Gas-liquid Chromatography-mass Spectrometry Analysis Research Articles

Antimicrobial Activities of European Propolis Collected from Various Geographic Origins Alone and in Combination with Antibiotics.

Background: Propolis consists of a complex mixture of resinous substances collected by honeybees from different plant sources. The objective of this study was to investigate the chemical composition, biological activities, and synergistic properties with antibiotics of propolis samples collected from various geographic origins (Germany, Ireland, and Czech Republic). Methods: The chemical composition of the propolis was analyzed by Gas Liquid Chromatography-Mass Spectrometry (GLC-MS) and High-performance liquid chromatography (HPLC). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were evaluated by the standard broth microdilution method, while synergistic interactions were assessed by checkerboard dilution and time-kill curve assays. Results: HPLC and GLC-MS analyses revealed that ethanol extract of propolis (EEP) and water extracts of propolis (WEP) contained more than 100 different phytochemicals. The most abundant compounds were aromatic alcohols, aromatic acids, cinnamic acid and its esters, fatty acids, and flavanone (chrysin). Czech propolis showed the highest phenolic content (129.83 ± 5.9 mg CAE/g) followed by Irish propolis and German propolis. Furthermore, Irish propolis exhibited the highest value of total flavonoid content (2.86 ± 0.2 mg QE/g) and antioxidant activity (IC50 = 26.45 µg/mL). All propolis samples showed moderate antibacterial effect against Gram-positive microorganisms with MIC ranging from 0.08 mg/mL to 2.5 mg/mL. Moreover, EEP exhibited moderate activity against Gram-negative bacteria with MIC between 0.6 mg/mL to 5 mg/mL. In addition, EEP displayed moderate antifungal activity (MIC values between 0.6–2.5 mg/mL). The results obtained from time kill-kinetic assay and checkerboard dilution test of two-drug combinations between EEP and antibiotics such as vancomycin, oxacillin, and levofloxacin indicate mainly synergistic interactions against drug-resistant microbial pathogens including MRSA and VRE. Conclusions: The propolis extract synergistically enhanced the efficacy of antibiotics, especially those acting on cell wall synthesis (vancomycin and oxacillin) against drug-resistant microorganisms.

Phytanic acid: measurement of plasma concentrations by gas–liquid chromatography–mass spectrometry analysis and associations with diet and other plasma fatty acids

Epidemiological data suggest that a diet rich in animal foods may be associated with an increased risk of several cancers, including cancers of the prostate, colorectum and breast, but the possible mechanism is unclear. It is hypothesised that phytanic acid, a C20 branched-chain fatty acid found predominantly in foods from ruminant animals, may be involved in early cancer development because it has been shown to up regulate activity of alpha-methylacyl-coenzyme A racemase, an enzyme commonly found to be over-expressed in tumour cells compared with normal tissue. However, little is known about the distribution of plasma phytanic acid concentrations or its dietary determinants in the general population. The primary aim of the present cross-sectional study was to determine circulating phytanic acid concentrations among ninety-six meat-eating, lacto-ovo-vegetarian and vegan women, aged 20-69 years, recruited into the Oxford component of the European Prospective Investigation into Cancer and Nutrition (EPIC-Oxford). Meat-eaters had, on average, a 6.7-fold higher geometric mean plasma phytanic acid concentration than the vegans (5.77 v. 0.86 micromol/l; P < 0.0001) and a 47 % higher mean concentration than the vegetarians (5.77 v. 3.93 micromol/l; P = 0.016). The strongest determinant of plasma phytanic acid concentration appeared to be dairy fat intake (r 0.68; P < 0.0001); phytanic acid levels were not associated with age or other lifestyle factors. These data show that a diet high in fat from dairy products is associated with increased plasma phytanic acid concentration, which may play a role in cancer development.

Whole body distribution of deuterated linoleic and α-linolenic acids and their metabolites in the rat

Little is known about the uptake or metabolism of essential fatty acids (EFAs) in various mammalian organs. Thus, the distribution of deuterated alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) and their metabolites was studied using a stable isotope tracer technique. Rats were orally administered a single dose of a mixture (20 mg each) of ethyl D5-18:3n-3 and D5-18:2n-6, and 25 tissues per animal were analyzed for D5-labeled PUFAs at 4, 8, 24, 96, 168, 240, 360, and 600 h after dosing. Plasma, stomach, and spleen contained the highest concentrations of labeled precursors at the earliest time points, whereas other internal organs and red blood cells reached their maximal concentrations at 8 h. The time-course data were consistent with liver metabolism of EFAs, but local metabolism in other tissues could not be ruled out. Brain, spinal cord, heart, testis, and eye accumulated docosahexaenoic acid with time, whereas skin accumulated mainly 20:4n-6. On average, approximately 16-18% of the D5-18:3n-3 and D5-18:2n-6 initial dosage was eventually accumulated in tissues, principally in adipose, skin, and muscle. Approximately 6.0% of D5-18:3n-3 and 2.6% of D5-18:2n-6 were elongated/desaturated and stored, mainly in muscle, adipose, and the carcass. The remaining 78% of both precursors was apparently catabolized or excreted.

Streptococcus iniae Capsule Impairs Phagocytic Clearance and Contributes to Virulence in Fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

Characterisation of the substituent distribution in hydroxypropylated potato amylopectin starch

The distribution of substituents in hydroxypropylated potato amylopectin starch (amylose deficient) modified in a slurry of granular starch (HPPAPg) or in a polymer ‘solution’ of dissolved starch (HPPAPs), was investigated. The molar substitution (MS) was determined by three different methods: proton nuclear magnetic resonance ( 1H NMR) spectroscopy, gas-liquid chromatography (GLC) with mass spectrometry, and a colourimetric method. The MS values obtained by 1H NMR spectroscopy were higher than those obtained by GLC–mass spectrometry analysis and colourimetry. The relative ratio of 2-, 3-, and 6-substitution, as well as un-, mono-, and disubstitution in the anhydroglucose unit (AGU) were determined by GLC–mass spectrometry analysis. Results obtained showed no significant difference in molar distribution of hydroxypropyl groups in the AGU between the two derivatives. For analysis of the distribution pattern along the polymer chain, the starch derivatives were hydrolysed by enzymes with different selectivities. Debranching of the polymers indicated that more substituents were located in close vicinity to branching points in HPPAPg than in HPPAPs. Simultaneous α-amylase and amyloglucosidase hydrolysis of HPPAPg liberated more unsubstituted glucose units than the hydrolysis of HPPAPs, indicating a more heterogeneous distribution of substituents in HPPAPg.

Occurrence of N-acetyl- and N-O-diacetyl-neuraminic acid derivatives in wild and mutant Crithidia fasciculata.

The cell-surface expression of sialic acids in wild-type Crithidia fasciculata and three drug-resistant mutants (FU(R)11, TR3, and TFRR1) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. Both N-acetyl- and N-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TFRR1 mutant strain confirmed the occurrence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).

Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus

The galactomannan (GM) produced extracellularly by Aspergillus fumigatus has been purified by a double sequential hydrazine-nitrous acid treatment of the ethanol precipitate of the culture filtrate. Nuclear magnetic resonance and gas-liquid chromatography-mass spectrometry analysis have been performed on intact GM, acid-hydrolyzed GM, and oligomers resulting from the acetolysis of the acid-hydrolyzed GM. Results show that A. fumigatus GM is composed of a linear mannan core with an alpha-(1-2)-linked mannotetraose repeating unit attached via alpha-(1-6) linkage. Side chains composed of an average of 4 to 5 beta-(1-5)-galactofuranose units are linked to C-6 and C-3 positions of alpha-(1-2)-linked mannose units of the mannan. The immunoreactivity of GM and HCl-hydrolyzed GM was studied by use of human sera from aspergillosis patients and an antigalactofuran monoclonal antibody. The alpha-(1-2) (1-6)-mannan core is not antigenic. The immunogenic galactofuran is found amongst several exocellular glycoproteins. According to a direct enzyme-linked immunosorbent assay with GM as the detector antigen, only 26% of the serum samples from aspergilloma patients (all positive by immunodiffusion assays) give optical density values superior to a cutoff estimated as the mean +/- 3 standard deviations of values obtained with control sera.

Methoxylated fatty acids reported in Rhizobium isolates arise from chemical alterations of common fatty acids upon acid-catalyzed transesterification procedures

We obtained from a phospholipid extract of wild-type Rhizobium leguminosarum bv. trifolii ANU843 methoxylated fatty acids that had been previously reported as constitutive unusual Rhizobium fatty acids. The use of deuterated reagents and subsequent gas-liquid chromatography-mass spectrometry analyses showed that these methoxylated fatty acid derivatives are the products of chemical alterations of common cyclopropane-containing and unsaturated fatty acids occurring during various acid-catalyzed transesterification treatments aimed at producing the methyl ester derivatives. Similar results were obtained from a phospholipid extract of Escherichia coli K-12. In contrast, these chemical alterations were not induced by an alkaline methanolysis method of transesterification. If an acidic treatment is needed to release the fatty acids from the source molecule, the finding of unusual methoxylated fatty acids should be carefully confirmed with deuterated reagents.

Chemical Characterization of Stress-Induced Vascular Coating in Tomato

Indirect evidence suggests that vascular coatings formed by plants in response to stress consist of suberin-like substances containing lipid and phenolic compounds. To provide more direct chemical evidence that coatings are suberin, we used a natural pathogen, Verticillium albo-atrum, or a stress-responsive hormone, abscisic acid, to induce coating in two isolines of tomato (Lycopersicon esculentum L. cultivar Craigella) that are resistant or susceptible to the pathogen. Using treated petioles that had been monitored cytologically, chemical depolymerization followed by combined gas-liquid chromatography-mass spectrometry analysis of alkane-alpha,omega-diol levels confirmed the presence of suberin after induction of coating and showed quantitative differences between the isolines that correlated with cytological measurements of the coating response. Northern analysis of suberization-associated anionic peroxidase mRNA showed corresponding increases, and tissue blot analysis further indicated that induction of the mRNA was localized in the responding vascular bundles, as determined by suberin histochemistry. Taken together, these results provide chemical evidence that the coatings are mainly suberin.

Purification, characterization and serological properties of a glycolipid antigen reactive with a serovar-specific monoclonal antibody against Leptospira interrogans serovar canicola.

A glycolipid antigen possessing a serovar-specific antigenic determinant of Leptospira interrogans serovar canicola was purified from a chloroform/methanol extract of the organism. The purification procedures included silicic acid column chromatography and preparative thin-layer chromatography (TLC). Antigenic activity was detected by a TLC-enzyme immunostaining technique using monoclonal antibody CT3, which specifically agglutinates serovar canicola and only weakly serovar sumneri but no other serovars of Leptospira. The purified glycolipid reacted with CT3 antibody, indicating that the glycolipid possessed a serovar-specific antigenic determinant. Infrared spectrum and proton nuclear magnetic resonance analyses showed that the glycolipid contained sugar and lipid moieties, which possessed amide linkages and an acetyl group. Gas-liquid chromatography-mass spectrometry analysis showed that the glycolipid contained two unknown sugars, one of which (unknown sugar II) appeared to be associated with the antigenic determinant specific for canicola. The serovar-specific antigenic determinant was destroyed by mild alkali treatment of the glycolipid. These findings suggested that the antigenic determinant was an alkali-labile moiety which may be related to the unknown sugar II.

Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species.

An analysis of the phospholipid ester-linked and the lipopolysaccharide (LPS) fatty acids and hydroxy fatty acids of six lactate-utilizing Desulfovibrio-type sulfate-reducing bacteria (SRB) has been performed using capillary gas-liquid chromatography-mass spectrometry (GLC-MS). The concentrations of normal fatty acids were essentially similar, with the possible exception of a high content of normal fatty acids in the LPS of Desulfovibrio gigas. Determination of monounsaturated acid double bond configuration was performed by GLC-MS analysis of the derivatized fatty acids. A total of nine branched chain and eight straight chain monounsaturated fatty acids was detected in the Desulfovibrio species analyzed. The major component detected in five Desulfovibrio was the 17-carbon iso-branched monoenoic acid which showed cis unsaturation [i17:1(n-7)c] seven carbons from the terminal methyl group of the fatty acid chain. D. gigas, in contrast, contained almost no unsaturated fatty acids and was greatly enriched in iso-branched 15:0. Major differences between strains were found in the phospholipid and LPS hydroxy fatty acids. These components, in addition to the i17:1(n-7)c and other characteristic branched chain unsaturated acids, can possibly be utilized as signatures of the lactate-utilizing SRB.

Synthesis and stereochemical determination of diunsaturated valproic acid analogs including its major diunsaturated metabolite.

Valproic acid, an antiepileptic drug, is transformed into diunsaturated metabolites in humans. Synthesis of the geometric isomers of 2-(1'-propenyl)-2-pentenoic acid and 2-(1'-propenyl)-3-pentenoic acid was attempted using known procedures. The final product, a mixture of isomers, was converted into tert-butyldimethylsilyl or ethyl derivatives. Capillary gas-liquid chromatography-mass spectrometry analysis of the derivatives showed at least three isomeric dienoic acids from synthesized products. Argentation thin-layer chromatography was effective in resolving the isomeric mixture into a single isomer or mixture of two isomers. Thin-layer chromatography and gas-liquid chromatography retention data, photochemical isomerization studies, and nuclear magnetic resonance spectrometry were used to characterize the dienoic acids. By comparison of the retention times of the diunsaturated metabolites with synthesized reference compounds, the structure assigned to the major diunsaturated metabolite is 2-[(E)-1'-propenyl](E)-2-pentenoic acid.

Overproduction of N-acetylneuraminic acid (sialic acid) by sialuria fibroblasts.

Fibroblasts from the original sialuria patient are shown to contain high levels of free sialic acid, i.e., an average of 87 versus a normal average of 2 nmol/mg of protein. Gas liquid chromatography-mass spectrometry analysis confirmed that the accumulated material is N-acetylneuraminic acid. Addition of D(+) glucosamine (0-5 mM) to the media of these cells increased the intracellular free sialic acid concentrations from 74 to 137 nmol/mg protein. In contrast, normal cells treated in an identical manner maintained their normal free sialic acid level of less than 4 nmol/mg protein. Addition of 20 mM N-acetylmannosamine in place of glucosamine resulted in a marked increase in free sialic acid in both the patient and the control, i.e., increases of 157 (from 95 to 252) and 120 (from 3 to 123) nmol/mg protein, respectively. Finally, while normal cells in the presence of glucosamine accumulated high levels of uridine diphosphate N-acetylhexosamine, the patient's cells accumulated much lower amounts of this compound. It is concluded that the elevated sialic acid level in sialuria is due, at least in part, to overproduction of free N-acetylneuraminic acid. Indirect evidence is presented that this may result from either hyperactivity or increased levels of the enzyme (uridine diphosphate N-acetylglucosamine 2-epimerase) that has been shown in other tissues to convert uridine diphosphate N-acetylglucosamine to N-acetylmannosamine.

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium.

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.

Chemical studies on proteinaceous attractants for fruit flies, including the identification of volatile constituents

The chemical characteristics of protein attractants for the Queensland fruit fly, Dacus tryoni, have been investigated. Highly effective commercial fruit fly attractants were found to contain a large proportion of high molecular weight material. Lists are provided of the amino acids present in free and bound form in these hydrolysates. The products found by gas liquid chromatography-mass spectrometry analysis of methylene chloride extracts and head-space volatiles were mainly the primary and secondary products of amino acid degradation, including a number of aldehydes and significant amounts of ethylene. None of these aldehydes nor any of a number of others that may have been present in smaller quantities was found to be active when tested individually for attractancy. Nor did ethylene show any evidence of attractancy under the conditions of these tests. Strecker reagents (such as isatin), which stimulate the formation of amino acid degradation products, caused a temporary and relatively small elevation in attractancy of the commercial hydrolysates. Three enzymatic hydrolysates of a relatively pure protein (bovine serum albumin) were prepared and studied; one, a tryptic digest, was exceptionally attractive to the fruit fly. Analysis of methylene chloride extracts and head-space of these 'model' preparations revealed a number of significant differences between them, but the most important difference in relation to attractancy was found to be the presence of ammonium salts in the trypsin hydrolysate. Subsequent experiments showed that high attractancies could be attained by combinations of amino acid mixtures and ammonium bicarbonate, even though neither had high attractancy (under the particular conditions of these experiments) when tested independently. This apparent synergism was probably due to the attractancy of the ammonia coupled with a feeding stimulus provided by the amino acids.

The use of [ 18O 4]phosphoric acid in the quantitation of phosphate by gas-liquid chromatography-mass spectrometry analysis

A procedure is described to quantitate inorganic phosphate in the form of the tris(trimethylsilyl) (TMS) phosphate by gas-liquid chromatography-mass spectrometry (glems) that increases the previously reported detection limit from the microgram to the nanogram range (i.e., 3 ng). The sensitivity for detecting TMS-phosphate by glc-ms analysis was shown to be limited by an increasing fractional loss with decreasing concentrations of TMS-phosphate analyzed due to its adsorption on different types of glc column supports. The method developed employs [ 18O 4]phosphoric acid which serves as both an internal standard to permit quantitation and as a carrier to minimize sample adsorption on the glc column support.

Identification of 15-keto-9, 11-peroxidoprosta-5, 13- dienoic acid as a hematin-catalyzed decomposition product of 15-hydroperoxy-9, 11-peroxidoprosta-5, 13-dienoic acid.

A labile prostaglandin was isolated as one of the products generated from [1-14C] eicosatetraenoic acid incubated with sheep vesicular gland microsomes. The eicosatetraenoic acid metabolite amounted to ca. 16% of the total radiolabeled products. Formation of this new prostaglandin was prevented when heat-denatured microsomes were employed or when incubation mixtures were supplemented with indomethacin or phenol. However, incubation of prostaglandin G2 (PGG2) with hematin in the presence or absence of catalytically active or heat-inactivated microsomes led to production of approximately the same quantity of the new prostaglandin. These results indicated that the new prostaglandin can be formed nonenzymically. The new prostaglandin was conclusively identified by gas liquid chromatography-mass spectrometry analysis as 15-keto-9,11-peroxidoprosta-5,13-dienoic acid (15-keto-PGG2) after chemical conversion to known prostaglandins. The effects of 15-keto-PGG2 and PGG2 were similar on canine lateral saphenous vein; both promoted contraction followed by prolonged relaxation, but 15-keto-PGG2 appeared to be 1/50 as potent as PGG2.

Cytology and quantitative cytochemistry of a poliferative atypical hemocytic condition in Mytilus edulis (Bivalvia, mollusca).

Proliferative lesions were found in 16 of 994 Mytilus edulis mussels obtained from the mouth of the River Lynher at Plymouth, England. These lesions were characterized by infiltration and replacement of the connective tissue by enlarged, atypical, mitotically active, basophilic, hemocyte-like cells. Cytologic examination indicated the involvement of two cell types in this disorder, both of which were similar in appearance to the much smaller, normal basophallic agranular hemocyte. The abnormal cells were rich in cytoplasmic RNA and had significantly higher DNA levels than normal hemocytes as determined by scanning microdensitometry. A few of the atypical cells contained small cytoplasmic granules that were positive for lysosomal hydrolases. This condition was observed sequentially from what is believed to be the earliest stage of the disease, in which only a few atypical cells were present, to the terminal stage, in which the connective tissue was almost completely replaced and the digestive gland cells were necrotized. Some potentially carcinogenic aromatic hydrocarbons have been identified in the substrate of the mussel bed by gas-liquid chromatography-mass spectrometry analysis.

Isolation, identification and quantitation of serum 5α-pregnane-3,20-dione and its relationship to progesterone in the pregnant mare

5α-pregnane-3,20-dione was isolated from pooled pregnant mare serum using Sephadex LH-20 column chromatography and identified by the use of radioimmunoassay, gas-liquid chromatography and gas-liquid chromatography-mass spectrometry analyses. 5β-pregnane-3,20-dione was not cross-reactive with the radioimmunoassay system and was not detected by gas-liquid chromatography. Peripheral blood levels of progesterone and 5α-pregnane-3,20-dione were determined by radioimmunoassay in four Quarter Horse mares for the first 150 days of gestation. Progesterone and 5α-pregnane-3,20-dione declined from a range of 6 to 15 ng/ml at day 10 to a range of 2 to 8 ng/ml at day 40. After day 40 there was an increase in progesterone and 5α-pregnane-3,20-dione concentration. Toward the end of 150 days of gestation progesterone tended to decline whereas, 5α-pregnane-3,20-dione levels were maintained or increased to levels as high as 35 ng/ml. Neither source nor function of 5α-pregnane-3,20-dione is known.