Analysis of fatty acid samples by hydrophilic interaction liquid chromatography and charged aerosol detector.

HILIC/CAD techniques were used in analysis of samples containing fatty acids. Amine base column appeared to be the more retentive stationary phase compared to zwitterionic and BEH silica. The retention decreased with pH mobile phases changing from 3 to 5. Acetonitrile and acetone organic modifier were compared. Acetone gave higher eluotropic strength and better peak symmetry whereas acetonitrile led to higher efficiency. The retention decreased when ammonium acetate concentration increased from 5 to 20mM. The use of sub-2µm column did not show flat Van Deemter curves at high flow rates. A rapid separation of PGI2 and its main degradation product, 6-keto prostaglandin F1α was obtained in 1.6min with a Hypersil GOLD, 50mm×2.1mm, 1.9µm with; acetonitrile/acetate ammonium pH 5 at 20mM (85/15; v/v at 0.7ml/min).

The occurrence of 15-keto-prostaglandins in the red alga Gracilaria verrucosa.

New 15-keto-prostaglandins (1-4) were isolated from the MeOH extract of the red alga, Gracilaria verrucosa. Their structures were determined to be prostaglandin B congeners (1-3) and a prostaglandin E congener (4) based on the NMR and MS data. Prostaglandins with a C-15 keto function are rare from natural sources. The presence of these metabolites in the alga is notable because 15-keto-prostaglandins (15-keto-PGs) are considered to be the metabolic products of regular prostaglandins in mammals. The occurrence of different prostaglandins in this alga might be due to the existence of different oxidative enzymes, as previously mentioned for oxygenated fatty acids of the red alga Gracilariopsis lemaneiformis. The antiinflammatory activity of these prostaglandins was examined by evaluating their inhibitory effects on nitric oxide production in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. These prostaglandins showed weak activity on nitric oxide production.

Evidence for the formation of dinor isoprostanes E1 from alpha-linolenic acid in plants.

The free radical oxidation of arachidonic acid is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins and exert potent receptor-mediated biological activities. In the present study, we show that alpha-linolenic acid can undergo a similar oxidation process, resulting in a series of isomeric dinor isoprostanes E1. E-ring dinor isoprostane formation from linolenate was found to be catalyzed by soybean lipoxygenase. The main enzymatic products were 13- and 9-hydroperoxylinolenate but in addition, two dinor isoprostane E1 regioisomers were formed with a yield of 0.31%. Identification and quantification of two dinor isoprostane E1 regioisomers in plant cell cultures was achieved by a negative chemical ionization gas chromatography-mass spectrometry method using [18O]dinor isoprostanes E1 as internal standards. Endogenous levels of these compounds were determined in four taxonomically distant plant species and found to be in the range of 4.5 to 60.9 ng/g of dry weight. Thus analogous pathways in animals and plants exist, each leading to a family of prostaglandin-like compounds derived from polyunsaturated fatty acids. It remains to be shown whether the dinor isoprostanes exert biological activities in plants as has been demonstrated for their C20 congeners in mammals.

Regulation of agonist-induced prostaglandin E1 versus prostaglandin E2 production. A mass analysis.

Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), derived by enzymatic oxidation of cellular dihomogammalinolenic acid (DHLA) and arachidonic acid (AA), respectively, have diverse and, at times, distinct biological actions. It has been suggested that PGE1 specifically inhibits a variety of inflammatory processes, and, in light of the potential therapeutic benefit of PGE1 and its fatty acid precursor in inflammatory disorders, there is growing interest in the biochemical mechanisms which determine the balance between PGE1 and PGE2 synthesis. Metabolic studies in this area have been hampered by the difficulties in measuring the extremely small masses of these prostaglandins which are generated in cell culture systems. We studied the regulation of PGE1 versus PGE2 synthesis using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Because EFD-1 cells contain no endogenous AA or DHLA, we were able to replete the cells with AA and DHLA of known specific activities; thus, the mass of both cellular AA and DHLA, and synthesized PGE1 and PGE2, could be accurately determined. The major finding of this study is that production of PGE2 was highly favored over production of PGE1 due to preferential incorporation of AA versus DHLA into, and release from, the total cellular phospholipid pool. Further, we correlated the selective release of AA versus DHLA from total cellular phospholipids with the selective incorporation of AA versus DHLA into specific phospholipid pools. In addition, we showed that conversion of DHLA to AA by delta 5 desaturase was enhanced by increasing the cellular mass of n-6 fatty acids and by increasing the cell proliferative activity. Together, these results indicate that the relative abundance of PGE2 versus PGE1 in vivo is not merely a function of the relative abundance of AA versus DHLA in tissues, but also relates to markedly different cellular metabolism of these two fatty acids.

Analytical and preparative resolution of enantiomers of prostaglandin precursors and prostaglandins by liquid chromatography on derivatized cellulose chiral stationary phases.

Analytical methods were developed for the separation of the enantiomers of four cyclopentenone precursors of prostaglandins. The resolution obtained is correlated with the chemical environment around the chiral center of the cyclopentenones. The analytical methods were scaled up to preparative loadings and the chromatographic parameters were varied to determine their effect on the preparative separations. The correlation between analytical resolution and preparative resolution was also investigated. In addition to the precursors, the preparative resolution of the enantiomers of a synthetic prostaglandin analogue was investigated.

Identification of 6-keto-prostaglandin E1 obtained from isolated perfused kidney of the rabbit.

We studied the release of 6-keto-prostaglandin (PG) E1-like material into urinary and venous effluents of the isolated perfused kidney of the rabbit. After high performance liquid chromatographic (HPLC) separation, material coeluting with authentic 6-keto-PGE1 was measured by radioimmunoassay, platelet antiaggregation bioassay and characterized further by gas chromatography/mass spectrometry-single ion monitoring (GC/MS-SIM). Injection of either arachidonic acid or ATP into the renal artery stimulated the release of prostacyclin [measured as immunoreactive (i)-6-keto-PGF1 alpha] and i-6-keto-PGF1. HPLC fractions containing i-6-keto-PGE1, and coeluting with 6-keto-PGE1 standard, exhibited potent inhibition of platelet aggregation. The presence of authentic 6-keto-PGE1 was verified by the GC/MS-SIM spectra in HPLC zones from which radioimmunoassayable and bioassayable 6-keto-PGE1-like material was recovered.

Isolation and biosynthesis of 18-hydroxyprostaglandins E1 and E2 in human seminal fluid.

18-Hydroxy-PGE1 and 18-hydroxy-PGE2 were identified in human seminal fluid by capillary gas-liquid chromatography-mass spectrometry. The levels of these prostaglandins was 1-2% of the corresponding 19-hydroxy-PGE compounds in human semen. 18-Hydroxy-PGE1 and 18-hydroxy-PGE2 are likely formed by cytochrome P-450 in seminal vesicles in analogy with the 19-hydroxy-PGE compounds. This was supported by the finding that microsomes of seminal vesicles of the cynomolgus monkey, Macaca fascicularis, supplemented with 1 mM NADPH, metabolized PGE1 to both 19-hydroxy-PGE1 (92%) and 18-hydroxy-PGE1 (8%). The hydroxylation of prostaglandins in seminal vesicles of primates may thus show a high but not absolute specificity for the penultimate carbon of prostaglandins.

[Complete synthesis and properties of prostaglandins. X. Hydroxyl-containing esters of 11-deoxyprostaglandin E1]. / Polnyi sintez i svoistva prostaglandinov. X. Gidroksilsoderzhashchieéfiry 11-dezoksiprostaglandina E1.

Hydroxyl-containing 11-deoxyprostaglandin E1 esters were synthesised by using the methods of mixed anhydrides and nucleophilic displacement.

Separation and quantification of prostaglandins E1 and E2 as their panacyl derivatives using reverse phase high pressure liquid chromatography.

Separation and quantification of prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were achieved using reverse phase high performance liquid chromatography (HPLC). Panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) (PAB) derivatives of PGE2 and PGE1 were prepared. Reverse phase HPLC using a linear gradient of 56% to 80% acetonitrile in water containing 0.10% acetic acid gave baseline resolution of the two derivatives. A 3 um diameter particle, C18 column provided good resolution and reproducible recoveries. Human synovial tissue cells were incubated with the precursor fatty acids for PGE1 or PGE2 and stimulated with a crude Interleukin 1 (IL-1) preparation. Cells grown in the presence of dihomogammalinolenic acid (DGLA), the precursor for PGE1, made significantly more PGE1 than cells grown in control medium or in the presence of arachidonic acid, precursor for PGE2. PGE2 synthesis was reduced when DGLA was added to cells (resting or IL-1-stimulated).

Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid.

Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.

Biosynthesis of a novel prostaglandin, delta 17-PGE1, in the ram.

The prostaglandin, delta 17-PGE1, was purified from extracts of ram seminal fluid by reversed phase high performance liquid chromatography (HPLC) and identified after conversion to delta 17-PGB1 by UV-analysis and by capillary column gas chromatography-mass spectrometry (GC-MS). It was also formed by incubation of a homogenate of ram vesicular glands. The amount of delta 17-PGE1 in the seminal fluid and in the homogenate averaged 12% and 25% of the amount of PGE2, respectively. The results show that cis-8,11,14,17-eicosatetraenoic acid can be metabolized to prostaglandins in vivo in the ram.

Isolation of two novel E prostaglandins in human seminal fluid.

cis-8,11,14,17-[1-14C]Eicosatetraenoic acid was incubated with microsomes of ram seminal vesicles and 1 mM glutathione for 3 min at 37 degrees C. The main metabolite was identified as 17,18-dehydroprostaglandin E1 by capillary column gas chromatography-mass spectrometry. Human seminal fluid was analyzed for the presence of 17,18-dehydroprostaglandin E1 and prostaglandin E3. Whereas prostaglandin E3 could be demonstrated by capillary gas chromatography-mass spectrometry, 17,18-dehydroprostaglandin E1 could not be found under these conditions. However, human seminal fluid contained two compounds with a similar polarity on reversed phase high performance liquid chromatography as 17,18-dehydroprostaglandin E1 and prostaglandin E3. The two compounds were identified as 18,19-dehydroprostaglandin E1 and 18,19-dehydroprostaglandin E2 by gas chromatography-mass spectrometry, by UV analysis after conversion to the corresponding prostaglandin B compounds, and by ozonolysis. The amount of each of the two prostaglandins in human seminal fluid seemed to be in the same order of magnitude as the amount of prostaglandin E3.