Tomas Cronholm

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

Abstract The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

Acetaldehyde as a substrate for ethanol-inducible cytochrome P450 (CYP2E1)

Liver microsomes from starved and acetone-treated rats catalyzed NADPH-supported metabolism of acetaldehyde at a rate 8-fold higher than corresponding control microsomes; the Vmax was about 6 nmol/mg microsomal protein/min and the apparent Km 30 microM. The reaction was efficiently inhibited by anti-CYP2E1 IgG, but not by control IgG. Reconstituted membranes containing rat CYP2E1 and cytochrome b5 metabolized acetaldehyde with a Vmax of 20 nmol/nmol/min and an apparent Km of 30 microM, whereas CYP2B4 containing vesicles or vesicles without b5 were ineffective. Gas chromatographic/mass spectrometric analysis of products formed from [2H4]-acetaldehyde with CYP2E1-containing reconstituted membrane vesicles revealed the formation of acetate as the only detectable product, although other water soluble products were also formed as evidenced from incubations with [1,2-14C]acetaldehyde. The results indicate that CYP2E1 is an aldehyde oxidase and thus metabolizes both ethanol and its primary oxidation product. This might have implications in vivo for acetaldehyde metabolism in liver and brain.

Quantitative analysis of unlabelled and polydeuterated compounds by gas chromatography-mass spectrometry

SummaryA system for computer evaluation of gas chromatographic-mass spectrometric analyses of unlabelled and polydeuterated compounds is described. The system has been in routine use for two years and has been found to fulfil most of the needs of the laboratory in connection with studies of steroid and bile acid metabolism. Complete or partial spectra are taken by repetitive magnetic scanning and are recorded on magnetic tape. Methods for qualitative evaluation have been described previously [6, 9]. Quantitative determinations of unlabelled compounds are based on areas of gas chromatographic peaks in selected specific fragment ion current chromatograms generated by the computer. Percentage distribution of molecular or fragment ion species containing 0–19 deuterium atoms is calculated from averaged spectra by comparison with spectra of the unlabelled reference compound analyzed on the same day. When small amounts of material are available and when ions occur in low abundance, repetitive accelerating voltage scanning over a short mass range is used. A slow scan rate, 1–5 s, and a high data sampling rate, 10 kHz, permit a 100–300 fold increase of amplification. After bunching of intensity readings, partial mass spectra are constructed which are treated in the same way as complete spectra.

Aldehyde dismutase activity of human liver alcohol dehydrogenase

Human alcohol dehydrogenases of class I and class II but not class III catalyse NAD+‐dependent aldehyde oxidation in addition to the NADH‐dependent aldehyde reduction. The two reactions are coupled, i.e. the enzymes display dismutase activity. Dismutase activity of recombinantly expressed human class I isozymes β 1 β 1 and γ 2 γ 2, class II and class III alcohol dehydrogenases was assayed with butanal as substrate by gas chromatographic‐mass spectrometric quantitations of butanol and butyric acid. The class I γ 2 γ 2 isozyme showed a pronounced dismutase activity with a high k cat, 1300 min−1, and a moderate K m, 1.2 mM. The class I β 1 β 1 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower k cat values, 60–75 min−1. 4‐Methylpyrazole, a potent class I ADH inhibitor, inhibited the class I dismutation completely, but cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, did not affect the dismutation. The dismutase reaction might be important for metabolism of aldehydes during inhibition or lack of mitochondrial aldehyde dehydrogenase activity.

Ethanol induced increase of the ratio between hydroxy- and ketosteroids in human pregnancy plasma

Ethanol was given to three young women in the third trimester of pregnancy and the concentrations of plasma steroid sulfates were measured by gas chromatography. The ratios between the concentrations of 20α-hydroxy- and 20-ketosteroid monosulfates increased during ethanol metabolism. There was also a pronounced increase in the concentration of androst-5-ene-3β, 17β-diol monosulfate. Consistent changes were not found in the disulfate fraction. Acetylation of the steroid monosulfate fraction followed by solvolysis and mass spectrometric analysis showed that the steroid diols were present in plasma as 3-sulfates. It is suggested that the changes are due, directly or indirectly, to an increase of the NADH/NAD+ ratio in the liver during ethanol metabolism.

Effects of ethanol on the levels of unconjugated and conjugated androgens and estrogens in plasma of men

The concentrations in plasma of estradiol, estrone, testosterone and 4-androstene-3,17-dione and their monosulphates and glucuronides were determined after oral administration of 0.3 g ethanol per kg body weight to four men. The levels of the unconjugated steroids did not change in a consistent way. In contrast, the concentrations of estradiol monosulphate and estradiol glucuronide increased markedly. The increase paralleled the blood alcohol concentrations and control levels were reached 3 h after the ethanol intake. A coupling of ethanol oxidation with reduction of dehydroepiandrosterone sulphate has previously been established, and it is suggested that the ethanol-induced change of the hepatic redox level affects the interconversion of conjugated forms of estradiol and estrone resulting in elevated levels of conjugated estradiol. This could have a feminizing effect and affect the feed-back regulation of gonadal hormone production.

Deuterium and carbon-13 tracer studies of ethanol metabolism in the rat by 2H, 1H-decoupled 13C nuclear magnetic resonance

STA1/sup -13/C, 1, 1-/sup 2/H/sub 2/! ethanol and STA2,2,2-/sup 2/H/sub 3/! ethan ol were administered to bile fistula rats. A new technique, /sup 2/H, / sup 1/H-decoupled /sup 1 3/C nuclear magnetic resonance, vas used in attempting to account for the distribution of the isotopic species along the steroid skeleton of 3 to 45 mg of isolated bile acids. The technique revealed /sup 2/H incorporation at many carbon sites unambiguously, but has limitations as a quantitative /sup 2/H assay at these levels of sample availability.

Position of the sulfate group in steroid sulfates from human plasma

Abstract The position of the sulfate group in plasma steroid sulfates from human male subjects has been studied. The steroids were acetylated prior to solvolysis and were then analyzed by gas chromatography-mass spectrometry after formation of trimethylsilyl ethers. In the monosulfate fraction androst-5-ene-3β, 17β-diol, pregn-5-ene-3β, 20α-diol and about 25 per cent of 5α-androstane-3α, 17β-diol were conjugated at C-3, whereas androst-5-ene-3β, 17α-diol and the major part of 5α-androstane-3α, 17β-diol were conjugated at C-17. The results indicate a different metabolic origin of the 17α and 17β isomers of androstenediol and show that the plasma pool of 5α-androstane-3α, 17β-diol monosulfate consists of two different compounds.

Analysis of aldehydic lipid peroxidation products in rat liver and hepatocytes by gas chromatography and mass spectrometry of the oxime-tert-butyldimethylsilyl derivatives☆

A method for analysis of aliphatic aldehydes in biological samples is described. Cyclohexanone is added as internal standard and the samples are treated with hydroxylamine and perchloric acid. The oximes are extracted and converted to the oxime-tert-butyldimethylsilyl derivatives, which are quantitated by capillary gas chromatography and identified by mass spectrometry. The characteristic M-57 fragment ions in the mass spectra enabled a rapid identification of the derivatives of the aldehydes, alkanals, alk-2-enals, alka-2,4-dienals, and 4-hydroxyalk-2-enals, which in addition gave rise to characteristic double peaks in the gas chromatographic analysis. The method was applied to analysis of autoxidized arachidonic acid, ADP-Fe3(+)-treated rat hepatocytes, and rat liver given a single dose of ethanol, 5 g/kg. The amounts of hexanal and 4-hydroxynon-2-enal were not increased 6 h after the administration of ethanol.

Changes in the metabolic profile of steroids in urine during ethanol metabolism in man

Three healthy men were given ethanol, 0.3 g/kg, and the excretion of steroids in urine was analyzed by capillary column gas chromatography and mass spectrometry, following group separation of conjugates. The ratio between the excretion rates of 17β-hydroxy- and corresponding 17-oxosteroid monosulphates increased 2–5 times during ethanol metabolism. This change is similar to that previously observed for the concentrations of these steroids in plasma, and is considered to be due to the changed redox state of hepatic pyridine nucleotides. Neither 20α (andβ) -hydroxy-/20-oxo-steroid couples formed from cortisol, nor various 11β-hydroxy-/11-oxosteroid couples in the glucuronide fraction changed towards a more reduced state during ethanol oxidation. The excretion of glucuronides of 11-oxygenated C19 steroids relative to that of cortisol metabolites of the C21 series decreased slightly, indicating an inhibition of side-chain cleavage. The rate of excretion of steroid disulphates was positively correlated to the urine volume.