Aronia melanocarpa fruit extract exhibits anti-inflammatory activity in human aortic endothelial cells.

BACKGROUND: Altered expression of cell adhesion molecules (CAMs) has been implicated in a variety of chronic inflammatory conditions, including atherosclerosis. Regulation of adhesion molecule expression by specific redox-sensitive mechanisms has been reported. Additionally, it has been observed that the extract of Aronia melanocarpa (A. Melanocarpa) fruits, rich in polyphenols, exhibits potent anti-oxidant properties and displays cardioprotective activity. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were pretreated with various concentrations (primarily 50 µg/mL) of Aronia Melanocarpa fruit extract prior to treatment with TNFα (10 ng/mL) for various periods of time. The surface protein and mRNA expression of ICAM-1 and VCAM-1 were determined using flow cytometry and real-time RT-PCR, respectively. Adhesion of peripheral blood mononuclear leucocytes (PBMLs) to TNFα-treated HAECs was evaluated by an adhesion assay. Activation of NF-κB was evaluated by measuring NF-κB p65 phosphorylation using flow cytometry. ROS production was determined by reduction in fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA). Tested A. Melanocarpa extract significantly inhibited the expression of ICAM-1 and VCAM-1, attenuated the phosphorylation of NF-κB p65 and decreased intracellular ROS production in TNFα-treated HAECs. CONCLUSION: We conclude that A. Melanocarpa fruit extract exhibits anti-inflammatory effects in HAECs by inhibiting the expression of endothelial CAMs, activation of NF-κB and production of ROS.

Trans fatty acids induce a proinflammatory response in endothelial cells through ROS-dependent nuclear factor-κB activation.

UNLABELLED: It has been shown that increased intake of trans fatty acids (TFAs) is associated with a higher risk of cardiovascular disease. In this study, we have investigated the effects of linoelaidic (LA) and elaidic (EA) acids on the proinflammatory response in endothelial cells, a key step in vascular disease. Human aortic endothelial cells (HAECs) were treated with different concentrations (100 µmol/l in most experiments) of LA or EA for different periods of time. The surface protein and mRNA expression of ICAM-1 and VCAM-1 were determined by flow cytometry and real time RT-PCR, respectively. Adhesion of leukocytes to TFA-treated HAECs was evaluated by an adhesion assay. Activation of nuclear factor-κB (NF-κB) was evaluated by measuring NF-κB p65 phosphorylation using flow cytometry. ROS production was determined by the reduction of fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA). LA treatment significantly increased protein and mRNA levels of ICAM-1 and VCAM-1, leukocyte adhesion to HAECs, phosphorylation of NF-κB and ROS generation. Similar effects were achieved for cells incubated with EA. Experiments with HAECs pretreated with pyrrolidine dithiocarbamate, an inhibitor of NF-κB, revealed that both LA and EA-mediated induction of ICAM-1 and VCAM-1 is mainly regulated by NF-κB. The ROS production induced by both of the studied acids was inhibited in the presence of diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, suggesting ROS production through the activation of NADPH oxidase. Furthermore, LA or EA-induced ICAM-1 and VCAM-1 expression, activation of NF-κB and adhesion of leukocytes to HAECs were abolished in the presence of DPI. CONCLUSION: TFAs present in our diet have a direct proinflammatory effect, which promotes leukocyte adhesion to the endothelium through ROS-dependent NF-κB activation.

[Mechanisms of drug metabolism--implications for drug interaction]. / Mechanizmy metabolizmu leków--implikacje dla interakcji leków.

Most drugs undergo biotransformation before excretion by renal, biliary or other routes. The main purpose of metabolism is to make the drug, which is usually lipophilic, more water soluble. Metabolic reactions, depending upon the end product formed, can be classified as functionalisation (phase I) or conjugation (Phase II) reactions. Phase I metabolic reactions include oxidation, reduction and hydrolysis; while phase II processes are glucuronidation, sulfation, methylation, acetylation and mercapture formation. Cytochrome P-450 isozymes play a central role in metabolism of great majority of xenobiotics, as well as some endogenous substances. Many drugs can inhibit, induce and alter relative amounts of different P-450 enzymes; therefore, possibilities of drug-drug interactions exist in that one drug can influence biodisposition of another with potential clinical implications. One drug can inhibit metabolism of another, leading to excessive accumulation and toxicity. Alternatively, one drug can stimulate or induce metabolism of another drug resulting in subtherapeutic plasma levels of the later.

Endothelin-1 inactivating peptidase in the human kidney and urine.

OBJECTIVE: Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology. METHODS: Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37 degrees C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [125I]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography. The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft RESULTS: ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155-1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (Rf 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (KM 180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (KM 11.5 micromol/l), endothelin 11 21 fragment (KM 15.3 micromol/l), endothelin antagonist Trp-Leu-Asp-Ile-Ile-Trp (KM 3.1 micromol/I), gastrin (KM 2.2 micromol/l) and cholecystokinin (KM 4.0 micromol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme. The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 micromol/l), cyclosporin (IC50 400 micromol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l). Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft CONCLUSIONS: Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system.

[Elevated levels of homocysteine in plasma as a risk factor for coronary artery disease]. / Podwyzszone stezenie homocysteiny w osoczu jako czynnik ryzyka choroby niedokrwiennej serca.

This study was performed to assess the significance of association between coronary artery disease (CAD) and circulating homocysteine concentrations. 100 consecutive CAD patients (78 men and 22 women, aged 31 to 79 years) qualified for PTCA were investigated. At the time of PTCA, the risk factors for CAD and plasma for homocysteine and vitamins were obtained. The controls were without clinical evidence of coronary artery disease and hypertension (90 men and 30 women aged 32 to 81 years). Homocysteine was assayed using ELISA test. Red cell folate and plasma vitamin B12 were assayed by immunofluoroscency (Delphia test). Homocysteine concentrations were higher in patients than in controls (13.61 +/- 4.5 vs 10.99 +/- 4.49 mumol/L, p < 0.001, adjusted for age). Male patients had nonsignificantly higher homocysteine levels than females (13.94 +/- 5.21 vs 11.46 +/- 5.16 mumol/L, p = 0.05, adjusted for age). Elevated homocysteine level--defined as one in the top fifth of the control distribution > or = 12.83 mumol/L--was seen in 46% of the patients compared with 20% of the control group (p = 0.001). The odds ratio (OR) for CAD in persons with elevated homocysteine level was 3.1 (95% Cl 1.6-5.8, p < 0.001, adjusted for age). The OR for CAD of 5 mumol/L increment in homocysteine level was 2.1 (95% Cl 1.4-3.1 p < 0.001, adjusted for age). After adjustment for conventional risk factors (age, smoking, hypertension, family history of CAD, hyperlipidemia), elevated homocysteine level remained independent risk factor for CAD (OR 2.88, 95% Cl 1.1-7.8, p < 0.05). We observed inverse correlation between homocysteine and folate level (r = -0.32, p = 0.005) and between homocysteine and vitamin B12 concentrations (r = -0.24, p = 0.03), especially in men. Patients with elevated homocysteine level had lower levels of folate (629.6 +/- 241.2 nmol/L vs 735.1 +/- 252.4 nmol/L, p < 0.05), and vitamin B12 (213.6 +/- 64.4 pmol/L vs 246.6 +/- 62.3 pmol/L, p < 0.05) than patients with normal level of homocysteine. Elevated plasma homocysteine level is a strong risk factor for coronary artery disease. A 5 mumol/L increment in total homocysteine level may be associated with twofold increase of risk for the disease.

[Effect of hydroxylation polymorphism on the concentration of propranolol in the blood of humans with hyperlipidemia]. / Wplyw polimorfizmu hydroksylacji na stezenie propanololu we krwi u ludzi z hiperlipidemia.

An influence of hydroxylation phenotype on the concentration of propranolol [corrected] was examined in 52 subjects with hyperlipidemia divided into 4 groups: 1--control, normolipemic, 2--hypercholesterolemic, 3--hypertriglyceridemic, and 4--mixed-form hyperlipidemic. Each study group included extensive metabolizers and one subject characterized by a poor hydroxylation phenotype. Propranolol was given intragastrically at a single dose of 80 mg [corrected]. Blood was sampled within 24 hours following the drug administration. HPLC method was used for determining blood serum concentrations of propranolol. In each study group mean blood serum concentrations of propranolol in poor metabolizers were at maximum in subject with hypertriglyceridemia, at minimum in the normolipemic one, and intermediate in hypercholesterolemic (upper) and mixed-form hyperlipidemic ones. Lipid metabolic disturbances also affected blood serum concentrations. They were the highest in hypertriglyceridemic patients, whereas in hypercholesterolemic were, in early stage of observation, even lower then in normolipemic subjects. Blood serum concentrations of propranolol [corrected] attained minimal values in patients with mixed form of hyperlipidemia. In the light of the present study we can state that hyperlipidemia modifies the blood serum concentrations of propranolol [corrected]. Although, the type of hyperlipidemia and lipophilic propranolol are not the only determinants affecting blood serum concentrations of propranolol, but also a genetic factor, i.e. hydroxylation phenotype may play an important role.

[Evaluation of the rate for reaching steady state during oral propafenone therapy in patients with ventricular arrhythmias]. / Ocena szybkosci uzyskiwania stanu stacjonarnego w trakcie doustnego leczenia propafenonem chorych z komorowymi zaburzeniami rytmu.

UNLABELLED: We prospectively evaluated reaching of steady state and clinical efficacy of propafenone (PPF), class Ic antiarrhythmic agent, in 16 patients (pts) (age 46-69, mean 57 years) with symptomatic ventricular arrhythmias (Low class II and IV). The majority (13 pts) had coronary artery disease. Drug was administered for 7 days (daily dose: 3 x 150 mg). Efficacy was defined as > 80% reduction of ventricular premature complexes (VPC) and class IV elimination in 24-hours Holter recording. Responders were continued on PPF for 3 weeks, in non-responders dose was titrated to 900 mg a day for the next 7 days. After second Holter evaluation the treatment was continued for 2 weeks in responders group. The non-responders were switched to other drug. After 4 weeks final Holter monitoring was performed. Serum concentration of PPF and its 2 metabolites: 5-hydroxy PPF and N-depropyl PPF were determined in 2, 3, 4, 5, 6, 7th day, just before the morning dose (3 x 150 mg/day) in 9 pts. RESULTS: Trough serum concentrations of PPF differed in high degree: 0-226 ng/ml (2nd day), 22-438 ng/ml (4th day), 42-614 ng/ml (6-7 day). An increasing tendency of serum concentrations of PPF was observed, so steady-state was not reached. This great dispersion of concentration values is because of non-linear metabolism and individual differences. Defined efficacy critetion was achieved in 62% pts, 56% for lower dose. Mean frequency of VPC was reduced by 86% in 24-hour Holter recording and per hour (p = 0.0011). Reduction of couplets/24 h was 87% (p = 0.0175). Significant prolongation of PQ (14%, p = 0.009) and QRS (13%, p = 0.0052) were observed. Changes of QT interval were not significant. One case of proarrhythmia was the cause of stopping the treatment. CONCLUSIONS: Serum concentrations' values undermine common opinion, that steady state can be reached after 1-2 days of treatment. High dispersion of serum levels is the result of nonlinear metabolism of PPF and individual differences. In spite of this the study showed defined antiarrhythmic efficacy in 62.5% pts. In this group 90% success rate was achieved after lower dose 3 x 150 mg.

N-acetylation and hydroxylation polymorphisms in type II diabetics with microvascular disturbances.

The N-acetylation and hydroxylation (CYP2D6) genetic polymorphisms were assessed in 43 healthy subjects and in 84 type II (non-insulin-dependent) diabetics. The proportions of slow and fast acetylators as well as poor and extensive metabolisers in a group of diabetics suffering from microvascular disturbances (nephropathy, retinopathy and neuropathy) were compared with the control group and with diabetics without such complications. Sulphadimidine was used as a probe for polymorphic acetylation and debrisoquine for CYP2D6. Debrisoquine and its 4-OH metabolite were assayed by means of HPLC, and sulphadimidine using a modified Bratton-Marshall procedure. The frequency of the slow phenotype (63%) was significantly higher in diabetics with microvascular disturbances than in patients without diabetic complications (P < 0.005). In patients with type II diabetes (84), only the extensive phenotype of hydroxylation was observed.

[Evaluation of vaccine potency against viral hepatitis B performed with HB-vax II vaccine in a group with an increased risk for hepatitis B]. / Ocena skutecznosci szczepien przeciwko wzw typu B przeprowadzonych szczepionka HB-vax II w grupie zwiekszonego ryzyka na zakazenie wirusem B.

The aim of this investigation was to established the rate of seroconversion against hepatitis B after vaccination of workers of the National Institute of Cardiology who are at increased risk. All participants were HBs-Ag, and anti-HBs negative and also had normal standard hepatic laboratory tests. Vaccination was carried out according to following scheme: the first vaccination at t = 0, the second after one month and the third after six months. Vaccine was given intramuscular. The success of vaccination was monitored by the quantitative determination of antibodies against HBs-Ag (Anti-HBs) by the ELISA method (Abbott). After each vaccination, anti-HBs formation above 10 iu/l protective level was observed in 40.7%, 85.9%, and 97.1% of immunocompetent subjects after first, second, and third vaccination respectively. The acquired anti-HBs titer varies from individual to individual but in immunocompetent subjects the level of antibodies against HBs after third vaccination was 170 fold higher then those after first vaccination. These results seems to suggest that vaccine HB-Vax-II is effective and may by recommended in pre-exposure prophylaxis against hepatitis B.

Analysis of diltiazem and desacetyldiltiazem in serum by high-performance liquid chromatography.

This paper describes a simple high-performance liquid chromatographic (HPLC) method for the determination of diltiazem and desacetyldiltiazem in human serum. After basic methyl-tert-butyl ether extraction and back-extraction with hydrochloric acid, the drug and its metabolite was injected into a Supelcosil LC-CN column and the absorbance of the eluate was measured at 240 nm. The sensitivity was 5 ng/ml and the obtained precision, selectivity and stability during storage were adequate for the performed clinical studies in patients therapeutically treated with diltiazem.

Debrisoquine hydroxylation in a Polish population.

The genetic polymorphism of drug oxidation mediated by cytochrome P450IID6 (CYP2D6) was determined in 154 Polish volunteers using debrisoquine as the test substance. The results showed a bimodal distribution of the debrisoquine metabolic ratio (MR). Nine persons (5.8%) with MR > 12.6 were classified as poor metabolisers (gene frequency 0.242), which is in substantial agreement with the data reported for other Caucasian populations.

Characterization of a novel, high-molecular weight, acidic, endothelin-1 inactivating metalloendopeptidase from the rat kidney.

OBJECTIVE: To characterize endothelin-1 inactivating peptidase (ET-1 peptidase) recently isolated from rat kidney. METHODS: ET-1 peptidase was purified from the membranes of whole Wistar-Kyoto (WKY) rat kidneys using differential centrifugation, detergent solubilization, ion-exchange chromatography, ultrafiltration and preparative electrophoresis. The enzyme activity in the presence of increasing concentrations of unlabelled peptides, inhibitors and other substances was determined at pH 5.5 and 37 degrees C using fixed amounts of [125I]-ET-1 as the substrate. RESULTS: On non-denaturing gels, the purified enzyme migrated in the form of a compact, low-mobility (Rf 0.07), high relative molecular mass (approximately 250,000) protein band. During denaturing polyacrylamide gel electrophoresis this protein separated into three fractions with apparent relative molecular masses 158,000, 110,000 and 61,000. Using different buffers, the optimum pH for this enzyme was found to be 5.5. Zinc (3.7 mmol/l), nickel (4.0 mmol/l), citrate (0.6 mmol/l), phosphate (1.3 mmol/l) and barbital ions (2.5 mmol/l) inhibited ET-1 peptidase activity by 50%, whereas magnesium, calcium, cobalt, manganous, sodium and borate ions were without effect. The most powerful inhibitors of the enzyme included: phenanthroline [median inhibitory concentration (IC50) 28 mumol/l], phosphoramidon (IC50 8.0 nmol/l), thiorphan (IC50 32 nmol/l) and N-carboxymethyl-Phe-Leu (IC50 12 mumol/l). Also, bacitracin (25 mumol/l), cyclosporine A (20 mumol/l) and sodium dodecyl sulphate (0.5%) inhibited enzyme activity by 50%, whereas bestatin, puromycin, aprotinin, phenylmethylsulphonyl fluoride, amanitin (50-100 mumol/l) and cardiotoxin (25 micrograms/assay) had no effect. The Michaelis constant (Km) values of 70 and 66 nmol/l were found towards ET-1 and the ET(16-21) fragment, respectively, whereas the Km values in respect to big-ET-1, sarafotoxin S6b, sulphated cholecystokin octapeptide, gastrin, glucagon, insulin, gastric inhibitory peptide and growth hormone ranged from 1.5 to approximately 50 mumol/l. The enzyme showed no apparent affinity for enkephalins, bradykinin, angiotensins, cholecystokinin tetrapeptides and kyotorphin. CONCLUSIONS: The present data suggest that the ET-1 peptidase that we isolated from rat kidney displays inhibitory characteristics similar to that of other known metalloendopeptidases. However, this enzyme exhibits several unique properties such as high molecular mass, an apparent complex subunits structure, pH optimum at 5.5, and very high substrate specificity towards ET-1 and the ET(16-21) fragment compared with other peptides either related or unrelated to endothelin.

Purification of endothelin-1-inactivating peptidase from the rat kidney.

OBJECTIVE: To identify and purify endothelin-1-inactivating peptidase from rat tissues. METHODS: Subcellular fractions of rat kidney, aorta, heart, lung, liver and blood cells were prepared by differential centrifugation. Kidney membrane-bound peptidase was solubilized with Triton X-100, chromatographed on the diethylaminoethyl-cellulose, ultrafiltered through a membrane of relative molecular mass 100,000 cutoff and subjected to electrophoresis on a non-denaturing polyacrylamide gel. The enzyme activity assay was performed at pH 5.5 using [125I]-endothelin-1 as the substrate. The trichloroacetic acid precipitation test, an endothelin-1 immunoreactivity assay, reverse-phase high-performance liquid chromatography and a receptor-binding assay were applied for the detection of degradation products. RESULTS: High-activity endothelin-1-degrading peptidase coincided with the fraction from the kidney membranes of both Wistar-Kyoto and spontaneously hypertensive rats, but not with any other of the tissues that were studied. The membrane (0.5 microgram protein/assay) degraded [125I]-endothelin-1 (5-100 pmol/l) within a half-time of about 10 min at 37 degrees C. The enzyme was purified to an apparent homogeneity with non-denaturing gel electrophoresis, by which it was identified as a low-mobility (Rf 0.07) protein fraction of high relative molecular mass (> 250,000). The optimum pH was 5.5, with a little activity found outside the range 5.0-7.0. The activity of the peptidase was inhibited by 0.5 mmol/l 1,10 phenanthroline (half-maximal inhibitory concentration 0.03 mmol/l), and by 1 mmol/l EDTA, implicating a metalloenzyme. Bestatin, puromycin, phenylmethylsulphonyl fluoride and thiorphan were without effect. Unlabelled endothelin-1 inhibited the degradation of [125I]-endothelin-1 (half-maximal inhibitory concentration 100 nmol/l), whereas 100 mumol/l methionine enkephalin or angiotensin I did not. High-performance liquid chromatography analyses of the [125I]-endothelin-1 incubated with purified peptidase revealed a time-dependent accumulation of one major radioactive fraction that was soluble in trichloroacetic acid. This product (or products) was not further hydrolysed. It did not react with the endothelin antibodies or with the specific, myocardial membrane receptors. CONCLUSION: Our data suggest that the rat kidney contains an acidic metalloproteinase of high relative molecular mass that is able to hydrolyse endothelin-1 rapidly and efficiently in vitro. The enzyme may participate in the inactivation of circulating or tissue endothelins, or both.

High-performance liquid chromatographic method for the simultaneous determination of propafenone and 5-hydroxypropafenone in human serum.

A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of propafenone and 5-hydroxypropafenone in human serum is described. Method involves a single-step extraction of the drug and its metabolite with dichloromethane:2-propanol (4:1 v/v) mixture from 0.2 ml of serum. Separation of the investigated compounds on deactivated Supelcosil LC18-DB column is accomplished by ultraviolet detection at 210 nm. The limit of detection is 10 ng/ml for propafenone and 4 ng/ml for 5-hydroxypropafenone. The method is useful for the routine monitoring of propafenone and its main metabolite in serum as well as for the pharmacokinetic studies.

[Time-resolved fluorescence of europium ions and its application for determination of biologically active substances]. / Mierzona w czasie fluorescencja jonow europu i jej zastosowanie do oznaczania substancji aktywnych biologicznie.

At present radioimmunoassay is still one of the most widely used analytical procedure. It is, however, continuously challenged by a number of non-isotopic techniques. This article reviews the measurement of europium chelates with high sensitivity using time-resolved fluorescence and its use as labels in immunoassays of protein and peptide hormones, haptens, virus antigens and for detection of inborn metabolic errors. It can be concluded that the time-resolved fluorescence detection of lanthanides and their chelates has been applied in a wide variety of both routine and research application. This technique is anticipated to gain wide use for measurement of numerous anylates of different origin. In immunoassays time-resolved fluorometry is one of the most promising alternatives available in the non-isotopic field.

Simultaneous determination of mexiletine and four hydroxylated metabolites in human serum by high-performance liquid chromatography and its application to pharmacokinetic studies.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of mexiletine and its four hydroxylated metabolites in human serum. The method involves a single-step extraction of mexiletine, hydroxymethylmexiletine, p-hydroxymexiletine and their corresponding alcohols with diisopropyl ether-dichloromethane-propan-2-ol (2.5:1.5:0.5, v/v). Separation of the compounds on a deactivated Supelcosil LC8-DB column is accomplished by high-performance liquid chromatography with ultraviolet detection at 203 nm. Overall the recovery of each compound is reproducible and greater than 75%. The lower limit of detection is 2 ng/ml for mexiletine and its metabolites. The application of the method is shown by measuring the concentrations in serum of mexiletine and its metabolites over 24 h in a healthy volunteer after a single intravenous injection of the drug and by monitoring serum concentrations in patients receiving long-term treatment by mouth of the drug.

The use of the firefly bioluminescent reaction for the rapid detection and counting of mycobacterium BCG.

A rapid test for the counting of Mycobacterium BCG, based on firefly luciferase assay of bacterial ATP has been evaluated. Three different methods for the extraction of ATP from mycobacterial cells were examined. Extraction with n-butanol proved to be the best method. The amount of ATP extracted correlated well with the number of colony forming units. It was found that the ATP content per colony forming particle of BCG varied only slightly after various periods of cultivation.

Modification of brain mitochondrial enzymes by the oxygen analogue of ronnel at various stages of development and aging.

This paper describes the effect of the organophosphorus compound, the oxygen analogue of ronnel (OAR), on the activity of some membrane-bound enzyme systems in the brain mitochondria of developing, young-adult, and old rats. Age-related changes were noted in the cholesterol-to-protein ratio, whereas the phospholipid content in mitochondria showed little change during development as well as aging. The results obtained suggest that development of brain succinate dehydrogenase may consist in a decrease of Km and increase of Vmax values. In aged rats an altered, perhaps inhibited form of the enzyme is produced. The oxygen analogue of ronnel caused a mixed-type inhibition of the succinate dehydrogenase derived from brains of 4-day-old, 16-day-old and 2-month-old animals. In the case of enzyme from the brain of 18-month-old rats, a typical competitive-type inhibition was observed. Mechanisms responsible for inhibition of the succinate: cytochrome c reductase from brains of developing animals are similar to those for succinate dehydrogenase. In aged rats (18 months old), however, a noncompetitive mechanism of inhibition of succinate: cytochrome c reductase was revealed. The experiments reported here provide evidence that lipid-soluble molecules of OAR may interact with membrane phospholipids and lead to modification of membrane architecture and also of enzyme kinetic behaviour. It may be also concluded, that the sensitivity of the enzyme systems studied to inhibition by OAR is an age-dependent phenomenon. Modification of membrane by development or aging alters the kinetics as well as the sensitivity of enzymes to inhibitors.