(-1) mg(-1)) or UGT2B7 (K(m) 438 microM, V(max) 10.9 pmol mg(-1) min(-1)) but not with UGT1A1 or UGT1A6. There was no significant metabolism of DHA by cytochrome-P450 oxidation or by cytosolic sulfotransferases. We conclude that alpha-DHA-G is an important metabolite of DHA in humans and that its formation is catalyzed by UGT1A9 and UGT2B7.