Use of ethylene oxide for the sterilization of thermoplastic articles]

The author applied ethylene oxide for sterilization of articles incompatible with thermal sterilization. This method has been widely accepted in many counteries. The tests have been made in the Warsaw Medical School Clinic of Pediatric Surgery, the Polish made "Rotanox" being the source of ethylene oxide. The following items served as sterilizing chambers in this order: Polyethylene foil bags, glass flask and pressurized chambers. The gas pressure of 1 at and temperature of 40-50 degrees C were maintained in the chamber. Sterilized material was closed in the polyethylene foil bags. The effectiveness of this method was proved by bacterial tests.     

Evaluating the formulae for integrated lethality in ethylene oxide sterilization using six different endospore forming strains of bacteria, and comparisons of integrated lethality for ethylene oxide and steam systems

Bacterial endospores from six different species of bacteria were exposed to a spectrum of ethylene oxide (EtO) sterilizing conditions. Temperature was varied from 40 to 60 degrees C and the ethylene oxide concentration was varied from 300 to 750 mg/L. Relative humidity was maintained at 60+/-10% RH. The fraction negative procedure was used to determine the D value for each of the test conditions. Bacterial species tested included Bacillus atrophaeus ATCC # 9372, Bacillus smithii ATCC # 51232, Bacillus subtilis "5230" ATCC # 35021, Bacillus subtilis, DSM # 4181, Bacillus pumilus ATCC # 27142, and Geobacillus stearothermophilus ATCC # 7953. All spore preparations were inoculated on filter paper strips packaged in blue, sterilizable glassine pouches. G. stearothermophilus was the least resistant organism tested. The most resistant organisms tested were B. atrophaeus and B. subtilis "5230". The B. subtilis "5230" strain was slightly more resistant than B. atrophaeus at conditions of 54C and EtO concentrations of 400, 600, and 750 mg/L, as well as at 60C/750mg/L EtO. The other species were between these extremes. This empirical data allowed the application of the recently published formula for converting D values from one set of conditions to another and evaluations of accuracy. The measured D values also allowed the determination of Z values based on temperature variations. These formulae, when applied to process temperatures independent of gas concentration, result in a Z value of approximately 32 degrees C that appears to be similar for all species tested. These data support the application of the previously published formulae 1-6 and allow the same approach to integrated lethality for ethylene oxide processes as is commonly applied to steam sterilization. A review of steam sterilization and related principles was conducted for comparison of integrated lethality for these two methods of sterilization. Errors associated with D values, Z values, extrapolation, and integrated lethality for both methods of sterilization are discussed.     

Influence of the sterilization process on alginate dispersions

Abstract— The influence of different sterilization procedures on alginate dispersions was studied by measuring viscosity and molecular weight changes. Autoclaving caused a 64% decrease in viscosity. Heating at a low temperature over several cycles was less efficient in sterilizing alginates and there was a progressive breakdown of the alginate chain over the succeeding cycles. Heating during ethylene oxide sterilization also resulted in reduced viscosity and breakdown. Membrane filtration yielded a sterile product with no significant reduction in viscosity or mol. wt.     

Paradoxical release of nitric oxide by an L-type calcium channel antagonist, the R+ enantiomer of amlodipine.

Amlodipine is a mixture of two enantiomers, one having L-type channel blocking activity (S-) and the other about 1,000-fold weaker activity and of little known other activity (R+). To determine whether the R+ enantiomer releases nitric oxide, the ability of amlodipine, its enantiomers, and nitrendipine to release nitric oxide in isolated coronary microvessels and to regulate cardiac tissue oxygen consumption via nitric oxide release was studied in vitro. Amlodipine and the R+ enantiomer released nitric oxide in a concentration-dependent fashion, increasing nitrite release from coronary microvessels by 57 +/- 12 and 45 +/- 5 pmol/mg/20 min at 10(-6) M (p < 0.05 from control). Nitrite release was entirely blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and HOE-140, a B2-kinin receptor antagonist. The S- enantiomer had no effect on nitrite release at any concentration. Amlodipine and the R+ enantiomer also reduced oxygen consumption in canine cardiac tissue in vitro and this was in an L-NAME-blockable manner. The S- enantiomer of amlodipine had no effect. This study shows that the R+ enantiomer of amlodipine is responsible for the release of nitric oxide and not the S- enantiomer (the L-type calcium channel blocking portion of amlodipine). Interestingly, nitric oxide release is dependent on the production of kinins because it is blocked by HOE-140. This study defines a potentially important property by which calcium channel blockers may release nitric oxide and may contribute to their use in the treatment of cardiovascular disease.     

Pharmacokinetics of ethylene in man; body burden with ethylene oxide and hydroxyethylation of hemoglobin due to endogenous and environmental ethylene

The inhalation pharmacokinetics and the endogenous production of ethylene has been determined in healthy volunteers with respect to the formation of the carcinogen ethylene oxide. Ethylene showed a low degree of accumulation in the body determined in six subjects, the thermodynamic partition coefficient body/air being 0.53±0.23 (mean ± SD) and the accumulation factor body/air at steady-state being 0.33±0.13 (mean ± SD). The rate of metabolism was directly proportional to the exposure concentration. Only 2% of ethylene inhaled was metabolized to ethylene oxide, whereas 98% of ethylene was exhaled unchanged. The rate of the endogenous production of ethylene was 32±12 nmol/h (mean ± SD), as calculated from exhalation data from 14 subjects. The resulting body burden was 0.44±0.19 nmol/kg (mean ± SD). By analyzing published data on ethylene oxide in man its half-life was estimated to be 42 min. Using the pharmacokinetic parameters of ethylene and ethylene oxide, the body burden of ethylene oxide due to the sum of the exposure to environmental ethylene of about 15 ppb and to endogenous ethylene exposure of 0.44 nmol/kg was predicted to be 0.25 nmol/kg. In the blood of five nonsmokers and one smoker the hemoglobin adduct resulting from the reaction of ethylene oxide with the N-terminal valine, N-(2-hydroxyethyl)valine, was quantified by gas chromatography/mass spectrometry. The value of 20±5 pmol/g Hb (mean ± SD) found in the non-smokers corroborated the steady-state level of 18±3 pmol/g Hb (mean ± SD) calculated from the pharmacokinetic approach.     

Structure-activity relationships of gamma-MSH analogues at the human melanocortin MC3, MC4, and MC5 receptors. Discovery of highly selective hMC3R, hMC4R, and hMC5R analogues

It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in alpha-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in gamma-MSH, two different series of gamma-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native gamma-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr-Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH(2) with Gly(4)) is more important for selectivity. The 2nd series is made of d-2-naphthylalanine (d-Nal(2')) scan of the gamma-MSH sequence at position 6 and 8 and the replacement of His(5) with Pro (analogue 13). Analogue 12, H-Tyr-Val-Nle-Gly-His-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr-Val-Nle-Gly-Pro-d-Nal(2')-Arg-Trp-Asp-Arg-Phe-Gly-NH(2), which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores.     

Carcinogenicity and genotoxicity of ethylene oxide: new aspects and recent advances

Long-term inhalation studies in rodents have presented unequivocal evidence of experimental carcinogenicity of ethylene oxide, based on the formation of malignant tumors at multiple sites. However, despite a considerable body of epidemiological data only limited evidence has been obtained of its carcinogenicity in humans. Ethylene oxide is not only an important exogenous toxicant, but it is also formed from ethylene as a biological precursor. Ethylene is a normal body constituent; its endogenous formation is evidenced by exhalation in rats and in humans. Consequently, ethylene oxide must also be regarded as a physiological compound. The most abundant DNA adduct of ethylene oxide is 7-(2-hydroxyethyl)guanine (HOEtG). Open questions are the nature and role of tissue-specific factors in ethylene oxide carcinogenesis and the physiological and quantitative role of DNA repair mechanisms. The detection of remarkable individual differences in the susceptibility of humans has promoted research into genetic factors that influence the metabolism of ethylene oxide. With this background it appears that current PBPK models for trans-species extrapolation of ethylene oxide toxicity need to be refined further. For a cancer risk assessment at low levels of DNA damage, exposure-related adducts must be discussed in relation to background DNA damage as well as to inter- and intraindividual variability. In rats, subacute ethylene oxide exposures on the order of 1 ppm (1.83 mg/m3) cause DNA adduct levels (HOEtG) of the same magnitude as produced by endogenous ethylene oxide. Based on very recent studies the endogenous background levels of HOEtG in DNA of humans are comparable to those that are produced in rodents by repetitive exogenous ethylene oxide exposures of about 10 ppm (18.3 mg/m3). Experimentally, ethylene oxide has revealed only weak mutagenic effects in vivo, which are confined to higher doses. It has been concluded that long-term human occupational exposure to low airborne concentrations to ethylene oxide, at or below current occupational exposure limits of 1 ppm (1.83 mg/m3), would not produce unacceptable increased genotoxic risks. However, critical questions remain that need further discussions relating to the coherence of animal and human data of experimental data in vitro vs. in vivo and to species-specific dynamics of DNA lesions.     

The effect of salts on the micellization temperature of aqueous poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) solutions and the dissolution rate and water diffusion coefficient in their corresponding gels.

Studies were performed to examine the effect of ionic salts on phase transitions, dissolution rates, and diffusion coefficients of water in gels of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) with polymer concentrations ranging from 22 to 32% w/w and salt concentrations ranging from 0 to 1.5% w/w. Salts tested include Na(3)PO(4), Na(2)SO(4), Na(2)HPO(4), NaH(2)PO(4), NaCH(3)CO(2), NaCl, and KI. Micellization transition temperatures were obtained using differential scanning calorimetry. The dissolution rates were obtained by measurement of the surface erosion rates, and diffusion coefficients were obtained by using a method to analyze the intrusion of water into the aqueous gels. It was found that salts had no effect on the dissolution rate of the polymer gels into deionized water. However, when the salt concentration in the aqueous dissolution media was adjusted to match the concentration in the gels, the dissolution rate of the polymer gel decreased with increasing salt concentration. The salts also had a profound effect on the critical micellization temperature (CMT) and the diffusion coefficient of water within the gel. The diffusion coefficient and CMT decreased in the presence of salts. The magnitude of these effects was comparable to their placement on the Hofmeister, or lyotropic series for salts. The effects of polymer and salt concentrations on the CMT were quantified, and a single correlation was proposed to predict the micellization temperatures for a wide range of salt and polymer concentrations.     

Kinetics and disposition in toxicology

Pharmacokinetic analysis of the behaviour of xenobiotics in living organisms is the basis for the understanding of dose (concentration-) response relationships. Risk estimates may lead to erroneous results if pharmacokinetic principles are neglected. The pharmacokinetic behaviour of a xenobiotic should be known before planning long-term experiments on toxicity and carcinogenicity. This is demonstrated using ethylene as an example.On the basis of the pharmacokinetics in rats of ethylene and ethylene oxide, its carcinogenic metabolite, we estimated the theoretical risk for the carcinogenic potential of ethylene. Our results demonstrate that exposure of rats to ethylene concentrations higher than 1000 ppm correspond to a (theoretical) exposure to 5.6 ppm ethylene oxide: exposures to ethylene at 40 ppm are equivalent to ethylene oxide exposures of 1 ppm, which is the (new) TLV for ethylene oxide.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Carcinogenicity and Genotoxicity of Ethylene Oxide: New Aspects and Recent Advances

ABSTRACT

Long-term inhalation studies in rodents have presented unequivocal evidence of experimental carcinogenicity of ethylene oxide, based on the formation of malignant tumors at multiple sites. However, despite a considerable body of epidemiological data only limited evidence has been obtained of its carcinogenicity in humans. Ethylene oxide is not only an important exogenous toxicant, but it is also formed from ethylene as a biological precursor. Ethylene is a normal body constituent; its endogenous formation is evidenced by exhalation in rats and in humans. Consequently, ethylene oxide must also be regarded as a physiological compound. The most abundant DNA adduct of ethylene oxide is 7-(2-hydroxyethyl)guanine (HOEtG). Open questions are the nature and role of tissue-specific factors in ethylene oxide Carcinogenesis and the physiological and quantitative role of DNA repair mechanisms. The detection of remarkable individual differences in the susceptibility of humans has promoted research into genetic factors that influence the metabolism of ethylene oxide. With this background it appears that current PBPK models for trans-species extrapolation of ethylene oxide toxicity need to be refined further. For a cancer risk assessment at low levels of DNA damage, exposure-related adducts must be discussed in relation to background DNA damage as well as to inter-and intraindividual variability. In rats, subacute ethylene oxide exposures on the order of 1 ppm (1.83 mg/m³) cause DNA adduct levels (HOEtG) of the same magnitude as produced by endogenous ethylene oxide. Based on very recent studies the endogenous background levels of HOEtG in DNA of humans are comparable to those that are produced in rodents by repetitive exogenous ethylene oxide exposures of about 10 ppm (18.3 mg/m³), Experimentally, ethylene oxide has revealed only weak mutagenic effects in vivo, which are confined to higher doses. It has been concluded that long-term human occupational exposure to low airborne concentrations to ethylene oxide, at or below current occupational exposure limits of 1 ppm (1.83 mg/m³), would not produce unacceptable increased genotoxic risks. However, critical questions remain that need further discussions relating to the coherence of animal and human data of experimental data in vitro vs. in vivo and to species-specific dynamics of DNA lesions.     

Solubilisation in aqueous micellar solutions of block copoly(oxyalkylene)s

The solubilisation capacities of micellar solutions of diblock and triblock copolymers composed of hydrophilic poly(ethylene oxide) and hydrophobic poly(styrene oxide) have been compared using the poorly water-soluble drug griseofulvin as a model solubilisate. Our results showed an increase of solubilisation capacity (expressed as mg griseofulvin per gram of hydrophobic block) with temperature and, for spherical micelles, with core volume before reaching limiting values. A change of micelle shape from spherical to cylindrical (or worm-like) resulting from an increase in micelle aggregation number was accompanied by a further enhancement of solubilisation capacity. Comparison with the solubilisation of the same drug in micellar solutions of block copolymers of poly(ethylene oxide) and poly(1,2-butylene oxide) showed that the solubilisation capacity of a poly(styrene oxide) block was approximately four times that of a poly(1,2-butylene oxide) block for spherical micelles. Solubilisation capacity at 25 degrees C was approximately doubled when griseofulvin was incorporated into a copolymer melt and micelles initially formed from the drug-loaded melt at 65 degrees C rather than by loading the drug into pre-micellised solution at 25 degrees C in the usual manner.     

Effect of molecular weight and end-group nature on the solubility of ethylene oxide oligomers in 2H, 3H-decafluoropentane and its fully fluorinated analogue, perfluoropentane

Fluorinated liquids possess high chemical and physical stability, are tolerated by the human body and, therefore, show great promise in biomedical fields; however, they require extensive formulation. Phase diagrams are reported here for a series of ethylene oxide oligomeric additives in 2H,3H-perfluoropentane (HPFP), a non-chlorofluorocarbon fluorinated liquid regarded as a model propellant for pressurized metered-dose inhalers. Over a wide range of temperatures and concentrations, dihydroxyl end-capped poly(ethylene glycols) (PEGs) exhibited a lower critical solution temperature (LCST) that was strongly molecular weight dependent. In contrast, monomethyl (and thus monohydroxy) and dimethyl end-capped poly(ethylene oxides) were fully miscible with HPFP over the same temperature and concentration ranges, suggesting that the phase behaviour was dominated by end-group/solvent interactions. By systematically substituting HPFP for the fully fluorinated analogue perfluoropentane, the ability of these end-groups to interact with the solvent was perturbed and LCST-type behaviour was induced in the previously fully miscible monomethyl and dimethyl end-capped PEGs. Concomitantly, with increasing perfluoropentane content, the LCST of the dihydroxyl end-capped PEGs was driven to lower temperatures. Therefore, the phase behaviour of these systems may be controlled by 'tuning' the end-group structure of the ethylene oxide oligomers, and varying the hydrogen bonding capabilities of the fluorinated solvents.     

Postfabrication encapsulation of model protein drugs in a negatively thermosensitive hydrogel

A postfabrication encapsulation technique was developed for loading model protein drugs into an intelligent and biodegradable hydrogel film, which exhibits negative thermosensitivity with a desirable phase transition temperature between refrigerator temperature and body temperature. The hydrogel comprises mainly poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer, and oligo(lactide). The model proteins Hemoglobin and Bovine Serum Albumin were loaded into the hydrogel films by soaking the gels at 4 degrees C, at which the hydrogel film was swollen. The loaded drug was released gradually in PBS at 37 degrees C, where the hydrogel film was shrunken. Because the hydrogel is biodegradable, the loaded drug could be released completely. It is confirmed that proteins can, in their native structures, be included in the hydrogel via the present technique, as characterized by FTIR, Raman spectrum, UV/VIS spectrum, and circular dichroism spectrum. The highlight of our approach is avoidance of high temperatures and organic solvents in encapsulation, making it ideal for protein drug delivery systems.     

Enantio-selective inhibition of (1R,9S)- and (1S,9R)-beta-hydrastines on dopamine biosynthesis in PC12 cells

The inhibitory effects of (1R,9S)- and (1S,9R)-enantiomers of beta-hydrastine (BHS) on dopamine biosynthesis in PC12 cells were investigated. (1R,9S)-BHS decreased the intracellular dopamine content with the IC50 value of 14.3 microM at 24 h, but (1S,9R)-BHS did not. (1R,9S)-BHS was not cytotoxic at concentrations up to 250 microM towards PC12 cells. In these conditions, (1R,9S)-BHS inhibited tyrosine hydroxylase (TH) activity mainly in a concentration-dependent manner (33% inhibition at 20 microM) and decreased TH mRNA level in PC12 cells. The inhibitory patterns of dopamine content and TH activity by (1R,9S)-BHS showed similar behavioral curves. (1R,9S)-BHS at 10-50 microM also reduced the intracellular cyclic AMP level and Ca2+ concentration. In addition, treatment of L-DOPA at 20-50 microM for 24 h increased the intracellular dopamine content to 198-251% compared with the control in PC12 cells. However, the increase in dopamine levels induced by L-DOPA (20-50 microM) was reduced when L-DOPA was combined with (1R,9S)-BHS (10-50 microM). These results indicate that (1R,9S)-BHS, but not (1S,9R)-BHS, reduced dopamine content and L-DOPA-induced increase in dopamine content, in part, through the inhibition of TH activity and TH gene expression in PC12 cells: thus, (1R,9S)-BHS proved to have a function to regulate dopamine biosynthesis.     

HPLC-electrospray mass spectrometric assay for the determination of (R,R)-fenoterol in rat plasma

A fast and specific liquid chromatography-mass spectrometry method for the determination of (R,R)-fenoterol ((R,R)-Fen) in rat plasma has been developed and validated. (R,R)-Fen was extracted from 125 microl of plasma using solid phase extraction and analyzed on Atlantis HILIC Silica 3 microm column. The mobile phase was composed of acetonitrile:ammonium acetate (pH 4.1; 20mM) (85:15, v/v), at a flow rate of 0.2 ml/min. The lower limit of detection (LLOD) was 2 ng/ml . The procedure was validated and applied to the analysis of plasma samples from rats previously administered (R,R)-Fen in an intravenous bolus.     

Stereoselective synthesis and structure-activity relationship of novel ceramide trafficking inhibitors. (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide and its analogues

New ceramide trafficking inhibitors, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) and a series of its analogues, were synthesized in diastereomerically and enantiomerically pure forms, and the structure-activity relationship was investigated. These analogues were stereoselectively synthesized via catalytic enantioselective Mannich-type reactions using a Cu(II)-chiral diamine 4 complex. Analysis of HPA-12 analogues having various lengths of the amide side chain showed that the optimal chain length for the inhibition of sphingomyelin biosynthesis is 13 with an IC(50) of approximately 50 nM. Masking of the hydroxy group at the 2'- or 3-position of HPA-12 was carried out by methylation, and it was revealed that these hydroxy groups were essential for the activity. Installation of another hydroxy group onto HPA-12 at the same position as that in the natural ceramide was also conducted, but no enhancement of the activity was observed.     

超临界二氧化碳制备布地奈德-聚氧乙烯固体分散体及体外评价

本文以水难溶性药物布地奈德为模型药物，研究超临界流体技术制备布地奈德-聚氧乙烯固体分散体的方法及其影响因素。采用超临界二氧化碳静态法制备布地奈德-聚氧乙烯固体分散体，用粉末X射线衍射法、差示扫描量热法、溶解度法和体外溶出实验进行固体分散体的物相鉴别。在40 ℃，20 MPa条件下，布地奈德-聚氧乙烯N750(1∶10)是形成固体分散体的最佳条件，布地奈德与聚氧乙烯载体形成氢键，以无定形状态存在于载体中，溶解度和体外溶出速率显著提高。超临界流体技术是制备固体分散体的一种可行方法。     

Development and characterization of micellar systems for application as insect repellents

N,N-diethyl-meta-toluamide (DEET) is a widely used insect repellent due to its high efficacy. In this work, micellar systems based on poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymer were developed and studied for the purpose of controlling the release and cutaneous permeation of DEET, using concentrated solutions of the copolymer Pluronic F127 to form thermoreversible gels. The formulations presented thermoreversible gelation above 5 °C and altered rheological behavior at 15 and 25 °C. The presence of the drug drastically changed the sol–gel transition temperatures. The micrographs suggest that DEET induced the formation of anisotropic structures, and Maltese Crosses were observed. The formulation containing 10 wt% DEET and 15 wt% Pluronic F127 presented sustained drug release for up to 7 h. DEET release profile followed the Higuchi kinetics model. There was a reduction of approximately 35% in the amount of DEET absorbed through the skin after 6 h. About 62% of DEET from the formulation consisting of Pluronic F127 and DEET remain retained on the skin. The anisotropic structure may constitute a barrier to diffusion and thereby controlling the drug release effectively. These tests suggest that the tested samples exhibit safety profile greater than some commercially available products.     

Comparative study of the pharmacokinetics and bioaccessibility of orthophen suppositories

The pharmacokinetic characteristics of 0.05 g orthophen rectal suppositories produced by the Semashko MosKhimFarmPreparaty company have been studied in comparison to analogous suppositories with a modified composition based on tallow with an additive of 5% of poly(ethylene oxide) (PEO-400), which have been developed at the Pyatigorsk State Pharmaceutical Academy in collaboration with the AltaiVitaminy joint-stock company. It is established that the two medicinal preparations are bioequivalent. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 10, pp. 12–13, October, 2005.     

Percentages of the deuterium retained afterpara-hydroxylation of (R) (+) 4-2H-phenytoin and (S) (−) 4-2H-phenytoin in rat

(R)(+) and (S)(-) 4-2H-phenytoin have been used as substrates for the determination of the percentage of deuterium retention (NIH shift) after para-hydroxylation of the substrates in rat. By using GC-MS analyses, the percentages of deuterium retention were found to be 69% and 70% for the (R) and (S) phenyl rings, respectively. The results add additional evidence for the involvement of arene oxide in the oxidation of the pro (R) and pro (S) phenyls of phenytoin. The oxidation process of each ring could be mediated by independent enzyme systems, a rapid oxidative enzyme for the pro (S) phenyl and a slow oxidative enzyme for the pro (R) phenyl.