Development of a GC-MS method for quantitation of 2,3-dinor-6-keto-PGF1 alpha and determination of the urinary excretion rates in healthy humans under normal conditions and following drugs

Tetradeuterated 2,3-dinor-6-keto-PGF1 alpha was used as internal standard in the development of a method for quantitation of 2,3-dinor-6-keto-PGF1 alpha in human urine based on gas chromatography - mass spectrometry. The urinary excretion rates of 2,3-dinor-6-keto-PGF1 alpha in twenty normal healthy males and females were 9.7 +/- 4.6 and 8.8 +/- 8.5 (mean +/- SD) ng/h respectively. A considerable inter- and intra-individual variation was found under normal conditions. It was also found that the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha was increased about fivefold during and shortly after 30 min of strenuous jogging. Any data about the effect of nonsteroidal antiinflammatory drugs on the excretion rate of 2,3-dinor-6-keto-PGF1 alpha are difficult to interpret when considering the above findings. However, oral administration of 500 mg of aspirin did not seem to reduce the excretion rate of 2,3-dinor-6-keto-PGF1 alpha.     

Neonatal urinary prostanoid excretion

Urinary excretion of prostanoids prostaglandin E2 (PGE2), PGE-M (7alpha-hydroxy-5,11-diketo-2,3,4,5,20-penta-19-carboxyprostano ic acid), 6-keto-PGF1alpha, 2,3-dinor-6-keto-PGF1alpha, thromboxane B2 (TxB2) 2,3-dinor-TxB2 and 11-dehydro-TxB2 was determined by gas chromatography/mass spectrometry in preterm and term infants to show that there is an age-dependent excretion rate of the above prostanoids in infants this young. Group I included premature children with normal postnatal development, Groups II and III included term children who were admitted in the neonatal period for observation because of feeding problems but who were subsequently found to be completely healthy. We present normal data of three primary prostanoids and four prostanoid metabolites. In Group I, excretion rates of 2,3-dinor-TxB2 were significantly lower than in Group II (P = 0.04) and in Group III (P = 0.05). Furthermore, the excretion rate of 11-dehydro-TxB2 in group I was significantly lower than in Group II (P = 0.05). We found no significant age-dependent differences between the three groups in excretion rates of PGE2, PGE-M, 6-keto-PGF1alpha, 2,3-dinor-6-keto-PGF1alpha, and TxB2.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.     

Urinary prostaglandin excretion in pregnancy: the effect of dietary sodium restriction

INTRODUCTION: Dietary sodium restriction results in activation of the renin-angiotensin-aldosterone-system. In the non-pregnant situation renin release in response to a low sodium diet is mediated by prostaglandins. We studied the effect of dietary sodium restriction on urinary prostaglandin metabolism in pregnancy. PATIENTS AND METHODS: In a randomized, longitudinal study the excretion of urinary metabolites of prostacyclin (6-keto-PGF(1 alpha)and 2,3-dinor-6-keto-PGF(1 alpha)) and thromboxane A(2)(TxB(2)and 2,3-dinor-TxB(2)) was determined throughout pregnancy and post partum in 12 women on a low sodium diet and in 12 controls. RESULTS: In pregnancy the excretion of all urinary prostaglandins is increased. The 6-keto-PGF(1 alpha)/ TxB(2)-ratio as well as the 2, 3-dinor-6-keto-PGF(1 alpha)/ 2,3-dinor-TxB(2)-ratio did not significantly change in pregnancy. CONCLUISION Prostacyclin and thromboxane do not seem to play an important role in sodium balance during pregnancy.     

Modulation of renal hemodynamics by renal eicosanoids during vasopressor infusions in man

Pressor doses of norepinephrine (NE) (n = 8) and angiotensin II (A II) (n = 5) were infused in normal volunteers to determine whether the systemic administration of vasopressor hormones influence renal eicosanoid production and whether, in turn, the eicosanoids produced could modulate renal hemodynamics and electrolyte excretion. At the doses administered, both pressor substances induced the expected rise in blood pressure, a significant decrease (P less than 0.05) in renal blood flow and a proportionally smaller fall in glomerular filtration rate, resulting in a consistent augmentation in filtration fraction. Fractional sodium excretion was concomitantly reduced. NE infusion produced only slight modifications in urinary prostaglandin (PG)E2, 2,3-dinor-6-keto-PGF1 alpha and thromboxane (TX)B2, while urinary 6-keto-PGF1 alpha and PGF2 alpha were increased by 38% and 176% respectively. The increase in urinary 6-keto-PGF1 alpha (the non-enzymatic degradation product of PGI2, predominantly of cortical origin) was proportional to the level of circulating NE (r = 0.78, P less than 0.05) and to the renal vascular resistance (r = 0.85, P less than 0.01), suggesting an immediate compensatory role for PGI2 in response to the NE-induced pressor stimulus. The renal production of PGE2 and PGF2 alpha (predominantly medullary) was inversely correlated with the filtration fraction: the greater the increase in PGE2 and PGF2 alpha the lower the elevation in filtration fraction or the decline in renal blood flow upon NE administration. All infusion variably stimulated the renal eicosanoid production: PGE2, 41%; PGF2 alpha, 102%; 6-keto-PGF1 alpha, 38%; 2,3-dinor-6-keto-PGF1 alpha, 38%; and TXB2, 25%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of icosanoids. Report of the Group for Standardization of Methods in Icosanoid Research

This statement from laboratories highly qualified in icosanoid analysis identifies the urgent need for the availability of the following compounds in labeled (deuterium and tritium) and unlabeled form: PGE2 PGF2 alpha PGD2 6-keto-PGF1 alpha Thromboxane B2 9 alpha,20-dihydroxy-11,15-dioxo-2,3- dinorprost -5-enoic acid 9 alpha-hydroxy-11,15-dioxo-2,3,18,19- tetranorprost -5-ene-1,20-dioic acid 15-keto-13,14-dihydro-PGE2 15-keto-13,14-dihydro-PGF2 alpha 5 alpha-7 alpha-dihydroxy-11- ketotetranorprosta -1,16-dioic acid 7 alpha-hydroxy-5,11-diketo- tetranorprosta -1,16-dioic acid 2,3 dinor-thromboxane B2 2,3 dinor-6-keto-PGF1 alpha 2,3 dinor-6,15-diketo 13,14 dihydro-20-carboxyl-PGF1 alpha 2,3 dinor-13,14-dihydro-6,15-diketo-PGF1 alpha LTB4 LTC4 LTD4 LTE4 LTF4 20-OH-LTB4 20-COOH-LTB4 5-HETE 12-HETE 15-HETE omega-OH-12-HETE 5S, 12S-di HETE 5S, 15S-di HETE HHT other hydroxylated polyunsaturated fatty acids and their epoxides.     

Monitoring of the thromboxane A2/prostacyclin ratio in the urine of patients with retinal vascular occlusion through the low-dose-aspirin therapy using the gas chromatography/selected ion monitoring method

We determined the levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) in patients with retinal vascular occlusion (RVO) to elucidate the change of the thromboxane A2/prostacyclin (TX/PGI) ratio with this disease and the effect of low-dose-aspirin therapy. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were converted to 1-methyl ester-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative and 1-methyl ester-6-methoxime-9,12,15-tris-dimethylisopropylsilyl ether derivative, respectively, and applied to a gas chromatography/selected ion monitoring. The average level of 11-dehydro-TXB2 in 30 patients with RVO was 1038 +/- 958 pg/mg creatinine. It was significantly higher than that of 27 healthy volunteers, which was 616 +/- 294 pg/mg creatinine (p < 0.05 with unpaired t-test). However, 2,3-dinor-6-keto-PGF1alpha levels were not significantly different between these two groups. The average ratio of TX/PGI in the RVO patients was 32 +/- 26 and it was significantly higher than that of healthy volunteers, 17 +/- 10 (p < 0.01). Patients with central retinal artery occlusion or branch retinal artery occlusion showed greatly high 11-dehydro-TXB2 levels and TX/PGI ratios, although the number of patients was limited in the current study. After the administration of low-dose aspirin (40 mg/day) for about 1 month, the TX/PGI ratio decreased to around the normal level. Following the levels for up to 10 months, they also remained at the normal level. These observations suggested that the 11-dehydro-TXB2 levels and the TX/PGI ratio reflect the pathological conditions of RVO and are useful markers of the treatment.     

Urinary thromboxane A2/prostacyclin balance reflects the pathological state of a diabetic

Levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) were measured in diabetics to elucidate the relation between the thromboxane A2/prostacyclin (TX/PGI) balance and pathological states of diabetes mellitus. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were derivatized to methyl ester-propylamide-dimethylisopropylsilyl ether and methyl ester-methoxime-dimethylisopropylsilyl ether derivatives, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of diabetics were higher than those of healthy volunteers, suggesting the hypercoagulative states of this disease. The ratios showed positive correlations with the levels of blood glucose. The levels of hemoglobin A1c and triglyceride were correlated weakly with the ratio. Some of the patients who had relatively low levels of blood glucose also showed high TX/PGI ratios. Furthermore, the ratio increased in the order of the groups 1, 2, and 3; group 1 contained patients who did not take medicine for diabetes, group 2 contained those who took oral hypoglycemic agents, and group 3 contained those who received insulin therapy. These observations indicate that the TX/PGI ratio reflects the pathological conditions of diabetes and is a useful marker, having few different features from other markers that are presently used.     

Reduced prostacyclin to thromboxane A2 ratio is correlated with central apneas in preterm infants

Prostacyclin has a vasodilating effect on pulmonary vessels, whereas thromboxane A2 results in vasoconstriction. This study was designed to test the hypothesis that recurrent central apneas in preterm infants are correlated with a reduced prostacyclin to thromboxane A2 ratio. Twelve preterm infants with clinical events of apneas were matched with 12 control infants. Urinary concentration of 2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-TxB2 was determined, and the ratio correlated with the number of central apneas (>20s) measured in overnight polygraphy. The number of central apneas >20s/12h was 97.4 (SE 7.8) in the study group, and 47.3 (SE 6.6) in the control group (p = 0.001). There was a significant correlation between the number of central apneas and the 2,3-dinor-6-keto-PGF1alpha/2,3-dinor-TxB2-ratio in all infants combined (r = -0.72, p < 0.0001) as well as in the two subject groups. Central apneas in premature infants are correlated with an decreased prostacyclin to thromboxane A2 ratio. The underlying pathomechanism may be increased intrapulmonary shunts with reflexive central apneas due to reduced pulmonary oxygenation.     

Thromboxane synthase inhibition: "endoperoxide shunt phenomenon" does not occur in healthy humans in vivo

The effects of the thromboxane synthase inhibitor CGS13080 on the in vivo synthesis of thromboxane and prostacyclin were determined in six healthy volunteers. Two different doses (0.08 and 0.25 mg/kg x h) were infused for six hours under strictly controlled conditions and 2,3-dinor-TxB2 and 2,3-dinor-6-keto-PGF1 alpha were measured in urine using gaschromatography--mass spectrometry. The in vivo synthesis of thromboxane was inhibited by 80-75% while there was no effect on the in vivo prostacyclin synthesis.     

In vivo biosynthesis of thromboxane and prostacyclin during exposure to physiological levels of epinephrine

The effects of 20-min epinephrine infusion (0.025 and 0.3 nmol/kg/min) on the in vivo synthesis of thromboxane A2 and prostacyclin were studied in ten healthy male volunteers. We assessed the in vivo biosynthesis of thromboxane A2 and prostacyclin by measurement of the urinary metabolites 2,3-dinor-TxB2 and 2,3-dinor-6-keto-PGF1 alpha, respectively. Epinephrine infusion did not cause any significant changes in the urinary excretion of the two metabolites. Thus, we conclude that physiological levels of epinephrine do not affect the in vivo biosynthesis of thromboxane A2 and prostacyclin.     

Pronounced reduction of in vivo prostacyclin synthesis in humans by acetaminophen (paracetamol)

The effect of a single dose of 500 mg acetaminophen (paracetamol) on the in vivo synthesis of prostacyclin was studied in healthy volunteers by measurements of the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha. Acetaminophen caused a marked reduction of prostacyclin synthesis for 6-8 hours without any obvious effect on the thromboxane synthesis. Thus, acetaminophen may at least theoretically be disadvantageous for patients suffering from diseases where prostacyclin mediated vascular defence mechanisms are activated, like myocardial infarction, deep vein thrombosis and following surgery.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF1 alpha, PGE-M, 2,3-dinor-TxB2 and 11-dehydro-TxB2) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE2 rose (p less than 0.05) from 14.2 +/- 4.0 to 86.2 +/- 20.7, PGF2 alpha from 60.0 +/- 4.9 to 119.8 +/- 24.0, 6-keto-PGF2 alpha from 7.2 +/- 1.3 to 51.5 +/- 17.0, and TxB2 from 11.2 +/- 3.3 to 13.6 +/- 3.6 ng/h/1.73 m2 (means +/- SEM) at the maximal urine flow. Except for 6-keto-PGF1 alpha and TxB2, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged period of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB2 one can assume that this prostanoid represents renal as well as systemic TxA2 activity.     

Prostacyclin metabolism in adults and neonates. Urinary profiles of 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha studied by gas chromatography-mass spectrometry

Metabolism of endogenous prostacyclin was studied in adults and neonates by measuring urinary levels of 6-ketoprostaglandin F1 alpha (spontaneous hydrolysis product) and 2,3-dinor-6-ketoprostaglandin F1 alpha (enzymatically formed by beta-oxidation). Quantification of prostanoids was achieved by capillary gas chromatography-mass spectrometry using the stable isotope dilution technique. Purification of the urinary lipid extract included silicic acid column chromatography and reverse- and straight-phase high-pressure liquid chromatographies. Accuracy of the method was proven by recovery experiments for both metabolites. Partial mass spectra of endogenous 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha were obtained from urine samples. In neonates (third day of life, n - 5 pooled urines) levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.28 +/- 0.18 ng/ml) were much lower than those of 6-ketoprostaglandin F1 alpha (2.13 +/- 1.10 ng/ml), indicating low beta-oxidation activity at high prostacyclin formation. In adults (n = 7), levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.27 +/- 0.21 ng/ml) and levels of 6-ketoprostaglandin F1 alpha (0.20 +/- 0.11 ng/ml) were about the same, indicating relatively high beta-oxidation at low prostacyclin formation. Values are expressed as mean +/- S.D.     

Metabolism of prostacyclin in blood vessels.

The activity of 15-hydroxyprostaglandin dehydrogenase has been shown to be high in both mesenteric arteries and veins; the present study suggests that it may be responsible for the inactivation of prostacyclin (PGI2). The cytoplasmic fractions of bovine mesenteric arteries and veins were incubated with radiolabeled PGI2 in the presence of NAD+ or NADP+. The substrate was rapidly converted to a product, which was isolated and identified as 6,15-diketo prostaglandin F1alpha, (6,15-diketo-PGF1alpha) by thin layer chromatography and gas chromatography-mass spectrometry. The initial reaction rate began to level off after less than 1 min of incubation at 37 degrees C. When radiolabeled 6-keto-PGF1alpha, the stable hydrolysis product of PGI2, was used as substrate under the same conditions, 97% was recovered unmetabolized after 2 min of incubation. Catabolism of PGI2 may be a major determinant of its levels in blood vessels and, therefore, may be of crucial importance to regulating the action of PGI2. Further, estimation of PGI2 generation by either tissues or organs may be misleading if only 6-keto-PGF1alpha is measured.     

Thromboxane (TX)A3 and prostaglandin (PG)I3 are formed in man after dietary eicosapentaenoic acid: identification and quantification by capillary gas chromatography-electron impact mass spectrometry

The low incidence of myocardial infarction in Greenland Eskimos has been related to their traditional marine diet rich in eicosapentaenoic acid. However, whether dietary eicosapentaenoic acid is indeed transformed in man to antiaggregatory PGI3 and weakly proaggregatory TXA3 has not been clarified. In our studies we ingested either cod liver oil or mackerel both rich in eicosapentaenoic acid. Formation of TXB3, the hydrolysis product of TXA3, in platelet-rich plasma stimulated ex vivo with collagen was traced by capillary GC/EIMS. Via external standard, TXB3 formation in platelets was estimated to be 5-15% of TXB2 formation. From urine we extracted dinor metabolites of PGI according to a selective method. We utilized delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) as an index of total body production of PGI3 in analogy to 2,3-dinor-6-keto-PGF1 alpha (PGI2-M), the major urinary metabolite of PGI2. We separated PGI2-M and PGI3-M as the Me, MO, Me3Si derivatives by capillary gas chromatography and identified PGI3-M by EI mass spectrometry. Excretion of PGI3-M, which was not detectable under control conditions, was 83 +/- 25 ng/24 h (SD) after ingestion of cod liver oil and 134 +/- 38 ng/24 h after mackerel ingestion, while excretion of PGI2-M was 162 +/- 52 ng/24 h and 236 +/- 32 ng/24 h, respectively. Our findings with diets rich in EPA show that it is possible in man to change in vivo the spectrum of biologically active prostanoids by nutritional means and alter it in a favourable direction.     

Influence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n-3) and 22:6(n-3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produce HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB2 and TxB3, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyclo-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB2 in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB2 formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGI3, as judged from the appearance of 2,3-dinor-delta 17-6-keto-PGF1 alpha in urine. The amount of the major metabolite of PGI2, 2,3-dinor-6-keto-PGF1 alpha was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.     

Quantitative analysis of two dinor urinary metabolites of prostaglandin I2

The synthesis of deuterium- and tritium-labeled analogs of 2,3-dinor-6-keto-prostaglandin F_1α and of 6,15-diketo-13,14-dihydro-2,3-dinor-prostaglandin F_1α is described. These analogs were used as internal standards in the assay of the corresponding unlabeled metabolites in human urine by stable isotope dilution and combined gas chromatography-mass spectrometry. In male subjects the 24-h urinary excretion of the two metabolites was found to be 719 ± 264 and 314 ± 115 ng, respectively. The method offers a noninvasive approach to the study of prostaglandin I₂ synthesis in man.     

Metabolism of prostacyclin in rat.

Following a single intravenous administration of [11-3H]prostacyclin in rat, 77% of the administered dose was excreted within 3 days with 33% in urine and 44% in feces. Urinary metabolites were accumulated by chronic intravenous infusions of [11-3H]prostacyclin for 14 days. The drug was extensively metabolized and the structures of seven metabolites were elucidated by combined gas chromatography and mass spectrometry. The urinary products include the dinor and 19-hydroxy dinor derivatives of 6-keto-PGF1alpha and 13,14-dihydro-6,15-diketo-PGF1alpha, omega-hydroxy and omega-carboxyl dinor derivates of dihydro-6,15-diketo-PGF1alpha, and a dihydrodiketotetranordicarboxylic acid. The metabolic pathways of PGI2 in rat are similar to that of PGF2alpha.     

In vivo formation of metabolites of prostaglandins I2 and I3 in the marmoset monkey (Callithrix jacchus) following dietary supplementation with tuna fish oil

Recent studies have shown that ingestion of eicosapentaenoic acid (EPA) in man results in the formation of 'trienoic' prostanoids which amy partly explain the potent antithrombotic/antiatherogenic properties of long-chain polyunsaturated n-3 fatty acids (PUFAs). However, endogenous formation of cyclooxygenase metabolites of EPA has not been demonstrated in an animal model, and in vitro studies indicate a clear species difference in the conversion of EPA to PGI3. Thus, in the present study, the in vivo formation of PGI3 following long-term dietary tuna fish oil supplementation was investigated in a small non-human primate - the marmoset monkey (Callithrix jacchus). The excretion of major urinary metabolites 2,3-dinor-6-keto-PGF1 alpha (PGI2-M) and delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) was estimated as an index of total body synthesis of PGI2 and PGI3, respectively. Following extraction, dinor prostanoid metabolites were separated by capillary gas chromatography and identified by negative ion chemical ionization mass spectrometry. Supplementation of the standard (reference) diet with either sheep fat or sunflower seed oil did not alter the body production of PGI2-M. However, following the tuna fish oil-enriched diet, there occurred not only an increase in urinary PGI2-M (reference 70.7 +/- 9.0; tuna fish oil 115.5 +/- 12.1 ng/g creatinine, P less than 0.05), but also a considerable formation of PGI3-M (62.9 +/- 5.3 ng/g creatinine), which was not seen in any other dietary group; in addition, the urinary level of immmunoreactive 2,3-dinor-thromboxane B2/3 was reduced after ingestion of tuna fish oil. These urinary changes were accompanied by a rise in plasma phospholipid-bound EPA and docosahexaenoic acid (DHA). In addition, tuna fish oil supplementation resulted in a significant reduction in plasma cholesterol (53%) and triacylglycerols (44%). The present study provides for the first time experimental evidence for the in vivo formation of PGI3 in an animal model and also confirms the earlier observations in man following dietary fish oil supplementation.