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.

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gg ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (k cat = 180 min 21 ) but saturation is reached first at high concentrations (K m = 30 mm) while dismutation of 5-HIAL is saturated at lower concentrations and is thereby more efficient (K m = 150 mm; k cat = 40 min 21 ). In a system where NAD + is regenerated, the oxidation of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid proceeds with concentration levels of the intermediary 5-HIAL expected for a two-step oxidation. Butanal and 5-HIAL oxidation is also observed for class-I ADH in the presence of NADH. The class-II enzyme is less efficient in aldehyde oxidation, and the ethanol-oxidation activity of this enzyme is competitively inhibited by acetate (K i = 12 mm) and 5-hydroxyindole-3-acetic acid (K i = 2 mm).Reduction of 5-HIAL is efficiently catalysed by class-I gg ADH (k cat = 400 min 21 ; K m = 33 mm) in the presence of NADH. This indicates that the increased 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD + ratio on the class-I ADH.Keywords: alcohol dehydrogenase; alcohol metabolism; aldehydes; sequential oxidation; serotonin metabolism.The alcohol dehydrogenase (ADH) family is the major enzyme system for metabolism of ingested ethanol [1,2]. In addition to ethanol, a range of substrates has been identified for these enzymes and a possible mechanism for ethanol-induced metabolic changes may be interference with other activities of ADH (Fig. 1). For example, ethanol affects human noradrenaline, dopamine and serotonin metabolism by increasing the relative formation of the alcohol products, 4-hydroxy-3-methoxyphenylglycol, 3,4-dihydroxyphenylethanol and 5-hydroxytryptophol (5-HTOL), while decreasing the formation of the carboxylic acid products, 4-hydroxy-3-methoxymandelic acid, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole-3-acetic acid (5-HIAA) [3,4]. Clinical monitoring of recent drinking takes advantage of the elevated 5-HTOL/5-HIAA ratio which can be detected in urine for several hours after ethanol is no longer measurable [5,6]. The ADH activities for noradrenaline and dopamine metabolites have been characterized, and alcohols and aldehydes in these catabolic pathways serve as good substrates for ADH of both class I and class II [7,8]. It has also been shown that the serotonin metabolite 5-HTOL is an ADH substrate [9].In addition to reversible alcohol oxidation/aldehyde reduction, horse class-I and human class-I and class-II ADH catalyse aldehyde oxidation and thereby mimic the activity of aldehyde dehydrogenase (ALDH) [10±12]. Incubation of ADH with aldehyde and N...

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 [~1[~1 and T2T2, class II and class III alcohol dehydrogenases was assayed with butanal as snbstrate by gas chromatographic-mass spectrometric quantitations of butanol and butyric acid. The class I Y2Y2 isozyme showed a pronounced dismutase activity with a high kcat, 1300 min -1, and a moderate Km, 1.2 mM. The class I ~1111 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower kcat 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.

Dolichols and polyprenols are polyisoprenoid lipids found in all cells. Polyisoprenoids have recently been found covalently bound to cellular proteins constituting a new type of post-translational modification. To study these compounds effectively in biological systems a sensitive mass spectrometric procedure giving molecular weight and structural information is required. In the present study an assessment has been made of possible mass spectrometric procedures. Dolichols and polyprenols have been analysed at the pmol level in their underivatized form and as ted-butyldmethylsiiyl ethers by electron ionization mass spectrometry. Sulphated dolichols and polyprenols have been analysed at a pmol level by fast-atom bombardment and at the sub-pmol level by electrospray mass spectrometry. Dolichol phosphates have also been analysed by electrospray mass spectrometry. Collision-induced dissociation spectra of the underivatized and derivatized polyisoprenoids have been recorded. These spectra provide structural information from only pmols of sample.

Dolichols and polyprenols are polyisoprenoid lipids found in all cells. Polyisoprenoids have recently been found covalently bound to cellular proteins constituting a new type of post-translational modification. To study these compounds effectively in biological systems a sensitive mass spectrometric procedure giving molecular weight and structural information is required. In the present study an assessment has been made of possible mass spectrometric procedures. Dolichols and polyprenols have been analysed at the pmol level in their underivatized form and as ted-butyldmethylsiiyl ethers by electron ionization mass spectrometry. Sulphated dolichols and polyprenols have been analysed at a pmol level by fast-atom bombardment and at the sub-pmol level by electrospray mass spectrometry. Dolichol phosphates have also been analysed by electrospray mass spectrometry. Collision-induced dissociation spectra of the underivatized and derivatized polyisoprenoids have been recorded. These spectra provide structural information from only pmols of sample.Polyisoprenoid lipids containing between 16 and 23 isoprene residues are present in all animal cells.' They consist mainly of dolichols in which the a-isoprene unit is saturated but polyprenols also occur (Scheme 1). The latter are dominant in plants and bacteria. Dolichols, polyprenols and other isoprenoid compounds are biosynthesized from mevalonic acid, the formation of which is closely linked to normal cell gr0wth.2.~ Recent studies have also demonstrated that mevalonate production may be linked to normal and tumorigenic cell growth?*' Although simple dolichols and polyprenols have not been found to be growth stimulating, complex analogues may have a stimulative effect on DNA synthesis.Polyisoprenoids are often found covalently linked to other types of compounds. Sugars linked to phosphorylated isoprenoids are intermediates in glycosylation reactions, e.g. the biosynthesis of glycoproteins. Recent interest has been focused on the isoprenylation of cellular proteins6-' which constitutes a new type of post-translational modification. In connection with studies of protein prenylation other than farnesylation and geranylgeranylation we have investigated a number of mass spectrometric approaches to the analysis of dolichols, polyprenols and their derivatives. Spectra of dolichols from rat liver were reported by Gough and Hemming in 19708 using electron ionization (EI) on a magnetic sector instrument. Weak molecular ions were observed and abundant fragment ions. Fast-atom bombardment (FAB) ionization was recently used in the analysis of dolichols and polyprenols? Protonated molecules were obtained with low sensitivity which was increased by derivatization to phosphates, allowing detection of 50-100 pmol of material. Polyisoprenoid phosphates also occur naturally and short-chain diphosphates have been analysed by FAB-MS with tandem mass spectrometry (MS/MS)'O as have glycosyl monophosphopolyprenols. "In the present study we have tried to evaluate sensitivity