Estimation of myocardial blood flow for longitudinal studies with 13N-labeled ammonia and positron emission tomography.

BACKGROUND: Although several modeling strategies have been developed and validated for quantification of myocardial blood flow (MBF) from 13N-labeled ammonia positron emission tomographic data, a comparison of noise characteristics of the various techniques in serial studies is lacking. METHODS AND RESULTS: Dynamic 13N-labeled ammonia positron emission tomographic imaging was performed at baseline and after pharmacologic stress in (1) single studies of four dogs with concomitant measurement of microsphere blood flow and (2) initial and follow-up studies of eight normal volunteers. Data were obtained from short-axis images for the blood pool and myocardial regions corresponding to the three arterial vascular territories. Indexes of MBF were obtained by four distinct techniques: (1) University of California, Los Angeles, two-compartment model, (2) Michigan two-compartment model, and (3) a one-compartment model with variable blood volume term. Coronary flow reserve (CFR) was measured as the ratio of stress/rest MBF. The estimated standard deviation of the measurement error for the relative change between studies of rest and stress MBF and CFR was determined for each technique. Estimates of MBF from all techniques showed good correlation with microsphere blood flow (r = 0.95 to 0.96) in canine myocardium. In human studies, similar mean estimates of MBF were found with all techniques. Techniques 1 and 3 showed the smallest interstudy variability in MBF and CFR. The estimated standard deviations for these techniques were approximately 20%, 30%, and 27% for rest MBF, stress MBF, and CFR, respectively. CONCLUSION: Noninvasive quantification of MBF and CFR from dynamic 13N-labeled ammonia positron emission tomography is most reproducible with technique 1 or 3. The ability to account for differences in myocardial partial volume gives preference to technique 3. However, substantial interstudy variability in regional MBF remains, suggesting the importance of procedural factors or real temporal fluctuations in MBF.

Lipoxin A4 metabolism by differentiated HL-60 cells and human monocytes: conversion to novel 15-oxo and dihydro products.

Lipoxins are tetraene-containing eicosanoids that possess biological activity in several organ systems. To determine their route of further metabolism, [11,12-3H]lipoxin A4 was prepared and incubated with human neutrophils, promyelocytic leukemia (HL-60) cells, and adherent monocytes. Intact neutrophils and undifferentiated HL-60 cells did not significantly metabolize [11,12-3H]LXA4, while HL-60 cells differentiated with PMA to monocyte/macrophage lineage rapidly (< 15 s) transformed this eicosanoid. The major radiolabeled LXA4-derived metabolites were characterized by physical methods and were shown to be 15-oxo-LXA4, 13,14-dihydro-15-oxo-LXA4, and 13,14-dihydro-LXA4. Substrate competition with cell-free supernatants from differentiated HL-60 cells suggests that lipoxins compete for 15-hydroxyprostaglandin dehydrogenase activity or an equivalent enzyme system. In addition, adherent monocytes exposed to [11,12-3H]LXA4 rapidly metabolized (> 60% within 30 s) the label to its oxo and dihydro derivatives. These results indicate that, unlike leukotrienes, LXA4 is subject to dehydrogenation and reduction of its conjugated tetraene to form triene-containing products. Moreover, they suggest that monocytes participate in lipoxin metabolism in their local milieu.

Effects on platelet aggregation and fibrinolytic activity during upright posture and exercise in healthy men.

The circadian variation of acute myocardial infarction suggests that daily activities such as assuming the upright posture and performing different daily activities may trigger the onset of coronary thrombosis. Such triggering may result from unfavorable alterations in the balance between the prothrombotic and antithrombotic properties of the blood. The present study compares the effects of 2 common daily activities, assuming the upright posture and exercise, on platelet aggregation and fibrinolytic activity. In healthy male subjects, assuming the upright posture in the morning significantly increased platelet aggregation and produced only a moderate increase in fibrinolytic activity within 10 minutes. These changes were still present after 90 minutes in the upright posture. Supine posture for 45 minutes resulted in levels of fibrinolytic activity and platelet aggregation comparable to that observed before initially assuming the upright posture in the morning. Return to the supine posture for 45 minutes resulted in levels of fibrinolytic activity and platelet aggregation comparable to that observed before the initial assumption of upright posture. The changes recurred when upright posture was taken later in the day. Exercise did not increase platelet aggregation to levels beyond that produced by the upright posture, but was associated with a marked increase in fibrinolytic activity. Thus, exercise and upright posture produce distinctive alterations in the thrombogenic potential of the blood that may influence the timing of clinical vascular events.

Angioplasty triggers intracoronary leukotrienes and lipoxin A4. Impact of aspirin therapy.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) is a widely used and important method of reperfusing coronary arteries. However, it is also associated with serious complications such as acute reocclusion and accelerated restenosis. The factors as well as the mechanisms involved in PTCA-associated complications remain to be fully elucidated. Because peptidoleukotrienes and lipoxins are potent vasoactive compounds, the formation of which is not inhibited by aspirin (ASA) treatment in vitro, it is possible that these eicosanoids are involved in PTCA-associated untoward events. To test this, we determined the intracoronary levels of peptidoleukotrienes and lipoxin A4 (LXA4) as well as thromboxane (TX) and 5S,12S-dihydroxyeicosatetraenoic acid (5S,12S-DiHETE; a product of double dioxygenation) after plaque rupture and evaluated the impact of ASA therapy. METHODS AND RESULTS: PTCA was performed on 12 patients with coronary artery disease, six undergoing ASA therapy and six without ASA therapy, for at least 2 weeks before PTCA. By means of a technique that permitted sampling of intracoronary blood at the plaque site in situ, samples were taken immediately before and 10 seconds after initiation of plaque rupture. Lipoxygenase (LO)-derived products, including LXA4 and 5S,12S-DiHETE, and a marker of cyclooxygenase activity, i.e., TXB2, were quantitated after extraction and chromatography using deuterium-labeled internal standards and electron capture negative ion chemical ionization mass spectrometry. Peptidoleukotrienes (LTC4 and LTD4) were quantitated after reverse-phase high-performance liquid chromatography coupled with radioimmunoassay. Intracoronary blood taken before PTCA showed no detectable levels of these eicosanoids (the minimum limits of detection were within the picomole range). In contrast, each of these LO products was detected after PTCA. Patients undergoing ASA treatment showed elevated levels of each LO product examined compared with those not receiving ASA. Eicosanoid levels were (mean +/- SEM): LTC4, 7.10 +/- 1.22 ng/ml (ASA) versus 0.48 +/- 0.10 ng/ml; LTD4, 4.92 +/- 0.56 ng/ml (ASA) versus 1.17 +/- 0.48 ng/ml; LXA4, 24.98 +/- 4.11 ng/ml (ASA) versus 15.83 +/- 2.43 ng/ml; 5S,12S-DiHETE, 19.47 +/- 3.98 ng/ml (ASA) versus 11.98 +/- 1.83 ng/ml; TXB2, complete blockage (ASA) versus 31.04 +/- 7.38 ng/ml (p less than 0.05 for LTC4 and LTD4). To distinguish between dilatation of whole blood versus dilatation of whole blood and atheroma for contribution of eicosanoids, we also monitored their formation in Gore-tex grafts. Upon balloon inflation, TXB2 was generated, but LO products were not detected. In contrast, injection of platelet- and leukocyte-directed agonists within the graft led to both peptidoleukotriene and lipoxin formation. CONCLUSIONS: The results indicate that PTCA triggers the intraluminal release of peptidoleukotrienes and LXA4 and that ASA therapy enhances their appearance in intracoronary blood. In addition, they provide direct evidence for LO products (LTC4, LTD4, and LXA4) in a local milieu in vivo. Moreover, the presence of the double dioxygenation product 5S,12S-DiHETE (a potential marker of 5- and 12-LO interactions) suggests that transcellular metabolic events can contribute to eicosanoid formation in vivo.

Platelet alpha 2 adrenoceptor characteristics during the morning increase in platelet aggregability.

The incidence of myocardial infarction and sudden cardiac death increases in the morning, as do platelet aggregability and sympathetic activity. We considered the possibility that increased platelet aggregability in the morning was due to an increase in platelet alpha 2 adrenergic receptor number or agonist binding affinity, or to a temporal disparity between the increase in sympathetic activity that accompanies arising and the anticipated decrease in platelet high affinity alpha 2 adrenoceptors. To evaluate these possibilities, we studied eight healthy male volunteers (20-35 years) before and 1-1.5 and 3-4 h after they arose at 08.00 h and performed routine morning activities. Platelet aggregability was determined in platelet-rich plasma; alpha 2 adrenoceptor density and agonist binding affinity were determined in intact platelets and in membrane preparations using the alpha 2 selective ligand [3H]-yohimbine. The threshold concentration of epinephrine required to produce biphasic aggregation decreased (i.e. platelet aggregability increased) from 3.6 +/- 1.2 to 0.9 +/- 0.3 microM (P less than 0.05) after arising. However, alpha 2 adrenoceptor density (380 +/- 71 to 365 +/- 51 sites per platelet) and agonist binding affinity assessed simultaneously did not change after arising, suggesting that the increase in platelet aggregability is due to factors extrinsic to the platelets or to an intra-platelet mechanism distal to the receptor level.

Characterization of lipoxins by combined gas chromatography and electron-capture negative ion chemical ionization mass spectrometry: formation of lipoxin A4 by stimulated human whole blood.

The lipoxins are a recent addition to the family of bioactive products derived from arachidonic acid. Here, we have prepared pentafluorobenzyl ester, trimethylsilyl ether derivatives of lipoxin A4, lipoxin B4 and pentadeuterolipoxin A4 and have characterized these products by electron-capture negative ion chemical ionization gas chromatography/mass spectrometry (NICI GC/MS). Lipoxin A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosa-tetraenoic acid; LXA4) was quantified following extraction from whole blood by stable isotopic dilution utilizing deuterium-labeled LXA4 as internal standard and selected ion monitoring of the [M--pentafluorobenzyl] anions. Studies with a second tritiated internal standard (e.g. [11,12-3H]LXA4) also showed that the recovery of LXA4 was greater than 80% following solid-phase extraction from whole blood, and greater than 90% from isolated cells. In addition, neither isolated neutrophils nor platelets oxidatively metabolized [11,12-3H]LXA4 when incubated in the presence or absence of stimuli. Whole blood incubated with either the ionophore of divalent cations (A23187), thrombin, or thrombin plus the chemotactic peptide formylmethionyl-leucine-phenylalanine generated both LXA4 and thromboxane, which were quantified by stable isotope dilution. The ratio of thromboxane to LXA4 formed by stimulated whole blood ranged from approximately 2:1 to 20:1. These results indicate that the lipoxins display suitable characteristics as their respective pentafluorobenzyl ester, trimethylsilyl ether derivatives for quantification by electron-capture NICI GC/MS. Moreover, they provide evidence that LXA4 can be generated from endogenous sources in whole blood following exposure to physiologically relevant stimuli.

Morning increase in platelet aggregability. Association with assumption of the upright posture.

The frequencies of onset of myocardial infarction and sudden cardiac death are increased between 6 AM and 12 noon. Platelet aggregability, which may play a role in the cause of these disorders, has been observed to increase after the normal morning activities of awakening, arising, and ambulating. To determine which morning activity or activities are responsible for this aggregability increase, we measured platelet aggregation in 16 normal subjects on a control day of delayed arising (i.e., subjects remained supine until 12:30 PM) and on a day in which normal morning activities were divided into three isolated components of awakening (8 AM), assumption of upright posture (9:30 AM), and ambulating (11 AM). Blood samples to assess platelet aggregability were drawn at 8 AM before activity and 90 minutes after the initiation of each activity (i.e., at 9:30 AM, 11 AM, and 12:30 PM). For the group, in vitro platelet responsiveness to adenosine diphosphate and epinephrine increased only after assumption of the upright posture. The lowest concentration of agonist required to produce biphasic platelet aggregation decreased (aggregability increased) between 9:30 and 11 AM (90 minutes after assumption of the upright posture) from 3.3 +/- 0.3 to 2.4 +/- 0.2 microM for adenosine diphosphate (p less than 0.05) and from 2.1 +/- 0.5 to 1.0 +/- 0.4 microM for epinephrine (p less than 0.05). During the same interval, plasma epinephrine increased from 34 +/- 7 to 55 +/- 9 pg/ml (p less than 0.05), and plasma norepinephrine increased from 169 +/- 19 to 298 +/- 25 pg/ml (p less than 0.01). There was no significant change in aggregability or catecholamine concentrations on the control day.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of rabbit nasal and hepatic cytochrome P-450-dependent hexamethylphosphoramide (HMPA) N-demethylase by methylenedioxyphenyl compounds.

Eighteen methylenedioxyphenyl (MDP) compounds, including some commonly inhaled by people, were tested for the ability to inhibit rabbit nasal microsomal cytochrome P-450-dependent hexamethylphosphoramide (HMPA) N-demethylase. For comparison, liver microsomes were also used. Nasal cytochrome P-450 from rabbits metabolized MDP compounds to form cytochrome P-450-metabolite (P-450-MI) complexes as indicated by difference spectra in the Soret region. Several of the MDP compounds were potent inhibitors of nasal P-450-dependent N-demethylase. If inhibition of nasal P-450 also occurs in vivo after inhibiting MDP compounds are inhaled, the metabolism of concurrently or subsequently inhaled compounds may be altered.