Cytochrome P450 isoenzymes--importance for the internist.

The studies with CYP isoenzymes give hope that in the near future it will be possible to predict the clinical pharmacokinetics of drugs, if their oxidation can be assigned to the activity of one or more well-characterized CYP isoenzymes (or if their metabolism is catalyzed by other well-studied enzymes). This strategy will reduce animal experimentation. Interindividual variability in drug metabolism translates into variability in drug efficacy and toxicity. Establishing the status of drug-metabolizing enzymes will therefore assist in making predictions of pharmacologic and toxicologic responses to drugs. Many clinically relevant drug-drug interactions can now be readily rationalized in terms of the substrate and inhibitor specificities of individual human CYP isoenzymes. The next 5 years should reveal whether selective inhibitors of xenobiotic-metabolizing CYP's can be used therapeutically in the treatment or prevention of cancer and various endocrine disorders, in analogy to the use of influencing steroidogenic CYP's.

[Ischemic hepatitis]. / Ischemische hepatitis.

Triggered by a case of ischaemic hepatitis (shock liver) in a patient with severe respiratory insufficiency, we tried to gather information about clinical characteristics and incidence. To our surprise, this information could be found neither in major critical care, medical or gastroenterology textbooks nor in textbook indices or works on differential diagnosis. From Sept. 1989 to May 1990 we studied all possible cases of ischaemic hepatitis in a 390 bed general hospital, to establish incidence. Using computerised data from the clinical chemistry laboratory, all patients with grossly abnormal liver function tests were identified. In this nine-month period 27 adult patients had a peak ALAT level of > 500 U/l: 8 of these suffered from ischaemic hepatitis, using the criteria described by Gibson et al. In another 5 this diagnosis was suspected but could not be ascertained before death (30% and 18% of all cases). In all these cases ASAT, ALAT, LDH levels were 8-100 times normal, but bilirubin, alkaline phosphatase, gamma-glutamyl transferase and prothrombin time were only slightly abnormal. With correction of the underlying disorder enzyme levels returned to normal very rapidly, in 5-10 days. Ischaemic hepatitis could easily be distinguished from other causes such as alcoholic, viral or drug-induced hepatitis. Ischaemic hepatitis was the most frequent cause of severely elevated ASAT, ALAT and LDH in hospitalised patients. The diagnosis can easily be made on clinical characteristics and the typical biochemical pattern. An elaborate work-up or invasive procedure is redundant. Prognosis per se is excellent but depends on the underlying disorder.

Human hepatic microsomal thiol methyltransferase. Assay conditions, biochemical properties, and correlation studies.

Thiol methyltransferase (TMT) catalyzes the S-methylation of aliphatic sulfhydryl drugs and xenobiotic compounds. As a first step in the determination of whether genetic control of TMT activity in an easily accessible human cell, the red blood cell (RBC), might reflect the regulation of TMT in the human liver, we determined optimal conditions for the assay of human hepatic microsomal TMT activity. We then used those assay conditions to study the biochemical properties and regulation of TMT in the human liver for comparison with the properties of TMT in the human RBC. Substrate kinetic studies of hepatic microsomes performed with 2-mercaptoethanol (2-ME) revealed biphasic kinetics similar to those found in the human RBC, with apparent "high-" and "low"-affinity forms of TMT activity. The high-affinity form had an optimal pH of 7.2-7.6 and apparent KM values of 9.0 microM for 2-ME and 7.5 microM for S-adenosyl-L-methionine. The low-affinity form had an optimal pH of 8.8 and apparent KM values of 20 mM for 2-ME and 44 microM for S-adenosyl-L-methionine. Both forms were inhibited by compounds that also inhibited human RBC membrane TMT. The two kinetic forms of hepatic microsomal TMT activity were inactivated approximately 50% by heating for 15 min at 53 degrees C and, as was found with RBC TMT, had very similar thermal stability profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse liver thiol methyltransferase. Assay conditions, biochemical properties, and strain variation.

Thiol methyltransferase (TMT) catalyzes the S-methylation of aliphatic sulfhydryl drugs and xenobiotic compounds. It would be useful if there were an experimental animal model in which the regulation and function of TMT could be studied. Therefore, TMT activity was measured in hepatic microsomes from A/J mice. Substrate kinetics for mouse liver microsomal TMT, like those for the enzyme in human red blood cell membranes and human kidney microsomes, were biphasic, with apparent "high" and "low" affinity forms of TMT with 2-mercaptoethanol as the methyl acceptor substrate. Apparent Michaelis (Km) constants of the high and low affinity activities for 2-mercaptoethanol were 30 microM and 12 mM, respectively. Apparent Km values of the high and low affinity activities for S-adenosyl-L-methionine, the methyl donor for the reaction, were 47 microM and 61 microM, respectively. The optimal pH for the high affinity activity was between 7.2 and 8.1, whereas the optimal pH for the low affinity activity was approximately 9.2. Differential centrifugation showed that more than 85% of both activities was associated with membrane fractions. SKF 525A, a potent inhibitor of TMT in human tissues, inhibited mouse liver high and low affinity TMT activities by 88% and 46%, respectively, at a concentration of 0.5 mM. TMT activities were then measured in hepatic microsomes from nine additional inbred strains of mice. High affinity TMT activities varied 1.7-fold, whereas low affinity activities varied 2.1-fold among these strains. Since the properties of TMT in mouse liver are similar to those of the enzyme in human tissue, the inbred mouse will be a useful experimental animal model in which to study the regulation and function of TMT.

Cephalosporin-induced hypoprothrombinemia: possible role for thiol methylation of 1-methyltetrazole-5-thiol and 2-methyl-1,3,4-thiadiazole-5-thiol.

Heterocyclic thiol metabolites of cephalosporin antibiotics may play an important role in the pathophysiology of hypoprothrombinemia and hemorrhage in patients treated with these drugs. A heterocyclic thiol metabolite of moxalactam, 1-methyltetrazole-5-thiol (MTT), inhibits the gamma carboxylation of glutamic acid that is required for the formation of active clotting factors. One possible pathway for the biotransformation of thiol compounds such as MTT is S-methylation catalyzed by either thiopurine methyltransferase (TPMT), a soluble enzyme, or by thiol methyltransferase, a microsomal enzyme. Therefore, MTT and 2-methyl-1,3,4-thiadiazole-5-thiol (MTD), a thiol "leaving group" structurally related to MTT that is present in cefazolin, were tested as possible substrates for S-methylation catalyzed by purified human kidney TPMT or by human liver microsomes, a source of thiol methyltransferase. MTT and MTD were methylated by both human kidney TPMT and human liver microsomes. The products of these reactions were shown by high-performance liquid chromatography to be S-methyl MTT and S-methyl MTD. Apparent Km constants for the methylation of MTT and MTD by TPMT were 0.26 and 0.068 mM, respectively. Apparent Km constants for the methylation of MTT and MTD by human liver microsomes were 0.60 and 0.20 mM, respectively. Maximal velocity (Vmax) values for the S-methylation of MTD catalyzed by TPMT and by human liver microsomes were 3.58- and 678-fold greater than were those for the thiol methylation of MTT. Finally, S-methyl derivatives of MTT and MTD were one to two orders of magnitude less potent as inhibitors of the in vitro gamma carboxylation of glutamic acid than were MTT and MTD themselves.(ABSTRACT TRUNCATED AT 250 WORDS)

S-Methylation of D- and L-penicillamine by human erythrocyte membrane thiol methyltransferase.

Human red blood cell (RBC) membranes contain a thiol methyltransferase activity that catalyzes the S-methylation of 2-mercaptoethanol (2-ME). These experiments were performed to determine whether human RBC membranes contain enzymes that can catalyze the S-methylation of D- and L-penicillamine, to determine whether those enzymes are similar to the RBC membrane thiol methyltransferase that catalyzes the S-methylation of 2-ME, and to determine whether lipophilic conjugates of the S-methyl metabolites of D- and L-penicillamine are formed by RBC membranes. Human RBC membranes were able to catalyze the S-methylation of D- and L-penicillamine. The apparent Michaelis (Km) constants for D- and L-penicillamine were 7.53 and 7.27 mM, respectively. However, the Vmax value for L-penicillamine was more than 2.5 times greater than the Vmax value for D-penicillamine. D- and L-Penicillamine methyltransferases and 2-ME thiol methyltransferase were similar with respect to their subcellular distributions, inhibitor sensitivities, and thermal stabilities. In addition, when methyltransferase activities for 2-ME and for D- and L-penicillamine were measured in RBC membranes from 19 individual subjects, there were highly significant correlations among all three activities (r greater than 0.98, p less than 0.001 for all three comparisons). These observations suggest either that a single enzyme in the human RBC membrane catalyzes the S-methylation of all three compounds, or, less likely, that these reactions are catalyzed by three separate enzymes that are regulated in parallel and have similar properties. Experiments were then performed to identify the products of the penicillamine methylation reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Furosemide kinetics and dynamics in aged patients.

Furosemide (F) is frequently used by elderly persons. We wanted to know whether age-related changes in the disposition or the effect of F occurred and whether these changes could be predicted or explained. Kinetics and diuretic effects were studied after intravenous F injection in geriatric patients (70 to 93 yr of age). The changes in kinetics were as follows: slightly enlarged volume of distribution (0.16 +/- 0.04 l) inversely related to serum albumin, diminished total body clearance (73 +/- 27 ml/min), decreased renal clearance (40 +/- 18 ml/min), and prolonged elimination t1/2 (180 +/- 87 min). F effects were unchanged only if the decreased renal delivery of F and the decreased glomerular filtration rate were taken into account. Renal F clearance correlated best with endogenous creatinine clearance (ECC) and with F effects. Only ECC and plasma creatinine turned out to be useful patient characteristics to predict all F effects. Many drugs have an increased effect in elderly persons if a standard dose is used, based on the attainment of higher free plasma levels. In the case of F, however, the effects are decreased because they depend on decreased glomerular filtration of electrolytes and water and on decreased renal F secretion. These decreases are in part age dependent and in part disease dependent.

Changes in pharmacokinetics and in effect of furosemide in the elderly.

In this paper the general changes in the pharmacokinetics of drugs in the elderly are summarized. The suggestion is proposed, that in the case of diuretics--which act after renal excretion--the results of these changes are totally different: contrary to other drugs the standard dose of a diuretic must have less saliuretic effect instead of more effect. This thesis is supported by the results of experiments with furosemide in old patients: the kinetics are changed as expected, leading to higher plasma levels and increased Area Under the (plasma level versus time) Curve (AUC), but the effects are--dependent on renal function--decreased. To a certain degree kinetics and dynamics of furosemide can be explained and predicted on the basis of simple patient parameters. If diuretics are used in old people to treat congestive heart failure, the normal adult dose has to be used, and occasionally a larger dose has to be given.

Specimen handling and high-performance liquid chromatographic determination of furosemide.

A simple high-performance liquid chromatographic method to measure furosemide in plasma and urine is described. Furosemide fluoresces best, but is unstable, at acidic pH and is subject to photochemical degradation. These factors were analysed and the results prompted changes in previously described methods. All specimens were very carefully protected from light; extraction and acidification were done with acetic acid instead of hydrochloric acid. With these precautions no 4-chloro-5-sulphamoylanthranilic acid was found in biological specimens. The main metabolite was furosemide glucuronide (20% of furosemide excretion). Sensitivity was 0.1 and 0.5 microgram/ml for plasma and urine, respectively. The applicability of our method for furosemide studies is demonstrated.

Pharmacokinetic and pharmacodynamic studies of tienilic acid in healthy volunteers.

A simple and reliable HPLC method for the determination of tienilic acid ((TA) Selacryn, Selcryn, Diflurex, Ticrynafen) and its alcoholic metabolite in plasma and urine has been developed. In 8 healthy adult volunteers the plasma and urinary levels of tienilic acid and its alcoholic metabolite, and plasma and urinary levels of sodium, creatinine and uric acid were measured after oral administration of tienilic acid 250 mg. The pharmacokinetic parameters found differed only slightly from those reported in the literature, as there was faster absorption and a shorter half-life. TA is probably excreted by a saturable renal tubular transport mechanism. The pharmacodynamic effects of tienilic acid developed quickly, the uricosuric effect being very impressive and the natriuretic effect moderate. These effects disappeared in about 8 h. An inverse relationship was found between the starting plasma uric acid level in an individual and the maximal uric acid clearance - the higher the plasma uric acid level, the lower was the maximum effect. Plasma tienilic acid level and natriuretic effect were correlative within individuals and intra-individually (p less than 0.05). Urinary tienilic acid level and natriuretic effect were correlated, too (p less than 0.05 to p less than 0.001), but only intraindividually. No correlation between drug level and uricosuric effect was found.