Sulphation and glucuronidation of ritodrine in human foetal and adult tissues

Summary Ritodrine is a ₂-adrenoceptor agonist used for the management of preterm labour. It is inactivated by conjugation with sulphate and glucuronic acid. There is more ritodrine sulphate than ritodrine glucuronide in urine from the newborn whereas equal amounts of ritodrine glucoronide and sulphate are excreted in maternal urine [Clin. Pharmacol. Ther 44, 634–641, 1988]. We show that, in the mid-gestational human fetal liver, ritodrine sulphotransferase is well expressed, whereas the glucuronidation of ritodrine is little developed compared to the adult liver. The average sulphotransferase activity was 308 pmol·min^–1 per mg protein in fetal (N=48) and 145 pmol·min^–1 per mg protein in adult (N=32) liver. The rates of ritodrine sulphation in fetal gut, lung and kidney were higher than in the corresponding adult tissues. The development and tissue distribution patterns of ritodrine sulphotransferase are consistent with those of dopamine sulphotransferase. Ritodrine and dopamine are sulphated by thermolabile enzymes. The activity of glucuronyl transferase was measurable in only 5 of the 48 foetal livers assayed, and in those in which could be assayed, the average activity was 44.6 pmol·min^–1 per mg protein, one-tenth of that in adult livers (524 pmol·min^–1 per mg protein).     

Minoxidil sulphation in human liver and platelets

Summary Minoxidil requires to be sulphated to exert its hypotensive effect. We report on interindividual variability in the rate of minoxidil sulphation in 118 specimens of human liver and in platelets obtained from 100 healthy subjects and 100 newborns.The frequency distribution histogram of the hepatic activity of minoxidil sulphotransferase was positively skewed; the mean was 631 pmol · min^–1 · mg^–1. After logarithmic transformation of the enzyme activity, the frequency distribution histogram became symmetrical and did not significantly deviate from normality. The rate of minoxidil sulphation was not different in platelets from adults (0.74 pmol · min^–1 · mg^–1) and newborns (1.16 pmol · min^–1 · mg^–1). The frequency distribution histograms were positively skewed and the results of normal equivalent deviation analysis was compatible with the presence of at least two subgroups of sulphotransferase in liver and platelets.Thus, two phenotypes of sulphotransferase exist in human liver and platelets, and the extensive sulphator phenotype contributes to skewing the frequency distribution. In platelets, the percentage of subjects that fall in the two subgroups is different at birth and in adulthood. This can explain the different shape of the frequency distribution in newborn and adult platelets and suggests that platelet minoxidil sulphotransferase undergoes modification after birth.     

(+) and (−) terbutaline are sulphated at a higher rate in human intestine than in liver

Summary The sulphation of (+) and (–) terbutaline was investigated in specimens of human intestinal mucosa isolated from the duodenum, ileum, ascending colon and sigmoid colon and in specimens of liver and lung. The lung specimens came from 8 current smokers and 11 ex-smokers, the latter having stopped at least 3 months before surgery.The rates (pmol·min^–1·mg protein^–1) of (+) and (–) terbutaline sulphation were 1195 and 948 (duodenum), 415 and 317 (ileum), 268 and 166 (ascending colon), 263 and 193 (sigmoid colon) and 45 and 34 (liver), respectively. Terbutaline sulphotransferase was more active in the small and large intestine than in the liver.In the lung, the rate of (+) terbutaline sulphation was 118 (ex-smokers) and 82 (smokers), and for (–) terbutaline it was 82 (ex-smokers) and 56 (smokers). In the gut, the activity of catechol sulphotransferase was significantly correlated with that of (+)- and (–)- terbutaline sulphotransferase whereas no correlation was found with phenol sulphotransferase.This correlation, the finding of the higher activity of terbutaline sulphotransferase in gut than in liver, and the pronounced thermal inactivation of the enzyme, are all consistent with the view that catechol sulphotransferase has a role in the sulphation of terbutaline.     

The effect of atrial natriuretic peptide on plasma renin activity,plasma aldosterone,and urinary dopamine in man

Summary The acute natriuretic effect of human atrial natriuretic peptide (ANP) has been well described in man. We have now studied possible hormonal mediators of this effect.We studied six healthy volunteers on two occasions when they received either an infusion of ANP of 1.5 pmol·kg^–1·min^–1 for 30 min followed by 15 pmol·kg^–1·min^–1 for a further 30 min, or matching vehicle infusions in a randomized single-blind fashion.On the placebo day, plasma renin activity (PRA) rose from 1.26±0.08 to 1.57±0.14 ng A1·ml^–1·h^–1, while on the ANP study day PRA fell from 1.45±0.15 to 1.28±0.05 ng A1·ml^–1·h^–1 (p<0.01). No significant changes were found in plasma aldosterone concentrations or in urinary dopamine excretion.These results provide evidence that ANP suppresses renin release in man.     

Development of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo

1. The sensitivity of the developing embryo to xenobiotics is highly dependent on the expression of metabolizing enzymes including cytochromes P450 (CYP). In the present study, therefore, the ontogeny of the CYP-dependent system in the chick was investigated with testosterone hydroxylase activity as a marker of CYP expression.
2. Chicken embryo livers were assayed for basal and phenobarbitone (PB)-induced regio- and stereo-selective testosterone hydroxylase activity, from the first appearance of the liver as a discrete organ at 5 days of incubation through day 10 posthatching. In addition, whole embryo preparations were assayed at 3 and 4 days of incubation.
3. Whereas testosterone 16β-hydroxylase and androst-4-ene-3,17-dione-linked activities were expressed during all stages of embryonic development, testosterone 6α-, 6β-, 7α- and 16α-hydroxylase activities were observed only in basal embryos from 8 days of incubation. Furthermore, testosterone 2α- and 2β- hydroxylase activities were detected exclusively from 10 days of incubation onward. All activities increased steadily throughout development as did the responsiveness of the embryonic liver to PB induction.
4. A typical pattern of development with a higher activity from 10 to 14 days of incubation (testosterone 16α-, 7α-, 6α- and 2β-hydroxylase activities; up to 4.1±0.3 pmol mg⁻¹ protein min⁻¹ at 13 days of incubation for testosterone 7α-hydroxylase) or shifted to 14 to 18 days of incubation (testosterone 6β-, 2α- and 16β-hydroxylase activities: up to 56.6±1.4 pmol mg⁻¹ protein min⁻¹ at 16 days of incubation for testosterone 6β-hydroxylase) was observed. There was a tendency towards an increased activity for all activities around hatching, specifically from 19 days of incubation to 4 days posthatching (up to 1,759.3±179.4 pmol mg⁻¹ protein min⁻¹ at 1 day posthatching for androst-4-ene-3,17-dione-linked activity).
5. The highest level of PB-induced enzyme activity was observed for testosterone 2α-hydroxylase activity (95.14±7.35 and 660.19±45.27 pmol mg⁻¹ protein min⁻¹) at 12 days of incubation and day 3 posthatching, respectively. Except for testosterone 2α- and 2β-hydroxylase activities at 3 to 4 days of incubation, all metabolites were detectable during the first period of organogenesis in the presence of PB.
6. The use of highly specific substrates, studies on the immunoinhibition of metabolism by polyclonal antibodies raised against highly purified rat CYPs, and the use of selective inhibitors seemed to reveal a wide pleiotropic response with the posssible presence in liver of PB-treated chickens of CYP1A together with CYP2H1/H2, CYP2E and CYP3A.

     

Interindividual variability in the rate of salbutamol sulphation in the human lung

The ₂-adrenergic agonist salbutamol is administered by inhalation to treat lung-obstructive disease. Salbutamol is metabolized by conjugation with sulphate, and the sulphation of salbutamol was investigated in human lung. Specimens of lung were obtained at lobectomy from 11 non-smokers, 39 smokers and 46 ex-smokers, the latter refraining from smoking at least 6 months before surgery. Neither sex nor ageing influenced the activity of sulphotransferase. The rate of salbutamol sulphation (pmol·min^-1·mg^-1) was greater in non-smokers (27.7) than in smokers (21.3), whereas it was similar in smoker and ex-smokers (22.8). The rate of salbutamol sulphation ranged up to six fold and its distribution did not deviate from normality. As the rate of formation of the inactive salbutamol sulphate varied in the lung, the availability of salbutamol and, in turn, the evoked pharmacological effect should vary in parallel. The activities of salbutamol and dopamine sulphotransferase correlated, suggesting that catechol sulphotransferase takes part in the sulphation of salbutamol. The sulphation of salbutamol is stereoselective in the human lung, the k _M estimate for (+)-salbutamol (1198 M) being greater than those for either (-)-salbutamol (190 M) and racemic salbutamol (142 M). These results are consistent with the view that (-)-salbutamol is a better substrate than (+)-salbutamol for sulphotransferase.     

Variable activation of lovastatin by hydrolytic enzymes in human plasma and liver

Lovastatin, widely used to lower cholesterol, is a pro-drug that requires metabolic activation through hydrolysis by carboxyesterases. There appear to be at least three distinct esterases in humans capable of catalysing this reaction, one in plasma and two in the liver.The rate of lovastatin hydroxy acid formation was measured as 15.8 pmol · ml^–1 · min^–1 in plasma, 2.13 pmol · mg^–1 protein · min^–1 in hepatic microsomes and 0.92 pmol · mg^–1 protein · min^–1 in cytosol. The data suggest that on average the three esterases together are capable of activating about 220 nmol (90 g) lovastatin per minute per person, to which the esterases of plasma, liver microsomes and liver cytosol contribute approximately 18, 15 and 67%, respectively.All three esterases showed evidence of inter-individual variability. In one of 17 livers, both cytosolic and microsomal esterase activity was completely missing, while two other liver specimens lacked one esterase.Such variability must be expected to influence the therapeutic efficacy of the drug, and they might be related to its occasional toxicity.     

Sex-difference and the effects of smoking and oral contraceptive steroids on the kinetics of diflunisal

Summary The single dose pharmacokinetics of diflunisal were studied in 4 groups of 6 young volunteers: control men, control women, women taking low estrogen oral contraceptive steroids (OCS), and women smokers (10–20 cigarettes/day).The plasma clearance of diflunisal was significantly higher in men (0.169 ml·min^–1·kg^–1) and in women on OCS (0.165 ml·min^–1·kg^–1) as compared to control women (0.108 ml·min^–1·kg^–1). Partial metabolic clearances of diflunisal by the three conjugative pathways (phenolic and acyl glucuronide formation, sulphate conjugation) were all increased in men and women OCS users as compared to control women. Statistically significant increases, however, were only observed for the partial metabolic clearance of diflunisal by phenolic glucuronidation between men and women (2.91 vs. 1.85 ml·min^–1 respectively), and for the partial clearance by acyl glucuronidation between OCS users and control women (4.81 vs. 3.01 ml·min^–1 respectively).Smoking resulted in a moderate increase (35%) in plasma diflunisal clearance. However, a significant reduction in total urinary recovery of diflunisal and its glucuronide and sulphate conjugates was found in smokers (70.5% in smokers as compared to 84.2–87.2% in the 3 other study groups). Consequently, smoking may have induced hydroxylation, a minor oxidative metabolic pathway of diflunisal recently discovered in man.     

Induction of cytochrome P4501A by smoking or omeprazole in comparison with UDP-glucuronosyltransferase in biopsies of human duodenal mucosa

Drug-metabolizing enzymes were investigated in duodenal biopsy specimens. Cytochrome P4501A (CYP1A) activity was determined by measuring 7-ethoxyresorufin O-deethylase (EROD) activity in biopsies from 20 smokers (3–30 cigarettes per day), 21 nonsmokers, and 10 nonsmokers receiving omeprazole treatment (20–60 mg/day for at least 1 week). Omeprazole is known to act as a polycyclic aromatic hydrocarbon (PAH)-type inducer in humans.EROD activity was found to be significantly induced in smokers and omeprazole-treated patients, with medians of 2.1 and 1.1 pmol· min^–1·mg protein^–1, respectively, compared with 0.5 pmol·min^–1·mg protein^–1 in nonsmokers. Immunoblot analysis substantiated that EROD activity was correlated with CYP1A protein. In contrast, UDP-glucuronosyltransferase (UGT) activity towards 4-methylumbelliferone (an overlapping substrate of several constitutive and inducible UGTs) was not significantly affected.The results demonstrate CYP1A induction by omeprazole and by constituents of cigarette smoke in the human duodenum and support the utility of duodenal biopsies to monitor CYP1A induction by PAH-type inducers.     

Calcitonin gene-related peptide (CGRP) causes redistribution of blood flow in humans

Summary In normal human subjects (n=6), blood flow in the common carotid artery, assessed with an ultrasonic duplex-scanning unit, was increased up to 152% of basal levels by 60-min infusions of human calcintonin gene-related peptide I (CGRP) 80 pmol·kg^–1·h^–1, but it was not affected by 20 pmol·kg^–1·h^–1 CGRP or 88 pmol·kg¹·h^–1 human calcitonin.In the superior mesenteric artery, on the other hand, blood flow was reduced by 80 pmol·kg^–1·h^–1 CGRP to 58% of the basal level, but not by 20 pmol·kg^–1·h^–1 CGRP or with 88 pmol·kg^–1·h^–1 calcitonin.Blood flow in the abdominal aorta remained largely unchanged under the same conditions.Skin blood flow, assessed by a laser Doppler unit, was increased up to 682% of the basal level by 80 pmol·kg^–1·h^–1 CGRP, but not by 20 pmol·kg^–1·h^–1 CGRP or calcitonin.Thus CGRP increased regional blood flow to the brain and the skin at the expense of the gastrointestinal tract.     

Sensitivity of residual nephrons to high dose furosemide described by diuretic efficiency

Ten haemodialysis (HD) patients with a median residual creatinine clearance (CL_CR) of 1.9 ml·min^–1·1.73 m^–2 (range 0.6–5.3) were treated with oral furosemide (F) 2.0 g. Overall-efficiency (O-E, daily sodium excretion/total urinary F) and total-efficiency (-E, increase in daily sodium excretion/total urinary F) were measured on the last 24 hours of each interdialysis interval. In addition, O-E was measured during the complete interdialysis interval in 10 HD patients with a median CL_CR of 5.6 ml·min^–1·1.73 m^–2 (range 0.7–6.8) treated for 1 year with a fixed oral dose of F between 250–1000 mg (median 625 mg).In the short study the median O-E was 10.6 mmol·mg^–1 (range 1.9–22.0) and -E 6.2 mmol·mg^–1 (range 1.3–11.2). The fractional excretion of sodium FE_Na was significantly increased from 9.6% (range 4.1–22.9) to 27% (range 14.6–56.2) during F treatment. A positive correlation was found between the basal FE_Na and -E. In the long-term study median O-E was 6.4 mmol·mg^–1. O-E and FE_Na showed no change over time although median RCC decreased from 5.6 to 1.9 ml·min^–1·1.73 m^–2 and median F excretion from 11.8 to 7.5 mg per day.It can be concluded that diuretic efficiency in haemodialysis patients is dependent on FE_Na and the state of hydration during the interdialysis interval.     

Acute effects of felodipine and nifedipine on hepatic and forearm blood flow in healthy men

Summary The acute effects of oral administration of felodipine 10 mg and nifedipine 10 mg on heart rate, blood pressure, forearm blood flow and hepatic blood flow were studied in nine healthy men.Both drugs caused an increase in heart rate of 16 and 7 beats · min^–1, respectively. Hepatic blood flow was significantly increased by 1.2 and 0.41 · min^–1 after felodipine and nifedipine. There was also a decrease in diastolic blood pressure, 10 and 5 mm Hg, respectively, after felodipine and nifedipine. The forearm blood flow was increased by about 30 ml · 100 ml^–1 · min^–1 after felodipine, but nifedipine had no effect.The haemodynamic effects were most pronounced 50 min after drug administration.     

Renal effects of atrial natriuretic peptide during dopa-decarboxylase inhibition in patients with essential hypertension

Summary To assess whether intrarenal dopamine synthesis could contribute to the renal response to ANP in essential hypertension, the effects of -human ANP influsion (50 ng·min^–1·kg^–1 b.w. for 30 min) on the urinary excretion of dopamine and sodium, urine flow rate and arterial pressure were evaluated in 7 patients with mild-moderate essential hypertension before (control period) and during DOPA-decarboxylase inhibition with carbidopa (carbidopa period).In the control period, urinary dopamine excretion was 400 pg·min^–1 in baseline conditions and 340 pg·min^–1 during ANP infusion. Carbidopa significantly decreased urinary dopamine excretion both before (210 pg·min^–1) and during ANP (99 pg·min^–1). In contrast, carbidopa did not affect sodium excretion (control from 184 to 460 Eq·min^–1; carbidopa period from 140 to 390 Eq·min^–1) or urine flow rate (control from 5.35 to 11.21 ml·min^–1; carbidopa period from 4.29 to 11.54 ml·min^–1).Arterial pressure fell significantly during ANP infusion in both periods, and no significant difference was observed between the two study days, i.e. in the absence of and during carbidopa administration.We conclude that DOPA-decarboxylase inhibition does not influence the diuretic and natriuretic response to -human ANP infusion in patients with essential hypertension.     

Polymorphic 2-hydroxylation of desipramine

Summary We have studied the clearance of monomethylaminoantipyrine (MMAAP), the pharmacologically active form of metamizol, in 46 patients in surgical intensive care with different degrees of renal dysfunction.In 23 patients without any renal impairment, mean clearance was 2.8 ml·min^–1·kg^–1. Twentyone patients with acute renal impairment had a significantly reduced clearance of MMAAP (0.83 ml·min^–1·kg^–1). There was also reduced clearance in four patients with septic shock (1.0 ml·min^–1·kg^–1).Kinetics of the metabolites of MMAAP (N-formylaminoantipyrine (FAAP), aminoantipyrine (AAP), and its secondary product N-acetylaminoantipyrine (AcAAP)) were calculated. FAAP and AcAAP showed delayed invasion, which can be explained by reduced hepatic metabolic activity. The product of N-demethylation, AAP, was not significantly altered.The delayed elimination of monomethylaminoantipyrine can be explained by reduced hepatic function in parallel with acute renal failure due to disturbed cardiovascular function caused by septic shock. This may also lead to disturbed hepatic macro- and microperfusion associated with altered oxygen supply and oxygen consumption.     

Dopamine pharmacokinetics in critically ill newborn infants

Summary Dopamine is frequently used in critically ill newborn infants for treatment of shock and cardiac failure, but its pharmacokinetics has not been evaluated using a specific analytical method. Steady-state arterial plasma concentrations of dopamine were measured in 11 seriously ill infants receiving dopamine infusion, 5–20 g · kg^–1 · min^–1, for presumed or proven sepsis and hypotensive shock.Steady-state concentrations of dopamine ranged from 0.013–0.3 g/ml. Total body clearance averaged 115 ml · kg^–1 · min^–1. The apparent volume of distribution and elimination half life averaged 1.8 1 · kg^–1 and 6.9 min, respectively.No relationship was observed between dopamine pharmacokinetics and gestational age, postnatal age or birthweight. Substantial interindividual variation was seen in dopamine pharmacokinetics in seriously ill infants, and plasma concentrations could not be predicted accurately from its infusion rate.Marked variation in clearance explains in part, the wide dose requirements of dopamine needed to elicit clinical response in critically ill newborn infants.VBM was a Fellow at Ohio State University and Children's Hospital at the time of study and is now at the Department of Pharmacy Services and College of Pharmacy, University of Michigan, Ann Arbor, MI, USA     

Intravenous infusion of iloprost in arterial occlusive disease: dose-dependent effects on skin microcirculation

Summary Transcutaneous oxygen pressure (tcPo₂), laser Doppler flux and capillary microscopy have been used to examine the forefoot skin in 5 healthy men and 8 patients with severe peripheral arterial occlusive disease in order to evaluate the dose dependent effects of iloprost on skin microcirculation. Iloprost was infused IV starting at 0.0625 ng·kg^–1·min^–1 and doubling the dose every 15 min up to 2 ng·kg^–1·min^–1.While tcPo₂ at an electrode core temperature of 44°C decreased in both patients and controls, there was a significant dose dependent increase in tcPo₂ (37°C) in the controls from 0.25 ng·kg^–1·min^–1. In the patients the reaction was variable: it was decreased in two and increased in 6, with a maximum either at 0.25–0.5 ng·kg^–1·min^–1 (n=3) or at the highest dose (1.0 or 2.0 ng·kg^–1·min^–1; n=3). Mean laser Doppler flux in both groups was increased, although the reaction was not consistent in the patients. Density of forefoot skin capillaries was reduced in 3 patients, and in the others the flow velocity was very low. During infusion of iloprost, both an increase in capillary density and blood cell velocity were observed. The effects were of variable intensity and occurred at varying doses, some appeared early and diminished as the dose was increased, and others were found only at 2 ng·kg^–1·min^–1.Adverse effects were numerous, extending from harmless skin flushing to mental changes and a quickly reversible attack of angina pectoris. It may be possible to divide patients into those with early effects on the microcirculation, at doses of 0.25–0.5 ng·kg^–1·min^–1, and those in whom the microcirculatory response is preceded and counteracted by the adverse effects.     

Pharmacokinetics and pharmacodynamics of thiazinamium in asthmatic patients

Summary The pharmacokinetics and pharmacodynamics of thiazinamium (Multergan) were studied after intravenous and intramuscular administration to 7 males with chronic reversible airways obstruction.Disposition after i.v. administration was described by a clearance of 0.54 l·min^–1, central compartment volume of 14.8 l, distribution rate constant 0.092 min^–1, and an elimination rate constant of 0.0044 min^–1. The corresponding estimates after i.m. administration were 0.324 l·min^–1, 34.1 l, 0.035 min^–1, and 0.0018 min^–1. The bronchodilator response (expressed as % predicted FEV1) after i.v. administration was characterized by maximum increase in FEV1 of 33.9%, with an EC₅₀ of 12.8 ng·ml^–1 and an equilibration half-time of 11 min. Corresponding parameter estimates after i.m. administration were 32.2%, 18.8 ng·ml^–1, and 9 min.Anticholinergic activity, measured by the change in heart rate after i.v. administration, showed maximum increase of 76 beats·min^–1, with an EC₅₀ of 176 ng·ml^–1 and an equilibration half-time of 1.3 min. After i.m. administration the corresponding values were 120 beats·min^–1, 250 ng·ml^–1, and 3 min.The optimal plasma concentration of thiazinamium was about 100 ng·ml^–1, which should give a near maximal bronchodilator response (over 80% of predicted normal) and a heart rate of about 100 beats·min^–1.     

Effect of loperamide on jejunal electrolyte and water transport,prostaglandin E 2-induced secretion and intestinal transit time in man

Summary Jejunal perfusion was performed in 12 healthy volunteers to evaluate the dose dependent effects of loperamide on intestinal absorption, stimulated secretion and transit.In 6 volunteers intestinal perfusion of the jejunal segment with isotonic NaCl solution was followed by addition of loperamide in increasing doses (2–8 mg·l^–1). The volunteers were pretreated with 1 mg·l^–1 prostaglandin E2 (PgE2) in the perfusate before addition of 4 mg·l^–1 loperamide. Phenolsulphonphtalein (PSP) boluses (2 ml) were given to measure mean transit time (MTT).Loperamide 2 mg·l^–1 converted the minor secretion after perfusion with the standard solution (water –1.45 ml·min^–1, Na –0.09 and Cl –0.04 mmol·min^–1) to absorption (water 0.93 ml·min^–1, Na 0.23, Cl 0.25 mmol·min^–1) within 15 min. Higher doses of loperamide did not increase absorption.The addition of PgE2 induced net secretion of water (–4.48 ml·min^–1) and electrolytes (Na –0.57, Cl –0.51 mmol·min^–1). Loperamide 4 mg·l^–1 significantly diminished the PgE2-induced net secretion by approximately 50%.Loperamide dose dependently increased the MTT from 6 (2 mg·l^–1) to 13.3 min (8 mg·l^–1). MTT was still delayed 60 min after a wash out period (10.5 min).It is concluded that loperamide had a dual effect or intestinal activities stimulating absorption and prolonging intestinal transit time with rising doses.     

Sulphation of resveratrol,a natural compound present in wine,and its inhibition by natural flavonoids

1. Resveratrol, a polyphenolic compound present in grape and wine, has beneficial effects against cancer and protective effects on the cardiovascular system. Resveratrol is sulphated, and the hepatic and duodenal sulphation might limit the bioavailability of this compound. The aim of this study was to see whether natural flavonoids present in wine, fruits and vegetables inhibit the sulphation of resveratrol in the human liver and duodenum. 2. In the liver, IC₅₀ for the inhibition of resveratrol sulphation was 12 ± 2 pM (quercetin), 1.0 ± 0.04 μM (fisetin), 1.4 ± 0.1 μM (myricetin), 2.2 ± 0.1 μM (kaempferol) and 2.8 ± 0.2 μM (apigenin). Similarly, in the duodenum, IC₅₀ was 15 ± 2 pM (quercetin), 1.3 ± 0.1 μM (apigenin), 1.3 ± 0.5 μM (fisetin), 2.3 ± 0.1 μM (kaempferol) and 2.5 ± 0.3 μM (myricetin). 3. The type of inhibition of quercetin on resveratrol sulphation was studied in three liver samples and was determined to be non-competitive and mixed in nature. K_m (mean ± SD; μM) was 0.23 ± 0.07 (control), 0.40 ± 0.08 (5 pM quercetin) and 0.56 ± 0.09 (10 pM quercetin). V_max (mean ± SD; pmol·min^?1·mg^?1) was 99 ± 11 (control), 73 ± 15 (5 pM quercetin) and 57 ± 10 (10 pM quercetin). K₁ and K_1es estimates (mean ± SD) were 3.7 ± 1.8 pM and 12.1 ± 1.7 pM respectively (p = 0.010). 4. Chrysin was a substrate for the sulphotransferase(s) and an assay was developed for measuring the chrysin sulphation rate in human liver. The enzyme followed Michaelis‐Menten kinetics and K_m and V_max (mean ± SD) measured in four livers were 0.29 ± 0.07 μM and 43.1 ± 1.9 pmol·min^?1·mg^?1 respectively. 5. Catechin was neither an inhibitor of resveratrol sulphation nor a substrate of sulphotransferase. 6. These results are consistent with the view that many, but not all, flavonoids inhibit the hepatic and duodenal sulphation of resveratrol, and such inhibition might improve the bioavailability of this compound.     

A Comparison of Peptidase Activities and Peptide Metabolism in Cultured Mouse Keratinocytes and Neonatal Mouse Epidermis

One of the barriers to transdermal delivery of peptides is the metabolic activity of the epidermis. To define this metabolic activity, aminopeptidase activity and Leu-enkephalin metabolism were measured in the epidermis obtained from neonatal mouse skin and in cultured mouse keratinocytes. Aminopeptidase activity was measured fluorometrically using leucine, tyrosine, lysine, and aspartic acid derivatives of -naphthylamine as substrates. Similarities in substrate kinetic values (K _m and V _max) and substrate specificity of the enzyme(s) in homogenates prepared from neonatal mouse skin epidermis and cultured mouse keratinocytes strongly suggest that the keratinocytes in culture express the same aminopeptidase(s) with the same relative activity as in neonatal skin. The K _m and V _max values for aminopeptidase(s) with different substrates in epidermis homogenates are as follows: leucine -naphthylamide (11 µM and 38 nmol · min^–1 · mg^–1), tyrosine (-naphthylamide (21 µM and 18 nmol · min^–1 · mg^–1), and lysine -naphthylamide (11 µM and 35 nmol · min^–1 · mg^–1). Aspartic acid -naphthylamide and glutamic acid -naphthylamide were not hydrolyzed by these homogenates at pH 7.4 (37°C). Leu-enkephalin hydrolysis by the homogenates from cultured mouse keratinocytes and neonatal mouse epidermic gave similar K _m (32 and 24 µM), V _max (9.77 and 7.55 nmol · min^–1 · mg^–1) and K _i (223 and 194 µM) values. In addition, the cellular homogenates gave similar metabolite profiles for Leu-enkephalin.