Macrocystis pyrifera source of nutrients for the production of carotenoids by a marine yeast Rhodotorula mucilaginosa.

AIMS: To evaluate an aqueous extract of Macrocystis pyrifera as a nutrient source for the production of carotenoids by a marine Rhodotorula mucilaginosa isolated from seaweed samples. MATERIALS AND RESULTS: The effect of different culture conditions on the concentration of biomass and total pigments was evaluated using a Box-Behnken experimental design. The seaweed extract contained 15% w w-1 of protein and 20% w w-1 of carbohydrate; the main sugar in this fraction was trehalose (78%). The culture conditions that maximize the total pigment concentration (1·84 ± 0·03 mg l-1 ) were initial pH equal to 7, yeast extract as nitrogen source at a concentration of 4 g l-1 , seaweed extract concentration at 25% v v-1 , incubation performed at 25°C and 150 rev min-1 during 6 days. Under optimal growth conditions, three carotenoids were identified among the pigments produced by R. mucilaginosa, lycopene (38·4 ± 9·4%), ß-carotene (21·8 ± 1·5%) and astaxanthin (1·8 ± 0·3%). CONCLUSIONS: Carotenoids of commercial interest (lycopene, ß-carotene and astaxanthin) can be produced using a marine R. mucilaginosa cultivated with an aqueous extract of M. pyrifera as nutrient source. The total pigment concentration in the culture ranged between 0·82 and 1·84 mg l-1 , and was significantly affected by the concentration of the seaweed extract, and yeast extract. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that M. pyrifera can be used as a nutrient source for the production of carotenoids by the marine yeast.

Effect of ultrasound in enantioselective hydrogenation of 1-phenyl-1,2-propanedione: comparison of catalyst activation, solvents and supports.

The enantioselective hydrogenation of 1-phenyl-1,2-propanedione was carried out over Pt/Al2O3, Pt/SiO2, Pt/SF (silica fiber), Pt/C catalysts modified with cinchonidine under ultrasonic irradiation. The initial rate, regioselectivity and enantioselectivity were investigated for different catalyst pretreatments, solvents and ultrasonic powers. The ultrasound effects were very catalyst dependent. The sonication significantly enhanced enantioselectivity and activity of the Pt/SF (silica fiber) catalyst. For the other Pt supported catalysts the reaction rate, enantioselectivity and regioselectivity increased moderately. The choice of solvent influenced the impact of ultrasound effect, namely in mesitylene, which has the lowest vapor pressure, the highest ultrasound enhancement was observed. The effect of sonication on catalysts surface was studied by transmission electron microscopy and scanning electron microscopy (SEM). No significant change in the metal particle size distribution due to sonication was observed. However, in the case of the Pt/SF catalyst, acoustic irradiation induced morphological changes on the catalyst particle surface (SEM), which might be the cause for enhancement of the initial reaction rate and enantioselectivity.

Effect of single doses of cimetidine and ranitidine on the steady-state plasma levels of midazolam.

H2-Receptor antagonists may interfere with the pharmacokinetics of concomitantly administered drugs. Our study was designed to investigate whether cimetidine or ranitidine influence the disposition and sedative effect of midazolam. The effect of single oral doses of 800 mg cimetidine, 300 mg ranitidine, or placebo on the steady-state concentrations of midazolam was examined in a randomized crossover study in eight healthy subjects. A midazolam steady-state concentration was achieved by an intravenous bolus (0.05 mg/kg)-infusion (0.025 mg/kg/hr) technique. Plasma concentrations of midazolam, cimetidine, and ranitidine and the pharmacodynamic response to midazolam (choice reaction time, sedation index) were monitored throughout the 10-hour infusion. Cimetidine significantly increased the mean (+/- SD) steady-state plasma concentration of midazolam from 56.7 +/- 7.8 to 71.3 +/- 19.6 ng/ml (P = 0.004). In contrast, the steady-state midazolam concentration after ranitidine dosing (61.8 +/- 6.8 ng/ml) did not differ significantly from that after placebo. No change in choice reaction time or sedation index was detected after cimetidine or ranitidine dosing. Nevertheless, in contrast to ranitidine, the recently advocated once-daily dosing of cimetidine has a potential for hepatic drug interaction that should be considered before its coadministration with drugs that have a narrow therapeutic index.

Effects of heme arginate on cytochrome P450-mediated metabolism of drugs in patients with variegate porphyria and in healthy men.

We investigated the effects of heme on metabolism of coumarin, debrisoquin, caffeine, and lidocaine in seven female patients with variegate porphyria and in 10 healthy men. During baseline conditions metabolism of the drugs was identical in the two groups. Compared with the results without heme, a single infusion of heme arginate (3 mg/kg heme) significantly decreased the debrisoquin/4-hydroxy-debrisoquin metabolic ratio in subjects with porphyria (p = 0.016) and in the control subjects (p = 0.016) and increased formation of monoethylglycinexylidide from lidocaine (p = 0.016 and p = 0.004, respectively). Metabolism of coumarin and caffeine was not affected by heme. Our results show that, in patients with porphyria and in healthy subjects, exogenous heme is able to accelerate the reactions mediated by the cytochrome isozymes CYP2D6 (debrisoquin) and CYP3A4 (lidocaine) but not reactions mediated by CYP1A2 (caffeine) and CYP2A6 (coumarin). This suggests that influence of heme on drug metabolism is P450 isozyme-specific.

Effect of five structurally diverse H2-receptor antagonists on drug metabolism.

Some H2-receptor antagonists can interact with the biotransformation of other drugs. This is due to their binding to cytochrome P-450. We tested the in vitro effects of 5 different H2-receptor antagonists cimetidine (C), oxmetidine (O), ranitidine (R), famotidine (F) and nizatidine (N) on arylhydrocarbon-hydroxylase, 7-ethoxycoumarin-O-deethylase and 7-ethoxy-resorufin-O-deethylase activity using liver microsomes from man as well as from untreated, phenobarbital and 3-methylcholanthrene treated rats. In addition their binding to human microsomal cytochrome P-450 was evaluated. The in vivo effects of these antagonists were investigated on the hepatic elimination of diazepam in healthy volunteers. In vitro O was found to be the most effective inhibitor of the enzyme activities studied. C showed a clear inhibitory effect only with rat liver microsomes whereas the remaining drugs were more than 10 times less potent. The binding affinities of these antagonists showed a similar tendency: the Ks-values for O, C and R were 0.2, 0.9 and 5.1 mM, respectively; for F and N no binding up to 4 mM could be observed. However, in man, only C inhibited the hepatic elimination of diazepam by about 45% while R, O, N and F did not affect the pharmacokinetics of diazepam. Thus, it could be concluded from our studies that one cannot extrapolate in vitro data of the inhibitory potency of H2-receptor antagonists in every case to human in vivo drug metabolism.

Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.

Comparison between cobalt and pyrazole in the increased expression of coumarin 7-hydroxylase in mouse liver.

The data in this report show that administration of both cobalt and pyrazole results in an elevation in the amount of hepatic mRNA encoding for microsomal P45015 alpha/P450Coh, an increase in the amount of P450Coh protein, and an activation of COH and to a lesser extent testosterone 15 alpha-hydroxylase in two inbred strains of mice. Considerable quantitative differences between the two compounds and the two mouse strains in the response suggest that the effects of cobalt and pyrazole are mediated, at least partly, through different mechanisms. It is of interest that human hepatic COH resembles very closely that in the mouse liver.

The cerium-induced liver injury and oxidative drug metabolism in DBA/2 and C57BL/6 mice.

The influence of the known hepatotoxic agent, cerium (Ce) on the activity of liver microsomal monoxygenases, especially coumarin 7-hydroxylase (COH) was investigated in two inbred strains of male mice, DBA/2N and C57BL/6N. Ce was injected intravenously in three doses (0.5, 1.0 and 2.0 mg/kg body wt) and the animals were killed 24 or 72 h later. On the basis of histological assessment of the liver, C57BL/6N mice are apparently more resistant to the hepatotoxic effect of Ce. At 24 h, COH activity was increased in a dose-dependent manner in DBA/2 animals, whereas no change was seen in C57BL/6N animals. A significant increase in all other enzymes studied, cytochrome P-450 (P450), ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase, was seen in DBA/2 mice injected with the highest dose of Ce. At 72 h Ce increased CON activity, as well as other enzymes, in C57BL/6N mice in a dose-dependent manner, whereas in DBA/2 mice the increase was only seen after the two lower doses, the highest dose causing severe morphological changes in the liver structure and a clear decrease in COH and other activities. The distribution studies with Ce-141 showed that C57BL/6N livers contained more Ce than DBA/2 livers after the highest dose.

Antipyrine, coumarin and glipizide affect n-acetylation measured by caffeine test.

The effect of various treatments on acetylation status measured by caffeine metabolites was investigated in 17 subjects with non-insulin dependent diabetes mellitus (NIDDM). The test drugs, caffeine (200 mg), antipyrine (20 mg/kg) and coumarin (5 mg), were given simultaneously, and urinary 5-acetylamino-6-formyl-amino-3-methyluracil/1-methylxanthine (AFMU/1X) molar ratio was measured before and after 8 weeks of therapy. The urinary AFMU/1X molar ratio decreased (p < 0.05) after 8 weeks of therapy with glipizide (2.5 mg), but remained unaltered in those treated with placebo or those who self-monitored blood glucose (SMBG) by glucometer. Antipyrine and coumarin decreased (p < 0.05) the AFMU/1X molar ratio both in diabetics and healthy volunteers. Our data demonstrate that glipizide, antipyrine and coumarin may interfere with the classification of acetylator status measured by caffeine metabolites.

Survey of acute poisoning cases in North-Finland during 1973-1982.

In a total of 2239 toxicological laboratory screenings requested from health centres and hospitals in North-Finland during 1973-1982, the most commonly found drug groups were benzodiazepines and phenothiazines. Alcohol was found in an average 45% of the patients screened during the years 1979-1982. Females accounted for 45% of all requests. No seasonal variation in the requests could be observed.

Effect of phenobarbital on hepatic injury induced by chronic carbon tetrachloride treatment.

Rats were given 1 ml CCl4 per kg body weight subcutaneously 2 times a week, for 16 weeks. The effects of simultaneous phenobarbital (PB) treatment (0.05% in drinking water) on the hepatotoxicity of CCl4 was studied during 16 weeks of treatment. The retardation of growth, the increase in liver weight and mortality were greater in animals receiving both PB and CCl4 than those given CCl4 alone. Cirrhosis was apparent only in animals treated by PB + CCl4. The potentiating effect of PB on CCl4 hepatotoxicity was also seen in hexobarbital sleeping time, the rate of hexobarbital metabolism, and the cytochrome P-450 content in liver microsomes. The inducing effect of PB alone decreased with time both in vivo and in vitro, which suggests an adaptation or some kind of exhaustion of liver to the effects of PB.

The influence of lanthanons on morphology and function of rat exocrine pancreas in vivo.

The intravenously administered light lanthanon praseodymium selectively destroys the Golgi complex of rat exocrine pancreas. This effect impairs the normal secretion of proteolytic enzymes which manifests in a reduced output of amylase and trypsin from rat pancreas and a decrease of the basal level of amylase in serum after pilocarpine stimulation.

HPLC method for rapid determination of acetylator phenotype by measuring urinary caffeine metabolites.

A validated reversed-phase high-performance liquid chromatographic (RP-HPLC) method is developed for the selective and rapid determination of two major metabolites of caffeine, namely 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (MX) from human urine. HPLC separation is achieved by means of a Supersphere-60 RP-Select B (4 microns) analytical column using a non-linear gradient elution programme of 70-95% solvent B (2.5% acetic acid-methanol, 60:40, v/v) in solvent A (water-acetonitrile, 80:20, v/v). A selective UV detection method is used for determination of AFMU, MX and internal standard with readings at 284, 268 and 248 nm, respectively. Urine samples are prepared for measurement by a simple chloroform-diethyl ether (80:20, v/v) extraction. The assay is validated with respect to linearity, sensitivity, accuracy, precision and system suitability. All validation parameters are found to be within the required limits. The limit of detection of AFMU and MX is found to be 50 ng/200 microliters urine. Calibration curves show good linearity between 0.1 and 5 micrograms/200 microliters urine concentration range for both metabolites. The assay is sufficiently sensitive and rapid (4.5 min chromatographic run) to be applied for routine monitoring of change in AFMU/MX molar ratio, indicating acetylation phenotype and change of caffeine metabolism in clinical cocktail studies.

Metabolites and DNA-binding of carbamazepine and oxcarbazepine in vitro by rat liver microsomes.

DNA-binding of carbamazepine (CBZ) and oxcarbazepine (OCBZ) catalysed by non-induced, phenobarbital-induced or methylcholanthrene-induced rat liver microsomes in vitro was studied. 14C-CBZ 200 nmol incubated with DNA, liver microsomes and cofactors led to the formation of a significant amount of CBZ-epoxide, which has been suspected as the cause of teratogenesis and other side-effects of CBZ, 1,2 but has not been reactive in any test systems for genotoxicity, including the Ames test.3 No enzyme-dependent DNA-binding of CBZ was found. Using the same conditions, however, OCBZ was bound to DNA. This binding was dependent on the presence of NADPH. 10-hydroxy-10, 11-dihydro-carbamazepine, which is known to be the major metabolite of OCBZ, and an unknown peak were demonstrated by HPLC. These results are the first indication of a higher level of covalent DNA binding of OCBZ than of CBZ. The nature of the unknown metabolite and the pathway leading to covalent binding remain to be studied.

Maternal, fetal and placental distribution of lidocaine-epinephrine and bupivacaine after epidural administration for cesarean section.

Bupivacaine and lidocaine are both lipophilic drugs, bupivacaine being more lipophilic and protein-bound. Our earlier studies, using human placenta perfused in vitro, showed that increased placental binding of bupivacaine restricts fetal transfer compared to the higher fetal transfer of lidocaine. However, placental tissue concentrations of local anesthetics have not been determined in the clinical context. Term parturients were randomized to receive either 2% lidocaine-epinephrine (n = 10) or 0.5% bupivacaine (n = 10) through a lumbar epidural catheter for elective cesarean section. Total drug concentrations of lidocaine and bupivacaine in maternal and umbilical plasma and placental tissue were determined. There was a higher incidence of maternal hypotension in the lidocaine-epinephrine group than in the bupivacaine group ( vs , P < 0.05). At delivery, fetal/maternal ratios for total concentrations of lidocaine and bupivacaine were similar (0.49 vs 0.42). The mean placental tissue/maternal plasma concentration ratio of lidocaine was higher than that of bupivacaine (1.45 vs 1.01, P < 0.05). The mean amount of the drug retained in the placenta per unit of dose (mg/kg) was also higher in the lidocaine-epinephrine group, although this difference did not reach statistical significance (0.46 mg/unit dose vs 0.40 mg/unit dose). Values for area under the concentration-time curves per unit of dose were similar. In conclusion, maternal plasma concentrations, fetal/maternal concentration ratios and placental tissue binding of lidocaine resembled those of bupivacaine after epidural administration. These findings are probably explainable by the effect of maternal hypotension on the distribution of lidocaine.

Age and CYP3A4 and CYP2A6 activities marked by the metabolism of lignocaine and coumarin in man.

The effect of age on human liver drug-metabolizing ability was investigated by using probe drugs, metabolized by specific isozymes in liver, as an index. Formation of monoethylglycinexylide (MEGX) after i.v. infusion of lignocaine (1 mg/kg), metabolized by CYP3A4, and excretion of 7-hydroxycoumarin (7-OHC) after oral coumarin (5 mg) administration, hydroxylated by CYP2A6, were investigated in healthy young (< 25 years) and elderly (> 65 years) women and men (n = 10 in each group). MEGX content in young subjects (men 57.8 +/- 11.3 and women 52.9 +/- 13.1 ng/ml) did not diverge significantly but was reduced in elderly subjects (men 43.57 +/- 15.8 and women 29.2 +/- 13.6 ng/ml, p < 0.05 and 0.01, respectively). 7-OHC excretion at 2 h averaged 68.1 +/- 13.1 per cent (men) and 65.0 +/- 18.3 per cent (women) of the dose given in young subjects and was delayed in elderly persons (men 46.5 +/- 16.3 per cent and women 44.8 +/- 18.3 percent, p < 0.01 and 0.05, respectively). The change in probe drug metabolism was related to age (MEGX, r = -0.473 (men) and -0.682 (women) and 7-OHC; r = -0.690 (men) and -0.565 (women)). MEGX formation was reduced by 0.92 microgram/L per year and 7-OHC excretion by 0.85 per cent per year. The results indicate a decrease of CYP3A4 and CYP2A6 metabolic activities with age.