Formation of phosphatidylethanol in frozen kidneys from ethanol-treated rats.

We recently identified phosphatidylethanol (Pet) in tissues from ethanol-treated rats. Since phosphatidyl esters are formed artefactually during freezing in plants we wanted to examine if PE was elevated during freezing in animal tissues. Rats were treated with 3 g/kg of ethanol, killed after 3 h and PE was isolated from kidneys at once or after storage at 0, -5, -10, -15, -20 and -80 degrees C for 7 days. Kidneys analyzed at once or after storage at -80 degrees C had Pet equivalent to 0.02 mumol Pet/g. Storage at -10 degrees C and -15 degrees C resulted in increases of Pet to 1.5 mumol Pet/g and 1.2 mumol Pet/g, respectively. Thus, Pet is artefactually elevated during storage of tissues from ethanol-treated rats at lower freezing temperatures, reflecting considerable changes in composition of acidic phospholipids.

Phosphatidylethanol formation in rat organs after ethanol treatment.

An abnormal acidic phospholipid was found in high concentration in kidney and brain, and also in other organs of rats exposed to ethanol by i.p. injection or by a liquid diet. The compound could be identified as phosphatidylethanol. Phosphatidylethanol is probably formed in cell membranes by a phospholipase D-catalyzed transphosphatidylation reaction.

Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin.

Plasma or whole venous or arterialized blood from healthy human donors was incubated with NO (50-300 microM), and the resulting formation of methaemoglobin (MetHb), nitrosyl haemoglobin (HbNO), and plasma nitrite and nitrate were measured. In plasma, NO was converted to nitrite and nitrate in a ratio of 5:1. In arterial blood (O2 sat. 94-99%) NO was almost quantitatively converted to nitrate and MetHb. No nitrite was detected and HbNO formation was low. In venous blood (O2 sat. 36-85%) more HbNO and less nitrate was formed, in comparison to arterialized blood. We propose that NO liberated from endothelium of conductance and resistance vessels is taken up by red blood cells and inactivated by HbO2 via stoichiometric conversion to MetHb and nitrate.

Conversion of inhaled nitric oxide to nitrate in man.

1. Nitric oxide (NO) is potentially useful as a selective vasodilator drug in infants and adults with pulmonary hypertension. In vitro and in vivo observations demonstrate that NO may be converted to nitrate in the blood, to be further excreted into the urine. The aim of the present study was to assess quantitatively the importance of this pathway for inhaled NO in human subjects. 2. Healthy subjects inhaled 15NO (25 p.p.m.) for 1 h. The plasma and urine levels of 15NO3- were followed for 2 and 48 h, respectively. 3. The measured retention of 15NO in the lungs was 224 +/- 13 mumol, corresponding to 90 +/- 2% of the inhaled amount. Plasma 15NO3- increased during the inhalation of 15NO, to about 15 mumol l-1, and fell when inhalation of 15NO was terminated. 4. Urinary excretion of 15NO3- during the first 24 h after inhalation was 154 +/- 12 mumol. During the following 24 h another 8 +/- 2 mumol of 15NO3- appeared in the urine. 5. We conclude that conversion of inhaled NO to nitrate is a major metabolic pathway in man, covering more than 70% of its inactivation. The metabolic fate of the remaining NO inhaled requires further study.

Urinary excretion of 2, 3-dinor-6-keto prostaglandin F1 alpha and platelet thromboxane formation during ethanol withdrawal in alcoholics.

The excretion of 2,3-dinor-6-keto prostaglandin F1 alpha, a major urinary metabolite of prostacyclin, and the formation of thromboxane B2, a stable metabolite of thromboxane A2, by platelets stimulated by adenosine diphosphate, were studied in alcoholics, who had been admitted for detoxification. Once prolonged heavy drinking had stopped, platelet count and thromboxane formation, calculated either per 10(7) platelets or per litre of blood, significantly increased (p less than 0.05), while the skin bleeding time and urinary excretion of the metabolite of prostacyclin decreased (p less than 0.05). The balance between prostacyclin and thromboxane therefore seemed to favour the excretion of prostacyclin while it shifted to favour thromboxane formation about a week later.

Effects of exercise and ethanol ingestion on platelet thromboxane release in healthy men.

We studied the effects of repeated bicycle exercises and ethanol ingestion (1.5 g/kg) on platelet aggregation and thromboxane (TxB2) release in 10 healthy male volunteers. After a bicycle exercise performed in the morning, the adenosine diphosphate (ADP)-induced platelet aggregation and the aggregation-associated thromboxane release were found to be decreased in fasting men. In contrast, after ingestion of fruit juice and a second exercise at noon, platelet aggregation and thromboxane release were increased. These latter changes were negligible when ethanol was ingested together with fruit juice. A third exercise, performed in the evening, again caused a decrease in the aggregation and associated thromboxane release during the control session, but provoked an increase during the ethanol session. Exercise increased the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha. Changes in the plasma arachidonic acid (AA) concentration probably influenced the platelet thromboxane release. The results suggest that both physical exercise and ingestion of ethanol in fruit juice influence the ADP-stimulated platelet thromboxane release.

Differential inhibition of thromboxane A2 and prostacyclin synthesis by low dose acetylsalicylic acid in atherosclerotic patients.

Differential inhibition of thromboxane A2 (TxA2) and prostacyclin (PGI2) biosynthesis has an antithrombotic potential, since it may change the TxA2/PGI2 formation ratio in a favourable direction. Very low doses of acetylsalicylic acid (ASA) have been demonstrated to elicit differential inhibition of TxA2 and PGI2 formation in healthy subjects; whether a similar effect can be obtained in patients with atherosclerosis is still an open question. We addressed this by analyzing the urinary excretion of the 2,3-dinor-metabolites of TxA2 (Tx-M) and PGI2 (PGI-M) in 10 patients with severe atherosclerosis during 10 consecutive days. The first three days were a basal period, under which no treatment was given. During the subsequent seven days a daily 50 mg oral dose of ASA was administered. In the basal state urinary Tx-M did not differ from that of PGI-M, the median excretion rates of the two eicosanoid metabolites being 526 (range 68-1490) and 562 (range 93-1970) pg/mg creatinine, respectively. During ASA treatment urinary Tx-M fell to a lower (p less than 0.001) level than PGI-M. Thus, during the last 5 days of ASA treatment the median excretion of Tx-M was depressed (p less than 0.001) to 148 (range 48-428) pg/mg creatinine, while that of PGI-M was decreased (p less than 0.01) to 313 (range 42-2658) pg/mg creatinine. These data indicate that a daily 50 mg dose of ASA inhibits cardiovascular formation of eicosanoids in patients with severe atherosclerosis and increased platelet TxA2 formation. Furthermore, this dose of ASA inhibits the formation of TxA2 more than that of PGI2.

Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn.

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, alpha-linolenic and gamma-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18:2n-6, 18:3n-3, and 18:3n-6 in a ratio of approximately 1:1-3 at high substrate concentrations. 8-Hydroxy-9(10)epoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.

Nitric oxide is not increased in alcoholic brain.

Nitric oxide (NO) metabolites nitrite and nitrate were measured in the cerebrospinal fluid in 12 alcohol-dependent subjects and in 16 healthy controls. The amounts of NO metabolites in alcoholics were not different from those in the controls. The results suggest that NO is not a major factor responsible for brain damage in these patients.

On the metabolism of epoxyeicosatrienoic acids by ram seminal vesicles: isolation of 5(6)epoxy-prostaglandin F1 alpha.

cis-5(6)Epoxy- and cis-14(15)epoxyeicosatrienoic acid are formed from arachidonic acid by monooxygenases. 5(6)Epoxyeicosatrienoic acid is metabolized by fatty acid cyclooxygenase of ram seminal vesicles and the major products were recently identified as 5(6)epoxy-PGE1 and two stereoisomers of 5-hydroxy-PGI1. The two isomers were likely formed from an unstable intermediate, 5(6)epoxy-PGF1 alpha. The isolation of 5(6)epoxy-PGF1 alpha is described here and 14(15)epoxyeicosatrienoic acid is shown to inhibit fatty acid cyclooxygenase of ram seminal vesicles, albeit less potently than eicosatetraynoic acid (IC50 0.18 and 0.05 mM, respectively).

Isolation of 19,20-dehydroprostaglandins E1 and E2 in human seminal fluid and further studies on 18,19-dehydroprostaglandins E1 and E2.

Human seminal fluid was recently found to contain 18,19-dehydroprostaglandins E1 and E2 (E. H. Oliw, H. Sprecher, and M. Hamberg, (1986) J. Biol. Chem. 261, 2675-2683). In the present study, the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2 were prepared by incubation of microsomes of ram vesicular glands and glutathione with the precursor fatty acids, 8(Z),11(Z),14(Z),18(E/Z)-eicosatetraenoic acids, and 5(Z),8(Z),11(Z),14(Z),18(E/Z)-eicosapentaenoic acids, and used as references to characterize the 18,19-dehydroprostaglandins of human seminal fluid. Based on separation by reversed-phase high-performance liquid chromatography, capillary gas chromatography-mass spectrometry, and ozonolysis of the (-)-menthoxycarbonyl derivatives and on comparison with the authentic compounds, human seminal fluid was found to contain both the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2. Furthermore, human seminal fluid contained two related compounds, viz. 19,20-dehydroprostaglandins E1 and E2. The structures of these compounds were established by conversion into the corresponding prostaglandin B compounds, by mass spectrometric analysis and by chemical degradation by oxidative ozonolysis, which afforded, inter alia, 2(S)-hydroxy-adipic acid.

Maintained hyperexcretion of thromboxane A2 metabolite in healthy young cigarette smokers: results from a prospective study in randomly sampled males with stable smoking habits.

Although several studies have identified cigarette smoking as a factor increasing platelet formation of thromboxane A2 (TxA2), no prospective data on this issue have been presented in a defined population with stable smoking habits. Therefore, we analysed the relation between smoking habits and urinary excretion of the 2,3-dinor metabolites of thromboxane A2 (Tx-M) and prostacyclin (PGI-M) in 87 males, randomly sampled from a population of 18-19-year-old men, at two different occasions separated by 31-49 months. The daily cigarette consumption among the smokers was unchanged between the study occasions (11 vs. 11 cigarettes day-1), but 9 of 43 initial smokers had quit. None of the initial non-smokers had begun smoking. Tx-M was higher in the smokers than in the non-smokers and correlated with the daily cigarette consumption both at the initial (176 vs. 123 pg mg-1 creatinine; P = 0.01) and the second (214 vs. 164 pg mg-1; P = 0.002) study occasion. Those who had quit smoking since the initial study did not differ in Tx-M from the non-smokers at the second study occasion. Urinary PGI-M did not differ between cigarette smokers, non-smokers and quitters at either of the study occasions. We conclude that cigarette smoking elicits an increased formation of thromboxane A2, indicating platelet activation, that is stable during an observation period of up to 4 years. The increased platelet activity is reversible upon quitting.

Paired analysis of thromboxane and prostacyclin metabolites in urine from healthy mothers and their children.

Eicosanoids have been implicated in the adaptation of the fetal to the neonatal circulation, but biochemical support for an activation of their synthesis in relation to birth has not been presented. We addressed this by assessing in pairs the excretion of two metabolites of thromboxane A2, 11-dehydro-TxB2 (dTx) and 2,3-dinor-TxB2 (Tx-M), and one metabolite of prostacyclin, 2,3-dinor-6-keto-PGF1 a (PGI-M), in the first voided urine from 13 healthy term neonates and in the immediate pre-delivery urine from their respective mothers. The excretion of dTx was higher (p less than 0.01) in the neonates than in their mothers (7430 and 1330 pg/mg creatinine, respectively), and so was the excretion of Tx-M (3730 and 900 pg/mg creatinine, respectively, p less than 0.001). Also the excretion of PGI-M was higher (p less than 0.05) in the neonates than in their mothers (2550 vs. 1510 pg/mg creatinine). Amniotic fluid contained detectable levels of both Tx-M and PGI-M. These data indicate that activation of platelets takes place in the neonate after separation of the fetal and maternal vascular circuits. The possible physiological implication of such activation requires further studies.

Increased urinary excretion of a major thromboxane metabolite in early alcohol withdrawal.

Urinary excretion of 2,3-dinor-thromboxane B2 as a marker of in vivo thromboxane A2 (TxA2) biosynthesis was measured in six alcoholics 1 and 14 days after the cessation of heavy drinking using gas chromatography/mass spectrometry. Six non-alcoholic healthy volunteers served as controls. One day after alcohol withdrawal the excretion of the dinor metabolite was significantly higher (P < 0.01) in the alcoholics (408 +/- 42 pg mg-1 creatinine) than in the controls (180 +/- 30 pg mg-1 creatinine) and was accompanied by a significantly reduced platelet count (103.0 +/- 20.2 x 10(9) l-1 vs. 194.0 +/- 13.9 x 10(9) l-1 in controls; P < 0.01). The metabolite excretion fell then significantly (P < 0.05) to 245 +/- 53 pg mg-1 creatinine 14 days after alcohol withdrawal and this was paralleled by an increase in platelet count to 453.5 +/- 72.0 x 10(9) l-1 (P < 0.05). The present results support the hypothesis that Tx-A2 biosynthesis is increased in early alcohol withdrawal and strongly suggest platelets as a cellular origin of the increased TxA2 formation.

Effects of a small dose of ethanol and calcium carbimide-induced acetaldehyde intoxication on human platelet aggregation, associated thromboxane formation and urinary excretion of 2,3-dinor-6-keto prostaglandin F1 alpha.

We studied ADP-induced platelet aggregation, associated thromboxane B2 (TXB2) formation, urinary excretion of the prostacyclin metabolite 2,3-dinor-6-keto prostaglandin F1 alpha (2,3-dinor-6-keto PGF1 alpha) and formation of malondialdehyde in 10 healthy volunteers after ingestion of a small dose of ethanol (0.25 g per kg of body weight) and calcium carbimide (50 mg). Platelet aggregation in platelet-rich plasma (PRP) was suppressed (p less than 0.05) by ethanol, but no change occurred in platelet TXB2 formation. Ingestion of calcium carbimide caused significant elevations in blood acetaldehyde (p less than 0.001) and ethanol (p less than 0.05) levels, but acetaldehyde did not influence platelet aggregability or the aggregation-associated TXB2 formation. However, calcium carbimide per se significantly (p less than 0.05) elevated TXB2 formation. No effects were found on plasma malondialdehyde levels and urinary excretion of 2,3-dinor-6-keto PGF1 alpha. These observations indicate that a small dose of ethanol attenuates platelet aggregation without any significant effect on aggregation-associated TXB2 formation. By contrast, ingestion of calcium carbimide per se may enhance TXB2 formation.

Platelet function during acetaldehyde intoxication in healthy male volunteers.

ADP-induced platelet aggregation, associated thromboxane B2 (TXB2) formation and urinary excretion of the prostacyclin metabolite 2,3-dinor-6-keto prostaglandin F1 alpha (PGI2-M) were studied in healthy male volunteers after ingestion of ethanol (0.25 g per kg of body weight) and calcium carbimide (50 mg). Platelet aggregation was suppressed (p less than 0.05) by ethanol, but no changes were observed in platelet TXB2 formation. Ingestion of calcium carbimide caused a significant elevation in blood acetaldehyde (p less than 0.001) and ethanol (p less than 0.05) levels, but acetaldehyde did not influence platelet aggregation or TXB2 formation. However, calcium carbimide per se elevated TXB2 production (p less than 0.05). Ethanol and acetaldehyde appeared not to have any significant effect on urinary excretion of PGI2-M.

2,3-Dinor metabolites of thromboxane A2 and prostacyclin in urine from healthy human subjects: diurnal variation and relation to 24h excretion.

1. Urinary levels of the 2,3-dinor metabolites of thromboxane A2 (2,3-dinor-thromboxane A2, Tx-M) and prostacyclin (2,3-dinor-6-keto-prostaglandin F1 alpha, PGI-M) are frequently analysed as indices of platelet and endothelial activity and interaction in vivo. Despite this, little is known about the possible diurnal variations in urinary Tx-M and PGI-M in healthy human subjects, and how the urinary levels of Tx-M and PGI-M in single samples reflect their respective 24 h excretion rates. We addressed this by determining Tx-M, PGI-M and creatinine in consecutive portions of urine collected during 24 h in 15 healthy non-smoking subjects. 2. The total 24 h excretion of Tx-M and PGI-M did not differ between men (223 +/- 31 and 132 +/- 27 ng, respectively) and women (215 +/- 44 and 127 +/- 29 ng, respectively). Neither the excretion of Tx-M nor that of PGI-M displayed any significant diurnal variation. 3. The excretion of Tx-M during a 3 h period and the Tx-M/creatinine ratio in a urine sample accurately reflected the 24 h excretion of Tx-M (correlation coefficient ranges 0.74-0.94 and 0.74-0.86, respectively). The excretion of PGI-M during a 3 h period and the PGI-M/creatinine ratio in a urinary sample were accurate measures of 24 h excretion of PGI-M (correlation coefficient ranges 0.76-0.94 and 0.72-0.83, respectively). Urinary Tx-M and PGI-M expressed as simple concentrations were poor indices of their respective 24 h excretion. 4. We conclude that time-related excretions of Tx-M and PGI-M may be the best indices ex vivo of the cardiovascular formation of thromboxane A2 and prostacyclin, but that urinary creatinine-related concentrations of Tx-M and PGI-M in a urine sample are accurate measures as well.

Release of endothelial mediators and sympathetic transmitters at different coronary flow rates in rabbit hearts.

1. The release of three endothelial mediators, namely, endothelial-derived relaxing factor (EDRF), prostacyclin (PGI2) and endothelin, and of two sympathetic neurotransmitters, noradrenaline and neuropeptide Y (NPY), from resting or sympathetically stimulated rabbit Langendorff hearts was investigated at normal or elevated coronary flow. The sympathetic nerves to the hearts were stimulated at 5 Hz for 30 s and the cardiac effluent was analysed for nitrite (metabolite of EDRF) with electron paramagnetic resonance spectrometry, for 6-keto-PGF1 alpha (metabolite of PGI2) with gas chromatography/mass spectrometry, for endothelin- and NPY-like immunoreactivity with radioimmunoassay, and for noradrenaline and purines with liquid chromatography. 2. During perfusion of the hearts at normal flow (35 +/- 1.4 ml min-1) the effluent concentration of nitrite was 0.15 +/- 0.02 microM, that of 6-keto-PGF1 alpha 0.74 +/- 0.08 nM, and that of endothelin-like immunoreactivity 0.18 +/- 0.01 pM. Nerve stimulation augmented the release of 6-keto-PGF1 alpha from 76 +/- 8 to 99 +/- 10 pmol (3 min)-1 (P less than 0.05), but did not affect the release of nitrite or endothelin-like immunoreactivity. Nerve stimulation also facilitated the outflow of noradrenaline and of NPY-like immunoreactivity by 52 +/- 11 pmol (3 min)-1 and 19 +/- 7 fmol (3 min)-1, respectively. 3. Elevation of the coronary flow to 79 +/- 3.2 ml min-1 did not affect the effluent concentrations of nitrite, 6-keto-PGF1 alpha and endothelin-like immunoreactivity, implying that their outflows were augmented. Sympathetic stimulation at elevated coronary flow did not further augment the outflow of endothelial mediators or of NPY-like immunoreactivity, but increased the outflow of noradrenaline by 62 +/- 12%, in comparison to stimulation at normal flow. Perfusion of the heart with the noradrenaline uptake blocker desipramine (5 microM) completely abolished the promoting effecting of elevated coronary flow on noradrenaline outflow during sympathetic stimulation. 4. These data indicate that an increase in coronary flow in perfused rabbit hearts is paralleled by a corresponding facilitation of the formation of the endothelial mediators, EDRF, prostacyclin and endothelin. Such an elevation of mediator formation does not affect nerve stimulation-induced release of sympathetic transmitters in the heart.

Relation between tobacco use and urinary excretion of thromboxane A2 and prostacyclin metabolites in young men.

BACKGROUND: Cigarette smoking is a risk factor for cardiovascular disease. The present study addressed the effect of tobacco use on the formation of two eicosanoids, thromboxane A2 and prostacyclin, which have been implicated in both acute and chronic cardiovascular disorders. METHODS AND RESULTS: In 577 randomly sampled 18-19-year-old men, the urinary excretion of the 2,3-dinor metabolites of thromboxane A2 and prostacyclin (Tx-M and PGI-M, respectively) was analyzed and related to the subjects' self-reported use of tobacco. Sixty-five percent of the subjects used no tobacco, 7.5% were cigarette smokers, 22% used wet (oral) snuff, and the rest reported a mixed use of tobacco. The urinary excretion of Tx-M was higher (p less than 0.001) in cigarette smokers than in those not using tobacco (180 versus 128 pg/mg creatinine) and was correlated (r = 0.35, p less than 0.05) with the daily cigarette consumption. Snuff users had no increase in their urinary excretion of Tx-M, despite urinary cotinine levels comparable to those in the cigarette smokers (1,210 and 1,560 ng/ml, respectively). The excretion of PGI-M did not differ between non-tobacco users, cigarette smokers, and snuff users. CONCLUSIONS: We conclude that cigarette smoking, but not the use of snuff, facilitates the formation of thromboxane A2. We propose that such an increased formation reflects platelet activation in the absence of vascular injury and that it may be of significance for the subsequent development of cardiovascular disease.