Incorporation of radioactive mevalonate into C50 and C55 prenols by Streptococcus mutans (Short Communication)

Growing cells of Streptococcus mutans Ingbritt incorporate radioactive mevalonate into unsaponifiable lipid. Of the radioactive lipid 40% was shown by chromatography and mass spectrometry to be C(50) and C(55) prenol.     

Stereochemical aspects of the formation of double bonds in abscisic acid

The stereochemistry of the hydrogen elimination that occurs during the formation of the Delta(4)- and Delta(2)'-double bonds of abscisic acid has been determined from the (14)C/(3)H ratios in abscisic acid biosynthesized by avocado fruit from [2-(14)C,(2R)-2-(3)H(1)]-, [2-(14)C,(2S)-2-(3)H(1)]- and [2-(14)C,(5S)-5-(3)H(1)]-mevalonate. Setting the (14)C/(3)H ratio at 3:3 for [2-(14)C,(2R)-2-(3)H(1)]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-(14)C,(2S)-2-(3)H(1)]mevalonate was 3:1.63. Removal of the 3'-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3'-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-(14)C,(2R)-2-(3)H(1)]-mevalonate and to 3:1.05 for [2-(14)C,(2S)-2-(3)H(1)]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one (3)H atom at the 6'-methyl position of abscisic acid from the [(2R)-2-(3)H(1)]- and [(2S)-2-(3)H(1)]-mevalonate. The presence of some label at positions 3' and 4 when [(2S)-2-(3)H(1)]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.     

The role of deoxycorticosterone in the biosynthesis of cardenolides in Digitalis lanata

The role of deoxycorticosterone in the biosynthesis of digitoxigenin was investigated by the simultaneous administration of deoxy[1,2-(3)H(2)]corticosterone and [4-(14)C]progesterone to a Digitalis lanata plant. The biosynthetically formed [(3)H,(14)C]digitoxigenin and deoxy[(3)H,(14)C]corticosterone were isolated and the distribution of the two isotopes in these products was determined. The transformation of progesterone into deoxycorticosterone in vivo was established. The biosynthetic route from progesterone via deoxycorticosterone to cardenolides was found to be of little significance.     

Stereochemistry of phytoene biosynthesis by isolated chloroplasts

The incorporation of [2-(14)C,(5R)-5-(3)H(1)]MVA* and [2-(14)C,5-(3)H(2)]MVA into geranylgeraniol and phytoene by a preparation of ;non-aqueous' bean leaf chloroplasts has been studied. In the formation of phytoene from two molecules of geranylgeranyl pyrophosphate, the loss of hydrogen is stereospecific, the hydrogen atom lost from C-1 of each molecule of geranylgeranyl pyrophosphate being that which was originally the pro-S hydrogen atom from C-5 of mevalonate. All the pro-R hydrogen atoms from C-5 of mevalonate are retained. These results with a cell-free system confirm and extend the observations made in previous work with tomato slices.     

The biosynthesis of β-amyrin. Mechanism of squalene cyclization

1. beta-Amyrin synthesized by pea seedlings in the presence of (3RS)-[2-(14)C,(4R)-4-(3)H(1)]mevalonic acid (for nomenclature see Cahn, Ingold & Prelog, 1956) was subjected to a series of degradations to locate the positions within the molecule of the incorporated tritium. 2. The location of five of the six labelled hydrogen atoms at C-3, C-9, C-18 and C-19 (two) confirms that the mechanism of cyclization of squalene expected from the biogenetic isoprene rule is functioning in vivo.     

Polyprenol phosphates and mannosyl transferases in Phaseolus aureus

The transfer of mannose from GDP[¹⁴C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [¹⁴C]polymer formed was glycoprotein in nature although small quantities of ¹⁴C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [¹⁴C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [¹⁴C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [¹⁴C] from GDP[¹⁴C]-mannoseto polymer and simultaneously increased (3-fold) the [¹⁴C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots.     

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.     

Incorporation of [2-14C,(5R)-5-3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1. Incubation of a rat liver homogenate with 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid gave cholesterol with (3)H/(14)C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3beta-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6alpha-hydrogen atom of a precursor sterol is eliminated during formation of the C-5-C-6 double bond. 4. Incorporation of 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and beta-sitosterol with (3)H/(14)C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled beta-sitosterol into either 3beta-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5-C-6 double bond of phytosterols also involves the elimination of the 6alpha-hydrogen atom of a precursor sterol. 6. The 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.     

Biosynthesis of trans,trans- and cis,trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.     

Neoglycolipids derived from phosphatidylethanolamine serve as probes in cell culture studies on glycolipid metabolism

The neoglycolipid (NeoGL) N-acetyl-1-deoxy-1-phosphatidylethanolamino lacto-N-tetraositol [Lc4Ose-PtdEtn(NAc)] and the radioactivly labeled analog [Lc4Ose-PtdEtn(N[14C]Ac)] were synthesized by coupling the corresponding oligosaccharide to phosphatidylethanolamine (dihexadecyl) via reductive amination and subsequent N-acetylation with unlabeled and [14C]acetic acid anhydride, respectively. Lc4Ose-PtdEtn(N[14C]Ac) was then incubated with homogenates of rat small intestine epithelial cells (IEC-6) at pH 4. The reaction products were shown to be the degradation products formed by glycosidases by fast atom bombardment mass spectrometry (FAB MS). On the other hand, incubation of Lc4Ose-PtdEtn(NAc) with IEC-6 cell homogenates in sialyltransferase assays yielded the corresponding sialylated product. When Lc4Ose-PtdEtn(N[14C]Ac) was fed to IEC-6 cells as BSA complex, up to 5% of the NeoGL administered were taken up by the cells. After extraction of the NeoGL and separation by thin layer chromatography (TLC) the catabolic products Lc3Ose-PtdEtn(N[14C]Ac), Lac-PtdEtn(N[14C]Ac), and Glc-PtdEtn(N[14C]Ac), as well as the main anabolic product NeuGc-Lc4Ose-PtdEtn(N[14C]Ac) could be identified by FAB MS. These results demonstrate that PtdEtn-derived NeoGL can be used as probes for studies on the metabolism of specific oligosaccharide structures in cell culture.     

Structure of a colitose-containing O-specific polysaccharide of the marine bacterium Pseudoalteromonas tetraodonis IAM 14160(T).

O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Pseudoalteromonas tetraodonis type strain IAM 14160(T) and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments. The polysaccharide was found to consist of hexasaccharide repeating units containing one residue each of D-Gal, D-GlcA, D-GalNAc and D-GlcNAc and two residues of 3,6-dideoxy-L-xylo-hexose (colitose, Col) and having the following structure:In common with the polysaccharides of some other bacteria, the polysaccharide studied contains a tetrasaccharide fragment alpha-Colp-(1-->2)-beta-D-Galp-(1-->3)-[alpha-Colp-(1-->4)]-beta-D-GlcpNAc, which is a colitose ('3-deoxy-L-fucose') analogue of the Lewis(b) blood group antigenic determinant.     

Dolichols, ubiquinones, geranylgeraniol and farnesol as the major metabolites of mevalonate in Phytophthora cactorum

Farnesol, geranylgeraniol, dolichols and ubiquinones were the main radioactive components of the unsaponifiable lipid recovered from Phytophthora cactorum grown in aerated cultures containing [2-¹⁴C]mevalonate. The ¹⁴C recovered in each of these components was in the approximate proportion 2:4:3:5. When the culture was not aerated no radioactive ubiquinone was recovered. Most of the ¹⁴C recovered in the dolichols was found in dolichol-15 (37%), with decreasing amounts in dolichol-14 (30%) and -13 (14%) and only a little (5%) in dolichol-16, whereas the major components, by weight, of the mixture (13μg/g of damp-dry tissue) were dolichol-14, -15 and -16 in the approximate proportion of 1:3:1. Radioautography of appropriate chromatograms indicated the presence also of traces of radioactivity in dolichol-9, -10, -11, -12 and -17. Most (80%) of the ¹⁴C recovered in the ubiquinones was associated with ubiquinone-9, the rest being in ubiquinone-8. Most (80%) of the weight of ubiquinones (19μg/g of damp-dry tissue) was also ubiquinone-9. The identification of these compounds was by chromatographic methods and, for the ubiquinones and dolichols, was confirmed by mass spectrometry. In addition, the incorporation of 4R- and/or 4S-³H from [4-³H]-mevalonates showed the expected stereochemistry of biosynthesis, namely that farnesol, geranylgeraniol and ubiquinones were biogenetically all trans and the dolichols each contained three biogenetically trans isoprene residues, the remaining residues being biogenetically cis. The distribution of ¹⁴C in the components of the whole lipid of the fungus was consistent with 97% of both the farnesol and geranylgeraniol being present as the fatty acid ester. The corresponding value for dolichols was 37%. The observation by other workers, that this fungus does not form either squalene or sterol, was confirmed.     

Polyprenols of Aspergillus niger. Their characterization, biosynthesis and subcellular distribution

A polyprenol complex of Aspergillus niger was shown, by using spectrometric methods, to consist of a family of exo-methylene-hexahydroprenols that contain between 18 and 24 isoprene residues per molecule. Each prenol contains two trans residues, three saturated residues (alpha, omega and psi) and an exo-methylene substituent on the carbon atom beta to the isopropyl group in each omega-residue. The ubiquinone complex consisted of 90% ubiquinone-9, 9% ubiquinone-8 and 1% ubiquinone-10. The amount of polyprenol complex present reached a maximum of 1.7mg/culture bottle after 9-10 days of growth, coincident with the maximum weight of mycelium. The amount of ergosterol (10mg/culture bottle) and ubiquinone (1mg/culture bottle) reached a peak at 8 days. By the 13th day of growth the yield of ergosterol had fallen by 20% and that of ubiquinone by 85%. A study of the incorporation of [2-(14)C]mevalonate over different time-intervals confirmed that there was a slow turnover of prenol, a more rapid turnover of ergosterol and a very rapid turnover of ubiquinone. At any one time each member of the prenol complex had essentially the same specific radioactivity as other members of the complex. A similar conclusion was made about the ubiquinone mixture. Just over half of the polyprenol present was esterified to fatty acids. Subcellular fractionation studies indicated that the unesterified prenol is associated primarily with a mitochondrial fraction, whereas the ester is more widely distributed.     

The biosynthesis of cyclic carotenes

1. The incorporation of (3RS)-[2-(14)C,(4R)-4-(3)H(1)]mevalonic acid into various cyclic carotenes in the fruit of the tomato mutant delta has been studied. The results confirm our previous view that the alpha-ionone ring of alpha-carotene does not arise by isomerization of a beta-ionone residue, and show that the same is also true for the alpha-ionone ring of delta- and in-carotene and alpha-zeacarotene. 2. The incorporation of (3RS)-[2-(14)C,2-(3)H(2)]mevalonic acid into alpha- and beta-carotene in carrot roots has been studied. The results show that the beta-ionone ring of beta-carotene does not arise by isomerization of the alpha-ionone residue of alpha-carotene. 3. These experiments show that alpha- and beta-ionone rings in cyclic carotenes are formed independently, probably by elimination of different protons from the same carbonium ion intermediates.     

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.     

Intestinal renal metabolism of L-citrulline and L-arginine following enteral or parenteral infusion of L-alanyl-L-[2,15N]glutamine or L-[2,15N]glutamine in mice

Previously, we observed increased plasma arginine (ARG) concentrations after glutamine (GLN)-enriched diets, in combination with clinical benefits. GLN delivers nitrogen for ARG synthesis, and the present study was designed to quantify the interorgan relationship of exogenous L-GLN or GLN dipeptide, by enteral or parenteral route, contributing to intestinal citrulline (CIT) and renal de novo ARG synthesis in mice. To study this, we used a multicatheterized mouse model with Swiss mice (n = 43) in the postabsorptive state. Stable isotopes were infused into the jugular vein or into the duodenum {per group either free L-[2,(15)N]GLN or dipeptide L-ALA-L-[2,(15)N]GLN, all with L-[ureido-(13)C-(2)H(2)]CIT and L-[guanidino-(15)N(2)-(2)H(2)]ARG} to establish renal and intestinal ARG and CIT metabolism. Blood flow was measured using (14)C-para-aminohippuric acid. Net intestinal CIT release, renal uptake of CIT, and net renal ARG efflux was found, as assessed by arteriovenous flux measurements. Quantitatively, more de novo L-[2,(15)N]CIT was produced when free L-[2,(15)N]GLN was given than when L-ALA-L-[2,(15)N]GLN was given, whereas renal de novo L-[2,(15)N]ARG was similar in all groups. In conclusion, the intestinal-renal axis is hereby proven in mice in that L-[2,(15)N]GLN or dipeptide were both converted into de novo renal L-[2,(15)N]ARG; however, not all was derived from intestinal L-[2,(15)N]CIT production. In this model, the feeding route and form of GLN did not influence de novo renal ARG production derived from GLN.     

The biosynthesis of a N,N'-diacetylchitobiose containing lipid by liver microsomes. A probable dolichol pyrophosphate derivative

Incubation of liver microsomes with dolichol monophosphate, Mg⁺⁺ and UDP-[¹⁴C] -N-acetylglucosamine leads to the appearance of radioactivity in the lipid fraction. Mild acid treatment results in the formation of N-acetylglucosamine and N,N′-diacetylchitobiose (2-acetamido-2-deoxy-0-β-D-glucopyranosyl-(1rarr4)-2-acetamido-2-deoxy-D-glucose). The formation of the disaccharide containing lipid was increased by incubation with crude liver lipids or by reincubation with unlabelled UDP-N-acetylglucosamine. The labelling in the latter compound varied according to whether one of the N-acetylglucosamyl residues arose from a crude lipid or from unlabelled UDP-N-acetylglucosamine. Evidence is presented indicating that the compounds are dolichol pyrophosphate derivatives.     

The stereospecific biosynthesis of phytoene and polyunsaturated carotenes

1. The incorporation of [2-(14)C,(5R)-5-(3)H(1)]mevalonic acid and [2-(14)C,5-(3)H(2)]-mevalonic acid into phytoene, phytofluene, zeta-carotene, neurosporene, alpha-, beta-, gamma- and delta-carotene and lycopene by slices of fruit from two tomato mutants (delta and tangerine) and into alpha- and beta-carotene by bean leaves has been studied. 2. In the formation of phytoene, all the pro-R-hydrogen atoms from C-5 of mevalonic acid are retained whereas two pro-S-hydrogen atoms are lost. 3. Possible mechanisms for the condensation of two molecules of all-trans-geranylgeranyl pyrophosphate are outlined. 4. In each dehydrogenation step from phytoene to the fully unsaturated carotenes, one pro-R-hydrogen atom from C-5 of mevalonic acid is lost, indicating that the sequential dehydrogenation is stereospecific and in the same sense at each step.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.