Uterine artery function in pregnant rats fed a diet supplemented with animal lard.

We hypothesised that maternal uterine artery vascular dysfunction could contribute to cardiovascular dysfunction in offspring of rats fed a diet rich in fat. Sprague-Dawley rats were fed for 10 days prior to pregnancy and throughout gestation either: (a) a control breeding diet, or (b) the same diet supplemented with 20 % w/w lard, vitamins, essential micronutrients and protein to control values. At 20 days gestation vascular function was assessed in uterine arteries and third-order mesenteric arteries. Vascular reactivity in response to application of potassium, noradrenaline, the thromboxane analogue U46619, acetylcholine and nitric oxide was assessed. Maternal plasma concentrations of factors likely to contribute to endothelial dysfunction were measured. Maximum acetylcholine-induced relaxation was impaired in the mesenteric arteries of the lard-fed dams (max % relaxation: lard-fed, 69.7 +/- 6.48; control, 85.37 +/- 2.69, P = 0.03). Uterine artery vascular function was similar in the two groups (max % acetylcholine-induced relaxation: lard-fed, 73.7 +/- 4.01; control, 77.5 +/- 4.72, P = 0.98). Concentrations of plasma lipids, 8-epi-PGF(2alpha) and leptin were normal, whereas insulin and corticosterone concentrations were raised in the lard-fed group (insulin (ng ml(-1)): lard-fed, 8.04 +/- 0.47; control, 1.35 +/- 0.37, P < 0.0001; corticosterone (ng ml(-1)): lard-fed, 1164.0 +/- 170.9; control, 541.9 +/- 96.3, P = 0.005). Fetal and placental weights were reduced in lard-fed dams (fetus (g): lard-fed, 4.27 +/- 0.38; control, 2.96 +/- 0.40, P = 0.025; placenta (g): lard-fed, 0.72 +/- 0.06; control, 0.57 +/- 0.04, P = 0.05). Cardiovascular dysfunction in offspring is not associated with reduced uterine artery endothelial function but is associated with activation of the hypothalamic-pituitary-adrenal axis, hyperinsulinaemia and fetoplacental growth retardation.

Effects of cisapride on gall bladder emptying, intestinal transit, and serum deoxycholate: a prospective, randomised, double blind, placebo controlled trial.

BACKGROUND: Octreotide inhibits gall bladder emptying and prolongs intestinal transit. This leads to increases in the proportion of deoxycholic acid in, and cholesterol saturation of, gall bladder bile, factors that contribute to the pathogenesis of octreotide induced gall stones. AIMS: To see if an intestinal prokinetic, cisapride, could overcome these adverse effects of octreotide and if so, be considered as a candidate prophylactic drug for preventing iatrogenic gall bladder stones. METHODS: A randomised, double blind, placebo controlled, crossover design was used to examine the effects of cisapride (10 mg four times daily) on gall bladder emptying, mouth to caecum and large bowel transit times, and the proportions of deoxycholic acid and other bile acids, in fasting serum from: (i) control subjects (n=6), (ii) acromegalic patients not treated with octreotide (n=6), (iii) acromegalics on long term octreotide (n=8), and (iv) patients with constipation (n=8). RESULTS: Cisapride had no prokinetic effect on the gall bladder. In fact, it significantly increased both fasting and postprandial gall bladder volumes. However, it shortened mouth to caecum (from 176 (13) to 113 (11) minutes; p<0.001) and large bowel (from 50 (3.0) to 31 (3.4) h; p<0.001) transit times. It also reduced the proportion of deoxycholic acid in serum from 26 (2.3) to 15 (1.8)% (p<0.001), with a reciprocal increase in the proportion of cholic acid from 40 (3.5) to 51 (3.8)% (p<0.01). There were significant linear relationships between large bowel transit time and the proportions of deoxycholic acid (r=0.81; p<0.001) and cholic acid (r=-0.53; p<0.001) in fasting serum. INTERPRETATION/SUMMARY: Cisapride failed to overcome the adverse effects of octreotide on gall bladder emptying but it countered octreotide induced prolongation of small and large bowel transit. Therefore, if changes in intestinal transit contribute to the development of octreotide induced gall bladder stones, enterokinetics such as cisapride may prevent their formation.

Colonic transit influences deoxycholic acid kinetics.

BACKGROUND & AIMS: Prolonged large bowel transit, and an increase in the proportion of deoxycholic acid (DCA), have been implicated in the pathogenesis of cholesterol gallstones-including those developing in acromegalics treated with octreotide. However, there are few data on the effects of intestinal transit on bile acid kinetics. METHODS: We therefore measured the kinetics of DCA and cholic acid (CA) using stable isotopes, serum sampling, and mass spectrometry. The results were related to mouth-to-caecum (MCTT) and large bowel transit times (LBTTs) in 4 groups of 8 individuals: (1) non-acromegalic controls, (2) acromegalics untreated with octreotide, (3) acromegalics on long-term octreotide, and (4) patients with constipation. Paired, before and during octreotide, studies were performed in 5 acromegalics. RESULTS: In the unpaired and paired studies, octreotide significantly prolonged MCTT and LBTT. In the paired studies, the octreotide-induced prolongation of LBTT caused an increase in the DCA input rate (6.4 +/- 2.8 to 12 +/- 2.6 micromol. kg. d, P < 0.05) and pool size (18 +/- 12 to 40 +/- 13 micromol/kg, P < 0.05), and a decrease in CA pool size (45 +/- 15 to 25 +/- 11 micromol/kg, P < 0.05). Furthermore, during octreotide treatment, the mean conversion of 13C-CA to 13C-DCA (micromoles) was greater (P < 0.05) on study days 3, 4, and 5. There were also positive linear relationships between LBTT and DCA input rate (r = 0.78), pool size (r = 0.82, P < 0.001), and a weak (r = -0.49) negative linear relationship between LBTT and CA pool size (P < 0.01). CONCLUSIONS: These data support the hypothesis that, by increasing DCA formation and absorption, prolongation of large bowel transit is a pathogenic factor in the formation of octreotide-induced gallstones.

Aldehyde analysis by high performance liquid chromatography/tandem mass spectrometry.

This paper describes the development of a high performance liquid chromatography/tandem mass spectrometric (MS/MS) procedure for the specific qualitative and quantitative analysis of lipid aldehydes in biological matrices. A derivatisation method, which results in molecules that exhibit a common product ion on MS/MS, permits informative precursor ion scans, at high sensitivity. This has been applied to the examination of plasma in order to examine the production of aldehydes consequent on in vitro lipid oxidation. Quantitative analysis of target molecules using multiple reaction monitoring has been developed to permit quantitation in the same matrices.

Consumption of flavonoids in onions and black tea: lack of effect on F2-isoprostanes and autoantibodies to oxidized LDL in healthy humans.

BACKGROUND: Oxidative damage to lipids in vivo may be involved in the development of atherosclerosis and cancer. Onions and black tea are foods rich in flavonoids, predominantly the flavonoid quercetin, which is a potent in vitro inhibitor of membrane lipid peroxidation and LDL oxidation. OBJECTIVE: Our objective was to investigate the effects of consuming a high-flavonoid (HF) diet enriched with onions and black tea on indexes of oxidative damage in vivo compared with a low-flavonoid (LF) diet. DESIGN: Thirty-two healthy humans were studied in a randomized crossover design. Indexes of oxidative damage used were plasma F2-isoprostanes (a biomarker of lipid peroxidation in vivo) and the titer of antibodies to malondialdehyde (MDA)-modified LDL. RESULTS: There were no significant differences in the intake of macronutrients or assessed micronutrients, plasma F2-isoprostane concentrations, and MDA-LDL autoantibody titer between the HF and LF dietary treatments. In the men, however, plasma concentrations of the F2-isoprostane 8-epi-prostaglandin F2alpha were slightly higher after the HF treatment phase than after the LF treatment [0.31 +/- 0.029 nmol/L (111 +/- 10.4 ng/L) compared with 0.26 +/- 0.022 nmol/L (92 +/- 7.8 ng/L); P = 0.041]. In all subjects, plasma quercetin concentrations were significantly higher after the HF treatment phase than after the LF treatment: 221.6 +/- 37.4 nmol/L compared with less than the limit of detection of 66.2 nmol/L. CONCLUSION: Flavonoid consumption in onions and tea had no significant effect on plasma F2-isoprostane concentrations and MDA-LDL autoantibody titer in this study and thus does not seem to inhibit lipid peroxidation in humans.

Increase of the isoprostane 8-isoprostaglandin f2alpha in maternal and fetal blood of rats with streptozotocin-induced diabetes: evidence of lipid peroxidation.

OBJECTIVE: Pregnancy complicated by diabetes is associated with maternal complications and fetal abnormalities. Animal models of diabetes suggest that heightened free radical production may be implicated in the pathogenesis of this condition. The purpose of this investigation was to evaluate oxidative stress in plasma from diabetic rats and their fetuses through measurement of concentrations of 8-isoprostaglandin F(2alpha), a stable marker of lipid peroxidation. STUDY DESIGN: Diabetes was induced in virgin and pregnant rats with streptozotocin. Blood samples were collected after 20 days of diabetes. Adult and fetal plasma 8-isoprostaglandin F(2alpha) concentrations were determined by gas chromatography-mass spectroscopy. RESULTS: Significantly higher plasma 8-isoprostaglandin F(2alpha) concentrations were observed in the virgin rats with diabetes and in both the pregnant dams with diabetes and their fetuses when compared with their respective control groups without diabetes (P <.001). CONCLUSION: Oxidative stress was induced in both mother and fetus in rodent pregnancy complicated by diabetes. This finding may have implications for fetal dysmorphogenesis and in fetal programming for adulthood disease.

Cytokine release and cytotoxicity in human keratinocytes and fibroblasts induced by phenols and sodium dodecyl sulfate.

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.

Isoflavone phytoestrogens consumed in soy decrease F(2)-isoprostane concentrations and increase resistance of low-density lipoprotein to oxidation in humans.

BACKGROUND: Oxidative damage to lipids may be involved in the etiology of atherosclerosis, cardiovascular disease in general, and cancer. The soy isoflavone phytoestrogens, genistein and daidzein, and equol (a daidzein metabolite produced by intestinal microflora) are antioxidants in vitro; equol is a particularly good inhibitor of LDL oxidation and membrane lipid peroxidation. OBJECTIVE: We sought to investigate the effects of a diet enriched with soy containing isoflavones on in vivo biomarkers of lipid peroxidation and resistance of LDL to oxidation, compared with a diet enriched with soy from which the isoflavones had been extracted. DESIGN: : A randomized, crossover design was used to compare diets enriched with soy that was low or high in isoflavones in 24 subjects. Plasma concentrations of an F(2)-isoprostane, 8-epi-prostaglandin F(2)(alpha) (8-epi-PGF(2)(alpha)), a biomarker of in vivo lipid peroxidation, and resistance of LDL to copper-ion-induced oxidation were determined. RESULTS: Plasma concentrations of 8-epi-PGF(2)(alpha) were significantly lower after the high-isoflavone dietary treatment than after the low-isoflavone dietary treatment (326 +/- 32 and 405 +/- 50 ng/L, respectively; P = 0.028) and the lag time for copper-ion-induced LDL oxidation was longer (48 +/- 2.4 and 44 +/- 1.9 min, respectively; P = 0.017). Lag time for oxidation of unfractionated plasma and plasma concentrations of malondialdehyde, LDL alpha-tocopherol, polyunsaturated fatty acids, and isoflavonoids did not differ significantly between dietary treatments. CONCLUSIONS: Consumption of soy containing naturally occurring amounts of isoflavone phytoestrogens reduced lipid peroxidation in vivo and increased the resistance of LDL to oxidation. This antioxidant action may be significant with regard to risk of atherosclerosis, cardiovascular disease in general, and cancer.

Biliary and urinary metabolic profiles of 1,2-diethyl-3-hydroxypyridin-4-one (CP94) in the rat.

This study compares the biliary and urinary metabolic profiles of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), an orally active iron chelator, in the normal rat. Surprisingly, CP94 was found to form two phase II metabolites, the 3-O- and 4-O-glucuronides. These glucuronides accounted for 38 and 28% of the administered CP94 dose, in bile and urine, respectively. Unchanged CP94 accounted for 5% of the CP94 dose in both bile and urine. The 2-(1'-hydroxy) metabolite of CP94 was found to be the dominant metabolite in urine. In addition, an unstable metabolite was detected in the bile although its structure remains unknown at the present stage. The excretion of iron in bile, after administration of CP94, was found to parallel the biliary elimination of CP94 together with its hydroxylated derivatives, indicating the importance of metabolites in iron excretion.

Cholesterol-independent endothelial dysfunction in virgin and pregnant rats fed a diet high in saturated fat.

1. Western diets high in saturated fat are associated with an increased incidence of cardiovascular diseases. In this study we have evaluated vascular endothelial function and oxidative stress in virgin rats fed a normal (VC) or high in saturated fat diet (VHF) (20 % lard and corn oil w/w) from weaning until adulthood, and throughout subsequent pregnancy (PC and PHF, respectively). 2. The saturated fat diet was associated with enhanced noradrenaline sensitivity in small mesenteric arteries from VHF rats (VHF vs. VC, P < 0.05) and blunted endothelium-dependent relaxation in VHF and PHF rats (VHF vs. VC, P < 0.001; PHF vs. PC, P < 0.05). Endothelial dysfunction was attributable to a reduced nitric oxide component of relaxation in VHF rats, and blunted prostacyclin and endothelium-derived hyperpolarizing factor components in PHF rats. 3. Other than plasma cholesterol, which was reduced in VHF and PHF rats, plasma lipids were normal. Fasting plasma insulin and glucose concentrations were raised in VHF rats (P < 0.05) and the plasma marker of oxidative stress, 8-iso PGF2alpha, was increased in PHF animals (P < 0.01). 4. These findings suggest that endothelial dysfunction induced by a saturated fat diet is cholesterol independent and likely to be of different mechanistic origin in virgin and pregnant rats.

Prolonged large bowel transit increases serum deoxycholic acid: a risk factor for octreotide induced gallstones.

BACKGROUND: Treatment of acromegaly with octreotide increases the proportion of deoxycholic acid in, and the cholesterol saturation of, bile and induces the formation of gallstones. Prolongation of intestinal transit has been proposed as the mechanism for the increase in the proportion of deoxycholic acid in bile. AIMS: To study the effects of octreotide on intestinal transit in acromegalic patients during octreotide treatment, and to examine the relation between intestinal transit and bile acid composition in fasting serum. METHODS: Mouth to caecum and large bowel transit times, and the proportion of deoxycholic acid in fasting serum were measured in non-acromegalic controls, acromegalic patients untreated with octreotide, acromegalics on long term octreotide, and patients with simple constipation. Intestinal transit and the proportion of deoxycholic acid were compared in acromegalic patients before and during octreotide. RESULTS: Acromegalics untreated with octreotide had longer mouth to caecum and large bowel transit times than controls. Intestinal transit was further prolonged by chronic octreotide treatment. There were significant linear relations between large bowel transit time and the proportion of deoxycholic acid in the total, conjugated, and unconjugated fractions of fasting serum. CONCLUSIONS: These data support the hypothesis that, by prolonging large bowel transit, octreotide increases the proportion of deoxycholic acid in fasting serum (and, by implication, in bile) and thereby the risk of gallstone formation.

A synthetic peptide adhesion epitope as a novel antimicrobial agent.

The earliest step in microbial infection is adherence by specific microbial adhesins to the mucosa of the oro-intestinal, nasorespiratory, or genitourinary tract. We inhibited binding of a cell surface adhesin of Streptococcus mutans to salivary receptors in vitro, as measured by surface plasmon resonance, using a synthetic peptide (p1025) corresponding to residues 1025-1044 of the adhesin. Two residues within p1025 that contribute to binding (Q1025, E1037) were identified by site-directed mutagenesis. In an in vivo human streptococcal adhesion model, direct application of p1025 to the teeth prevented recolonization of S. mutans but not Actinomyces, as compared with a control peptide or saline. This novel antimicrobial strategy, applying competitive peptide inhibitors of adhesion, may be used against other microorganisms in which adhesins mediate colonization of mucosal surfaces.

Delayed production of biologically active O-glycosylated forms of human eotaxin by tumor-necrosis-factor-alpha-stimulated dermal fibroblasts.

Since a number of inflammatory skin diseases are characterized by selective eosinophil infiltration preferentially in the dermis, we speculated that dermal fibroblasts might represent a potential cellular source of eosinophil-selective attractants. Cultivated dermal fibroblasts treated with tumor necrosis factor alpha secreted, not before day 3 of stimulation, eosinophil-specific chemotactic activity. Purification of this activity revealed a heparin-binding protein with an apparent molecular mass of 13 kDa in SDS/polyacrylamide gel electrophoresis. Peptide mapping with subsequent amino acid sequence analyses revealed it to be human eotaxin. Natural eotaxin preparations contain 50% N-terminally truncated forms missing two or three amino acids. It is O-glycosylated at Thr71, resulting in at least two sialylated O-glycosylated variants. Electrospray ionization mass spectrometric analyses revealed the natural eotaxin preparation to be heterogeneous with principal masses of 9033 Da and 9317 Da. Natural eotaxin stimulated eosinophil chemotaxis with identical potency and efficacy as recombinant human eotaxin. Neither neutrophils, monocytes or lymphocytes responded towards natural eotaxin preparations indicating that N-terminal truncation and O-glycosylation did not affect the cell-specificity of chemotactic activity. Treatment of eosinophils with natural eotaxin desensitizes chemotactic responses towards eotaxin, regulated an normal T-lymphocyte expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), whereas RANTES and MCP-3 were unable to desensitize natural eotaxin-dependent responses.

IL-4 induces eotaxin: a possible mechanism of selective eosinophil recruitment in helminth infection and atopy.

A common feature of some parasitic infections and allergic and atopic skin diseases is the involvement of Th2 lymphocytes and the dermal appearance of eosinophils (Eos). Because Th2 lymphocytes apparently do not release Eo attractants, we addressed the question of whether the Th2 cytokine IL-4 induces its production in dermal fibroblasts. We therefore stimulated fibroblasts with IL-4. HPLC investigation of supernatants revealed a single Eo chemotactic protein, which was purified to homogeneity giving a single 13-kDa band upon SDS-PAGE analyses. Peptide mapping with subsequent amino acid sequencing revealed an Eo-selective chemotaxin, which consists of a mixture of N-terminally truncated and O-glycosylated forms of the chemokine eotaxin. Other chemokines such as RANTES, MCP-3, MCP-4, or MIP-1alpha were not detected as Eo chemotaxins under these conditions. Using reverse transcriptase-PCR techniques, we found that IL-4 dose and time dependently induces eotaxin mRNA in dermal fibroblasts. Stimulation with IL-4 and TNF-alpha caused a 10- to 20-fold increase of the release of three biochemically different eotaxin forms, each consisting of a mixture of N-terminally truncated and O-glycosylated variants having the same backbone amino acid sequence but different specific activities. Our findings support the hypothesis that eosinophil recruitment seen in IL-4-mediated skin reactions, at least in part, may be due to Th2 cytokine-mediated induction of eotaxin in dermal fibroblasts.

Rapid simultaneous analysis of prostaglandin E2, 12-hydroxyeicosatetraenoic acid and arachidonic acid using high performance liquid chromatography/electrospray ionization mass spectrometry.

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.

Capillary gas chromatography with chemical ionization negative ion mass spectrometry in the identification of odorous steroids formed in metabolic studies of the sulphates of androsterone, DHA and 5alpha-androst-16-en-3beta-ol with human axillary bacterial isolates.

The products of metabolism of the sulphates (0.5 micromol/l) of androsterone, dehydroepiandrosterone (DHA) and 5alpha-androst-16-en-3beta-ol have been investigated after incubation with 72 h cultures of human axillary bacterial isolates for 3 days at 37 degrees C. The medium used, tryptone soya broth (TSB), contained yeast extract and Tween 80. The isolates used were Coryneform F1 (known previously to metabolize testosterone and to be involved in under-arm odour (UAO) production, i.e. UAO +ve), Coryneform F46 (inactive in both the testosterone metabolism and UAO tests, i.e. UAO -ve) and Staphylococcus hominis/epidermidis (IIR3). Control incubations of TSB alone, TSB plus each of the steroid sulphates and TSB plus each of the bacterial isolates were also set up. After termination of reactions and addition of internal standards, 5alpha-androstan-3beta-ol and 5alpha-androstan-3-one (50 ng each), extracted and purified metabolites were subjected to combined gas chromatography-mass spectrometry with specific ion monitoring. Steroidal ketones were derivatized as their O-pentafluorobenzyl oximes; steroidal alcohols (only androst-16-enols in this study) were derivatized as their tert-butyldimethylsilyl ethers. Analysis was achieved by negative ion chemical ionization mass spectrometry for the pentafluorobenzyl oximes at [M-20]- and electron impact positive ion mass spectrometry for the tert-butyldimethylsilyl ethers at [M-57]+. The incubation broth contained two compounds which had gas chromatographic and mass spectrometric properties identical to those of DHA and 4-androstenedione. It was not possible, therefore, to show unequivocally that DHA sulphate (DHAS) was converted microbially into DHA, although this is implied by the finding of small quantities of testosterone and 5alpha-dihydrotestosterone in incubations with F1. With androsterone S, no free androsterone was recorded and only very small (5 pg or less) amounts of testosterone. Two odorous steroids, androsta-4,16-dien-3-one and 5alpha-androst-2-en-17-one (Steroid I) were formed (mean quantities 40 and 45 pg, respectively). The sulphate of 5alpha-androst-16-en-3beta-ol was metabolized with F1 into large quantities of the odorous steroids, 5alpha-androst-16-en-3-one and Steroid I. In addition, much smaller quantities of androsta-4,16-dien-3-one were formed. In contrast, incubations of DHAS with F46 resulted in no metabolites except, possibly, DHA, but the sulphate moiety of androsterone S was also cleaved to yield the free steroid together with large amounts of Steroid I. In incubations of DHAS and androsterone S with F1, no 16-unsaturated steroids were formed, although 5alpha-androst-16-en-3beta-yl S was de-sulphated and the free steroid further metabolized. No evidence was obtained for androst-16-ene metabolism in incubations with F46. In incubations with S. hominis/epidermidis (IIR3), androsterone S was converted into androsterone and, in high yield, to Steroid I plus some 5alpha-androst-16-en-3-one. Both DHAS and androsterone S were converted into androst-16-enols. Sulphatase activity was also manifested when 5alpha-androst-16-en-3beta-yl S was utilized as substrate with IIR3, large quantities of Steroid I and 5alpha-androst-16-en-3-one being formed, together with further metabolism of androst-16-enes. In view of the fact that both DHAS and androsterone S occur in apocrine sweat, the metabolism of these endogenous substrates by human axillary bacteria to several odorous steroids may have important implications in the context of human odour formation.

Quantitative measurement of 3-O-methyl-D-glucose by gas chromatography-mass spectrometry as a measure of glucose transport in vivo.

Existing methods of measuring glucose kinetics are subject to errors. There is considerable interest in improved methods of measuring glucose kinetics to allow the development of new regimes for the treatment of diabetes mellitus. 3-O-Methyl-D-glucose is transported but not metabolized and therefore allows independent estimation of transport parameters. We describe a method by which 3-O-methyl-D-glucose in plasma samples can be measured in protocols during which glucose flux is assessed with simultaneous use of two isotopically labeled glucoses to quantitate and validate measurements of the rate of glucose appearance and disappearance. Quantitative gas chromatographic/mass spectrometric (GC/MS) analysis of 3-O-methyl-D-glucose, D-glucose, D-[U-13C] glucose and D-[6,6-2H2] glucose in human plasma using methoxime-trimethylsilyl ether derivatives is described. Measurements of all four derivatives were performed together in a small sample volume (50 microliters) with high precision. The intra-assay (inter-assay) coefficients of variation at an isotope content of 0.25 atom% excess for D-[6,6-2H2] glucose, D-[U-13C] glucose and 3-O-methyl-D-glucose were 0.8% (1.0%), 0.5% (4.0%) and 0.1% (3.7%), respectively. This method provides the basis for quantitative estimation of parameters of glucose kinetics in man and the rates of glucose flux across the cell membrane.

Identification of an N-terminally truncated form of the chemokine RANTES and granulocyte-macrophage colony-stimulating factor as major eosinophil attractants released by cytokine-stimulated dermal fibroblasts.

Eosinophil (Eo) granule proteins and, rarely, intact Eos represent a characteristic histopathologic feature of the dermal part of affected tissue in atopic dermatitis and some allergic reactions. Dermal fibroblasts are a rich source of cytokines and inflammatory mediators; therefore, we have investigated whether these cells release Eo chemoattractants when stimulated with different stimuli. Eo-chemotactic activity was detected after stimulation of cells with TNF-alpha and IL-1, but not when phorbol ester, PHA, or medium alone was used. Biochemical characterization of Eo-chemotactic activity in supernatants of NF-alpha-stimulated cells revealed both heparin-binding and nonbinding activity. HPLC purification with subsequent N-terminal sequencing and mass spectrometric analysis showed that the heparin-binding Eo-chemotactic peak corresponded to the chemokine [Tyr-RANTES]66 that also contained [Ser-RANTES]68 as contaminant, whereas the nonheparin-binding activity was identified as granulocyte-macrophage CSF (GM-CSF) by the use of neutralizing Abs. [Tyr-RANTES]66 was found to show identical behavior in the chemotaxis assay system with respect to potency and efficacy as natural [Ser-RANTES]68. These findings support the hypothesis that dermal fibroblasts can play an important role in the recruitment of Eo by release of the chemokine RANTES together with GM-CSF.

Measurement of partition coefficients in waterless biphasic liquid systems by countercurrent chromatography.

Countercurrent chromatography (CCC) is a chromatographic separation technique that uses a liquid as a stationary phase. Centrifugal forces are used to immobilize the liquid stationary phase when the liquid mobile phase is pushed through it. In CCC, the solutes are separated according to their liquid-liquid partition coefficients. The solutes studied were the alkylbenzene homologues from benzene to hexylbenzene and some polyaromatic hydrocarbons (PAHs) from naphthalene to coronene. Their liquid-liquid partition coefficients were measured in the five waterless biphasic systems formed by heptane, as the apolar liquid phase of the five biphasic systems, and four dipolar aprotic solvents, dimethyl sulfoxide, dimethylformamide, furfural, and N-methylpyrrolidone, and the polar proton-donor solvent methanol. The coefficients were compared to the corresponding capacity factors obtained by classical liquid chromatography on octadecyl-bonded silica. For the five biphasic solvent systems studied, linear relationships were found between the partition coefficients and the sp(3) and sp(2) hybridized carbon atom number for the alkylbenzene and PAH series, respectively. The sp(2) and sp(3) transfer energies were estimated, and their ratio was used to quantify the solvent selectivity toward aromatic extraction.