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D N Kirk

Publications and source records attributed to D N Kirk.

At least 37 records · Page 2Linked to original sources
PubMed1988

The measurement of vitamins D2 and D3 and seven major metabolites in a single sample of human plasma using gas chromatography/mass spectrometry.

Selected ion monitoring of vitamin D metabolites has previously been described but there has been only one detailed description of the measurement by gas chromatography/mass spectrometry (GC/MS) of a number of metabolites in a single plasma sample. We describe here a GC/MS method, using stable isotope labelled internal standards, which allows the estimation of vitamins D2 and D3, and their 25-hydroxy, 24,25-dihydroxy and 25,26-dihydroxy metabolites in a single 2 ml sample of plasma, although more is needed for the measurement of 1,25-dihydroxyvitamin D3. Plasma was extracted on Bond Elut C18 cartridges and initial fractionation carried out on Sep-Pak SIL. Straight-phase high-performance liquid chromatography was required for separation of polyhydroxylated metabolites prior to GC/MS using an LKB 2091 mass spectrometer with conventional packed columns. n-Butylboronate esters were formed across vicinal hydroxyls, followed by formation of trimethylsilyl ethers using trimethylsilylimidazole. The [M - 90 - 15]+ ion for each compound was monitored. Deuterated internal standards were not available for all metabolites and it was necessary to use (2H6)D3 and (2H6)25OHD3 as standards for the measurement of D2 and D3, and 25OHD3 and 25OHD2, respectively, and (2H6)24,25(OH)2D3 as a standard for 24,25(OH)2D3 and 25,26(OH)2D2. Although the [M - 90 - 15]+ ion of 24,25(OH)2D and 25,26(OH)2D has the same mass: charge ratio, derivatives of these compounds are completely separated in the GC system used. The intra-assay precision for all these assays is usually less than 5%.

Cholecalciferol↗
PubMed1988

Microbial transformations of steroids--I. Rare transformations of progesterone by Apiocrea chrysosperma.

When Apiocrea chrysosperma is incubated with progesterone for 7 days in a peptone, yeast-extract medium, eight major metabolites are produced. Each compound has been purified and its structure determined by high-field 1D and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. A clear synthetic pattern is recognisable. The products have been formed by multiple transformation reactions, usually double hydroxylations. Seven compounds are tertiary alcohols in which the hydroxyl group is located on the underside of the progesterone skeleton at either the axial 9 alpha- or the axial 14 alpha-site. One compound has hydroxyl groups at both these sites. Five metabolites are also secondary progesterone alcohols, the hydroxyl groups being at the 6 beta-, 15 alpha- or 15 beta-sites. Two compounds are monohydroxy metabolites; one is dehydrogenated in ring B and the other has lost the pregnane side-chain. The structures of the eight metabolites are 6 beta, 9 alpha-dihydroxyprogesterone; 6 beta, 14 alpha-dihydroxyprogesterone; 9 alpha, 14 alpha-dihydroxyprogesterone; 9 alpha, 15 beta-dihydroxyprogesterone, 14 alpha, 15 alpha-dihydroxyprogesterone; 14 alpha, 15 beta-dihydroxyprogesterone; 14 alpha-hydroxypregna-4,6-diene-3,20-dione and 15 alpha-hydroxyandrostene-3,17-dione. All compounds, except the last one, are biologically rare because they are not products of mammalian progesterone or androstenedione metabolism. They would be difficult to synthesise chemically. We believe that the compounds, 9 alpha, 15 beta-dihydroxyprogesterone; 14 alpha, 15 alpha-dihydroxyprogesterone and 14 alpha-hydroxypregn-4,6-diene-3,20-dione, have not been reported previously as microbial transformation products of progesterone.

Basidiomycota↗
PubMed1986

Enzymatic synthesis and chromatographic purification of lignan glucuronides.

Enterolactone, enterodiol and secoisolariciresinol were conjugated with glucuronic acid by solubilized rabbit liver microsomal UDP-glucuronyltrasnferase. The monoglucuronide conjugate of all three substrates was formed and its identity confirmed by nuclear magnetic resonance (NMR) spectroscopy. Analytical high pressure liquid chromatography (HPLC) and NMR spectroscopy indicated conjugation with glucuronic acid to occur at several positions in the molecule. The enzymatic conjugation was monitored by analytical capillary isotachophoresis (ITP). The Km-values for enterlactone, enterodiol, and secoisolariciresinol were calculated to be 0.30, 0.23, and 0.22 mmol/l respectively.

Animals↗
PubMed1986

HPLC isolation and GC-MS characterization of a compound strongly cross reacting with tetrahydroaldosterone antiserum.

Urines from patients with hypertension and elevated aldosterone levels, i.e. primary aldosteronism due to adrenal adenoma or hyperplasia or carcinoma were extracted, paper chromatographed and thereafter chromatographed repeatedly with normal phase and repeatedly with reversed phase HPLC systems in an attempt to find new metabolites of aldosterone. Specific 3 alpha-hydroxy-5 beta-tetrahydroaldosterone antiserum was used in a radioimmunoassay system to detect possible aldosterone metabolites in the HPLC fractions after each isolation step. The immunoactive HPLC fractions were derivatized and analysed by GC-MS. A relatively nonpolar compound, 11 beta:18(S),18:20 alpha-diepoxy-5 beta-pregnan-3 alpha-ol, was isolated and identified in this manner. This material was originally described by Kelly et al., in 1962 after loading human subjects with huge amounts (25-160 mg) of exogenous aldosterone. This material has not yet been described from endogenously produced aldosterone. Very small amounts, if any, were similarly isolated from the urine of a control subject. Therefore, this compound could prove to be a new marker for hypertension due to hyper-production of aldosterone.

Aldosterone↗
PubMed1985

18-Substituted steroids: synthesis of 18-hydroxycortisol (11 beta,17 alpha,18,21-tetrahydroxy-4-pregnene-3,20-dione) and 18-hydroxycortisone (17 alpha,18,21-trihydroxy-4-pregnene-3,11,20-trione).

The isolation of 18-hydroxycortisol from the urine of patients with primary aldosteronism was recently described and no synthetic procedure was available for its preparation. The C-13 angular methyl group of prednisolone-17 alpha,21-acetonide-11 beta-nitrite was functionalized by photolysis in the presence of oxygen to give the product 18-hydroxy-prednisolone-17 alpha,21-acetonide-18-nitrate. The 18-nitrate was reduced with zinc and ammonium acetate to the corresponding 18-hydroxy compound, 18-hydroxy-prednisolone-17 alpha,21-acetonide. Homogeneous hydrogenation with Tris(triphenyl-phosphine)rhodium (I) chloride as catalyst resulted in the formation of 18-hydroxy-cortisol-17 alpha,21-acetonide. Acid hydrolysis of the latter compound gave 18-hydroxycortisol. Oxidation of 18-hydroxycortisol-17 alpha,21-acetonide with pyridinium dichromate followed by acid hydrolysis gave 18-hydroxycortisone. The 18-hydroxylated steroids exist as the 18,21-hemiacetals. Catalytic reduction with tritium gas with Tris(triphenyl-phosphine)rhodium (I) chloride of 18-hydroxyprednisolone-17 alpha,21-acetonide and acid hydrolysis gave [1,2(3)H]18-hydroxycortisol.

Chemical Phenomena↗
PubMed1984

Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease.

Equol, a nonsteroidal estrogen of dietary origin, was recently identified in human urine, and is excreted in amounts comparable to the classical steroidal estrogens. We confirm here that phytoestrogens which are abundant in dietary soya protein are converted by human gastrointestinal flora to this weak estrogen. After the ingestion of meals containing cooked soya protein the urinary excretion of equol in four of six subjects studied increased by up to 1000-fold and this compound was the major phenolic compound found in the urine. These data also indicate that some subjects are unable to either produce or excrete equol despite the challenge of a diet containing soya. In view of the increasing use of commercial soya products in the diet and the capacity of human bacterial flora to synthesize this weak estrogen from the abundance of phytoestrogens in soya, the potential relevance of these observations to the diseases implicating steroid hormones is discussed.

Adult↗
PubMed1984

Specific estimation of 24,25-dihydroxyvitamin D in plasma by gas chromatography-mass spectrometry.

This paper describes a specific mass-fragmentographic method, involving a stable-isotope-labeled internal standard, for measurement of 24,25-dihydroxyvitamin D in human plasma. Vitamin D metabolites were rapidly extracted from plasma by using Sep-Pak C18 cartridges and separated into fractions on Sep-Pak SIL cartridges. The polar fraction, containing the dihydroxylated metabolites, was further purified by "high-performance" liquid chromatography on Zorbax SIL. The fraction containing 24,25-dihydroxyvitamin D was collected, evaporated, and converted to the 24:25-cyclic n-butyl boronate-3-trimethylsilyl ether derivative before analysis by gas chromatography-mass spectrometry. The intensity of the mass fragment (m/z 449, m/z 455 for the hexadeuterated internal standard) arising from the loss of one of the angular methyls and the 3-silanol group [( M-90-15]+) was monitored. The minimum limit of detection for this method is about 0.1 microgram/L. Inter- and intra-assay reproducibility was acceptable, and analytical recovery of added 24,25-dihydroxyvitamin D3 over the concentration range 1.0 to 5.0 micrograms/L was quantitative. Concentrations of 24,25-dihydroxyvitamin D3 in plasma of 21 apparently healthy volunteers were between 0.55 and 5.39 micrograms/L, higher values being obtained after prolonged exposure to the sun. No 24,25-dihydroxyvitamin D2 could be detected in any plasma sample examined.

24,25-Dihydroxyvitamin D 3↗
PubMed1983

Measurement of enterolactone and enterodiol, the first mammalian lignans, using stable isotope dilution and gas chromatography mass spectrometry.

Methods are described to extract and measure 2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone (enterolactone) and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol (enterodiol) from physiological fluids. They are based on the use of stable labelled analogues of each compound as internal standards with end point assay being by gas chromatography mass spectrometry. The methods were developed to quantify enterolactone and enterodiol in experiments designed to investigate the significance and source of these compounds in man and animals. Examples of studies on human ovarian blood and investigations of their excretion in urine following dietary manipulation are described.

4-Butyrolactone↗
PubMed1983

Recent applications of N.M.R. to the investigation of steroid structures.

A detailed and clear understanding of the solution state structure and conformation of steroids is now possible. This is achieved using high field super conducting magnets and sophisticated computer control systems in nuclear magnetic resonance (n.m.r.) spectroscopy. The combination of greater signal dispersion and sensitivity obtained at high magnetic fields (cf. greater than 9.4 T--1H resonant frequency 400 MHz) has increased the utility of traditional assignment techniques such as nuclear Overhauser enhancement (nOe) and spin decoupling measurements. It has also permitted the application of various two dimensional (2D) n.m.r. experiments and resulted in complete unambiguous analysis of the proton spectra of various steroids [1-3]. The power of the various methods is illustrated by their application to the solution conformation of 17 alpha-acetoxy-6 alpha-methylpregn-4-en-3,20-dione (Medroxyprogesterone acetate, MPA).

Contraceptive Agents, Female↗
PubMed1982

18-substituted steroids--9[1]. Studies on the stability of aldosterone in dilute alkali.

In the presence of dilute alkali at room temperature aldosterone undergoes rearrangement to form an isomeric compound identified as 11 beta, 18;18,21-diepoxy-20,21-dihydroxypregn-4-en-3-one. The presence of dissolved oxygen causes simultaneous degradation to form 11 beta, 18-epoxy-18-hydroxy-3-oxo-17(beta H)-androst-4-ene-17 alpha-carboxylic acid. Under similar alkaline conditions at reflux temperature in aqueous methanol aldosterone undergoes rearrangement to form (20S)-20,21-dihydroxy-3-oxo-pregn-4-eno-18,11 beta-lactone accompanied by an intramolecular aldol type condensation to form 11 beta, 21-dihydroxy-18,21-cyclopregna-4,18(21)-diene-3,20-dione. Identical products were formed from 17-isoaldosterone. The mechanisms of these reactions are discussed.

Aldosterone↗
PubMed1981

The definitive identification of the lignans trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol in human and animal urine.

The definitive identification of the first lignans to be found in humans and animals is described. Gas chromatography--mass spectrometry, n.m.r. spectroscopy, i.r. spectroscopy and chemical techniques were employed to establish the structures of two lignans as trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol. Both compounds are essetially racemic. Evidence was also found for several methoxy analogues of these lignans in the vervet monkey.

4-Butyrolactone↗
PubMed1981

Microbial synthesis of 1 beta- and 15 beta-hydroxylated bile acids.

Lithocholic, deoxycholic, and cholic acids were incubated with the molds Absidia coerulea Bain (CBS 105.08, I), three strains of Rhizoctonia solani Kühn (CBS 130.14 (II), CBS 341.36 (III), CBS 325.47 (IV)), and Penicillium species ATCC 12556 (V). The products were analyzed by gas-liquid chromatography and partially characterized by gas-liquid chromatography-mass spectrometry. Lithocholic acid was most extensively hydroxylated while cholic acid yielded only traces of hydroxylated metabolites. I, II, III, and V hydroxylated at position C-1; I at C-6; I and II at C-7; III at C-12; and I, II, IV, and V at C-15. Hydroxylations at unidentified positions were observed for all molds, and oxidoreductions at C-7 and C-12 were carried out by I, II, and III. The two major trihydroxycholanoates formed by V from deoxycholic acid were characterized by chemical, mass spectrometric, and nuclear magnetic resonance techniques as 1 beta, 3 alpha, 12 alpha- and 3 alpha, 12 alpha, 15 beta-trihydroxy-5 beta-cholanoic acids. The former bile acid was identical with the 1,3,12-trihydroxycholanoic acid present in human urine.

Biotransformation↗
PubMed1980

Lignans in man and in animal species.

In our laboratories, for several years, two phenolic compounds have been detected during gas chromatographic-mass spectrometric analysis of urinary steroid extracts from human and animal species. Although features of the mass spectra of their trimethylsilyl (TMS) ether derivatives resembled those of oestrogens, they were atypical of steroids. The possibility that they were artefacts of the isolation procedures was discounted after careful studies with blanks, by varying the extraction method and because they were present almost exclusively as conjugates of glucuronic acid. Several of the general characteristics of the unknown compounds were reported after one (referred to as compound 180/442) was found to have a cyclic pattern of excretion during the menstrual cycle of an adult vervet monkey (Fig. 1). An investigation of the nature and distribution of the compounds has shown them to be urinary constituents in humans, baboons, vervet monkeys and rats, and further related compounds have been detected, so far only in vervet monkey urine. We now report spectroscopic and chemical studies that show the two original compounds to be lignans, which have a 2,3-dibenzylbutane skeleton as their basic structure. Unlike all previously known natural lignans, invariably of plant origin, the two mammalian compounds carry phenolic hydroxy groups only in the meta position of the aromatic rings.

Adult↗
PubMed1979

18-substituted steroids. Part 4. A chemical synthesis of 3 alpha,18,21-trihydroxy-5 beta-pregnan-20-one 18--20-hemiacetal (18-hydroxy-tetrahydro-DOC).

3 alpha,18,21-Trihydroxy-5 beta-pregnan-20-one 18 leads to 20-hemiacetal (18-hydroxy-tetrahydro-DOC) has been prepared from 3 alpha-acetoxy-5 beta-pregnan-20-one by reduction to the 20 beta-alcohol, application of the 'hypoiodite' reaction [Pb(OAc)4-I2-hv] with subsequent steps leading to the 18-hydroxy-20-ketone (as hemiacetal), and C-21 acetoxylation [Pb(OAc)4] followed by hydrolysis.

18-Hydroxydesoxycorticosterone↗
PubMed1979

Improved preparation of pregn-5-ene-3 beta,16 alpha,20-triols and 5 alpha-pregnane-3 alpha,16 alpha,20-triols.

The compounds named in the title were prepared by routes which included the reduction of suitable 16 alpha,17-epoxypregnan-20-ones with aluminium amalgam to give 16 alpha-hydroxypregnan-20-ones, and reduction of the 20-oxo function either with sodium borohydride to obtain the 3,16 alpha,20 beta-triols or with lithium-liquid ammonia to obtain the 3,16 alpha,20 alpha-triols.

Hydroxysteroids↗