Martin L. Ogletree

Effect of N-acetylcysteine on the pulmonary response to endotoxin in the awake sheep and upon in vitro granulocyte function.

Oxygen free radicals released during endotoxemia may contribute to the lung injury of the adult respiratory distress syndrome (ARDS). As this syndrome occurs frequently after gram-negative sepsis in humans, we studied the effect of intravenous N-acetylcysteine (NAC), a free radical scavenger, upon the endotoxin (E)-induced model of ARDS in awake sheep. In vivo studies demonstrated that NAC attenuates the endotoxin-induced rise in pulmonary artery pressure (62 +/- 3 torr with E control vs. 43 +/- 3 torr for E + NAC), and markedly diminishes the rise in lymph flow at 1 h (8.5 +/- 1.2 vs 4.5 +/- 0.6 ml/15 min) and 4 h (5.0 +/- 0.6 vs. 3.3 +/- 0.4 ml/15 min), respectively, for E control vs. E + NAC. NAC also markedly attenuated the alterations in lung mechanics after endotoxemia. Dynamic compliance at 2 h after endotoxemia was 44 +/- 6% of base line for E vs. 76 +/- 10% of base line for E + NAC. Resistance to airflow across the lung at 1 h postendotoxin was 811 +/- 280% of base line for E vs. 391 +/- 233% of base line for E + NAC. NAC substantially reduced the 1 h postendotoxin rise in lymph concentrations of thromboxane B2 (8.29 +/- 3.28 vs. 2.75 +/- 1.93 ng/ml for E vs. E + NAC) and 6-keto-prostaglandin-F1 alpha (0.91 +/- 0.27 vs. 0.23 +/- 0.12 ng/ml for E vs. E + NAC). In addition, in vitro studies were performed which revealed NAC to be a potent free radical scavenger in both biologic and nonbiologic free radical generating systems. NAC decreased phorbol-stimulated granulocyte aggregation in a concentration-dependent manner in vitro. Minimal effects were observed, however, upon leukocyte degranulation at the concentrations of NAC achieved during the in vivo tests. Thus, NAC significantly attenuated all monitored pathophysiologic changes in the endotoxin model of ARDS in sheep, possibly by its ability to scavenge toxic oxygen free radicals. A direct impairment of the ability of inflammatory cells to generate oxygen radicals cannot be ruled out.

Effects of cyclooxygenase inhibitors on the alterations in lung mechanics caused by endotoxemia in the unanesthetized sheep.

The effects of Escherichia coli endotoxin on lung mechanics, hemodynamics, gas exchange, and lung fluid and solute exchange were studied in 12 chronically instrumented unanesthetized sheep. A possible role for cyclooxygenase products of arachidonate metabolism as mediators of the endotoxin-induced alterations in lung mechanics was investigated by studying sheep before and after cyclooxygenase inhibition with sodium meclofenamate and ibuprofen. Sheep were studied three times in random order: (a) sodium meclofenamate (or ibuprofen) infusion alone; (b) E. coli endotoxin alone; and (c) meclofenamate (or ibuprofen) and endotoxin. Meclofenamate alone had no effect on any of the variables measured. Endotoxin alone caused early marked changes in lung mechanics: resistance to airflow across the lungs (RL) increased 10-fold, dynamic lung compliance (Cdyn) decreased 80% and functional residual capacity (FRC) decreased by greater than 30%. The alveolar-to-arterial oxygen difference (delta AaPO2) increased markedly following endotoxemia. In the presence of sufficient meclofenamate to inhibit accumulation of thromboxane-B2 and 6-keto-prostaglandin F1 alpha in lung lymph, endotoxin caused no increase in RL, Cdyn decreased by less than 40%, and FRC decreased by only 6%. Meclofenamate significantly attenuated the hypoxemia and early pulmonary hypertension caused by endotoxemia but had no effect on the late increases in lung fluid and solute exchange. Ibuprofen had similar effects to those observed with meclofenamate. We conclude that both the pulmonary hypertension and changes in lung mechanics observed after endotoxemia may be mediated, at least in part, by constrictor prostaglandins or thromboxanes and that gas exchange may be improved by preventing endogenous synthesis of these mediators.

Evidence for pulmonary release of 5-hydroxyeicosatetraenoic acid (5-HETE) during endotoxemia in unanesthetized sheep☆

Leukocyte trapping in the pulmonary circulation may be an important component of the lung vascular injury response to endotoxin, but mediators of the pulmonary leukostasis and increased lung vascular permeability are unknown. The leukocyte 5-lipoxygenation pathway of arachidonic acid metabolism yields highly biologically active products including leukotrienes C4 and D4 (formerly slow reacting substance of anaphylaxis) and the potent chemotaxin, leukotriene B4. A major product of 5-lipoxygenation is 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), for which a sensitive, stable isotope dilution assay employing combined gas chromatography-mass spectrometry is available. This assay was used to test the hypothesis that 5-lipoxygenation products might participate in pulmonary vascular responses to endotoxin. We measured 5-HETE concentrations in lung lymph at three intervals during endotoxemia in unanesthetized sheep. Concentrations of 5-HETE in lung lymph exceeded those in aortic blood plasma. Lymph 5-HETE concentrations increased from 1.7 +/- 0.3 (mean +/- SEM, N = 7) ng/ml during baseline to peak values of 6.1 +/- 1.8 ng/ml (p less than 0.05) during the 2 1/2 hours after endotoxemia and preceeding the steady state increased lung vascular permeability response. During the increased permeability steady state from 240 to 270 minutes after endotoxin, lymph 5-HETE concentrations (1.4 +/- 0.3 ng/ml) and lymph 5-HETE flow (i.e., 5-HETE concentration x lung lymph flow rate) returned to baseline values. Although these observations are consistent with the hypothesis that 5-lipoxygenation products participate in the pulmonary vascular injury response to endotoxin, lymph 5-HETE concentrations did not correlate with any of the other experimental measurements. It may be only coincidence that the increase in lymph 5-HETE concentrations appeared contemporaneous with the onset of lung vascular injury.

Effects of platelet depletion on the unanesthetized sheep's pulmonary response to endotoxemia.

The effect of platelet depletion on the unanesthetized sheeps pulmonary response to endotoxemia was studied in eight unanesthetized sheep. Platelets were depleted with rabbit anti-sheep platelet antibodies (APA). Bolus injections of APA alone caused marked pulmonary hypertension (PPA increased from 21 +/- 2 to 62 +/- 5 cm H2O +/- SE) and alterations in lung mechanics (dynamic compliance of the lung [Cdyn] decreased to 38.5 +/- 4.6% and resistance to air flow across the lung [RL] increased to 705 +/- 162% +/- SE of control), which were attenuated by pretreatment with meclofenamate. It was possible to deplete platelets before endotoxemia through a slow continuous infusion of APA without altering base-line values of the measured variables. Platelet depletion did not significantly attenuate the alterations in pulmonary hemodynamics, lung mechanics, lung fluid and solute exchange, or the normal increase in lung lymph concentrations of thromboxane B2 or 6-keto-PGF1 alpha observed following endotoxemia in the sheep. We conclude that normal circulating platelet counts are not required for the full expression of the sheeps response to endotoxemia.

Effects of cyclooxygenase inhibition on the response to group B streptococcal toxin in sheep.

Summary: The effects of cyclooxygenase inhibition on the reaction to a toxin isolated from group B β-hemolytic streptococci, type III, were studied in seven sheep instrumented for chronic measurement of pulmonary lymph flow, pulmonary artery and left atrial pressures. Each sheep was infused with toxin alone on one day and with indomethacin plus toxin on a different day in random order. The toxin alone caused a two-phased reaction. After the infusion of toxin, alone, in the initial phase, pulmonary artery pressure increased from 16.5 ± 1.2 mmHg to 47.1 ± 4.8 mmHg and the rectal temperature rose from 39.7 ± 0.13°C to 40.9 ± 0.16°C. During the second phase, the granulocyte count decreased to less than 10% of baseline values and the lymph protein clearance increased from 4.8 ± 1.2 ml/h to 10.02 ± 1.4 ml/h, suggesting increased pulmonary vascular permeability. Indomethacin pre-treatment prevented the initial phase of pulmonary hypertension, the increases in thromboxane and prostacyclin metabolites in lung lymph, and the febrile response to toxin infusion but did not modify the granulocytopenia or the increased pulmonary vascular permeability. It appears that the hemodynamic changes are independent from the pulmonary vascular changes, and that prostaglandin endoperoxides or their metabolites are necessary for the fever and the acute pulmonary hypertension.

PHARMACOLOGY OF PROSTAGLANDINS IN THE PULMONARY MICROCIRCULATION

In unanesthetized sheep, arachidonate infusion increases lung microvascular pressure and augments pulmonary transvascular filtration without altering lung vascular permeability. Pulmonary vascular effects of arachidonate are caused by its conversion to highly active cyclooxygenase products, including PGH2 and, perhaps, thromboxane A2. Cyclooxygenase blockers inhibit pulmonary vascular actions of arachidonate. Most prostaglandins derived from PGH2 [i.e., PGB2, PGD2, PGE, and PGF2 alpha] are vasoconstrictors that exhibit potency intermediate between PGH2 and arachidonate. Vasoconstrictor prostaglandins cause increased flow of protein-poor lung lymph with no indication of altered lung vascular permeability. Vasodilator prostaglandins [i.e., PGE1, PGI2] cause changes in lung lymph formation that are not explained by microvascular hydrostatic-pressure effects alone. Whether lung lymph responses to PGE1 and PGI2 resulted from changes in perfused pulmonary vascular surface area or vascular permeability effects was tested with infusions during near-maximal pulmonary vascular recruitment with increase pressure. Maintaining mechanically increased left atrial pressure constant should minimize changes in lung lymph formation due to pulmonary vascular recruitment and derecruitment, and exaggerate lymph responses due to changes in vascular permeability. When infused during mechanically increased pressure, PGE1 and PGI2 produce similar pressure-mediated decreases in steady-state lung lymph flow, despite increases in pulmonary blood flow, heart rate, and respiratory rate with PGI2 and not with PGE1. In conclusion, PGE1 and PGI2 cause hemodynamic changes that can increase [PGI2] or decrease [PGE1] lung lymph flow. Prostaglandins do not directly alter lung vascular permeability.

Plasma 6-keto prostaglandin F1α and thromboxane B2 in sick preterm neonates

To determined if vascular abnormalities in preterm neonates might be related to vasoactive prostaglandins, stable prostacyclin(6-KPGF1α)andthromboxaneA2(TxB2) metabolites in arterial blood were measured at < 6 hours after birth and at 24, 48, and 72 hours using a radioimmunoassay. Neonates of <32 weeks gestation (N=26) were diagnosed as having either the idiopathic respiratory distress syndrome (IRDS, N=15) or pulmonary edema (PE, N=11), and were also grouped according to the presence or absence of intracranial hemorrhage (ICH, N=11) or patent ductus arteriosus (PDA, N=10). Initial plasma 6-KPGFla was greater in neonates with ICH (0.23 ± 0.04 ng/ml, mean + SE) than without ICH (0.11 ± 0.04, p < 0.05). Neonates with both ICH and IRDS (N=8)had significantly elevated TxB2 at all sampling times compared to neonates with IRDS and no ICH (N=7). Both TxB2 and 6-KPGFla increased with time in those with major ICH. Among neonates without ICH, 7 with IRDS had higher initial 6-KPGFla (0.19 + 0.07 ng/ml) and lower TxB2(0.15 + 0.04 ng/ml) than 8 with PE (0.04 +0.01 and 0.37 + 0.09 ng/ml, respectively).The initial 6-KPGFlα (0.024 + 0.003 ng/ml), measured in neonates with PE and without PDA or ICH (N=6), was significantly less than the corresponding value in the other neonates (0.201 + 0.036 ng/ml) (N=20).

Understanding the role of prostaglandin E2 in regulating human platelet activity in health and disease.

The platelet thrombus is the major pathologic entity in acute coronary syndromes, and antiplatelet agents are a mainstay of therapy. However, individual patient responsiveness to current antiplatelet drugs is variable, and all drugs carry a risk of bleeding. An understanding of the complex role of Prostaglandin E2 (PGE2) in regulating thrombosis offers opportunities for the development of novel individualized antiplatelet treatment. However, deciphering the platelet regulatory function of PGE2 has long been confounded by non-standardized experimental conditions, extrapolation of murine data to humans, and phenotypic differences in PGE2 response. This review synthesizes past and current knowledge about PGE2 effects on platelet biology, presents a rationale for standardization of experimental protocols, and provides insight into a molecular mechanism by which PGE2-activated pathways could be targeted for new personalized antiplatelet therapy to inhibit pathologic thrombosis without affecting hemostasis.

Effects of Methylprednisolone on the Response to Group B Streptococcal Toxin in Sheep

ABSTRACT. The effects of pretreatment with methylprednisolone on the reaction to a toxin isolated from group β-hemolytic streptococci, type III, were studied in seven sheep instrumented for chronic measurements of pulmonary lymph flow and pulmonary artery and left atrial pressure. Each sheep was infused with toxin alone on one day and with methylprednisolone plus toxin on a different day in random order. The toxin alone caused a two-phase reaction. After the infusion of toxin, alone, in the initial phase, pulmonary artery pressure increased from 16 ± 1 to 45 ± 5 mm Hg and the rectal temperature rose from 39.5 ± 0.14 to 40.8 ± 0.18° C. During the second phase, the peripheral blood granulocyte count decreased to 10% of baseline values and the lung lymph protein clearance increased from 5.1 ± 1.1 to 11.2 ± 1.8 ml/h, suggesting increased pulmonary vascular permeability. Methylprednisolone pretreatment did not alter the initial phase of pulmonary hypertension or the febrile response but completely abolished the granulocytopenia and the increased pulmonary vascular permeability. These effects are unlikely to be related to inhibition of prostaglandin synthesis. Prevention of the lung vascular injury by methylprednisolone may be related to inhibition of granulocyte accumulation in the lung.

[72] Measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) in biological fluids by GCMS

Publisher Summary This chapter presents the highly sensitive and selective assay for 5-hydroxyeicosatetraenoic acid (5-HETE) employing racemic octadeuterated 5-HETE for stable isotope dilution measurements with quantitation via combined gas-liquid chromatography-mass spectrometry (GC–MS). Measurement of 5-HETE is employed as an index of 5-1ipoxygenation of arachidonic acid. Octadeuterated arachidonic acid is prepared from 5,8,11,14-eicosatetraynoic acid. Two independent methods are employed for standardization of ds-5- HETE, because the tendency of 5-HETE to form δ-lactone. Ultraviolet spectrophotometry at , λ MeOH max = 235 nm and gas–liquid chromatography with tricosanoic acid as internal standard should give agreement within ±5%. In purification by reversed-phase high performance liquid chromatography (HPLC), solvent is evaporated from the sample extract under a stream of nitrogen, and the residue is dissolved in a small volume of methanol for purification by reversed-phase HPLC. 5-HETE is eluted from μBondapak C18 column with a solvent of methanol-water -glacial acetic acid delivered at a flow rate of 1-2 ml/min.