Effects of infusion rate of intravenously administered morphine on physiological, psychomotor, and self-reported measures in humans.

Although the rate of onset of a drug effect is commonly believed to contribute to a drug's abuse liability, only a few systematic experimental studies have been conducted examining this notion. The present study determined the profile of physiological, psychomotor, and self-reported effects of infusion rate (a key means of manipulating onset of drug action) of intravenously administered morphine, the prototypical analgesic with a known abuse liability in human participants. Two doses of morphine sulfate (5 and 10 mg/70 kg, i.v.) and a placebo dose (0 mg/70 kg, i.v.) were administered to healthy volunteers under three infusion rates (2 min bolus, 15 min, and 60 min). Faster infusions of morphine produced greater positive subjective effects than slower infusions on visual analog scale measures of good drug effect, drug liking, and high. Faster infusions also resulted in greater self-reported drug effects and opioid agonist effects, without producing significant physiological or psychomotor impairment. Importantly, faster rates of drug infusion produced significantly higher morphine plasma levels than slower rates, and morphine plasma levels followed a similar pattern and timing of peak effect as the self-reported effects of the drug. Moreover, morphine produced dose-dependent increases in self-reported drug effects, opioid agonist effects, and morphine plasma levels in the study. Results suggest that the pharmacokinetic properties of a drug, including the dosage administered and the rate of at which it is administered may function to jointly affect the abuse liability of the drug.

Determination of sameridine in blood plasma by nitrogen-selective gas chromatography.

A gas chromatographic method was developed and validated for the determination of sameridine in human plasma. Sameridine is a new type of compound with both local anaesthetic and analgesic properties, when administered intrathecally. The method is based on liquid-liquid extraction of sameridine from 1.0 ml of plasma, followed by gas chromatography with nitrogen-phosphorus detection. Method validation results showed that this method is very sensitive, selective and robust. The limit of quantification was 1 nM for 1.0 ml of human plasma in the low-level range (1.00-75.0 nM) and the between-day accuracy and precision were measured at 99-104% of nominal values and 3.4-5.6% (RSD), respectively.

Determination of ropivacaine and [2H3]ropivacaine in biological samples by gas chromatography with nitrogen-phosphorus detection or mass spectrometry.

Bioanalytical methods for determining the total concentration of the new local anaesthetic drug ropivacaine in blood plasma, urine and tissues are presented. Ropivacaine is a drug mainly used in connection with surgery and for post-operative pain relief. The biological samples were prepared using liquid-liquid extraction and analysed using capillary gas chromatography with nitrogen-phosphorus detection or mass spectrometry. The methods are highly selective and reliable with a between-day precision, given as the relative standard deviation, generally below 6%. More than 20000 samples have been analysed using the methods described.

Determination of free concentration of sameridine in blood plasma by ultrafiltration and coupled-column liquid chromatography.

Sameridine is a new candidate drug with both local anaesthetic and analgesic properties. The free concentration of sameridine in blood plasma was determined by coupled-column liquid chromatography. Following adjustment of the pH and the temperature of the plasma samples, the free fraction was prepared by ultrafiltration. The coupled-column liquid chromatographic system consisted of a reversed-phase column, a cation-exchange extraction column and a cation-exchange analytical column. Sameridine was detected by UV determination at 205 nm and the system showed high selectivity. The limit of quantification was 1 nM and the within-day precision was 4.6% (R.S.D., n = 10).

Dehydrogenase-dependent metabolism of alcohols in gastric mucosa of deer mice lacking hepatic alcohol dehydrogenase.

Deer mice (Peromyscus maniculatus) lacking hepatic alcohol dehydrogenase (ADH) have been used as a model for studies of ethanol elimination catalysed by non-ADH systems like catalase and cytochrome P450. However, in an in vivo study on these animals (ADH- deer mice), we detected reversibility in the oxidation of [2H]ethanol, indicating that a major part of the ethanol elimination was due to a dehydrogenase (Norsten et al., J Biol Chem 264: 5593-5597, 1989). In the present investigation, we found significant ethanol oxidizing activity in the gastric mucosa of the deer mice. Reversibility was demonstrated by the use of [2H]acetaldehyde and gas chromatography-mass spectrometry of the products. The kinetic 2H isotope effect of the gastric system was about 3.0 and the system was comparatively insensitive to inhibition by 4-methylpyrazole. The behavior of the deer mice gastric ADH in isoelectric focusing and its higher activity with longer alcohols as substrates indicated similarity with the previously described human class IV enzymes. Our data are in agreement with results obtained in vivo and indicate that ethanol is oxidized extrahepatically in ADH- deer mice. This has to be taken into account when deer mice are used to study non-ADH-dependent ethanol oxidation in vivo.

Transfer of deuterium from [1,1-2H2]ethanol to steroids and organic acids in the rat testis.

Rats were given [1,1-2H2]ethanol in a single dose, and the 2H content was determined in testicular steroids and in organic acids of low molecular mass in the testis, liver and blood. The acids were quantified by capillary gas chromatography/mass spectrometry of t-butyldimethylsilyl derivatives with [2H4]lactate as internal standard. In addition to lactate, pyruvate, 3-hydroxybutyrate and acids of the tricarboxylic acid cycle, the testis was shown to contain 2-hydroxybutyrate, 2-hydroxy-2-methylbutyrate, 2-hydroxyisohexanoate and glycerate. No 2H was found in pregnenolone, 5-androstene-3 beta,17 beta-diol or testosterone, whereas the abundance of monodeuterated molecules of 5 alpha-androstane-3 alpha,17 beta-diol and its 3 beta-isomer were 7.6% and 11.2% respectively. The abundance of monodeuterated lactate was 7.0% in the testis and 5.3% in the blood. The other acids were less labelled but 3-hydroxybutyrate had a higher 2H content in the testis (3.1%) than in the liver. These results support the contention that ethanol is oxidized in an alcohol dehydrogenase-catalysed reaction in testis in vivo and that the acute inhibition of the testosterone production is due at least partly to a redox effect. The labelling and increased concentration of 3-hydroxybutyrate in the testis indicate that a change in the mitochondrial redox state might be involved.

Oxidoreduction of butanol in deermice (Peromyscus maniculatus) lacking hepatic cytosolic alcohol dehydrogenase.

In view of conflicting information in the literature regarding enzyme systems responsible for alcohol oxidation in deermice previously reported to lack hepatic alcohol dehydrogenase (ADH) activity, the reversibility of butanol oxidation was studied in vivo and in liver-perfusion systems. Mixtures of [1,1-2H2]ethanol and butanol were given intraperitoneally to deermice lacking (ADH-) or possessing (ADH+) ADH activity, followed by analysis of alcohols in blood by GC/MS. 2H exchange between the two alcohols was seen in all experiments. In ADH- deermice, the 2H excess of butanol increased steadily and reached 18 +/- 5% after 2.5 h. In ADH+ deermice, butanol was rapidly eliminated and the 2H excess was about 7% after 0.5 h. In similar experiments with rats, the 2H excess was about 40% for 2 h. Perfusions of livers from ADH- deermice with mixtures of unlabelled and 1-[2H]butanol showed significant but slow intermolecular hydrogen transfer at C1, indicating oxidoreduction catalyzed by a dehydrogenase. Slow reduction of butanal was observed in mitochondria from ADH- deermice. ADH activity with a pH optimum of 10 and Km for ethanol of 6 mM was detected in the inner mitochondrial membranes from rats and deermice. However, low rates of oxidation observed in experiments carried out with perfused livers and in vitro suggest that this enzyme system does not contribute significantly to alcohol oxidation in vivo. Thus, perfused liver from ADH- deermice appears to be a useful system for studies of ADH-independent oxidation of alcohols. The 2H exchange between the alcohols seen in vivo indicates that both ethanol and butanol are substrates for a common extrahepatic dehydrogenase in ADH- deermice.

Acetaldehyde as a substrate for ethanol-inducible cytochrome P450 (CYP2E1).

Liver microsomes from starved and acetone-treated rats catalyzed NADPH-supported metabolism of acetaldehyde at a rate 8-fold higher than corresponding control microsomes; the Vmax was about 6 nmol/mg microsomal protein/min and the apparent Km 30 microM. The reaction was efficiently inhibited by anti-CYP2E1 IgG, but not by control IgG. Reconstituted membranes containing rat CYP2E1 and cytochrome b5 metabolized acetaldehyde with a Vmax of 20 nmol/nmol/min and an apparent Km of 30 microM, whereas CYP2B4 containing vesicles or vesicles without b5 were ineffective. Gas chromatographic/mass spectrometric analysis of products formed from [2H4]-acetaldehyde with CYP2E1-containing reconstituted membrane vesicles revealed the formation of acetate as the only detectable product, although other water soluble products were also formed as evidenced from incubations with [1,2-14C]acetaldehyde. The results indicate that CYP2E1 is an aldehyde oxidase and thus metabolizes both ethanol and its primary oxidation product. This might have implications in vivo for acetaldehyde metabolism in liver and brain.

Analysis of aldehydic lipid peroxidation products in rat liver and hepatocytes by gas chromatography and mass spectrometry of the oxime-tert-butyldimethylsilyl derivatives.

A method for analysis of aliphatic aldehydes in biological samples is described. Cyclohexanone is added as internal standard and the samples are treated with hydroxylamine and perchloric acid. The oximes are extracted and converted to the oxime-tert-butyldimethylsilyl derivatives, which are quantitated by capillary gas chromatography and identified by mass spectrometry. The characteristic M-57 fragment ions in the mass spectra enabled a rapid identification of the derivatives of the aldehydes, alkanals, alk-2-enals, alka-2,4-dienals, and 4-hydroxyalk-2-enals, which in addition gave rise to characteristic double peaks in the gas chromatographic analysis. The method was applied to analysis of autoxidized arachidonic acid, ADP-Fe3(+)-treated rat hepatocytes, and rat liver given a single dose of ethanol, 5 g/kg. The amounts of hexanal and 4-hydroxynon-2-enal were not increased 6 h after the administration of ethanol.