High-performance Liquid Chromatography Retention Times Research Articles

Identification of O-methyldopa in the ventricular fluid of patients with Parkinson's disease.

A newly developed liquid chromatographic system with a multiple electrode detector was used for quantification of neurochemical substances in ventricular fluid of patients with Parkinson's disease. During the analysis, an unknown peak was observed at almost the same high-performance liquid chromatography retention time as 5-hydroxytryptophan. Through the use of ion suppression techniques, voltammographic analysis and fast atom bombardment mass spectrometry, the compound was identified as O-methyldopa, the major metabolite of L-dopa.

Characterization of neuropeptide Y-like immunoreactivity in vertebrate retina

We have determined the endogenous levels of neuropeptide Y-like immunoreactivity (NPY-LI) in retinal extracts from pigs, cats, rabbits, chickens, and frogs by radioimmunoassay (RIA). The NPY-LI levels varied among the species. The highest concentration was found in frog retina, where seasonal variations were seen, 861 ± 31 pmol g −1 wet weight in the autumn and 334 ± 26 pmol g −1 wet weight in the spring. Lower levels were demonstrated in chicken and pig retina, 4·1 ± 0·4 and 3·6 ± 0·3 pmol g −1 wet weight. respectively. The lowest concentration was demonstrated in rabbit retina, 2·0 ± 0·3 pmol g −1 wet weight. (All values are expressed as mean ± s.e.m.) The NPY-LI in pig, rabbit, chicken and frog retina was characterized by high-performance liquid chromatography (HPLC). The main part of the extracted NPY-LI had an elution volume close to that to the porcine NPY. We have also analysed the evoked release of endogenous NPY from frog retina, induced either with light flashes (3 Hz, 300 lx), or with potassium depolarization of the neurons (40 m m). Light flashes and potassium induced an increased release of NPY-LI of 61 and 77%, respectively. NPY-LI in the efflux had the same HPLC retention time as that extracted directly from the retina.

Characterization of .beta.-apo-13-carotenone and .beta.-apo-14'-carotenal as enzymic products of the excentric cleavage of .beta.-carotene

Two new products from the incubation of beta-carotene with intestinal mucosa homogenates of human, monkey, ferret, and rat were isolated using high-performance liquid chromatography (HPLC). Identification by comparing retention times in HPLC, by monitoring ultraviolet/visible spectra, by reduction to corresponding alcohol, by oxime formation, and by mass spectrometry demonstrated that they are beta-apo-13-carotenone and beta-apo-14'-carotenal. These compounds were not found in incubations done without intestinal homogenates or with disulfiram as an inhibitor. Under standard incubation conditions, these products increased linearly for 60 min and up to a protein concentration of 1.5 mg/mL and increased along with increasing concentrations of beta-carotene. Therefore, they are enzymatic cleavage products from beta-carotene. The formation of the beta-apo-13-carotenone and beta-apo-14'-carotenal provides direct evidence for an enzymatic excentric cleavage mechanism.

Comparison of high-performance liquid chromatography with capillary gel electrophoresis in single-base resolution of polynucleotides

High-resolution separations of polynucleotides were performed using capillary gel electrophoresis (cGE) and high-performance liquid chromatography (HPLC) with reversed-phase, ion-exchange and mixed-mode columns. Electropherograms showing cGE separations of single-stranded homopolynucleotides were presented and compared with HPLC separations according to the chain length of the polynucleotides. The resolving power of cGE is much higher than that of any HPLC mode. The chain length limits for complete separation within 60 min by cGE, reversed-phase, ion-exchange and mixed-mode HPLC are ca. 250, 30, 40 and 40 nucleotides, respectively. The plate number which is achieved for cGE of 3 · 10 6-7 · 10 6 plates/m is about ten times larger than those of all HPLC modes of 4 · 10 4-8 · 10 5 plates/m. The reproducibility of the migration time in cGE (2–4%) is comparable to those of retention times in HPLC in several separation modes (1–3%).

Association of microcystin‐LR and its biotransformation product with a hepatic‐cytosolic protein

Microcystin-LR (MCYST-LR), a cyclic peptide hepatotoxin, associates with high-molecular-weight, liver cytosolic components. Repetitive cycles of heat denaturation and pronase digestion released 80 +/- 6% of the bound radiolabel from these components, parent toxin (22%), and two biotransformation products, with high-performance liquid chromatography (HPLC) retention times of 6.7 (52%) and 5.6 (13%) min. Both parent and the biotransformed (6.7 min) toxin appeared to be covalently bound to a monomeric protein of molecular weight 40,000 (protein plus radiolabeled toxin). Binding and biotransformation reactions were time- and temperature-dependent and did not require endogenous molecules less than 6,000 daltons. The binding appeared to be saturable with a maximum of 20 pmol MCYST-LR bound per mg protein. The binding protein(s) and biotransformation activity were present in rat liver, brain, kidney, heart, lung, small intestine, large intestine, testes, skeletal muscle, and to a lesser extent, in fat. Okadaic acid, a specific protein phosphatase inhibitor, showed a concentration-dependent inhibition of [3H]MCYST-LR binding to hepatic cytosol. The molecular weight and organ distribution of the binding protein(s), and inhibition of binding by okadaic acid were consistent with one of the binding sites being the catalytic subunit of protein phosphatase type 2A.

Chromatographic characterisation of six human metabolites of the new anticancer drug GR63 178A

GR63178A is the second pentacyclic pyrroloquinone to enter clinical trials as an anticancer drug. We developed a reversed-phase, gradient-elution high-performance liquid chromatography (HPLC) method along with a Bond Elut C2 mini-column sample-preparation technique for the analysis of GR63178A, its 9-hydroxy-metabolite GR54374X and internal standard GR70440A in human plasma and urine. The limit of detection is 2 ng/ml for both GR63178A and GR54374X. Analysis of GR63178A is complicated by its light instability, whereby a number of chromatographically distinct, stable degradation products can form. These can be practically eliminated if clinical specimens are frozen immediately and all subsequent sample preparation is performed in a darkroom. Using this methodology, a total of six metabolites (including GR54374X) were detected in human plasma and urine specimens. The five new metabolites were characterised according to polarity (HPLC retention time), UV-visible absorption maxima and the effect of incubation with beta-glucuronidase and aryl-sulphatase. Application of this methodology to the analysis of GR63178A will aid in the development of this novel synthetic anticancer drug.

Isohematoporphyrin-diesters and diethers

1. 1. A series of diesters of isohematoporphyrin (isoHp), from dimethyl to dioctyl were prepared according to Rimington et al. (1989b). Their optical absorption, fluorescence spectra and high performance liquid chromatography (HPLC) retention times were recorded. 2. 2. A plot of HPLC retention time against number of C atoms in the alcohol used for esterification was approximately linear at first then rising steeply from diamyl to diocyi ester, whether a gradient elution was used or only methanol: water, 95/5, at pH 7.5. 3. 3. Preparation of the diethers of isoHp was much more difficult than that of the corresponding derivatives of hematoporphyrin (Hp). Several different methods were investigated, varying both times and temperatures. 4. 4. These methods included reaction of isoHp or its demethyl ester with 4.1. (i) a bromoalkane in presence of anhydrous K 2CO 3; 4.2. (ii) reaction with bromoalkane and Ag 2O; 4.3. (iii) reaction of brominated-isoHp, prepared by using thionylbromide, with the selected alcohol, or corresponding sodium alcoholate; 4.4. (iv) heating of isoHp alone with an alcohol containing 20% (w/v) H 2SCO 4 (temp. range from 45° to 118°C), 4.5. (v) refluxing as in (iv) at the b.p. of the alcohol; and 4.6. (vi) carrying out this reaction in refluxing ethyleneglycoldimethyl ether (b.p. 85°C) or diethyleneglycoldimethyl ether (b.p. 155°C). 5. 5. Some diether formation was observable by all these methods but yields were small, a considerable quantity of unreacted isoHp and other products remaining. 6. 6. Examined by HPLC, the diethers consistently afforded a forked peak which on thin layer chromatography was only resolved into two very closely associated bands by a solvent mixture carefully selected for development. 7. 7. On elution these materials had virtually identical optical absorption and fluoresence spectra. 8. 8. The nature of the association is discussed, atropisomers (Gottwald and Ullman, 1969) and possible stacked monomer: dimers (Abraham et al., 1963) being considered as possibilities.

Development of a high-performance liquid chromatographic method for fluroxypyr heerbicide and metabolites using computer simulation with drylab software

The herbicide fluroxypyr 1-methylheptyl ester was separated from its acid form (fluroxypyr) and two soil metabolites (a pyridinol and a methoxypyridine) by high-performance liquid chromatography (HPLC) with the acid of Drylab G. A difference in retention time of at least 6 min between each compound was achieved, to allow complete separation of radiolabeled components during collection of 1-min fractions. The total elution time was 31 min. Actual HPLC retention times differed from DryLab G predictions by 0.5 min or less.

HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY‐BASED PURIFICATION OF FIREFLY LUCIFERASES

Abstract— A simple procedure is described for purifying luciferases from firefly lanterns in greater than 50% yield. The key step in the method is high‐performance liquid chromatography (HPLC) using a Bio‐Gel TSK DEAE‐5PW analytical (75 times 7.5 mm) anion exchange column. In separate experiments, partially purified protein solutions obtained from Photinus pyralis and Photinus macder‐motti lanterns by solubilization, ammonium sulfate fractionation and Sephadex G‐25 gel filtration were chromatographed. Luciferases from both species had relative molecular masses (Mg) equal to 61 ± 2 times 103 and were strongly reactive to anti‐P. pyralis luciferase antibody. Isoelectric focusing (IEF) experiments, HPLC retention times (Rt), and bioluminescence emission spectra demonstrated that the luciferases from the different Photinus species were very similar, but not identical. The purification procedures described are most suitable for the isolation of 2–10 mg of protein; however, the use of a preparative column should enable the convenient isolation of larger amounts of protein. Also, this method should be readily adaptable to the isolation of luciferases from additional genera and species of fireflies.

Isolation of 11β-hydroxylated androgens from testicular vein blood of the mature boar

The isolation of 11β-hydroxyandrostenedione and 11β-hydroxytestosterone from testicular vein blood of the mature male domestic pig is described. Blood was collected from veins and arteries on the surface of the testes of mature boars. Steroids were extracted from plasma with SEP-PAK C 18 cartridges and recovered with acetonitrile. A separation of steroids was made by high performance liquid chromatography (HPLC) using acetonitrile/water ( 37 63 ; v/v ), and fractions were collected manually with detection at 254 nm. Preliminary identification was based on comparison with the HPLC retention time of an authentic steroid standard. Final characterization was achieved by means of capillary gas chromatography-mass spectrometry (GC-MS). The findings record the first evidence for the secretion of C 19-11-hydroxylated steroids by normal testes in a mammalian species.

High-performance liquid chromatographic separation of ring-oxidized metabolites of nitro-polycyclic aromatic hydrocarbons

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread genotoxic environmental pollutants, which require metabolic activation to exert their biological activities. Metabolism of nitro-PAHs generates ring-oxidized metabolites including epoxides, phenols, dihydrodiols and tetrahydrotetrols. Separation of the oxidized metabolites and related compounds of a series of isomeric nitro compounds derived from anthracene, benz[ a]anthracene, benzo[ a]pyrene and benzo[ e]pyrene was studied by high-performance liquid chromatography (HPLC) of different types of columns (monomeric and polymeric; reversed-phase and normal-phase). In the reversed-phase HPLC system, the general elution order of these compounds is: parent nitro-PAHs > phenolic derivatives > epoxides > dihydrodiols > tetrahydrotetrols. Among the geometric isomers, trans-dihydrodiols with both hydroxyl groups at the quasiaxial positions were eluted earlier than those with the hydroxyl groups at the quasiequatorial positions. Orientation of the nitro substituent has also been found to be an important structural feature of determining the relative retention order. Among the geometric isomers of nitro-PAHs and trans-dihydrodiols, the isomers with their nitro groups perpendicular or nearly perpendicular to the aromatic rings were eluted faster than the analogues with their nitro groups parallel or nearly parallel to the aromatic rings. Normal-phase HPLC gave opposite retention order, but with different separability among some of the compounds. Therefore, combination of both reversed-and normal-phase HPLC provides efficient separation of the ring-oxidized derivatives of nitro-PAHs. Results are also presented to compare the separation efficiency among different types of columns used. The results suggest that the polarity of solutes in the principal factor for determining their HPLC retention time.

Accumulation of regulatory oxysterols, 32-oxolanosterol and 32-hydroxylanosterol in mevalonate-treated cell cultures.

In a previous publication (Saucier, S.E., A.A., Taylor, F.R., Spencer, T.A., Phirwa, S., and Gayen, A.K., J. Biol. Chem. (1985) 260, 14571-14579), we demonstrated that cultured Chinese hamster lung (Dede) cells contain 24(S),25-epoxycholesterol and 25-hydroxycholesterol in cellular concentrations within the range required to repress 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. In this paper, we show that the addition to the culture medium of a concentration of mevalonate high enough to repress the reductase by 90% resulted in the appearance of two new regulatory oxysterols. The two new sterols are believed to be 32-oxolanosterol and 32-hydroxylanosterol on the basis of high performance liquid chromatography (HPLC) retention times and mass spectrometric and nuclear magnetic resonance spectroscopic data and by NaBH4 reduction of the putative aldehyde to material which had the HPLC retention time of the putative alcohol. The cellular concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholesterol were not significantly changed by the presence of mevalonate. However, there was approximately a 30% increase in total HMG-CoA reductase repressor units which can be ascribed to the 32-oxolanosterol and 32-hydroxylanosterol, where 1 unit equals the repressor activity of 1 ng of 25-hydroxycholesterol. These results support the idea that the level of HMG-CoA reductase activity in growing cell cultures is determined by intracellular oxysterol metabolites and that relatively small changes in their numbers or concentrations can alter the level of HMG-CoA reductase activity.

Formation of mutagens, 2-amino-3,8-dimethylimidazo[4,5-# f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-# f]quinoxaline (4,8-DiMeIQx), in heated fish meats

Fish meats were heated under conditions close to those used for cooking and processing. The mutagenic activity of the heated fish meats was estimated toward Salmonella typhimurium TA98 with metabolic activation after extraction with boiling water and adsorption to blue cotton. The numbers of His + revertant colonies/5 g of the meat heat-dried without charring at 220°C for 15 min were about 3000 for bonito, about 1000 for tunny, less than 500 for mackerel, salmon, swordfish, sardine, horse mackerel and cod, and 0 for cuttlefish. The mutagens in the heat-dried bonito meat were purified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were identified as 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline (4,8-DiMeIQx) by comparison with the authentic specimen with respect to R f values in TLC, retention times in HPLC, ultraviolet absorption spectra and mass spectra. The contents of MeIQx and 4,8-DiMeIQx in the bonito meat were estimated to be 5.2 and 5.4 ng/g, respectively. The major mutagens produced in the bonito, tunny and mackerel meats heated without charring at 100°C for 48 h and at 220°C for 15 min were found to be MeIQx and 4,8-DiMeIQx. It is interesting to note that the bonito and sardine meats grilled with charring for 15 min contained MeIQx and 4,8-DiMeIQx but higher mutagenicity was observed in the fraction that may contain 2-amino-3-methylimidazo[4,5- f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5- f]quinoline (MeIQ) and/or 2-aminodipyrido[1,2- a : 3′,2′- d]imidazole (Glu-P-2).

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C(18) high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [(13)C]-(C(6))-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [(3)H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [(13)C]-C(6)-IAA as an internal standard.

3′-Oxolutein, a Metabolite of Lutein in Chickens ,

3′-Oxolutein (3-hydroxy-3′-oxo-β,ε-carotene) was isolated using high performance liquid chromatography (HPLC) from the tissues and egg yolks of chickens fed a diet high in lutein and free of detectable 3′-oxolutein. It was identified by HPLC retention time, absorption spectrum identical to lutein and its esters, disappearance under alkaline conditions without giving rise to lutein, formation of an alkali labile palmitate, formation of a 2,4-dinitrophenylhydrazone, and reduction to lutein from which the dipalmitate was prepared. In each instance the isolated compound behaved identically with authentic 3′-oxolutein prepared by nickel peroxide oxidation of lutein. The order of ratios of lutein to 3′-oxolutein in the various tissues from laboratory and field birds was, in general, intestinal contents > intestinal mucosa > serum > liver ~ toe web ~ egg yolk > bile. This order was consistent with the hypothesis that lutein was oxidized to 3′-oxolutein in the liver, which, at least in part, was excreted via the bile into the intestinal lumen where it was diluted with dietary lutein. The remainder of the 3′-oxolutein formed in the liver presumably went into the serum where it was transported to depot sites. The ratio of lutein to 3′-oxolutein in the toe webs of broilers varied with their diet and pigmentation status. Amounts of 3′-oxolutein were found in quail and turkey egg yolks. This previously unreported lutein oxidative capability of liver extends the previously known metabolic acylation and deacylation reactions in poultry.

Metabolism of leukotriene B4 in isolated rat hepatocytes. Identification of a novel 18-carboxy-19,20-dinor leukotriene B4 metabolite.

Isolated rat heptocytes were found to metabolize leukotriene B4 (LTB4) to a number of products which could be separated by reverse phase high performance liquid chromatography (HPLC). After incubation of LTB4 with hepatocytes for 15 min, the known omega-oxidized metabolites, 20-hydroxy- and 20-carboxy-LTB4, were identified by HPLC retention time and gas chromatography-mass spectrometry. An early fraction corresponding to 15% of the initial LTB4 was structurally characterized as a novel metabolite, 18-carboxy-19,20-dinor-LTB4, by ultraviolet spectroscopy and gas chromatography-mass spectrometry of the derivatized and derivatized, reduced metabolite. The short HPLC retention time of this metabolite was consistent with its reduced lipophilicity. An additional minor metabolite was tentatively identified as 3-hydroxy-LTB4. These two novel metabolites provide evidence for beta-oxidation as an important route of hepatic biotransformation of LTB4 and 20-hydroxy-LTB4.

Antheraxanthin, a Light Harvesting Carotenoid Found in a Chromophyte Alga

The pigments of the chromophyte freshwater alga, Chrysophaera magna Belcher were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) to reveal the presence of chlorophylls a and c, beta-carotene, fucoxanthin, and antheraxanthin. The presence of antheraxanthin was verified by comparison of TLC R(F) values, HPLC retention times, and absorption features to those of authentic, synthetic antheraxanthin. Antheraxanthin accounted for about 15% of the total carotenoid content of C. magna. The molar ratio of the major carotenoids was antheraxanthin:fucoxanthin:beta-carotene, 1:2.3:3.3. The whole-cell absorption spectrum revealed a broad band between 470 and 520 nanometers which was attributed to fucoxanthin and antheraxanthin in vivo. Upon extraction in hydrocarbon, this broad absorption region was lost. The in vivo fluorescence excitation spectrum for 680 nm emission revealed the energy transfer activities and light harvesting roles of chlorophylls a and c, and fucoxanthin. In addition, an excitation band was resolved at 487 nanometers which could be attributed only to antheraxanthin. Comparison of whole-cell fluorescence excitation spectra of C. magna with the diatom Phaeodactylum tricornutum, which possesses fucoxanthin but not antheraxanthin, supports the assignment of the 487 nm band to antheraxanthin. This is the first report of a photosynthetic light harvesting function of the xanthophyll, antheraxanthin. This carotenoid broadens the absorption cross-section for photosynthesis in C. magna and extends light harvesting into the green portion of the spectrum.

Identification of Endogenous Gibberellins from Salix pentandra

Gibberellins A(1), A(19), A(20), and A(29) have been identified by sequential high-performance liquid chromatography retention time (Rt) and combined gas chromatography-mass spectrometry (Rt and characteristic mass spectra) from elongating shoots of Salix pentandra L. Gibberellins A(1) and A(19) were also detected in purified extracts from male and female flowers (catkins) of S. pentandra.

Intracellular acetylation of desacetyl αMSH in the xenopus laevis neurointermediate lobe

High performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA) of the chromatographic fractions were used to separate and quantify, respectively, the αMSH-like peptides stored in the neurointermediate lobe (NIL) of the Xenopus laevis (X. laevis) pituitary gland and released from the X. laevis NIL, in vitro. Immunoreactive (IR) material eluting with a similar HPLC retention time as desacetyl αMSH was the major IR peptide in the NIL. Material with a retention time similar to αMSH and immunological properties equivalent to αMSH was also present in the NIL. However, the retention times of the X. laevis and mammalian αMSH-like peptides were not identical, suggesting species difference in these peptides. Following incubation of NILs in the presence of [ 3H]-acetyl CoA, the X. laevis variant of αMSH was the major [ 3H]-labeled, immunoprecipitable material present. Following an incubation of NILs in the presence of [ 3H]-amino acids for 21 hours, immunoprecipitable [ 3H]-αMSH was detected in the NILs and the ratio of [ 3H]-desacetyl αMSH to [ 3H]-αMSH was similar to the ratio of IR-desacetyl αMSH to IR-αMSH. The X. laevis variant of αMSH was the major αMSH-like peptide released from the NILs into the incubation medium. Dopamine (50 μM) significantly inhibited the release of IR-αMSH but not IR-desacetyl αMSH. No net increase in total αMSH (sum of release and NIL content) was observed in the actively secreting (control) NIL group versus the dopaminetreated group. These results indicate that acetylation of desacetyl αMSH occurs intracellularly.

Formation of 3-Hydroxy-2,3-dihydrovitamin K1 In Vivo: Relationship to Vitamin K Epoxide Reductase and Warfarin Resistance

Hydroxy vitamin K [3(2)-hydroxy-2,3-dihydrovitamin K1] has been identified as a quantitatively important metabolite of injected vitamin K epoxide in vivo. The metabolite has been isolated and identified by comparison of its UV, mass spectra and high-performance liquid chromatography (HPLC) retention times with those of synthetic standards, and by its characteristic conversion to vitamin K quinone on treatment with the base triethylamine. This metabolite is formed from the vitamin K epoxide, not from the vitamin K quinone and can represent up to 3.5% of dose and 13% of hexane-extractable metabolites present in liver 1 hour after injection of 330 µg vitamin K1 epoxide per kilogram body weight. It is formed in both normal and warfarin-resistant rat strains, but to a significantly greater extent in the latter. Unlike the hydroxy vitamin K formed by warfarin-resistant rat liver microsomes in vitro, the metabolite formed from racemic vitamin K epoxide in vivo was not optically active, nor was its formation inhibited by coumarin anticoagulants under conditions that completely blocked vitamin K epoxide reduction in vivo. On this basis, hydroxy vitamin K formation in vivo differs from its formation in vitro; it is not a product of vitamin K epoxide reductase in vivo, but of some other possibly non-enzymatic reaction.