8-epi-prostaglandin (PG) F2 alpha, a major F2 isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF2 alpha as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF2 alpha in vivo is mostly COX-independent and urinary 8-epi-PGF2 alpha is therefore an accurate marker of 'basal' oxidative stress in vivo. 2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF2 alpha vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg-1 day-1; indomethacin: 4 mg kg-1 day-1) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF2 alpha was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry. 3. Naproxen reduced urinary excretion of 2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha but, unexpectedly, also that of 8-epi-PGF2 alpha (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF2 alpha in liver and plasma remained unchanged after indomethacin. 4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF2 alpha in man. We gave naproxen (1 g day-1) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF2 alpha remained unchanged in the two groups (9.63 +/- 0.99 before vs 10.24 +/- 1.01 after and 20.14 +/- 3.00 vs 19.03 +/- 2.45 ng h-1 1.73 m-2), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB2 and 2,3-dinor-TXB2; > 50% for 2,3-dinor-6-keto-PGF1 alpha). 5. To investigate whether rat COX-1 produces 8-epi-PGF2 alpha more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF2 alpha and TXB2 simultaneously in whole clotting blood. Serum levels of 8-epi-PGF2 alpha and TXB2 were similar in rats and man. 6. We conclude that a significant amount of COX-dependent 8-epi-PGF2 alpha is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF2 alpha cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.
摘要
8-表前列腺素（PG）F2α是一种主要的F2异前列腺素，在体内由脂质酯化花生四烯酸的自由基依赖性过氧化反应产生。两种环氧化酶同工型（COX-1和COX-2）也可能作为次要产物形成游离的8-表PGF2α。最近在人类志愿者中发现，体内8-表PGF2α的总体基础形成大多不依赖于COX，因此尿8-表PGF2α是体内“基础”氧化应激的准确标志物。2. 为了在大鼠中测试该标志物的有效性，我们在体内评估了COX抑制对8-表PGF2α与前列腺素形成的影响。将两种结构不相关的COX抑制剂（萘普生：30mg/kg/天；吲哚美辛：4mg/kg/天）腹腔注射给饲养在代谢笼中的大鼠。通过测量其尿排泄量来评估8-表PGF2α的体内形成。通过测量血栓素（TX）和前列环素的主要代谢产物（2,3-二去甲TXB1和2,3-二去甲-6-酮-PGF1α）的尿排泄量来评估前列腺素生物合成。所有化合物均通过免疫纯化/气相色谱-质谱法进行选择性测量。3. 萘普生降低了2,3-二去甲TXB1和2,3-二去甲-6-酮-PGF1α的尿排泄量，但出乎意料的是，也降低了8-表PGF2α的尿排泄量（分别抑制82%、49%和52%）。吲哚美辛有类似的效果（抑制77%、69%和55%）。吲哚美辛处理后，肝脏和血浆中酯化的8-表PGF2α保持不变。4. 这些发现促使我们重新评估COX活性对人体8-表PGF2α全身产生的贡献。我们给健康受试者（四名非吸烟者和四名吸烟者）服用萘普生（1g/天）。两组尿8-表PGF2α均保持不变（服药前9.63±0.99，服药后10.24±1.01；以及20.14±3.00 vs 19.03±2.45ng/h/1.73m²），而血栓素和前列环素的主要尿代谢产物显著减少（11-脱氢-TXB2和2,3-二去甲-TXB2均约减少90%；2,3-二去甲-6-酮-PGF1α减少>50%）。5. 为了研究大鼠COX-1是否比人COX-1更有效地产生8-表PGF2α，我们在全凝血血液中同时测量了体外培养的8-表PGF2α和TXB2的形成。大鼠和人血清中8-表PGF2α和TXB2的水平相似。6. 我们得出结论，在正常条件下，大鼠尿液中存在大量依赖COX的8-表PGF2α，而人尿液中则不存在。这意味着尿8-表PGF2α不能用作大鼠近基础氧化应激的指标。另一方面，我们的数据进一步证实了该标志物在人体中的有效性。
