Phenolic compounds used in pharmaceutical and industrial products can cause irritant contact dermatitis. We studied the effects of resorcinol, phenol, 3,5-xylenol, chloroxylenol, and 4-hexyl-resorcinol on normal human epidermal keratinocytes and dermal fibroblasts for cytotoxicity and cytokine release, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide methodology and enzyme-linked immunosorbent assay, respectively. An inverse correlation between phenol concentrations causing a 50% reduction in keratinocyte and fibroblast viability at 24 h and their octanol water-partition coefficients (i.e., hydrophobicity) was observed. 3,5-xylenol, chloroxylenol, hexyl-resorcinol, and sodium dodecyl sulfate, but not resorcinol or phenol, induced release of interleukin-1alpha from keratinocytes at cytotoxic concentrations. Variable release of tumor necrosis factor-alpha and interleukin-8 from keratinocytes occurred only at toxic threshold concentrations of the phenols or sodium dodecyl sulfate. Subtoxic concentrations of phenols or sodium dodecyl sulfate did not induce cytokine release from keratinocytes. Neither the phenols nor sodium dodecyl sulfate induced release of the chemokines interleukin-8, growth-related oncogene-alpha or monocyte chemotactic protein-1 from fibroblasts. Conditioned media from keratinocytes treated with cytotoxic concentrations of 3,5-xylenol, chloroxylenol, hexyl-resorcinol, or sodium dodecyl sulfate stimulated further release of the chemokines from fibroblasts above that obtained with control media. Rabbit anti-interleukin-1alpha serum inhibited keratinocyte-conditioned media induction of chemokine release. We have shown a structure-cytotoxicity relationship for a series of phenols as well as an association of interleukin-1alpha release with a cytotoxic effect. We demonstrated a cytokine cascade amplification step by the actions of stimulated keratinocyte media on cultured dermal fibroblasts, identifying interleukin-1alpha as the principal initiator of chemokine synthesis.

Arachidonic acid (AA) can be metabolized to a variety of lipid mediators including prostaglandins (PGE), and hydroxyeicosatetraenoic acids (HETE) by cyclooxygenase, lipoxygenase and cytochrome P450-dependent monooxygenase enzymatic pathways. Traditional experimental procedures to quantify these lipid mediators require purification, often by high performance liquid chromatography (HPLC), prior to derivatization for gas chromatography/mass spectrometry (GC/MS) analysis. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE2, 12-HETE, and AA by HPLC/electrospray ionization mass spectrometry on cultured human dermal fibroblast supernatants. Extension of the method to analyse 5-HETE and 15-HETE was investigated. The advantages of this method include minimal sample preparation and elimination of the problem associated with thermal stability for GC/MS analysis. A detection limit of 20pg on column for PGE2 and 5pg on column for 12-HETE and AA was determined.

Conference Article| November 01 1995 Characterization of chemokine proinflammatory proteins by combined liquid chromatography-mass spectrometry A. I. Mallet; A. I. Mallet * 1St John's Institute of Dermatology, UMDS, University of London, St Thomas‘ Hospital, London SE1 7EH, U.K. *To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar I. Kay I. Kay 1St John's Institute of Dermatology, UMDS, University of London, St Thomas‘ Hospital, London SE1 7EH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (4): 911–913. https://doi.org/10.1042/bst0230911 Article history Received: June 12 1995 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation A. I. Mallet, I. Kay; Characterization of chemokine proinflammatory proteins by combined liquid chromatography-mass spectrometry. Biochem Soc Trans 1 November 1995; 23 (4): 911–913. doi: https://doi.org/10.1042/bst0230911 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ESI-MS, electrospray ionization MS This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Recently, it has been reported that a series of prostaglandin F2-like compounds (F2-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF2 alpha is shown to be a potent vasoconstrictor. We describe an improved method for analysing F2-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C18) and an aminopropyl (NH2) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F2-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF2 alpha as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF 2 alpha. However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF2 alpha when analyzed for the total (sum of free and esterified) F2-isoprostanes. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.

Locusta-diuretic peptide (Locusta-DP) is a potent stimulant of fluid secretion and cyclic AMP production by locust Malpighian tubules. In this study, a polyclonal antiserum raised to the C-terminus of Locusta-DP reveals a wide distribution of immunoreactive cell bodies and processes throughout the CNS, and endings in two important neurohemal release sites: the corpora cardiaca and the perivisceral organs. HPLC fractionation of CNS, neurohemal structures, and hemolymph reveals immunoreactive material that coelutes with synthetic Locusta-DP and stimulates cyclic AMP production by locust tubules. The identity of the immunoreactive and biologically active material is confirmed as authentic Locusta-DP by mass spectrometry.

Phosphoramidon, a potent inhibitor of endopeptidase‐24.11 (E‐24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin‐1 (big ET‐1) in rats. To examine whether E‐24.11 or thermolysin convert big ET‐1 to endothelin‐1 (ET‐1) and C‐terminal fragment (CTF), the effects on porcine and human big ET‐1 of each of the purified enzymes were compared in vitro. For E‐24.11, the relative rates of hydrolysis were ET‐1 > CTF >> big ET‐1. The relative half‐lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET‐1 > 24 h; ET‐1, 37 min; CTF, 57 min. For comparison, the half‐life for hydrolysis of substance P under similar conditions was 2.1 min. For thermolysin the relative rates of hydrolysis were found to be big ET‐1 > CTF > ET‐1. The relative half‐lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET‐1, 25 min; ET‐1, 56 min; CTF, 47 min. Because the low rate of conversion of big ET‐1 to ET‐1 by E‐24.11 did not yield sufficient ET‐1 for h.p.l.c. quantification a RIA specific for ET‐1 (16–21) was used to study further the hydrolysis of big ET‐1 by E‐24.11. Incubation of big ET‐1 (0.2–2 nmol) with E‐24.11 (4–400 ng) generated ET‐1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET‐1 (2 nmol) with E‐24.11 (40 ng) for 8 h showed that steady state levels of ET‐1 were achieved after 4 h indicating that the rate of ET‐1 degradation was then equal to the formation of new ET‐1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET‐1 had been produced, but the yield was insufficient for verification by mass spectrometry. Both ET‐1‐like and CTF‐like peaks were detected at 214 nm when the products of big ET‐1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET‐1 with amounts in the range 120–160 pmol. The hydrolysis profile of ET‐1 by E‐24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E‐24.11 and could be useful for the design of ECE inhibitors. Since E‐24.11 can both synthesize and hydrolyse ET‐1, the presence of E‐24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.

Fast atom bombardment mass spectra were successfully recorded for intact glycosylphenolphthiocerol dimycocerosates (phenolic glycolipids, PGLs) from Mycobacterium kansasii, M. leprae, M. tuberculosis, M. marinum, M. bovis and M. haemophilum. Characteristic fragment ions from the loss of the oligosaccharide moiety and one of the long-chain multimethyl-branched mycocerosic acids were observed in most cases. A tandem mass spectrometric experiment was carried out on the PGL from M. tuberculosis, revealing the type of mycocerosic acids esterified to individual homologues. Mass spectra of homologues separated by reversed-phase high-performance liquid chromatography gave information on the substitution pattern in certain cases. The potential of matrix-assisted laser desorption ionization spectroscopy was demonstrated by a successful analysis of the PGL from M. tuberculosis.

It has been recently reported that a series of prostaglandin F2-like compounds (PGF2-isoprostanes) are produced in vivo by non-enzymatic peroxidation of arachidonic acid. Of these, 8-epi-PGF2α is the major component and has been shown to be a potent vasoconstrictor. Here we investigated the formation of PGF2-isoprostanes during copper-mediated oxidation of low density lipoprotein (LDL). Free and total (sum of free and esterified) levels of PGF2-isoprostanes were measured at times 0, 3, 6, 8 and 24 h, using a solid-phase extraction procedure and gas chromatography - mass spectrometry. In native LDL, free levels of PGF2-isoprostanes were between 0.06 and 0.10 ng/mg protein (n = 4), and the total levels ranged from 0.027 to 0.057 ng/mg protein (n = 4). Free levels of the isoprostanes were found to increase throughout the oxidation, whereas total levels reached a maximum after 3 h then gradually decreased. 8-epi-PGF2α was the major isoprostane formed (free concentration after 24 h 1.8 ± 0.1 ng/mg protein (n = 4); total concentrations of 8.8 ± 1.8 and 6.1 ± 3.6 ng/mg protein (n = 4) after 3 and 24 h, respectively). The levels of isoprostanes correlated well with other indices of lipid peroxidation (conjugated dienes, hydroperoxides, thiobarbituric reactive substances) measured at similar time points. The release of PGF2-isoprostanes from oxidised LDL in macrophages could be a contributory factor in the development of atherosclerosis.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

The difference in relative stereochemistry of the 1.3-diol unit of mycobacterial phthiocerols can be determined by simple thin-layer chromatographic analysis of pentafluorobenzylidene acetal derivatives. The threo phthiocerol acetals from Mycobacterium tuberculosis are composed of equal amounts of two axial-equatorial stereoisomers but the erythro phthiocerols from Mycobacterium marinum form only one acetal with diequatorial substituents. The two acetals formed from a threo phthiocerol can be separated by thin-layer chromatography and mass spectra of the two stereoisomers allowed assignment of their structures.

Previous work has shown that a peptide related to arginine vasopressin is present in the suboesophageal ganglion of the locust, Locusta migratoria. This peptide was determined to be an anti-parallel dimer of the nonapeptide Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2 and was reported to stimulate cyclic AMP production and fluid secretion in a combined Malpighian tubules and midgut preparation from locusts. For these reasons the peptide has been called the arginine-vasopressin-like insect diuretic hormone (AVP-like IDH). Recently, a second diuretic peptide (Locusta-DP), which is related to corticotropin releasing factor, has been identified: this is a potent stimulant of fluid secretion and cyclic AMP production by isolated locust tubules. Because water balance in insects is likely to be controlled by a cocktail of hormones acting on both Malpighian tubules and hindgut, this study directly compares the activity of these two peptides in fluid secretion and cyclic AMP production bioassays on one target organ, the isolated Malpighian tubule of Locusta migratoria. Locusta-DP was synthesised directly, whereas the dimeric AVP-like IDH was obtained by oxidation of a synthetic nonapeptide monomer. Products were separated by RP-HPLC and their structures unequivocally confirmed by enzymatic digestion, sequence analysis and electrospray mass spectrometry. We show that Locusta-DP causes strong stimulation of fluid secretion and cyclic AMP production, whereas the AVP-like IDH has no effect in either assay. These findings are discussed in the light of recent work on the anatomy and physiology of the vasopressin-like immunoreactive (VPLI) neurones in the suboesophageal ganglion of Locusta migratoria, the proposed source of the AVP-like peptide.

An integrated method is described for the sensitive detection of tuberculostearic, mycocerosic and mycolic acids in infected materials from tuberculosis patients. Tuberculostearic acid is analysed by two-dimensional gas chromatography of pentafluorobenzyl esters, the key component being switched from a short non-polar column to a high resolution polar column with final electron capture detection. Mycocerosic acids are identified by simple one-dimensional electron capture gas chromatography of pentafluorobenzyl esters, with the use of negative-ion chemical ionisation gas chromatography in indecisive cases. Conversion of mycolic acids to anthrylmethyl esters produces compounds which are suitable for sensitive detection by fluorescence high-performance liquid chromatography. Application of all three of these methods to sputum samples from tuberculosis patients gave profiles characteristic of Mycobacterium tuberculosis.

A number of reagents, commonly used in protein chemistry and purification, suppress the electrospray ionization signal response of proteins in a concentration-dependent manner. An on-line trapping column device has been developed which enables the purification of protein samples containing these reagents, prior to their introduction into the mass spectrometer ion source.

The production of mycotoxins from Fusarium species has been demonstrated in isolates cultured from patients suffering from keratomycosis. The method employed a combination of thin-layer chromatography directly performed on gel plugs taken from the growth medium, cartridge column chromatography, silylation and gas chromatography on a non-polar stationary phase capillary column linked to mass spectrometry. The sensitivities of detection obtained for a signal-to-noise ratio of 33:1, were 200 pg for single stage GC-MS and 20 pg using tandem GC-MS-MS. Two mycotoxins, diacetoxyscirpenol and T-2 toxin were identified in three cultures.

Members of the phthiocerol dimycocerosate family of waxes were extracted from Mycobacterium bovis BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans and a skin biopsy from a leprosy patient. The waxes were degraded by alkaline hydrolysis and the mycocerosic acids converted to pentafluorobenzyl ester. Profiles of the esters, recorded using electron-capture gas-chromatography, gave characteristic profiles for the mycocerosates from M. leprae but those from M. bovis, M. tuberculosis and M. kansasii were superficially similar. The mycocerosate profiles from M. marinum and M. ulcerans were similar, but distinct from the others. Selected ion monitoring negative ion-chemical ionisation gas chromatography-mass spectrometry of of the pentafluorobenzyl esters allowed the analysis of mycocerosate isomers not revealed on gas chromatography alone. M. bovis and M. tuberculosis had similar profiles of C29, C30 and C32 mycocerosates; and additional C33 component was also present in M. kansasii. The mycocerosates from M. marinum and M. ulcerans were C27, C29 and C30 and those from M. leprae were distinct in having C29, C30, C32, C33 and C34 components. These methods have excellent potential for use in the detection of mycobacterial disease by direct analysis of infected tissue without prior cultivation of the causative agent.

Human semen was examined for the presence of 16-androstenols, 16-androstenones and androgens. Extracts were analysed by gas chromatography-mass spectrometry after derivatization of steroids under study. In a qualitative study, 5alpha-androst-16-en-3alpha- and 3beta-ols, 5,16-androstadien-3beta-ol and 5alpha-androstan-3beta-ol were detected in a semen pool A. Hydroxyl groups were converted to tert-butyldimethylsilyl ethers, the ions selected for monitoring being [M-57]+, consistent with loss of the tert-butyl group. For a more detailed quantitative study, a second semen pool B was used. In this case, all hydroxyl groups were converted to trimethylsilyl ethers, while oxo groups were not derivatized. As with semen pool A, separation of steroids was achieved using capillary gas chromatography with appropriate temperature programming. Quantification was carried out by mass spectrometry using selected ion monitoring of two significant ions and appropriate internal standards. The following steroids were identified at the concentrations indicated: 5alpha-androst-16-en-3alpha- and 3beta-ols and 5,16-androstadien-3beta-ol (concentration range, 0.5-0.7 ng/ml). 5alpha-Androst-16-en-3-one and 4,16-androstadien-3-one were also present at levels of 0.7-0.9 ng/ml. Two androgens, testosterone and 5alpha-dihydrotestosterone were found at concentrations of 0.5 and 0.3 ng/ml, respectively. These data, showing the presence of 16-androstenes and androgens in human semen, appear to be consistent with testicular formation of these steroids. The possible significance of the odorous 16-androstenes is discussed.