Molecular mechanisms controlling the rate and specificity of catechol O-methylation by human soluble catechol O-methyltransferase.

Molecular mechanisms determining the turn-over rate and specificity of catechol O-methylation were studied by combining enzyme kinetic measurements, computational modeling of substrate properties and fitting ligands in a 3D model of the active site of the enzyme. Enzyme kinetic measurements were carried out for 46 compounds, including most clinically used catechol drugs, by using recombinant human soluble catechol O-methyltransferase (COMT). The most important mechanism decreasing the turnover rate and increasing affinity was the electron withdrawing effect of substituents. Several other mechanisms by which substituents affected reactivity and affinity were identified. Highest turnover rates were determined for unsubstituted catechol and pyrogallol. Pyrogallol derivatives generally seemed to be more specific substrates than catechols. Catecholestrogens were the most specific endogenous substrates, whereas catecholamines were rather poor substrates. Among the catechol drugs used in the L-DOPA treatment of Parkinson's disease, the COMT inhibitors entacapone and tolcapone were not methylated, whereas the DOPA decarboxylase inhibitor benserazide was 15 times more specific substrate than L-DOPA, the target of COMT inhibition. The structure-activity relationships found allow the prediction of reactivity, affinity, and specificity with useful accuracy for catechols with a wide range of structures and properties. The knowledge can be used in the evaluation of metabolic interactions of endogenous catechols, drugs and dietary catechols, and in the designing of drugs with the catechol pharmacophore.

Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the Semliki Forest virus expression system.

Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT 1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. No UGT signal was detected in noninfected cells. In addition, SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzyme. The measurement of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.

Nephrectomy in severe hypertension.

We describe a 12-year-old girl who died due to hypertensive encephalopathy and a 7-year-old boy with a favorable outcome after bilateral nephrectomy. Both had end-stage renal disease. Nephrectomy should be considered as a possible treatment of severe hypertension even without end-stage renal disease, if the patient has symptoms of hypertensive encephalopathy.

The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.

The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.

Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates.

A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl-L-[methyl-14C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showed very similar substrate selectivities. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and it was shown that accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters and can probably be further used for gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT.

Glucuronidation of entacapone, nitecapone, tolcapone, and some other nitrocatechols by rat liver microsomes.

PURPOSE: Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. METHODS: The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltransferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. RESULTS: Apparent K(m) values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold Vmax values compared with 4-nitrophenol (68.6 nmol min-1 mg-1), while all disubstituted catechols exhibited much lower glucuronidation rate. Vmax/K(m) values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K(m) nitecapone > > entacapone > tolcapone; Vmax nitecapone > entacapone > tolcapone; Vmax/K(m) tolcapone > nitecapone > entacapone. CONCLUSIONS: Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.

The state of young adults with juvenile onset diabetes.

Childhood diabetes is most common in Nordic countries and its incidence is rising. In order to evaluate the efficacy of health care follow-up units we investigated physical and psychosocial health status, mode of coping with adult health care and medical treatment in 82 young adults (46 males, 36 females, average age 20.9 yr. and average disease duration 12.7 yr.) who had had diabetes since childhood. All but three of them made regular visits to a health care facility but only 27% monitored blood glucose reasonably well. Only eight percent had a HbA1 concentration within the optimal range, and half had a inappropriate level. Half of the subjects with high HbA1 in adolescence had managed to improve it since leaving the paediatric unit. The most common clinical findings were lipohypertrophy and depressed patellar and achillar reflexes. Up to 70% had background retinopathy and 10% proliferative retinopathy, while two thirds (62%) had depressed conduction velocity of the peroneal nerve. Clinically significant psychiatric problems were found in 17% of the patients, depression being the most prominent feature. Among the social characteristics, delayed social maturation and lack of vocational education were found to be more common than in age-matched controls. One in three exhibited a major overt physical problem and one in five a major psychosocial problem. In conclusion, whatever the health care follow-up unit attended by young adults with diabetes since childhood, the teams face health problems that differ totally from one individual to another. It is important at this transitional age to focus attention in a broad-minded manner on the many factors complicating diabetes or affecting good compliance with treatment.

Characterization of 1-hydroxypyrene as a novel marker substrate of 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase(s).

Rats were treated with acetone, pyrazole, phenobarbital, 4,4'-methylenebis-(2-chloroaniline) (MOCA), 3-methylcholanthrene, creosote oil, or a mixture of polychlorinated biphenyls (Aroclor 1254) to study the inducibility and enzyme kinetics of UDP-glucuronosyltransferases towards 1-hydroxypyrene, which is a human metabolite and a urinary biomarker of exposure to pyrene. The rate of 1-hydroxypyrene glucuronidation was analyzed in rat liver microsomes by a fluorometric HPLC assay of the formed glucuronide. The apparent K(m) and Vmax values in untreated controls (K(m) = 0.27 mM; Vmax = 31 nmol/min./mg protein) did not differ markedly from those in rats treated with acetone, pyrazole or phenobarbital, whereas the significantly decreased K(m) and increased Vmax values of the rats treated with the carcinogenic chemicals, MOCA (0.11; 51), creosote (0.06; 137), 3-methylcholanthrene (0.07; 141) or the Aroclor-1254 polychlorinated biphenyl (PCB) mixture (0.08; 226), implicated major changes in the hepatic expression of UDP-glucuronosyltransferases. 1-Hydroxypyrene proved to be a high affinity substrate and a sensitive marker of the polycyclic aromatic hydrocarbon (PAH) metabolizing UDP-glucuronosyltransferase(s). Catalytically, the most efficient isoforms were induced in creosote, 3-methylcholanthrene and PCB-treated rats showing Vmax/K(m) ratios which were 22-27 times greater than in untreated controls. Our findings suggest the existence of a 3-methylcholanthrene type inducible and a functionally efficient low-K(m)/ high-Vmax form(s) of UDP-glucuronosyltransferase(s) that detoxify 1-hydroxypyrene and probably other polycyclic aromatic hydrocarbons as well.

Medical student abuse: an international phenomenon.

OBJECTIVE: To evaluate the prevalence of physical and psychological mistreatment of medical students at two medical schools in Finland. STUDY DESIGN AND SETTING: To enable comparison between Finnish and American students, we used the American Medical Association's Office of Education Research questionnaire. RESULTS: Three of every four students surveyed reported experiencing some kind of mistreatment during their medical education. Students most commonly reported sexual mistreatment, usually as slurs and sexual discrimination, from classmates, preclinical teachers, clinical teachers, clinicians, nurses, and patients. Other forms of verbal abuse, psychological mistreatment, and physical threats were also reported. CONCLUSIONS: All forms of mistreatment were reported occurring less frequently than in the United States; still, the level of such behavior was high. The results suggest the need for more international awareness and debate regarding the habits and behavior of teaching staff in medical schools.

Association of recurrent acute otitis media with nasopharynx dimensions in children.

The purpose of this study was to evaluate the hypothesis that the nasopharyngeal anatomy has influence in the risk of recurrent acute otitis media (AOM) attacks. We analysed the occurrence of acute otitis media in 238 healthy schoolchildren who were X-rayed for orthodontic purposes. Six measurements reflecting the size and shape of the bony nasopharynx were recorded from lateral cephalograms. The means for almost all the dimensions of the bony nasopharynx measured were smaller in the children with two or more attacks of AOM in their history than in those with no attack or only one attack. Logistic multivariate modelling showed the distance from the posterior nasal spine to the sella-basion line to be a significant risk factor for recurrent otitis media in girls (difference 1.0 mm; 95 per cent confidence interval 0.1-2.0 mm; p = 0.04) and the shape of the nasopharynx (roundness) in boys (difference 1.9 mm; 95 per cent confidence interval 0.1-4.0 mm; p = 0.01). Measuring the nasopharyngeal bony dimensions may help to identify those children with a risk of recurrent otitis media, at whom prophylactic therapies should be targeted.

Ocular complications in young adults with insulin-dependent diabetes mellitus since childhood.

A cross-sectional study in 80 insulin-dependent diabetic patients born 1963-1968 who experienced the onset of diabetes before 15 years of age showed that at a mean age of 21.6 (range 17-25) years and after a mean duration of diabetes of 13.3 (range 6-24) years, 80% of the patients had retinopathy: 70% had background and 10% proliferative changes. Retinopathy correlated with the duration of the diabetes and poor glucose control at 15 years of age but not with the actual level of glycated haemoglobin. The severity of retinopathy was worse in women than in men. One patient (1.2%) was blind. Two patients had had cataract operations and 66% had myopic refraction in one or both eyes. In 61 patients a further period of ophthalmological follow-up of 3-4 years was included. After 20 years of diabetes, all had retinopathy and 29% had proliferative changes: 33% had received laser treatment after 8-27 (mean 16.1) years of diabetes. Altogether, 2 patients (2.5% of the original series) were blind. For prevention of diabetic retinopathy and blindness, good glucose control from puberty and careful ophthalmological follow-up after transfer of the patient from paediatric to adult diabetes care play major roles.

Social maturation in juvenile onset diabetes.

We re-examined a group of 82 (36F, 46M) patients with juvenile onset diabetes at the age of 19-25 years and compared their social outcome in early adulthood with that of a group of 211 randomly selected controls. Their basic school achievements and vocational or higher education were covered in detail in an interview which also included data on their employment status. Various descriptors of social situation and social maturation assessed in a separate interview formed a social maturation index. Relations between factors probably affecting social maturation and this index were analysed. The average marks scored by the diabetics in Finnish comprehensive schools were significantly lower than those of the controls. High school, vocational and commercial schools were discontinued more often by the patient group. Diabetics (27%) and controls (35%) continued their studies equally often in various vocational high schools or universities. Presently, 17% of the diabetics and 11% of the controls had no vocational education or were not on their way to gaining it. Working experience, employment status and unemployment were similar in both groups, but diabetics were more often employed in public service and commerce. At the time of the study the diabetics were significantly more often unmarried and living in the same household as their parents compared with the controls. Other parameters also indicated difficulties in the diabetic group in separating from parents. The overall social maturation index showed poor social maturation in diabetics more often than in the controls. Neither social background, education nor sex were related to poor social maturation. It is concluded that having diabetes delays social maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemic nephropathy in children.

Epidemic nephropathy, a form of hemorrhagic fever with renal syndrome, caused by the Puumala serotype of hantaviruses and occurring endemically in northern Scandinavia, was studied in 13 children. The clinical symptoms and signs were somewhat different from those reported in adults; none of our patients had hemorrhagic manifestations despite low thrombocyte counts. The most common presenting symptoms were fever, abdominal pain, and renal tenderness with oliguria followed by polyuria. The predominant laboratory findings were proteinuria and/or hematuria and elevated serum creatinine levels. Thrombocytopenia was a constant finding in the children in whom thrombocyte count was obtained. Most children had a decreased serum sodium concentration during the oliguric phase of the disease. All the children recovered, with no long-term renal disease. Epidemic nephropathy is an important alternative for differential diagnosis in children with findings suggesting nephritis, especially in endemic areas. An awareness and knowledge of this syndrome and an ability to diagnose it by means of a specific antibody measurement will probably improve our understanding of its epidemiologic features in children.

Serum insulin and other cardiovascular risk indicators in children, adolescents and young adults.

We wanted to determine the levels of fasting serum insulin during growth, the tracking of serum insulin, and the correlation of serum insulin with other coronary heart disease risk indicators in children and young adults. In 1986 2433 subjects, aged nine to 24 were studied, and insulin data were available from the same population in 1980 and 1983. Serum insulin levels showed a peak during puberty in both sexes and the decline in insulin continued after the age of 21. Tracking of serum insulin was only moderate, especially in females and young boys. Serum insulin correlated positively with body mass index, concentrations of serum triglycerides, and blood pressure, and inversely with the concentration of high density lipoprotein cholesterol. High triglycerides, high systolic blood pressure, and low level of high density lipoprotein cholesterol clustered among subjects within the highest insulin quartile. Our results suggest that the insulin resistance phenomenon, caused mainly by obesity and leading to unfavourable levels of other coronary heart disease risk indicators, is already developing in children and young adults. This suggests that preventing obesity in early life is important.

Effect of once-a-week training program on physical fitness and metabolic control in children with IDDM.

To examine whether a physical activity program could improve physical fitness and glycemic control, 32 children and adolescents with insulin-dependent diabetes mellitus (IDDM) were examined before the program and 3 mo later. Fifty percent of the subjects (n = 16) participated in the training for 1 h/wk (exercise group), whereas the remaining subjects were engaged in nonphysical activities for an equal amount of time (nonexercise group). Age of the subjects ranged from 8.2 to 16.9 yr, (mean 11.9 yr), with mean duration of diabetes 0.6-13.1 yr (5.2 yr). During the 3-mo program peak oxygen consumption (VO2) rose from 40.0 to 43.8 ml.min-1.m-2 (P less than .01) in the exercise group but only by 1.3 ml.min-1.m-2 in the nonexercise group (NS). Metabolic control did not improve in either group, with glycosylated hemoglobin level rising from 9.8 to 10.5% (P less than .01) in the exercise group and from 9.4 to 9.7% (NS) in the control group. When subjects were stratified according to their participation, metabolic control was significantly better among diabetic subjects participating frequently (greater than or equal to 11 of 13 sessions) than among those participating infrequently (less than 11 of 13 sessions), regardless of the type of activity. It was concluded that a training program of 1 h/wk for 3 mo does improve physical fitness but not the metabolic control of diabetes. On the other hand, glycemic control appears to be best among diabetic subjects who are motivated to participate in any kind of program related to the treatment of their disease.

Nocturnal release of immunoreactive growth hormone-releasing hormone and growth hormone in normal children.

To evaluate the role of growth hormone-releasing hormone (GHRH) in the physiologic release of growth hormone (GH) we studied the nocturnal secretion of immunoreactive GHRH (ir-GHRH) and its relationship to GH release and various stages of sleep in six prepubertal (three boys) and six pubertal children (two boys) with normal stature. Their ages ranged from 8.1 to 14.9 yr and their bone ages from 6.8 to 14.8 yr. Blood was withdrawn continuously between 2200-0600 h at a constant rate of 5 mL/20 min. The EEG was simultaneously registered. The ir-GHRH and GH data were analyzed by a discrete-pulse detection algorithm (Pulsar). The number of nocturnal ir-GHRH pulses varied from 0-8 (median 7) and the number of GH peaks from 2-6 (median 3). Pubertal children had significantly more (p less than 0.05) ir-GHRH pulses and the pulse amplitude was higher (p less than 0.05) than in the prepubertal children. There were no significant differences in the GH parameters between the two groups. The ir-GHRH peaks were not significantly related to any specific sleep stage. The majority of the GH pulses (71%) were associated with slow wave sleep (p less than 0.001). Two-thirds (69%) of the GHRH peaks preceded closely or coincided with GH pulses (p less than 0.02). Pubertal subjects had more isolated ir-GHRH peaks than prepubertal children (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)