Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Hydroperoxide-dependent epoxidation of 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene by ram seminal vesicle microsomes and by hematin

Addition of arachidonic acid to ram seminal vesicle microsomes oxidizes 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene (BA-3,4-diol) to five more polar products. Four of the products are identified by chromatographic and spectroscopic analysis as tetrahydrotetraols, which are solvolysis products of dihydrodiolepoxides. The fifth product is a 10-methyl ether formed by methanolysis of the anti-diolepoxide. Quantitation of the individual products indicates that anti-diolepoxides predominate over syn-diolepoxides by approximately 2:1. Identical product profiles are detected from the reaction of BA-3,4-diol with hematin and 13-hydroperoxy-octadecadienoic acid in the presence of Tween 20. No other products are detected in either system, which indicates that peroxyl radicals oxidize BA-3,4-diol exclusively by epoxidation of the 1,2-double bond. The stereochemical and regiochemical differences between oxidation of BA-3,4-diol by peroxyl radicals and cytochrome P-450 are dramatic and suggest that BA-3,4-diol is uniquely suited as a probe to quantitate peroxyl radical-dependent epoxidation in vitro and in vivo.     

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.     

Calcium-dependent oxidation of 5,8,11-icosatrienoic acid by the cyclooxygenase enzyme system

Mead (5,8,11-icosatrienoic) acid was found to be metabolized by the cyclooxygenase enzyme system of ram seminal vesicle microsomes in a calcium-dependent manner. Although the enzyme converted Mead acid to products more slowly and less completely than the isomeric 8,11,14-icosatrienoic acid, both oxidations were inhibitable by indomethacin. Experiments using purified cyclooxygenase confirmed the participation of this enzyme system in the calcium-dependent oxidation. The products of the oxidation were separated by high performance liquid chromatography and analyzed by ultraviolet and gas chromatography-mass spectrometry. The spectra obtained were consistent with the products having the structures 13-hydroxy-5,8,11-icosatrienoate (the major product), 11-hydroxy-5,8,12-icosatrienoate, 9-hydroxy-5,7,11-icosatrienoate, and two isomeric 8,11-dihydroxy-5,9,12-icosatrienoates. No prostaglandin-like, cyclized products could be identified. This report is only the second to illustrate a calcium-dependent oxidation of a polyunsaturated fatty acid by a cyclooxygenase enzyme system and further extends the metabolic potential of Mead acid.     

Bilirubin diglucuronide formation by rat liver microsomes: demonstration by affinity and thin layer chromatography of bile pigment tetrapyrroles

The conjugates formed in vitro by bilirubin UDP-glucuronyl transferase were studied by examining reaction products as intact tetrapyrroles, rather than as dipyrrolic azoderivatives. Bile pigments were extracted from conventional microsomal enzyme reaction mixtures by affinity chromatography over albumin-agarose, eluted with 50% ethanol, and separated by a silica gel thin layer chromatographic system. In the presence of UDPGA, native and activated microsomal preparations all formed both bilirubin mono- and diglucuronides from unconjugated bilirubin, and bilirubin diglucuronide from bilirubin monoglucuronide. No significant non-enzymatic conversion of mono- to diglucuronide occurred without UDPGA, or in the presence of denatured enzyme. Hence, bilirubin diglucuronide is a major product of bilirubin-UDP-glucuronyl transferase.     

Prostaglandin synthase-catalyzed metabolic activation of some aromatic amines to genotoxic products

Cultured human fibroblasts were incubated with different aromatic amines in the presence of different activation systems and the induction of strand breaks in fibroblast DNA was studied. In the presence of ram seminal vesicle microsomes and arachidonic acid, DNA strand breaks were induced by 2-naphthylamine, 2,4-diaminotoluene and 4-methoxy-m-phenylenediamine. This effect was decreased when the prostaglandin synthase of the ram seminal vesicle microsomes was inhibited. The data suggest that metabolic activation catalyzed by prostaglandin synthase may be of importance in the formation of genotoxic products by certain urinary tract carcinogens.     

The phospholipid-binding protein of the reproductive tract of the bull — Effect on the removal of spermatozoal cytoplasm droplets in other species and influence of antibodies on its reactivity

The phospholipid-binding protein (PBP) isolated from bull seminal vesicle fluid removed cytoplasm droplets not only from bull, but also from ram, boar and rabbit epididymal spermatozoa. However, the presence of a protein cross-reacting with anti-PBP antisera was demonstrated by immunofluorescent staining in ram seminal vesicles and ampullae. In contrast to PBP from bull, the ram PBP-like protein did not lyse bull or ram erythrocytes. Rabbit antiserum against PBP only negligibly reduced the ability of PBP to remove cytoplasm droplets from bull epididymal spermatozoa, but it inhibited the haemolytic effect of the protein.     

Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.     

A carbon-centered free radical intermediate in the prostaglandin synthetase oxidation of arachidonic acid. Spin trapping and oxygen uptake studies

The ESR spin-trapping technique has been used to identify a free radical involved in the oxygenation of arachidonic acid by ram seminal vesicle microsomes. The ESR spectrum of the radical adduct indicates that a carbon-centered arachidonic acid free radical has been observed. The formation of this species is inhibited by indomethacin, but not by phenol, and it is probably the first intermediate formed during the prostaglandin synthetase-catalyzed oxidation of arachidonic acid. The chemical identity of the trapped radical was substantiated with an independent synthesis of a closely related radical adduct.     

A test for the intermediacy of 11-hydroperoxyeicosa-5,8,12,14-tetraenoic acid [11-HPETE] in prostaglandin biosynthesis

11-Hydroperoxy-eicosa-5,8,12,14-tetraenoic acid [11-HPETE] was prepared by chromatographic separation of the hydroperoxides formed from the singlet oxygen oxidation of arachidonic acid [20:4]. 1-[¹⁴C]-HPETE was incubated with prostaglandin endoperoxide synthetase preparations from ram seminal vesicles. No prostaglandins products deriving from 11-HPETE were detected in any of the incubations. 11-Hydroxy-eicosa-5,8,12,14-tetraenoic acid [11-HETE], formed by the action of the hydroperoxidase component of prostaglandin endoperoxidase synthetase was the major product formed. The peroxidase activity was absolutely dependent on epinephrine and was stimulated by hematin. 11-HPETE does not appreciably effect the extent of conversion of arachidonic acid into prostaglandin.     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene.     

Effect of processing methods on colouration of human serum albumin preparations

Human serum albumin is a well tolerated therapeutic for the treatment of hypovolemia. Despite all commercial human albumin preparations being derived from plasma, these products can have a highly variable colour. Albumin samples derived from ethanol precipitation and chromatographic fractionation procedures were evaluated for bilirubin and biliverdin levels and by spectrophotometry. It was shown that albumin derived from a chromatographic process, which had a bilirubin:albumin ratio similar to that observed in plasma, had a vibrant yellow appearance. The albumin derived from ethanol precipitation had undetectable levels of bilirubin, and the amber colour of this product was attributed mainly to residual haem. The presence of bilirubin during pasteurisation led to oxidation to biliverdin, with a resultant colour change from yellow to yellow/green. Given that the antioxidant properties of bilirubin are well established, it is possible that bilirubin helps protect albumin from oxidation during the pasteurisation step.     

Haemolytic factor in bovine seminal vesicle fluid

The haemolytic factor of bovine seminal fluid has a protein nature; it is present in the precipitate obtained by treating the fluid with alcohol-ether, ammonium sulphate and rivanol; it occurs in three out of five protein fractions obtained by chromatographic separation on Sephadex G 100.
of Enzymatic treatment of seminal vesicle fluid by pronase caused total inactivation of the haemolytic factor. Chymotrypsin caused considerable damage, while a number other enzymes did not affect the activity of the haemolytic factor.     

Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems

Centrifugal counter-current distribution (CCCD) in a dextran, Ficoll, poly(ethylene glycol) two-phase system was used to study the effect of seminal plasma proteins on the partition behaviour of ram spermatozoa exposed to thermal shock. Ram spermatozoa freed from seminal plasma by a ‘swim-up’ procedure were submitted to thermal shock and fractionated by CCCD. Cell viability decreased from 68% to 18% after the treatment, showing a slight displacement of the cells from the right (where a higher enrichment of live cells is found) to the centre of the profile. A change of the distribution profile was shown in the presence of either ram or bull seminal plasma. Bull seminal plasma was able to move the profile to the right, whereas ram seminal plasma increased the proportion of cells with enhanced affinity for the lower dextran-rich phase. Plasma proteins isolated from both seminal plasmas moved the profile to the right. In addition, cell viability rose to 48% after the CCCD run in the presence of ram plasma proteins. This restoring effect was lost when ram plasma proteins were thermally denatured. Bovine serum albumin was not only unable to move the profile to the right but even promoted displacement of the profile to the left. This negative effect was also observed when proteins from bull seminal plasma were in the presence of protein-free ram seminal plasma. However, proteins isolated from ram seminal plasma still restored the profile in the presence of bull seminal plasma freed from proteins. The results presented here strongly suggest that seminal plasma proteins are absorbed by a spermatozoal surface previously exposed to thermic shock. These proteins would exert a highly specific protective effect on ram spermatozoa. In addition, in the ram seminal plasma there must be some factor which avoids this adsorption.     

Identification and biological activity of a novel natural prostaglandin, 5,6-dihydro-prostaglandin E3

A novel natural E-prostaglandin was detected by HPLC among the endogenous prostaglandins extracted from ram seminal vesicles. The corresponding precursor - all-cis-eicosa-8, 11, 14, 17-tetraenoic acid was isolated from bovine liver lipids and the preparative biosynthesis with the microsomal fraction of ram seminal vesicles was performed. The isolated product was purified by HPLC and identified by GC-MS as 5,6-dihydro-PGE3. The results of in vitro tests demonstrate that 5,6-dihydro-PGE3 is 14 times less active uterine stimulant than PGE1, at the same time retaining 75% of the anti-aggregatory potency of PGE1. Thus, 5,6-dihydro-PGE3 meets the requirements of a selective antithrombotic agent more than PGE1.     

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.     

Isolation and culture of rat seminal vesicle epithelial cells : The use of the secretory protein SVS IV as a functional probe

A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.     

An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.     

Metabolism of phospholipids by spermatozoa and seminal plasma

1. The hydrolysis of added (32)P-labelled phospholipids by whole ram and bull semen and the separated spermatozoal and plasma components was examined. 2. The ethanolamine phosphoglycerides were rapidly attacked by washed spermatozoa, forming predominantly glycerylphosphorylethanolamine, but with whole semen and seminal plasma a lysophosphatidylethanolamine was also detected. 3. The hydrolysis of lecithin by spermatozoa and plasma was very slow, and glycerylphosphorylcholine was the sole product detected. 4. Ram testicular spermatozoa were comparatively inactive in metabolizing both phospholipids, but ampulla contents showed the same activity as ejaculated semen. 5. Phosphatidylinositol was metabolized by spermatozoa obtained from any portion of the ram reproductive tract and also by seminal plasma. With testicular components, ampulla contents and washed ejaculated spermatozoa, inositol monophosphate, an unidentified phosphate ester and inorganic phosphate were the main products. In contrast, with whole semen and seminal plasma, glycerylphosphorylinositol was the predominant water-soluble phosphate ester. 6. Accessory-gland secretion obtained from vasectomized rams showed a pronounced phospholipase A activity towards ethanolamine phosphoglyceride. 7. On aerobic incubation of whole ram semen there was a decrease in the concentration of all phospholipid classes, although cardiolipin showed the greatest percentage decrease. In the choline phosphoglyceride fraction, this loss was confined to the plasmalogen component. This breakdown of phospholipids was decreased considerably when the spermatozoa were washed, and was not observed when whole bull semen was incubated under similar conditions.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.