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.     

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.     

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.     

Isolation of leukotriene C4 from human seminal fluid

LTC4 was isolated and characterized from seminal fluid of seven human volunteers. A compound with a similar retention time to that of synthetic LTC4 was obtained using reverse-phase high performance liquid chromatography. The ultraviolet absorbance of the extracted substance was identical to synthetic LTC4. Furthermore this compound contracted the guinea pig ileum and lung parenchymal strip. Its effects were antagonized by the leukotriene antagonist FPL55712. It was concluded that LTC4 is present in human seminal fluid in very small amounts (about 100 ng/ejaculate). The possible physiological functions of LTC4 in the reproductive tract are discussed.     

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. 18Hydroxy-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 microsome of seminal vesicles of the cynomolgus monkey, Macaca fascicularis, supplemented with 1 nM 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.     

On the biosynthesis of 19,20-dehydroprostaglandin E2

19,20-Dehydro-PGE1 and 19,20-dehydro-PGE2 were recently identified in human seminal fluid. These prostaglandins might be formed by dehydration of 19(R)-hydroxy-PGE1 and 19(R)-hydroxy-PGE2 or, conceivably, by biosynthesis from precursor fatty acids with a terminal double bond. To examine the latter possibility, 5(Z), 8(Z), 11(Z), 14(Z), 19-eicosapentaenoic acid was prepared by chemical synthesis and incubated with microsomes of ram vesicular glands and glutathione (1 mM). The fatty acid was converted to 19,20-dehydro-PGE2, which was identified by GC-MS, by UV analysis after alkali treatment and by oxidative ozonolysis. The semisynthetic 19,20-dehydro-PGE2 and the corresponding compound in human seminal fluid showed the same polarity on reversed phase HPLC and virtually identical mass spectra. The described method might be used to generate 19,20-dehydro-PGE2 for evaluation of its biological effects.     

Isolation of leukotriene C4 from human seminal fluid

LTC₄ was isolated and characterized from seminal fluid of seven human volunteers. A compound with a similar retention time of that of synthetic LTC₄ was obtained using reverse-phase high performance liquid chromatography. The ultraviolet absorbance of the extracted substance was identical to synthetic LTC₄. Furthermore this compound contracted the guinea pig ileum and lung parenchymal strip. Its effects were antagonized by the leukotriene antagonist FPL55712. It was concluded that LTC₄ is present in human seminal fluid in very small amounts (about 100 ng/ejaculate). The possible physiological functions of LTC₄ in the reproductive tract area discussed.     

Quantification of prostaglandins in human seminal fluid

A method is described for measurement of the prostaglandins present in human seminal plasma. The PGEs and the 19-hydroxy-PGEs are converted to the respective PGB-compounds. They are determined with UV-absorbance after purification with two chromatography steps. The PGFs and the 19-hydroxy-PGFs are determines by gas chromatography, which also allows measurement of the 8β-isomers. Recovery of added PG was in the average 99.2% (range 89.9–107.7). Mean variation between duplicate analyses was 5.6% (range 0.9–9.1). The method has been simplified to allow at least limited clinical use.     

Creatine kinase and ATPase in human seminal fluid and prostatic fluid

A creatine kinase assay based on estimation of creatine liberated from creatine phosphate was accurate and reproducible for use with seminal or prostatic fluid, after allowance was made for acid phosphatase interference. Comparison of this method with one which relies on enzymic coupling of ATP formation to NADP+ oxidation shows that the latter under-estimates creatine kinase activity by a factor of about 3. This discrepancy could be due to the high ATPase activity found in prostatic and seminal fluid. Uncritical use of the NADP+ assay might account for different seminal creatine kinase values reported in the literature. Interrelationships between ATPase, creatine kinase and zinc suggest that seminal ATPase is a prostatic secretory product while creatine kinase may be multiglandular in origin.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.     

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma

A method is described which measures the four main prostaglandins of human semen (PGE₁, E₂, 19-hydroxy PGE₁, and 19-hydroxy PGE₂). For routine measurements E₁ and E₂ are measured together as are 19-OH E₁ and 19-OH E₂. These are measured by forming oximes in aqueous solution. extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 μg/ml of 19-hydroxy Es and 30–200 μg/ml of Es.     

The measurement of E and 19-hydroxy E prostaglandins in human seminal plasma

A method is described which measures the four main prostaglandins of human semen (PGE₁, E₂, 19-hydroxy PGE₁, and 19-hydroxy PGE₂). For routine measurements E₁ and E₂ are measured together as are 19-OH E₁ and 19-OH E₂. These are measured by forming oximes in aqueous solution. extraction, methylation and trimethyl silylation followed by gas chromatography. The method has sufficient sensitivity to measure the levels found in the majority of semen samples. The normal range in men with proven fertility was 90 to 260 μg/ml of 19-hydroxy Es and 30–200 μg/ml of Es.