Max T. Baker

Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required

Nitric oxide (NO) was produced from sodium nitroprusside in the presence of vascular tissue but was not released spontaneously from the nitroprusside anion. In the absence of tissue in the dark nitroprusside did not release NO. When solutions of nitroprusside alone were irradiated with visible light, nitric oxide was released at rates linearly proportional to nitroprusside concentration and light intensity. Nitric oxide was produced from solutions of nitroprusside in the dark after the addition of vascular tissue, including lengths of rabbit aorta, subcellular fractions of aorta, and human plasma. NO was also released from nitroprusside after reaction with various reducing agents including cysteine and other thiols, ascorbic acid, sodium dithionite, ferrous chloride, hemoglobin, myoglobin, and partially purified cytochrome P450 with an NADPH-regenerating system. HCN was simultaneously produced in these solutions, and addition of KCN blocked NO release. Iodine oxidized intermediate cyanoferrates and blocked nitric oxide release. KCN or iodine also blocked NO production by tissue, but had no effect upon photochemical NO release. These results show that, apart from photolysis which makes no physiological contribution, release of nitric oxide from nitroprusside, in simple solutions and in biological tissue, occurs after nitroprusside has undergone reduction and lost cyanide.

Propofol: the challenges of formulation.

Propofol is a potent lipophilic anesthetic that was initially formulated in Cremophor El for human use. Because of the occurrence of Cremophor EL anaphylaxis and improvements in the quality of lipid emulsions, it was ultimately brought to market as 1% propofol formulated in 10% soybean oil emulsion. Emulsions represent complex formulation compositions whose suitability for intravenous administration is dependent on a number of factors. Despite the success of propofol emulsions, drawbacks to such formulations include inherent emulsion instability, injection pain, a need for antimicrobial agents to prevent sepsis, and a concern of hyperlipidemia-related side effects. Efforts to overcome such drawbacks have involved the development of propofol emulsions with altered propofol and lipid contents, the addition of different excipients to emulsions for antimicrobial activity, and study of nonemulsion formulations including propofol–cyclodextrin and propofol–polymeric micelle formulations. In addition, a number of propofol prodrugs have been made and evaluated.

Inhibitory effects of propofol on cytochrome P450 activities in rat hepatic microsomes

The effects of propofol on cytochrome P450 activity in rat hepatic microsomes were evaluated to determine the potential influence of this anesthetic on the metabolism of coadministered agents. In microsomes from untreated and isoniazid-treated rats, propofol was a weak inhibitor of enflurane metabolism, inhibiting activity only at 0.35 mM propofol. In contrast, toluene, a related compound, effectively impaired enflurane de-fluorination in microsomes from untreated, and isoniazid- and phenobarbital-treated rats at concentrations as low as 0.025 mM. Propofol, in contrast to toluene, was an effective inhibitor of benzphetamine demethylation where it inhibited this activity at propofol concentrations as low as 0.025 mM in microsomes from phenobarbital-treated rats. In microsomes from phenobarbital-treated rats, propofol potently inhibited the metabolism of aniline. Sixty-four percent inhibition was achieved at 0.03 mM propofol, whereas toluene had no effect at 1 mM. These data demonstrate that propofol does not effectively inhibit enflurane metabolism performed by the isoniazid-inducible cytochrome P450IIE1 but effectively impairs activities of the phenobarbital-inducible cytochrome P450 isozymes. (Anesth Analg 1993;76:817–21)

Metabolism-dependent binding of the chlorinated insecticide DDT and its metabolite, DDD, to microsomal protein and lipids☆

Dichlorodi[U-14C]phenyltrichloroethane ( [14C]DDT), incubated with rat hepatic microsomes and NADPH, produced reactive intermediates which covalently bound to microsomal protein and lipids. In atmospheric oxygen, DDT bound to microsomal protein; however, binding was increased up to approximately 70% by oxygen depletion. Low levels of [14C]DDT binding to microsomal lipids occurred under atmospheric oxygen but, in contrast to protein binding, DDT-phospholipid binding was increased up to 20-fold by oxygen depletion. Dichlorodiphenyldichloroethane (DDD) was rapidly formed from DDT under anaerobic conditions, although when DDD was utilized as substrate, binding to microsomal protein occurred only in the presence of oxygen. Sodium dithionite, added to microsomes, produced [14C]DDT phospholipid and protein binding, and DDD formation, but failed to support DDD metabolism or binding. The data are consistent with the reductive formation of a DDT free-radical intermediate that led to the formation of DDD and that was bound preferentially to microsomal lipids.

The formation of chlorobenzene and benzene by the reductive metabolism of lindane in rat liver microsomes.

The major metabolite produced by incubating [14C]lindane with rat liver microsomes under anaerobic conditions was determined to be chlorobenzene, with lesser amounts of benzene also being formed. Using relatively high lindane concentrations (250 microM), four nonvolatile metabolites of lindane were also produced anaerobically, the predominant one being identified by mass spectrometry as tetrachlorocyclohexene (TCCH). TCCH, likewise, was reduced to chlorobenzene and benzene in microsomes under anaerobic conditions. Binding of [14C]lindane to microsomal protein occurred under aerobic as well as anaerobic incubation conditions; however, lindane protein binding was greatest in anaerobic incubations compared to those containing an atmosphere of air or 100% oxygen. Hemin reduced by dithionite also readily produced chlorobenzene and benzene from lindane. These results indicate that lindane interacts readily with heme and heme proteins, including cytochrome P-450, in the absence of oxygen to undergo multiple chloride eliminations forming chlorobenzene and benzene as end products.

Inhibition of Volatile Sevoflurane Degradation Product Formation in an Anesthesia Circuit by a Reduction in Soda Lime Temperature

BackgroundSevoflurane reacts with carbon dioxide absorbents, such as soda lime, to release the volatile products compounds A and B. These two products, which have been detected in anesthesia circuits, are among five formed when sevoflurane is degraded by soda lime at increased temperature; the others, compounds C, D, and E, have been detected only in heated sealed systems. The current study attempted to determine the influence of soda lime temperature on compounds A and B generation in an anesthesia circuit and whether a decrease in soda lime temperature could eliminate product formation in the circulating gases. MethodsSevoflurane (1.5% in oxygen) was circulated (6 1/ min) in a partially closed, low-flow (215 ml/min fresh gas) anesthesia circuit that included a canister containing 1.2 kg fresh soda lime. Carbon dioxide was introduced into the circuit at 200 ml/min, and gas samples for analysis of sevoflurane, compounds A, B, C, and D, and carbon dioxide were taken at the opening of an attached artificial lung. The circuit was operated for 8 h under conditions whereby the soda lime temperature could increase freely or the soda lime was chilled with ice. ResultsA maximum core soda lime temperature of about 46°C was measured when the experiment was run under conditions whereby the soda lime temperature was allowed to increase. Compounds A and B Increased with time to a maximum of 23 and 9 ppm, respectively. At 4.5 h of circuit operation, compound C/D was found. Chilling of the soda lime canister, which produced a maximum core soda lime temperature of 26°C, resulted in neither compound B nor C/D being detected during the 8-h period. Compound A was present in the circuit at all times at approximately 10 ppm; however, its concentration did not Increase as occurred when the experiment was conducted under nonchilled conditions. Carbon dioxide levels at the opening of the lung remained at a constant 5% for 8 h with or without soda lime chilling. ConclusionsThis study demonstrates that the release of volatile sevoflurane degradation products in an anesthesia circuit is highly dependent on soda lime temperatures. A reduction of the temperature of soda lime may be a feasible method of preventing the release of significant levels of sevoflurane degradation products without interfering with carbon dioxide absorption or altering the sevoflurane concentration.

Effects of ketamine on outcome from temporary middle cerebral artery occlusion in the spontaneously hypertensive rat

This experiment evaluated the potential for ketamine HCl, a non-competitive glutamate antagonist, to minimize injury resulting from temporary focal cerebral ischemia. Male spontaneously hypertensive rats were randomly assigned to receive either ketamine (n = 13) or halothane anesthesia (n = 12) during 2 h of reversible middle cerebral artery occlusion (MCAO). Ketamine was administered as a 50 mg/kg i.v. loading dose followed by a continuous 1.25 mg/kg/min i.v. infusion beginning 25 min prior to ischemia and continued until 30 min after reperfusion. An additional group of rats (ketamine-shams, n = 8) underwent craniectomy and ketamine administration (as above) but the middle cerebral artery was not ligated. Physiologic values were similar between groups with the exception of plasma glucose which was elevated in the halothane-MCAO group. After 4 days recovery, rats in all groups were neurologically evaluated. There were no differences between the two groups undergoing MCAO for neurologic grading or open field behavior, although both groups performed worse than did ketamine-shams (P less than 0.05). In contrast, motor performance revealed more severe deficits in the ketamine-MCAO rats vs either the halothane-MCAO or ketamine-sham groups (P less than 0.05). Cerebral infarct volume was then planimetrically measured after triphenyl tetrazolium chloride (TTC) staining of fresh brain sections. Mean +/- S.D. infarct volume was not different between the halothane-MCAO (134 +/- 51 mm3) and ketamine-MCAO (131 +/- 64 mm3) groups. Seven of 8 sham rats were free of TTC demarcated injury and in the remaining rat injury was minimal.(ABSTRACT TRUNCATED AT 250 WORDS)

The Hypnotic and Analgesic Effects of 2-Bromomelatonin

2-Bromomelatonin is an analog of melatonin with a higher melatonin receptor affinity. We tested the hypnotic and analgesic properties of 2-bromomelatonin and compared them with those of propofol. Sprague-Dawley rats were assigned to receive 2-bromomelatonin or propofol IV, or morphine intraperitoneally. Righting reflex and response to tail clamping were assessed. Both 2-bromomelatonin and propofol caused a dose-dependent increase in the percent of rats displaying loss of both the righting reflex and the response to tail clamping. 2-Bromomelatonin was comparable to propofol in terms of its rapid onset and short duration of hypnosis. The 50% effective dose (95% confidence interval) for loss of righting reflex for propofol and 2-bromomelatonin were 3.7 (3.4–4.0) and 38 (35–41) mg/kg, respectively. Corresponding values for loss of response to tail clamp were 2.9 (3.5–4.0) and 21 (15–30) mg/kg, respectively. 2-Bromomelatonin is approximately 6–10 times less potent than propofol depending on the end-point used. Intraperitoneal 30 mg/kg morphine did not affect the righting reflex, but resulted in loss of response to tail clamping in all animals. 2-Bromomelatonin can exert hypnotic and antinocifensive effects similar to that observed with propofol. Unlike propofol, the reduced nocifensive behavior persisted after the animals had regained their righting reflex. This study provides evidence that 2-bromomelatonin has properties that are desirable in anesthetics or anesthetic adjuvants.

Sulfite Supported Lipid Peroxidation in Propofol Emulsions

Background Sodium metabisulfite is added to a commercial propofol emulsion as an antimicrobial agent. The sulfite ion (SO3−2) is capable of undergoing a number of reactions, including autooxidation and the promotion of lipid peroxidation. This study evaluated sulfite reactivity in propofol emulsions by determining thiobarbituric acid reacting substances (TBARS), sulfite depletion, and emulsion pH in emulsions containing sulfite or EDTA. Methods Commercial EDTA and sulfite propofol emulsions were compared, and 10% soybean oil emulsion containing various additives were evaluated for TBARS, sulfite, and pH. TBARS were analyzed with a modified thiobarbituric acid method. Sulfite was analyzed by the reaction of sulfite with 5,5′-dithiobis(2-nitrobenzoic acid). pH was measured by glass electrode methodology. Results Thiobarbituric acid reacting substances were detectable in commercial sulfite propofol emulsions in concentrations ranging from 0.02 to 0.22 &mgr;g/ml based on malondialdehyde. No TBARS were detected in EDTA propofol emulsions. Incubation (37°C, up to 6 h) of sulfite propofol emulsions in air resulted in further increases in TBARS (35–160%). No increases occurred in incubated EDTA propofol emulsions. Metabisulfite (0.25 mg/ml) alone added to 10% soybean oil resulted in large increases in TBARS that were inhibited in part by propofol (10 mg/ml) and completely by ascorbic acid (0.05 mg/ml). Soybean oil emulsion pH declined rapidly on the addition of metabisulfite (0.25 mg/ml). The addition of propofol (10 mg/ml) partially inhibited the decline in pH and ascorbic acid (0.05 mg/ml) completely inhibited it. Conclusion These results show that sulfite supports the peroxidation of lipids in soybean oil emulsions and propofol functions to partially inhibit these processes.

Free radical and drug oxidation products in an intensive care unit sedative: Propofol with sulfite*

ObjectivesSome propofol emulsion formulations contain EDTA or sodium metabisulfite to inhibit microbe growth on extrinsic contamination. EDTA is not known to react with propofol formulation components; however, sulfite has been shown to support some oxidation processes and may react with propofol. This study compared the oxidation of propofol and the formation of free radicals by electron paramagnetic resonance analysis in EDTA and sulfite propofol emulsions during a simulated intensive care unit 12-hr intravenous infusion. DesignControlled laboratory study. SettingUniversity laboratory. Measurements and Main ResultsPropofol emulsions (3.5 mL) were dripped from spiked 50-mL vials at each hour for 12 hrs. Two propofol oxidation products, identified as propofol dimer and propofol dimer quinone, were detected in sulfite and EDTA propofol emulsions; however, sulfite propofol emulsion contained higher quantities of both compounds. After initiation of the simulated infusion, the quantities of propofol dimer and propofol dimer quinone increased in the sulfite propofol emulsion, but the lower levels in the EDTA propofol emulsion remained constant. Sulfite propofol emulsion began to visibly yellow at about 6–7 hrs. The EDTA propofol emulsion remained white at all times. The absorbance spectra of the propofol dimer and propofol dimer quinone extracted from sulfite propofol emulsion showed that propofol dimer did not absorb in the visible spectrum, but the propofol dimer quinone had an absorbance peak at 421 nm, causing it to appear yellow. Electron paramagnetic resonance analysis of the propofol emulsion containing metabisulfite revealed that the sulfite propofol emulsion yielded a strong free radical signal consistent with the formation of the sulfite anion radical (SO3·−). The EDTA propofol emulsion yielded no free radical signal above background. ConclusionSulfite from the metabisulfite additive in propofol emulsion creates an oxidative environment when these emulsions are exposed to air during a simulated intravenous infusion. This oxidation results in propofol dimerization and emulsion yellowing, the latter of which is caused by the formation of propofol dimer quinone. These processes can be attributed to the rapid formation of the reactive sulfite free radical.