Gian Maria Pacifici

Morphine glucuronidation in human fetal and adult liver

SummaryThe glucuronyltransferase activity towards morphine was measured in microsomes isolated from liver specimens obtained from human fetuses and cancer patients. All the fetal livers investigated had measurable UDP-glucuronyltransferase activity towards morphine. There was no correlation between the gestational age (15 to 27 weeks) and the glucuronidation rate. The mean value of the enzymatic activities was higher in fetal livers obtained by hysterotomy (0.20 nmoles×min−1×mg−1) than in livers obtained after induced abortion (0.11 nmoles×min−1×mg−1). The average rate of glucuronidation in microsomes from adult liver (mean 1.15 nmoles×mint-1×mg−1) was 6 to 10 times higher than in the fetal liver microsomes. Together with previous investigations on human adult and fetal liver glucuronidation, the present results support the theory of heterogeneity of human UDP-glucuronyltransferase.

Acetyltransferase in Humans: Development and Tissue Distribution

Acetyltransferase with p-aminobenzoic acid (PABA) as substrate was investigated in the cytosolic fraction of the placenta, liver, adrenals, lungs, kidneys, intestine from human fetuses and the liver, lungs, kidneys and intestinal mucosa from adult subjects. All tissue specimens assayed catalyzed the acetylation of PABA at a significant rate. The activity (expressed as nmol of product formed/min/mg protein; mean +/- SE) was 1.10 +/- 0.59 in the fetal liver, 0.66 +/- 0.04 in the placental and 3.87 +/- 0.53 in the adult liver cytosol. Among the fetal tissues, the adrenals had the highest (2.36 +/- 0.78) and the gut the lowest activity (0.71 +/- 0.11). The acetyltransferase activity (mean +/- SE) in the lungs, kidneys and intestinal mucosa from adult subjects was 1.19 +/- 0.15; 1.34 +/- 0.04 and 3.80 +/- 0.34, respectively.

Effects of Development, Aging, and Renal and Hepatic Insufficiency as well as Hemodialysis on the Plasma Concentrations of Albumin and α1-Acid Glycoprotein: Implications for Binding of Drugs

The concentrations of total proteins, albumin, alpha 1-acid glycoprotein (AGP), and nonesterified fatty acids (NEFA) were measured in the serum of 21 newborn infants and 13 children, aged 1-13 months, and in the plasma of 31 volunteers, 25 patients with renal failure, 27 patients with cirrhosis, 39 uremic patients undergoing hemodialysis, and 20 elderly subjects. The concentration of albumin in the volunteers was higher than in all other groups. The concentration of AGP in the volunteers was higher than in newborn infants but lower than in elderly subjects, patients with renal failure, and those with chronic uremia. The concentration of NEFA in volunteers was higher than in newborn infants and patients with renal failure and lower than in elderly subjects and patients with cirrhosis.

Thiopurine methyltransferase in humans : development and tissue distribution

Thiopurine methyltransferase (EC 2.1.1.67, TPMT) was studied with 6-mercaptopurine as substrate in the cytosolic fraction from 18 human fetal liver, 16 placental and 22 adult liver specimens. TPMT activity (pmol x min-1 x mg-1; mean +/- SD) was 33.2 +/- 15.8 (fetal liver), 19.5 +/- 11.1 (placenta) and 105 +/- 57.1 (adult liver). Fetal liver activity of TPMT is one third that in adult liver suggesting that this enzyme is well developed in the mid-gestational human fetus. The distribution of TPMT seems to be ubiquitous both in the fetus and adult subject. The kidney is an important site of methylation as suggested by the renal activity of TPMT (197 +/- 70 pmol x min-1 x mg-1) which is twice as high as the hepatic one. Fetal and adult hepatic TPMT obey nonmichaelian kinetics. Two phases, one with lower and one with higher affinity for 6-mercaptopurine, were observed. The average Km for the high affinity phase was 0.12 mmol/l (fetus) and 0.13 mmol/l (adult), whereas the Km for the lower affinity phase was 1.79 mmol/l (fetus) and 1.42 mmol/l (adult). This paper shows that TPMT develops before the second trimester of gestation in human fetus, that it has an ubiquitous distribution in the human fetus and adult subjects and the kinetic pattern of this enzyme is consistent in fetal and adult liver.

Glucuronidation of Morphine in Human Liver and Interaction with Oxazepam

Morphine is primarily metabolized through glucuronidation by a microsomal UDP‐glucuronyltransferase. With the use of 14C‐morphine the activity of this enzyme was measured in hepatic microsomes from ten kidney transplant donors with total cerebral infarction and four icteric patients with pancreatic carcinoma. In the former livers the rate of glucuronidation varied from 1.08 to 8.67 nmol per mg microsomal protein per min, with a mean value of 3.83. These values were somewhat higher than in the liver biopsies from the four cancer patients. Oxazepam, at 1/10 the concentration of morphine, inhibited the morphine glucuronidation by 35%. The inhibition was competitive. Salicylamide also inhibited the morphine glucuronidation but only at concentrations considerably higher than morphine. The relevance of the in vitro data for the in vivo situation is unclear, since the concentrations employed in this study are several‐fold higher than those encountered in the plasma of patients treated with these drugs.

Human liver morphine UDP-glucuronyl transferase enantioselectivity and inhibition by opioid congeners and oxazepam

1 Morphine uridine diphosphate glucuronyl transferase (UDP‐GT) was studied in human liver microsomes. The (−)− and (+)‐morphine enantiomers were used as substrates and inhibitors, such as oxazepam and various opioid congeners were employed to characterize the different glucuronidation pathways. The kinetics of the oxazepam inhibition were studied in the rat liver. 2 The overall glucuronidation of (+)‐morphine was higher than that of (−)‐morphine. The morphine congeners tested, potently inhibited the formation of (−)‐morphine‐3‐glucuronide ((−)‐M3G), except for normorphine and codeine. The formation of (+)‐morphine‐6‐glucuronide ((+)‐M6G) was potently inhibited by only dextromethorphan and (+)‐naloxone. All drugs except normorphine inhibited the formation of (+)‐M3G by 18–50%. 3 The metabolism of (−)‐morphine to (−)‐M3G was more sensitive to oxazepam inhibition than the formation of (+)‐M3G from (+)‐morphine in the rat liver. 4 The glucuronidation of natural morphine is subject to in vitro interaction with oxazepam and several opiate drugs. Our study supports the theory of more than one type of UDP‐GT being involved in morphine glucuronidation.

Profile of drug-metabolizing enzymes in the cortex and medulla of the human kidney.

The cortex and medulla were isolated from kidneys whose donors (5 men and 1 woman, aged between 44 and 68 years) were undergoing nephrectomy to remove a tumor. Kidneys with normal architecture for at least two thirds of the organ were included in the study. Tissue specimens used in our experiments were free from pathological changes. The activities of the following enzymes of phase I NADPH cytochrome c reductase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, microsomal and cytosolic epoxide hydrolases, glutathione reductase and glutathione peroxidase, and those of the following enzymes of phase II glutathione transferase, glucuronyl transferase, sulphotransferase, acetyltransferase, thiomethyltransferase, thiopurinemethyltransferase, thioltransferase and glyoxalase were measured. The activity in renal cortex was significantly higher than in medulla for NADPH cytochrome c reductase, cytosolic epoxide hydrolase, glutathione reductase and glutathione peroxidase (phase I enzymes), and glutathione transferase, acetyltransferase, thiomethyltransferase, thiopurinemethyltransferase, thioltransferase and glyoxalase (phase II enzymes). The other enzymes had similar activity in cortex and medulla. The distribution pattern of drug-metabolizing enzymes in the human kidney cannot be considered as a single pattern because of the observed enzyme-dependent differences between cortex and medulla.

Profile of Drug-Metabolizing Enzymes in Human Ileum and Colon

Six patients (4 women and 2 men, age between 60 and 90 years), subjected to right hemicolectomy, were gut donors. The mucosa was isolated from the last portion of the ileum and the first portion of the colon. Tissue specimens were free from pathological changes. The activities of the enzymes of phase I (NADPH cytochrome c reductase, ethoxycoumarin O-deethylase, aminopyrine N-demethylase, microsomal epoxide hydrolase, cytosolic epoxide hydrolase, glutathione reductase and glutathione peroxidase) and the enzymes of phase II (glutathionetransferase, glucuronyltransferase, acetyltransferase, thioltransferase, sulphotransferase and glyoxalase) were measured in the microsomal or cytosolic fractions obtained from ileum and colon mucosa. The activity in the ileum was higher than in the colon for NADPH cytochrome c reductase (p less than 0.05) and cytosolic epoxide hydrolase (p less than 0.001) (phase I enzymes), and glutathionetransferase (p less than 0.02), sulphotransferase (p less than 0.05) and glyoxalase (p less than 0.02) (phase II enzymes). The other enzymes had similar activities in two mucosa. The distribution pattern of drug metabolizing enzymes cannot be considered as a single pattern in human ileum and colon because of the observed enzyme-dependent differences.

Clonazepam serum protein binding during development

Clonazepam protein binding was investigated in sera from five different umbilical cords, 45 children (aged 2 mo to 12 yr), and five adults (aged 27 to 40 yr). The unbound fraction (X̄ ± SE) of clonazepam was 17.3% ± 0.7% in umbilical cord serum and 13.9% ± 0.2% in adult serum (P < 0.01). In children, the unbound fraction of clonazepam reached the adult values during the first year of life. The kinetics of clonazepam serum protein binding were studied in three umbilical cord serum and three adult serum specimens. The number of binding sites (n, reported as moles per gram protein) and the association constant (K, reported as M−1) were estimated from double reciprocal plots of 1 /r against 1 /D (r is the number of moles bound per gram plasma protein; D is the molar concentration of unbound drug). In umbilical cord sera, the mean values (± SE) of n and K were 8.4 ± 0.6 × 10−7 mol/gm and 8.3 ± 0.9 × 104 M−1. In adult serum samples the corresponding values were 3.0 ± 0.8 × 10−7 mol/gm and 2.7 ±0.6 × 105 M−1, which indicated lower binding capacity but higher affinity for clonazepam of plasma proteins in adults than in children.

Thiol methyltransferase in humans: development and tissue distribution.

Thiol methyltransferase (EC 2.1.1.9, TMT) activity was measured with 2-mercaptoethanol in the microsomal fraction of 12 placenta and 31 fetal and 33 adult liver specimens. TMT activity (nmol/min incubation/mg protein; mean +/- SD) was 0.61 +/- 0.25 (placenta), 0.74 +/- 0.45 (fetal liver), and 4.51 +/- 2.29 (adult liver). TMT activity was also measured in extrahepatic tissues and it was about one order of magnitude lower in fetal lungs, kidney and intestine as compared with the fetal liver. A similar distribution pattern was also observed in adult tissues except that in the kidney TMT activity was one third of the hepatic one. Studies of enzyme kinetics showed that fetal and adult hepatic TMT obeyed non-Michaelis-Menten kinetics when 2-mercaptoethanol was the varying substrate. Average values of Km for the higher and lower affinity phases were 0.03 and 14.05 mmol/l, respectively (fetal liver) and 0.005 and 14.57 mmol/l, respectively (adult liver). This paper shows that TMT develops prenatally and its distribution pattern is consistent with that of other microsomal enzymes, being preferentially associated with the liver both in the human fetus and in adult subject.