G. Heinemeyer

[35] Hydrogen peroxide in hepatic microsomes

Publisher Summary This chapter talks about the hydrogen peroxide in hepatic microsomes. H 2 O 2 determination in microsomes is dependent on a method that fulfills some requirements. Those requirements are (1) it is sensitive enough to permit determination of 1 μM H 2 O 2 without interfering with monooxygenase-dependent hydroxylation reactions, (2) allows to eliminate the influence of various substrates or products of mixed-function oxidation reactions, (3) permits inhibition of contaminating catalase so that during an incubation period measurements not only of small steady-state concentrations, but also of rate and extent of H 2 O 2 formation are possible, and (4) allows to determine possible degradation of H 2 O 2 by residual catalase-, peroxidase-, or NADPH-dependent mixed-function oxygenase activity. The reason that in the presence of sodium azide H 2 O 2 accumulates and is subjected to further metabolism by pathways other than catalase, for example NADPH-dependent mixed-function oxygenase, the trapping of H 2 O 2 as HCHO by the addition of exogenous catalase and methanol avoids such degradation. Under circumstances where H 2 O 2 accumulates, the measurements of rate and extent of H 2 O 2 formation in microsomes can vary depending on the method applied.

Clinical course and outcome in class IC antiarrhythmic overdose.

120 cases of class IC antiarrhythmic overdose, including propafenone, flecainide, ajmaline and prajmaline overdose, were evaluated with respect to clinical course, therapy and outcome. Whereas drug overdose in general has an overall mortality of less than 1%, intoxication with antiarrhythmic drugs of class IC was associated with a mean mortality of 22.5%. Nausea, which occurred within the first 30 minutes after ingestion, was the earliest symptom. Spontaneous vomiting probably led to self-detoxication in about half the patients. Cardiac symptoms including bradycardia and, less frequently, tachyrhythmia occurred after about 30 minutes to 2 hours. Therapeutic measures included administration of activated charcoal, gastric lavage and a saline laxative, catecholamines, and in some patients, hypertonic sodium bicarbonate, insertion of a transvenous pacemaker and hemoperfusion. Fatal outcome was mainly due to cardiac conduction disturbances progressing to electromechanical dissociation or asystolia. Resuscitation, which had to be performed in 29 patients, was successful in only two of them. No correlation was found between fatal outcome, the type of antiarrhythmic, and ingested dose. Since a specific treatment is not available and resuscitive procedures including sodium bicarbonate and insertion of a pacemaker are of limited therapeutic value, early diagnosis and primary detoxification are most important for prevention of fatal outcome.

Quantitative determination by HPLC of urinary 6β-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs

SummaryA method for determination of 6β-hydroxycortisol in urine by means of high performance liquid chromatography is described. After extraction of 10–30 ml aliquots of urine with ethylacetate, separation is accomplished on a silica gel column (30 cm, Lichrosorb Si 100) with a special two-phase four-component eluent of methylene chloride, n-hexane, ethanol and water. Complete separation of α- andβ-isomers requires 15 to 20 min. For routine determinations precolumn cleaning by backflush permits injections of samples at minimum time intervals. For quantitative determinations, each injection should contain at least 0.05–0.5 µg of 6β-hydroxycortisol, depending on the detector employed. The mean excretion rate in healthy male adults (26–40 years) was 273 µg/day (SD=74.5; n=12). In patients on long term mono-therapy with rifampicin, 6β-hydroxycortisol excretion had risen fourfold (1166 µg/d; SEM=248; n=7), paralleling the known enzyme-inducing effect of rifampicin. The relatively smaller increase to 498 µg/d observed in patients receiving triple therapy with rifampicin, isoniazid and ethambutol points to possible inhibition by isoniazid. The greatest stimulation of 6β-hydroxycortisol excretion (2352 µg/d) was found in patients receiving antiepileptic therapy (phenytoin and/or carbamazepine and other drugs). The HPLC technique for 6β-hydroxycortisol proved to be a tool routinely applicable to non-invasive evaluation of drug metabolizing enzyme activity in man.

Pharmacokinetic interaction between cyclosporin and diltiazem

SummaryPrevious reports have indicated that administration of the calcium antagonist diltiazem results in major changes in the pharmacokinetics of cyclosporin A (CyA). A new clinical trial was undertaken in 22 renal transplant patients receiving a constant dose of cyclosporin to further explore this interaction. Coadministration of diltiazem for one week produced an increase in the blood concentration of CyA and its metabolites 17 and 18 in almost all patients, but no increase in CyA metabolites 1 and 21. The mean whole blood CyA trough level determined by HPLC rose from 117 ng·ml−1 to 170 ng·ml−1 after one week on diltiazem, and the mean trough level of metabolite 17 rose similarly from 184 ng·ml−1 before to 336 ng·ml−1.Based on experiments with microsomes from human liver the effect of diltiazem was due to noncompetitve inhibition of CyA-metabolism by diltiazem, and the increased concentration of metabolite 17 might have been due to stronger inhibition of its secondary metabolism steps.

Oxidation and glucuronidation of valproic acid in male rats ― influence of phenobarbital, 3-methylcholanthrene, β-naphthoflavone and clofibrate

The influence of phenobarbital, clofibrate, 3-methylcholanthrene and beta-naphthoflavone on omega- and beta-oxidation as well as on glucuronidation of valproic acid (n-dipropylacetic acid) was evaluated in male Sprague-Dawley rats by determination of urinary excretion of its metabolites by GC-MS after administration of 100 mg/kg. In controls 12% of the dose was excreted within 24 hours, primarily as glucuronides; metabolites formed by oxidation amounted to about 4%. Phenobarbital treatment led to stimulation of 4-hydroxyvalproic acid [(omega-1)-oxidation], 5-hydroxyvalproic acid and n-propylglutaric acid (omega-oxidation) excretion. Clofibrate enhanced the excretion of 4-hydroxyvalproic acid and 3-keto-valproic acid, a product of peroxisomal beta-oxidation. beta-Naphthoflavone slightly increased the excretion of 5-hydroxyvalproic acid. The most specific effect was found for 3-methylcholanthrene, which was effective in stimulating the formation of 3-hydroxyvalproic acid which might be formed by (omega-2)-oxidation. The addition of fatty acids (olive oil in which 3-methylcholanthrene and beta-naphthoflavone were suspended) led to increased excretion of 3-keto-valproic, 4-hydroxyvalproic and n-propylglutaric acid. The excretion of 3-hydroxyvalproic acid was completely suppressed by olive oil. Such specific effects were not observed for glucuronidation of valproic acid and its metabolites, although stimulation was attained after phenobarbital, clofibrate and 3-methylcholanthrene treatment, because of instability of glucuronide conjugates. Stimulation of valproic acid metabolism was also shown by increased plasma clearance after treatment with phenobarbital. In contrast, clofibrate given once 1 hr before valproic acid inhibited excretion of valproic acid, possibly by competition during renal tubular secretion. Determination of valproic acid metabolites in urine provides a useful tool for evaluation of inducer specificity of short chain fatty acid metabolism and differentiation between microsomal and peroxisomal enzyme activity.

Clinical Pharmacokinetic Considerations in the Treatment of Increased Intracranial Pressure

SummaryLife-threatening increased intracranial pressure can be reversed by a variety of drugs. These compounds all have some disadvantages, producing rebound effects, severe coma or cardiovascular depression and electrolyte imbalance. However, reduction of intracranial pressure is a prerequisite for recovery and the benefits of treatment outweigh the risks.Dexamethasone is rapidly eliminated, the short half-life (about 3 hours) indicating that dosage intervals should be kept small. As yet, however, its therapeutic efficacy has not been clearly demonstrated. Therefore, an association between pharmacokinetics and pharmacodynamics cannot be established.Osmotic diuretics are the most widely used agents for reduction of intracranial pressure. Pharmacokinetics show a very close relationship to changes in serum osmolality, but there are large variations in the clearance. For the use of osmotics, the blood-brain barrier must be intact. Osmotic diuretics may lead to intracerebral oedema or to acute renal failure as serum osmolality increases. Considering the pharmacokinetics of each drug, and the dynamics of intracerebral pressure and osmolality, an intermittent, individually titrated dosage should be administered, with simultaneous monitoring of intracranial pressure.Frusemide (furosemide) can be used as an adjunct, to enhance the effect of osmotic diuretics. Its pharmacokinetics are limited by renal function, depending on age as well as on the extent of renal impairment. Altered renal elimination of concomitantly administered drugs, and electrolyte imbalances should be anticipated when diuretics are used.Barbiturates are certain to decrease intracranial pressure in humans by an as yet unknown mechanism. Their administration is recommended for patients that do not respond to conventional therapy.As barbiturates can result in deep coma, knowledge of their pharmacokinetics is of great importance for recovery. Following single doses, pentobarbitone has a relatively long elimination half-life (about 22 hours). However, after repeated administration for several days, its elimination may be enhanced due to autoinduction. Thiopentone kinetics are characterised by distribution and redistribution into deep peripheral compartments. Administration of high and frequent doses leads to considerably delayed recovery. This is not true for methohexitone, which shows comparable pharmacokinetics after single and multiple dose administration. Elimination depends on liver blood flow. Thus, recovery from methohexitone-coma is rapid. Rapid elimination is also an important characteristic of etomidate and alphaxolone/alphadolone, two non-barbiturate hypnotics.Patients requiring intracranial pressure-lowering medication are usually critically ill and on multiple drug therapy, leading to great variations in pharmacokinetics and pharmacodynamics. Drug interactions based on changes in plasma protein binding, drug metabolism activity and renal function have to be considered. Monitoring of plasma concentrations is therefore necessary to prevent side effects and to differentiate symptoms caused by intracranial pressure-reducing medication from those induced by coma due to brain damage.

Monitoring of pentobarbital plasma levels in critical care patients suffering from increased intracranial pressure.

Pentobarbital plasma levels were determined in 16 critical care patients receiving a dose of approximately 30 mg/kg/day and suffering from severe head injury. In 10 patients monitored more than six times, a continuous decrease in plasma concentrations, caused by a mean increase in pentobarbital total plasma clearance from 0.81 to 1.06 ml/min/kg, was found. This effect might be due to autoinduction of hepatic drug-metabolizing enzymes. As clearance values showed marked inter- and intraindividual variability, it is necessary to monitor pentobarbital plasma levels frequently to adapt the dosage to the changes in clearance. Infrequent determinations are of little clinical value, as the necessary changes in pentobarbital dosage will not be predicted precisely enough.

Hexobarbital-binding, hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig, rat and rabbit

SummaryCytochrome P-450 dependent oxygenase (3′-hydroxy-hexobarbital) and oxidase activities (hydrogen peroxide) have been measured in hepatic microsomes from guinea pigs, rats and rabbits. A sensitive gas-chromatographic assay was developed to measure the hydroxylated product 3′-hydroxy-hexobarbital. The kinetics of its formation were determined and correlated to hexobarbital type I binding and compared with oxidase activity: in the rat, Vmax for 3′-hydroxy-hexobarbital formation and for hexobarbital-dependent H2O2 formation was 5.1 and 2.6 nmoles/mg/min, resp. This was increased by phenobarbital tratment to 15.2 and 13.4 nmoles/mg/min. In phenobarbital treated rabbits, Vmax was 15.0 nmoles/mg/min for hydroxylation and 40.8 for H2O2 formation. Spectral affinity constants (Ks) in control animals were 0.12 mM (rats) and 0.14 mM (rabbits). Phenobarbital treatment decreased these affinity constants, which were similar for each activity measured. In guinea pigs, however, hydroxylation of hexobarbital was low (3.1 nmoles/mg/min) and hexobarbital-dependent formation of H2O2 was higher than hydroxylation (Vmax:7.0 nmoles/mg/min). Phenobarbital treatment led here to two affinity constants for each activity measured, which however, were alike. The existence of low in addition to high affinity constants observed here might explain the difficulties seen hitherto in correlating hexobarbital binding and metabolism in this species. Total oxidase activity was higher than oxygenase activity in all species tested.It is suggested that oxygenase activity of cytochrome P-450 is not limited by binding but by a competition with oxidase activity for a common intermediary species. This might be peroxy-P-450 (substrate-Fe3+O22−), rendering either substrate-Fe3+O for hydroxylation reaction, or oxidized cytochrome P-450-substrate and hydrogen peroxide as product of oxidase function.

Stoichiometric cooperation of NADPH and hexobarbital in hepatic microsomes during the catalysis of hydrogen peroxide formation.

Abstract The interaction of NADPH and hexobarbital during catalysis of microsomal mixed function oxidase-dependent hydrogen peroxide formation has been investigated in hepatic microsomes from phenobarbital-treated rabbits. The application of Jobs method (25) of continuous variation revealed optimal conditions for the rate and extent of hydrogen peroxide formation when hexobarbital and NADPH were in equimolar amounts. The formation of a complex of 1 mol NADPH with cytochrome c -reductase and 1 mol hexobarbital with cytochrome P -450 seems to be responsible for limitation of hydrogen peroxide formation. Rate and extent of hydrogen peroxide formation are directly proportional to the amount of hexobarbital and NADPH present and are governed by the mass action equation in a manner similar to that reported for interaction of purified enzymes ( G. T. Miwa, S. B. West, M. T. Huang, and A. H. Lu, 1979, J. Biol. Chem. 254 , 5695–5700). Depending on either the NADPH concentration maintained by a generating system or the hexobarbital concentration, the extent of hydrogen peroxide formation could be shown to be a function of either compound alone, as long as the other one is in excess. The question whether the formation of hydrogen peroxide depends on the availability of two independent one-electron transfer reactions forming O 2 − or of one simultaneous two-electron transfer forming O 2 2− might thus become rather a matter of association of substrate and cosubstrate to a catalytically active complex in which the substrate augments the availability of reducing equivalents.

Kinetics of hexobarbital and dipyrone in critical care patients receiving high-dose pentobarbital

SummaryThe effect of pentobarbital treatment in a mean dose of 30 mg/kg/day on the clearance of hexobarbital (Evipan) and dipyrone (Novalgin) has been evaluated in critical care patients receiving a large number of drugs as comedication. Eleven patients treated with pentobarbital showed a hexobarbital half-life of 2.79 h and a total plasma clearance of 9.80 ml·min−1·kg−1 as compared to 10 patients without pentobarbital administration in whom there was a significantly longer half life (6.92 h) and lower clearance (2.97 ml·min−1·kg−1). The kinetics of hexobarbital were correlated with the urinary excretion of D-glucaric acid, a non-invasive parameter of drug metabolising activity. In 10 patients on pentobarbital, the total plasma clearance of N-4-methylaminoantipyrine, the active form of dipyrone, did not differ from that in 8 patients not receiving pentobarbital. As drug kinetics show great variability in these patients, it is difficult to discriminate enzyme induction from other mechanisms, for example competitive inhibition or changes in volume of distribution. In the presence of pentobarbital, however, induction of drug metabolising enzymes should be considered as a possible reason for the higher clearance of hexobarbital.