Effect of pressure on oxidative metabolism of drugs by mouse hepatic microsomes.

The metabolism of antipyrene and delta-9-tetrahydrocannabinol (delta-9-THC) was studied in vitro with mouse liver supernatant and 105,000 g microsomes at 450 atm pressure. No significant change in rate of antipyrene metabolism was detected, whereas a slight (5.6%) but nonsignificant reduction in the rate of metabolism of delta-9-THC was found. Pressure did, however, produce a significant (23%) reduction in the formation of the 11-hydroxy metabolite from delta-9-THC, but at the same time had no effect on the production of the other major metabolite, 8-alpha-hydroxy-delta-9-THC. This is possibly due to a differential effect on cytochrome P-450 isozymes. Mechanisms for the possible cause of the inhibition of metabolism are discussed.

Pharmacokinetics of delta 9-tetrahydrocannabinol in rabbits following single or multiple intravenous doses.

The pharmacokinetics of delta 9-tetrahydrocannabinol (delta 9-THC) has been studied in rabbits under conditions of single or multiple administration. Measurement of the drug was by a sensitive GC/MS technique using metastable ion monitoring and capable of measuring 0.005 ng/ml of the drug in plasma. This method enabled measurements of the drug to be made for 8-10 days after a single iv dose of 1 mg/kg of delta 9-THC and for 26 days following treatment for 22 days with the same dose. Plasma concentration-time data were analyzed by least squares nonlinear regression with the program NONLIN and terminal half-lives in the range 30-66 hr [mean 47.1 +/- 3.5 (SE), n = 8] were found after acute drug treatment. These increased to 74.6 +/- 1.7 hr (n = 4) after an 8-day treatment and to 83.0 hr after the 22-day treatment. Plasma clearance values were high, but the large amount of drug sequestered in tissues, reflected by large volumes of distribution, was the main determinant of terminal half-life. Measurements of the drug in several tissues indicated that fat was the major site of sequestration. The relative concentrations of delta 9-THC in the various tissues were similar to those observed for other lipophilic compounds, suggesting that distribution of delta 9-THC is governed by its physicochemical properties and not by any active transport process or specific barriers.

Species variation and the mechanism of pressure-anaesthetic interactions.

The mechanism of general anaesthesia has proved difficult to elucidate (see ref. 1 for a review), although the relative potencies of anaesthetic agents have been used to establish that the site at which anaesthetics act is hydrophobic in nature. One further clue to their mode of action is that the effects of anaesthetics on vertebrates can be eliminated by pressures of approximately 100 atm (refs 3, 4). However, the effects of anaesthetics are not always reversed in model systems, where there is evidence that the pattern of pressure reversal varies significantly. We now find that pressure fails to reverse the effects of anaesthetics on the freshwater shrimp (Gammarus pulex), although the sensitivity of these crustaceans to anaesthetics is comparable with that of higher animals. This is hard to reconcile with traditional bio-physical mechanisms and indicates that anaesthetics may act at a specific protein site rather than having a general effect on cell membranes. The pharmacology of pressure in mammals seems to be more similar to that of strychnine than of any other central stimulant. As glycine, whose action is blocked by strychnine, is absent as a neurotransmitter in the arthropod central nervous system, we believe that this substance may be involved in determining pressure-anaesthetic interactions in vertebrates.

The partitioning of delta 1-tetrahydrocannabinol into erythrocyte membranes in vivo and its effect on membrane fluidity.

delta 1-Tetrahydrocannabinol (delta 1-THC) has been quantified directly in erythrocyte membranes from drug-treated mice using gas chromatography/mass spectrometry. Concentrations of approximately 6 ng delta 1-THC/mg membrane protein (10(-5) M) were found when effects of the drug on behavior were prevalent. At these concentrations the drug produced a decrease in membrane order as measured by ESR.

Changes in membrane lipid content after chronic ethanol administration with respect to fatty acyl compositions and phospholipid type.

Changes in the relative proportions of the phospholipid fatty acids of erythrocyte membranes in mice after chronic ethanol treatment (4.5 g/kg, i.p. twice daily for one week) were shown to vary with the differing control profiles observed. It is suggested that certain changes in membrane lipid composition after ethanol administration may not be interpreted simply in terms of an adaptation to a disordering effect of the drug. The fatty acid changes were, in addition, distributed asymmetrically within the individual phospholipid classes. Depending on the control profile, the effects varied from being mainly in phosphatidylethanolamine (PE; 80%) and phosphatidylserine-inositol (PS + PI; 10%), phospholipids primarily located on the inner half of the membrane bilayer, to being more evenly distributed between PE and phosphatidylcholine (PC) and probably, therefore, between the two halves of the bilayer. Changes in the monounsaturated acid remained primarily with PE, suggesting a specific functional role for this species. The remaining results are discussed in the light of possible effects on cell morphology and their potentially similar consequences of increasing cell volume.

Effects of chronic ethanol administration on the composition of membrane lipids in the mouse.

The relative proportions of the phospholipid fatty acids of erythrocyte membranes in mice were changed by chronic ethanol treatment and were not related to effects of the drug on nutrition, body temperature or experimental stress. Similar changes were observed using two different routes of ethanol administration and they did not reflect the metabolic effects of ethanol seen in the phospholipid fatty acids of whole liver. The observed increased content of saturated fatty acids and decreased content of polyunsaturated acids support the concept of adaptive changes taking place in the membrane during tolerance development to compensate for an increased membrane fluidity caused by ethanol. However, an increased content of the mono-unsaturated acid, octadecenoic (oleic), was found and there was no change in the cholesterol/phospholipid ratio. Other contrasting types of plasma membrane in mice showed different patterns of change in their phospholipid fatty acids during chronic ethanol administration. It is suggested that changes in membrane lipid composition could only partly account for an adaptation to ethanol-induced membrane disordering.

Gas chromatographic and mass spectrometric studies on the metabolism and pharmacokinetics of delta 1-tetrahydrocannabinol in the rabbit.

Gas chromatography-mass spectrometry has been used to investigate the in vivo hepatic metabolism of delta 1-tetrahydrocannabinol (delta 1-THC) in the New Zealand white rabbit. Sixteen metabolites were identified and shown to be present in different relative amounts compared with the hepatic metabolites of delta 1-THC produced by other species. The metabolic profile was also different from that reported from rabbit urine particularly with regard to the lower relative concentrations of acidic metabolites in the liver. The pharmacokinetics of delta 1-THC has been studied in the rabbit using the recently developed GC-MS method based on metastable ion monitoring. This revealed a terminal plasma delta 1-THC half life ranging from 34.16 to 59.30 h (mean 46.75 h) after a single dose and THC fat/plasma ratio of 10(3)-10(4):1.

Effects of halothane on the incorporation of [14C]-serine into phospholipid in the guinea-pig ileum.

1 The effects of halothane on the incorporation of L-[3-14C]-serine into phospholipid were studied in the resting, innervated longitudinal muscle preparation of the guinea-pig ileum. 2 The anaesthetic, at clinical concentrations, caused a dose-dependent, partial inhibition of incorporation. The effect was rapid and reversible, and did not show characteristics of competitive inhibition. 3 The incorporation was reduced by a decrease in the Ca2+ concentration of the Krebs incubation buffer. Part of the activity persisted in the absence of added Ca2+ and this was most susceptible to inhibition by halothane. Sensitivity to external Ca2+ was not influenced at the halothane concentrations studied. 4 Evidence in support of the 14C-incorporation being due to L-[3-14C]-serine-phospholipid base-exchange activity included: (a) the rapid appearance of radioactivity in phosphatidylserine; (b) the kinetics of this incorporation in relation to that in phosphatidylethanolamine; (c) its dependence on Ca2+, and (d) its sensitivity to 2,4-dinitrophenol and its sensitivity to temperature. 5 It is concluded that this preparation makes it possible for a membrane-bound lipid-dependent activity (L-serine-phospholipid base-exchange) to be studied in conditions of cellular integrity under which the normal functional effects of lipophilic drugs can be simultaneously tested. 6 A rapid gas-chromatographic assay for halothane from an aqueous medium is also described.

Measurement of delta 1-tetrahydrocannabinol in plasma to the low picogram range by gas chromatography-mass spectrometry using metastable ion detection.

A method for the assay of delta 1-tetrahydrocannabinol (delta 1-THC) in plasma using combined gas chromatography-mass spectrometry with metastable ion monitoring is described. delta 1-THC was extracted with hexane and the extracts were methylated with diazomethane to shift the peaks produced by endogenous plasma constituents away from the cannabinoid region. The delta 1-THC was then converted into its trimethylsilyl derivative and quantitated using the metastable ion at m/z 371 formed in the M+ leads to [M - CH3]+ transition with [1",1",2",2"-2H4]cannabinol as the internal standard. delta 1-THC could be measured to 5 pg/ml in plasma. This assay is 20-100 times more sensitive than existing assays and has the advantage of not needing the usual extensive purification step.

The detection of gas bubbles in guinea-pigs after decompression from air saturation dives using ultrasonic imaging.

1. Bubble formation in the hind limb of anaesthetized guinea-pigs, after decompression from two different saturation exposures to air, 0.69 and 0.83 MPa gauge, has been studied using an ultrasonic pulse--echo imaging technique. 2. A qualitative analysis of the bubble formation, observed over a 30 min period after decompression, showed that profuse, largely stationary bubble formation occurred within 3 min of the decompression from 0.83 MPa gauge but that extensive stationary bubble formation was not observed until 17 min after decompression from 0.69 MPa gauge. Electrocardiogram changes appeared coincidently with the appearance of major bubble formation after the 0.83 MPa decompression but after the 0.69 MPa decompression changes were not observed until the end of the 30 min surveillance period, considerably later than the occurrence of a large number of bubbles. 3. A quantitative analysis of the echo patterns recorded during the 60 sec decompression and for 60 sec after the decompression demonstrated that the increase in severity of the decompression corresponded to an increase of 152% in the number of bubbles observed. The echoes observed during this period have been identified as either transient or persistent and their distribution of size, location and times of appearance and duration have been described. 4. From the quantitative analysis approximate estimates of the contribution by mobile, intravascular gas bubbles to the elimination of the excess gas have been made. These estimates range from 0.01 to 0.9% after the 0.69 MPa decompression and from 0.06 to 6% after the 0.83 MPa decompression. 5. It is concluded that the pulse--echo ultrasonic imaging technique provides a powerful means of analysing the distributions of bubble formation, both qualitatively and quantitatively, after decompression; it has the important attribute of being able to monitor both moving and stationary bubbles simultaneously in a variety of tissue types.

Identification of the in vivo liver metabolites of (--)-delta 7-tetrahydrocannabinol produced by the mouse.

The major in vivo metabolites of (--)-delta 7-tetrahydrocannabinol were extracted from the livers of mice after a single intraperitoneal dose and were examined by combined gas-liquid chromatography-mass spectrometry. Twenty-six metabolites were identified; most of these appeared to be produced via epoxidation of the double bond, although the epoxides themselves were not detected. Major metabolites involved hydroxylation of the double bond, to give the isomeric 1 alpha- and 1 beta, 7-dihydroxyhexahydrocannabinols. Of these, the 1 alpha, 7-isomer was the major product and appeared to be produced by hydrolysis of 1 alpha, 7-epoxyhexahydrocannabinol. It was further oxidized to 1 alpha-hydroxyhexahydrocannabinol-7-oic acid. Rearrangement of the epoxide to the 7-aldehyde and subsequent reduction to either the 7-alcohol or oxidation to the 7-acid was another major metabolic route. Derivatives of all these metabolites, hydroxylated in the 2"-, 3"- and 4"-positions of the sidechain were also identified. Allylic hydroxylation, predominantly at position 6, was also observed, but this was a relatively minor route. Reduction of the double bond yielded the isomeric axial and equatorial hexahydrocannabinols in low yield.

Identification of in vivo liver metabolites of delta 6-tetrahydrocannabinol produced by the mouse.

Metabolites of delta 6-tetrahydrocannabinol (THC) were extracted from livers, separated from lipids by chromatography on Sephadex LH-20, and identified by combined gas chromatography and mass spectrometry. Three alcohols, delta 6-THD-7-oic acid, five diols, six triols, five monohydroxy acids, six dihydroxy acids, two substituted ketones, an epoxide, three diols produced by hydrolysis of the epoxide, three dihydro metabolites, and a glucuronide conjugate were identified. Several other metabolites were detected but not identified. The metabolites were similar to those produced by delta 1-THC in that allylic and side-chain hydroxylation coupled with carboxylic acid formation at C7 were the major biotransformation pathways. delta 6-THC was found to be metabolized more extensively via the epoxide-diol pathway than the delta 1-isomer.