A role for nitric oxide in X-ray contrast material toxicity.

RATIONALE AND OBJECTIVES: We assessed the role that nitric oxide (NO) plays in contrast media (CM) toxicity, using 100% lethal dose (LD100) studies in hyperimmune Brown Norway (BN) rats. METHODS: Ninety-two BN rats and 41 Sprague-Dawley (SD) rats underwent CM LD100 tail vein injections with methylglucamine iothalamate or sodium iothalamate to the point of cessation of respiration. Methylglucamine hydrochloride also was injected. The injections were accompanied by L-arginine (L-Arg) or D-arginine (D-Arg) analogues or by an H1 blocker. L-Arg analogues inhibit NO formation, and D-Arg analogues do not. RESULTS: An L-Arg analogue, but not a D-Arg analogue, increased the tolerance of BN rats (p < .005) for methylglucamine iothalamate but not for sodium iothalamate. The L-Arg analogue also protected BN rats against methylglucamine chloride injections (p < .002). H1 blockade protected BN rats against methylglucamine iothalamate (p < .0005) and methylglucamine chloride (p < .005) injections. None of these measures altered the CM tolerance of SD rats. In SD rats, injections of either methylglucamine iothalamate or sodium iothalamate along with a D-Arg analogue or normal saline were better tolerated than similar injections in BN rats (p < .01 and .002 for methylglucamine iothalamate and sodium iothalamate, respectively). In SD rats but not BN rats, sodium iothalamate was better tolerated than was methylglucamine iothalamate (p < .0005). CONCLUSION: NO appears to play a significant role in BN rats LD100 CM toxicity and has been implicated by others in the blood pressure fall characterizing some forms of antigen-induced anaphylaxis [1, 2]. The results of the current study and the literature suggest that methylglucamine-modulated release of histamine from mast cells may underlie the NO production.

Effects of probucol on plasma lipids, lipoproteins and parameters of high density lipoprotein metabolism.

Eight patients with primary hypercholesterolemia were treated with probucol for 17 weeks. Plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, and high density lipoprotein (HDL)-cholesterol decreased by 16.6, 15.0 and 25.7%, respectively, in response to probucol treatment. Plasma levels of apolipoprotein B and apolipoprotein A-I also decreased, while apolipoprotein A-II concentrations were unchanged. The decrease in HDL-cholesterol levels was associated with a reduction in HDL particle size. No changes in the plasma lecithin:cholesterol acyltransferase activity or mass occurred in response to probucol treatment. In contrast, a significant 25% increase in plasma cholesteryl ester and triglyceride transfer activity occurred following probucol treatment. There was a positive correlation (R = 0.94) between cholesterol ester and triglyceride transfer. We propose that the increase in lipid transfer activity may in part explain the changes in HDL concentration and size, as well as the previously reported effect probucol has on reducing atherosclerosis in animal models.

Protein kinase C inhibitors block the enhanced expression of intercellular adhesion molecule-1 on endothelial cells activated by interleukin-1, lipopolysaccharide and tumor necrosis factor.

Interleukin 1 (IL-1), bacterial lipopolysaccharide (LPS) and tumor necrosis factor (TNF alpha) enhance the adherence properties of endothelial cells (EC) for neutrophils (PMN). This is mediated in part by the up-regulation of Intercellular Adhesion Molecule 1 (ICAM-1) on EC. Phorbol esters, which activate protein kinase c (PKC) and enhance the adherence properties of EC for PMN also up-regulate the ICAM-1 expression on EC. We investigated the effect of PKC inhibitors on ICAM-1 expression of human umbilical vein EC (HUVEC). Staurosporine (STS) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented inflammatory mediator-dependent stimulation of both ICAM-1 expression and PMN adherence by HUVEC (ID50 for STS = 2.7-2.9 microM; for H-7 = 7.6-8.8 microM). Inhibition was dose and time-dependent and was not due to HUVEC toxicity. The STS analog K252a and the H-7 analog W-7 were less potent inhibitors of ICAM-1 up-regulation and adherence promotion. Prolonged exposure of HUVEC to phorbol myristate acetate down-regulated PKC activity and inhibited subsequent ICAM-1 up-regulation by this agent and by IL-1. We conclude that inflammatory mediator induced stimulation of HUVEC expression of ICAM-1 and promotion of adherence properties are mediated in part by activation of PKC.

Modulation of endothelial cell expression of intercellular adhesion molecule 1 by protein kinase C activation.

Inflammatory stimuli enhance the adherence properties of human umbilical vein endothelial cells (HUVEC) for neutrophils (PMN). This is mediated in part by the up-regulation on HUVEC of Intercellular Adhesion Molecule 1 (ICAM 1). Phorbol esters, which activate protein kinase c (PKC), have also been reported to enhance the adherence properties of HUVEC for PMN. We investigated the effect of agents which activate PKC on the expression of ICAM 1 by HUVEC. Both phorbol myristate acetate (PMA) and Mezerein, a non-phorbol which also stimulates PKC, enhanced both the expression of ICAM 1 on HUVEC and the adherence of HUVEC for PMN. The PKC inhibitors staurosporine and H-7 prevented both PMA and Mezerein-induced stimulation of HUVEC expression of ICAM 1 and adherence for PMN. We conclude that activation of PKC in HUVEC is associated with increased expression of ICAM 1 on HUVEC. PKC-mediated up-regulation of ICAM 1 may be responsible, in part, for the promotion of endothelial cell adherence properties toward PMN.

Chemotaxin receptor cycling in fresh and stored granulocytes.

The authors reported previously that stored granulocytes (PMN) had decreased receptor affinity (Kd) for and chemotaxis (CTX) to the chemotactic peptide F-Met-Leu-Phe (FMLP), but the evidence did not favor a significant role for altered FMLP receptor affinity in causing diminished CTX of stored PMN. Since recruitment and/or recycling of FMLP receptors is required for normal CTX, the hypothesis that stored PMN might have abnormal FMLP receptor cycling was tested. The effect of storage on the proportion of high- and low-affinity FMLP receptors was also investigated. Units of PMN were tested within 4 hours of collection and after 24 and 48 hours of storage at 22 degrees C, unagitated, in 150-ml transfer packs. In comparison to fresh PMN, there was no alteration in the Kd of the high-affinity FMLP receptor of PMN stored for 24 to 48 hours; however, the Kd of the low-affinity receptor increased (fresh PMN = 36 +/- 5 nM; 24 hours = 107 +/- 19; 48 hours = 121 +/- 22; p less than 0.01 for both 24 and 48 h versus fresh PMN). Likewise, while the number of high-affinity receptors increased (fresh PMN = 25,000 +/- 6,000 receptors/PMN; 24 hours = 95,000 +/- 21,000; 48 hours = 161,000 +/- 40,000; p less than 0.01 for both 24 and 48 hours versus fresh PMN). No abnormality was found in the ability of stored PMN to down-regulate FMLP receptors in the presence of ligand or to reexpress FMLP receptors after a 15-minute incubation in the absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Adherence of fresh and stored granulocytes to endothelial cells. Effect of storage temperature.

Granulocyte (PMN) concentrates collected for transfusion to septic, neutropenic patients are stored in the blood bank for various periods of time before they are given. Current methods of blood bank storage of PMN concentrates are associated with impaired in vitro PMN chemotaxis (CTX) and in vivo recovery and circulation kinetics after 24 hours of storage. This suggested the possibility that PMN may become hyperadherent during storage. To test this hypothesis, PMN concentrates were harvested and stored at both 22 and 6 degrees C and their adherence properties to relevant biologic surfaces, endothelial cell (EC) monolayers, and extracellular matrix (ECM) derived from endothelium were measured. Adherence was measured within 4 hours of collection and after 24 and 48 hours of storage. The aggregation properties of fresh and stored PMN were also studied. The adherence of fresh, unstimulated PMN to EC and ECM (31 +/- 5% and 34 +/- 4%, respectively) increased significantly after storage for 24 hours (EC = 41 +/- 8%; ECM = 43 +/- 4%) at 22 degrees C. F-Met-Leu-Phe (FMLP) stimulated the adherence of fresh PMN (EC = 37 +/- 4%; ECM = 42 +/- 4%; p less than 0.05). The adherence of PMN stored at 22 degrees C was further stimulated by FMLP (EC = 46 +/- 6%; ECM = 50 +/- 4%). PMN stored at 6 degrees C had significantly higher adherence than PMN stored at 22 degrees C, and the percentage of increase in adherence induced by FMLP was attenuated in PMN stored at 6 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulus-response coupling in fresh and stored granulocytes.

Granulocytes stored in the blood bank prior to transfusion undergo progressive decrements in their ability to circulate and migrate in vivo and to migrate in vitro (chemotaxis). The pathogenesis of granulocyte (PMN) chemotaxis (CTX) dysfunction after room-temperature storage of PMN is unclear. Previous work in the authors' laboratory and others led to the hypothesis that intracellular transmission of chemotactic signals, referred to as stimulus-response coupling (SRC), might be abnormal in stored PMN. This report presents an investigation of the ability of fresh and stored PMN to generate and respond to leukotriene-B4 (LTB4), the chief intracellular amplifier of SRC for CTX. PMN were sampled from concentrates within 4 hours of collection and after 24 and 48 hours of storage in transfer packs at room temperature (RT). Fresh, stimulated PMN synthesized 202 +/- 51 ng of LTB4 and 110 +/- 11 ng of 5-hydroxyeicosatetraenoic acid (HETE) per 10(7) PMN. Synthesis of LTB4, but not HETE was significantly decreased after 24 hours' storage, and LTB4 and HETE synthesis decreased after 48 hours. The incubation of stored PMN with arachidonic acid (AA) maintained levels of LTB4 synthesis in PMN stored for 24 but not 48 hours. Also, the CTX defect of stored PMN to F-Met-Leu-Phe (FMLP) was not improved by the supplementation of PMN with exogenous sources of LTB4 or AA.(ABSTRACT TRUNCATED AT 250 WORDS)

Lovastatin (mevinolin) in the treatment of heterozygous familial hypercholesterolemia. A multicenter study.

STUDY OBJECTIVE: To evaluate the efficacy and tolerability of lovastatin under controlled conditions in heterozygous familial hypercholesterolemia. DESIGN: Randomized, double-blind, placebo-controlled, multicenter trial. SETTING: Five lipid clinics with a central laboratory and coordinating center. PATIENTS: 101 adult patients with heterozygous familial hypercholesterolemia. INTERVENTIONS: Patients were on a lipid-lowering diet throughout the study. After a 4-week placebo baseline period, patients were randomized to five equal treatment groups. Each group received a different sequence of placebo or lovastatin 5 to 40 mg twice daily or 20 to 40 mg once daily in the evening, during three consecutive 6-week periods. MEASUREMENTS AND MAIN RESULTS: The mean reductions in total plasma cholesterol and low-density lipoprotein cholesterol across the dosage ranges were 14% to 34% and 17% to 39%, respectively (p compared with zero and placebo less than 0.01). High-density lipoprotein cholesterol and apolipoproteins AI and AII rose slightly. Apolipoprotein B fell substantially at the higher dosage levels (-23% at 40 mg twice daily, p less than 0.01), indicating a reduction in the concentration of circulating low-density lipoprotein particles. Maximum response was achieved in 4 to 6 weeks. Twice-daily dosing was slightly more efficient than once-daily dosing. Of those patients receiving 40 mg twice a day, 89% had a fall in low-density lipoprotein cholesterol of at least 20%, and 61% had a fall of at least 40%. Adverse effects attributable to lovastatin were minimal, and no patient was withdrawn from the study. CONCLUSION: Lovastatin was well tolerated and effective in the treatment of familial hypercholesterolemia.

Effects of fenofibrate on lipids, lipoproteins, and apolipoproteins in 33 subjects with primary hypercholesterolemia.

The effects of fenofibrate on lipids, lipoproteins, and apolipoproteins in 33 subjects with primary hypercholesterolemia were assessed in a 6-month parallel group study, placebo (n = 15) versus fenofibrate 300 mg/day (n = 18), followed by an open label 6-month treatment period. After stabilization on an isocaloric low fat (less than 35% total calories) diet with less than 250 mg cholesterol/day and a P/S ratio of 1, and maintenance of LDL-cholesterol (LDL-C) levels greater than or equal to 175 mg/dl, subjects received placebo for 6 weeks and were then randomized into placebo or fenofibrate groups for 6 months, followed by open label treatment for 6 months. During the 6-month double-blind period, compared to the placebo group, the treatment group had significant reductions in total cholesterol, LDL-C, total apo B, and triglyceride, and increments in HDL-cholesterol, apolipoprotein A-I and apolipoprotein A-II (P less than 0.01 for all comparisons). Compared to placebo baseline, therapy with fenofibrate resulted in a reduction of LDL-C, apo B, and the LDL-C/HDL-C ratio of 15%, 13%, and 18% respectively; HDL-C, apo A-I, and apo A-II increased respectively 12%, 13% and 30% (P less than 0.01 for all comparisons). Mean adherence during the double blind phase of the trial was 95% in the drug group and 96% in the placebo group. An additional 6 months of open label fenofibrate therapy maintained the reduced total and LDL-C as well as the elevated HDL-C, apo A-I and apo A-II in the drug-drug group.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased infection mortality and decreased neutrophil migration due to a component of an artificial blood substitute.

We previously showed that an artificial blood substitute containing perfluorocarbons, Fluosol-DA, inhibited both neutrophil migration and adherence, due to its detergent component, Pluronic F-68. The purpose of the studies we report here was to determine if Fluosol or Pluronic might also reduce in vivo neutrophil migration and impair host resistance to bacterial infection. We studied in vivo PMN migration by injecting mice intraperitoneally (IP) with glycogen, followed by intravenous (IV) infusion of saline, Fluosol, or Pluronic. Peritoneal lavage after eight hours showed a significant decrease in the accumulation of PMN in lavage fluids of animals given either Fluosol or Pluronic (control--.19 +/- .03 X 10(6) PMN/mL, glycogen--1.35 +/- .14; glycogen/Fluosol--0.63 +/- .12; glycogen/Pluronic--0.69 +/- .07). We ascertained the effect of Fluosol and Pluronic on infection mortality by injecting mice IV with saline, Fluosol, or Pluronic, followed by a quantity of E coli (0.6 X 10(7] IP shown in preliminary studies to kill 20% to 50% of the mice in 24 hours. The 24-hour mortality was 14/45-saline, 24/32-Fluosol (chi 2 = 17.1; P less than .001) and 17/23 - Pluronic (chi = 11.2; P less than .001). Neither Fluosol nor Pluronic caused mortality without E coli. The increase in infection mortality occurred when Fluosol was given either two hours before, or simultaneously with E coli, but only with the simultaneous administration of bacteria and Pluronic. Pluronic did not alter reticuloendothelial system (RES) clearance function. These studies indicate that, in an animal model, Fluosol-DA, due to its detergent component Pluronic F-68, impaired neutrophil delivery to an inflammatory locus, and resulted in an increased rate of infection mortality. Since Pluronic did not result in RES blockade, but did impair the delivery of PMN to an inflammatory locus, our results suggest that the latter effect is responsible for the increase in infection mortality.

Glycogen metabolism in stored granulocytes.

Optimal function of transfused granulocytes (PMNs) requires adequate glycogen metabolism. Previous studies in our laboratory suggested that stored PMNs had decreased glycogen. We report here the glycogen content and chemotaxis of stored PMNs, and the ability of fresh and stored PMNs to use glycogen as the fuel source for chemotaxis. PMNs were prepared from 8 fresh units of blood drawn into citrate-phosphate-dextrose-adenine, suspended at 2 or 8 X 10(7) PMN per ml in autologous plasma with or without 15 mM sodium bicarbonate, and stored at 22 to 24 degrees C in transfer packs for 48 hours. Glycogen was measured on resting PMNs, and after challenge with opsonized zymosan and F-Met-Leu-Phe (FMLP). The chemotaxis of fresh and stored PMNs was measured in the presence or absence of extracellular glucose. Fresh PMNs contained 10.3 +/- 0.5 (mean +/- SEM) micrograms of glycogen per 10(6) PMN. Glycogen decreased by 4.2 +/- 0.9 micrograms per 10(6) PMN after challenge with opsonized zymosan and by 1.1 +/- 0.6 micrograms per 10(6) PMN after FMLP. After 48 hours of storage, neutrophil glycogen increased by 18 percent, except in units stored at a concentration of PMN of 8 X 10(7) per ml without sodium bicarbonate. In PMNs from these units stored without bicarbonate, glycogen decreased by 9 percent (p less than .05), and there was a 19 and 55 percent decrease in the ability of PMN from these units to metabolize glycogen after exposure to opsonized zymosan and FMLP, respectively (p less than 0.05). In addition, in PMNs from units stored at a concentration of PMN of 8 X 10(7) per ml without bicarbonate, there was a 47 and 70 percent decrease in chemotaxis at 24 and 48 hours, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of storage on degranulation by human neutrophils.

We investigated the possibility that the functional impairment in neutrophil (PMN) chemotaxis which occurs during granulocyte concentrate storage might be due to autotoxicity from the release of neutrophil granule contents during storage. Preliminary experiments confirmed that the exposure of fresh PMNs to the intracellular contents of disrupted PMNs, decreased the subsequent chemotaxis of the fresh PMNs by 63 +/- 5 percent compared to control PMNs (p less than .01). Freshly harvested neutrophils were stored at low (2 X 10(7) PMN/ml) or high cell concentration (8 X 10(7) PMN/ml) with or without 15 mM sodium bicarbonate (in order to maintain pH). Prior to storage, and 24 and 48 hours after storage at 22 to 24 degrees C, we measured the cell and unit plasma content of lactate dehydrogenase (LDH), beta-glucuronidase, and lysozyme. These enzymes served as markers for cell lysis, and primary and specific neutrophil granule contents, respectively. We also measured the effect on neutrophil chemotaxis of adding a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), to the storage medium. In addition, we measured the ability of PMNs to degranulate in response to an inflammatory stimulus before and after storage. The cell content of granule markers was largely unchanged during storage, except in the case of the units at a concentration of 8 X 10(7) PMN per ml stored without bicarbonate. In these units, lysozyme activity decreased by 15 +/- 7 percent after 48 hours of storage (p less than 0.02 vs. fresh PMNs). Likewise, the plasma content of LDH, beta-glucuronidase, and lysozyme increased significantly during storage, especially in units of high cell concentration stored without bicarbonate.(ABSTRACT TRUNCATED AT 250 WORDS)

The substitution of sucrose polyester for dietary fat in obese, hypercholesterolemic outpatients.

Our aim was to determine the effects of the substitution of sucrose polyester (SPE) for dietary fat in a 16-week outpatient study in 36 obese subjects with primary hypercholesterolemia. The subjects were randomized into three groups who followed a 16-week treatment period where all subjects received hypocaloric diets which provided approximately 7 kcal/lb body weight, a polyunsaturated/saturated (P/S) fat ratio of 0.9, and 180 mg cholesterol/day. The percentages of calories as fat in the 3 groups were as follows: a low fat diet group (n = 12) received 27% of dietary calories as fat, a low fat plus SPE group (n = 13) received 25% of calories as fat plus 27 g SPE/day as a bread spread and salad dressing, and a third group (placebo, n = 11) received 37% of calories as fat with a 27 g/day conventional fat placebo (bread spread and salad dressing). Mean weight loss from baseline in the 16 week treatment period was 2.6, 3.9, and 3.4% respectively in the placebo, diet, and SPE groups, p less than .05 for each group, without significant differences between the groups. There was a mean reduction of low density lipoprotein cholesterol (LDL-C) of 16% in the SPE group (p less than .05), more than twice the reductions in the placebo and diet groups, 5% and 6%, respectively. There was a mean 20% reduction in the SPE group in triglyceride and very low density lipoprotein cholesterol (p less than .05), compared to 7 and 10% reductions in the placebo and diet groups respectively. The degree of weight loss was correlated with the degree of reduction in LDL-C in the low fat diet group, and in the low fat diet group plus SPE (r = 0.59 for both groups). Without confounding by different levels of dietary cholesterol or P/S, SPE induced significant reductions in LDL-C in hypercholesterolemic obese subjects beyond the effects of weight loss alone. The effects of SPE were significantly greater than those achieved by the use of a diet which severely limited conventional dietary fat intake (to 40 g/day). SPE in the form of a bread spread and a salad dressing is a practical formulation for outpatient hypocholesterolemic low fat diets and provides the lubricity and organoleptic benefits of authentic foods without the dense caloric content of digestible fats.

A reassessment of the energy requirements for neutrophil migration: adenosine triphosphate depletion enhances chemotaxis.

In view of previous studies demonstrating a significant correlation between adenosine triphosphate (ATP) depletion and impairment of chemotaxis (CTX) during granulocyte (PMN) storage, we sought to quantitate the relationship between CTX and PMN energy metabolism. We incubated PMNs at 37 degrees C with 2-deoxyglucose (2-dg) in the presence of 5 mmol/L glucose. As expected, ATP inhibition by 2-dg was time-dependent (T 1/2, 18 minutes) and dose-dependent, with half-maximal inhibition of ATP (ID50) with 1.3 +/- .3 mmol/L 2-dg. Similar concentrations of 2-dg inhibited lactate generation, phagocytosis, superoxide anion generation, and degranulation. The random migration of PMNs was inhibited by somewhat higher concentrations of 2-dg (ID50, 12 mmol/L). In contrast, up to 40 mmol/L 2-dg did not inhibit CTX toward synthetic peptides or activated serum. In fact, 2-dg consistently increased the CTX of PMNs toward 10(-8) mol/L f-Met-Leu-Phe (fMLP), to a maximum of 450% of control CTX using 15 mmol/L 2-dg. Half-maximal stimulation (ED50) of CTX occurred at 6.3 +/- 1.0 mmol/L 2-dg. Although maximal CTX toward optimal concentrations of fMLP was consistently increased with 2-dg, the ED50 of CTX to fMLP was unchanged (ED50 with glucose, 2.0 +/- 0.6 nmol/L fMLP; ED50 with 2-dg 2.2 +/- 0.7 nmol/L fMLP), and 2-dg did not increase fMLP receptors. In the absence of glucose, 2-dg exerted similar effects on ATP and CTX, but at doses 30- to 50-fold lower than in the presence of glucose. Other glycolytic inhibitors (iodoacetamide and sodium fluoride) exerted similar effects. Additional studies indicated that CTX enhancement by 2-dg (a) required Mg++ but not Ca++, (b) occurred with PMNs from a patient with chronic granulomatous disease, (c) was unaltered in the presence of inhibitors of proteolysis, (d) was not due to generation of a soluble agent, (e) was not due to alterations in PMN adherence, and (f) was not due to inhibition of glycosylation. We conclude that the chemotaxis, but not the random migration, of PMNs is surprisingly resistant to inhibition of energy metabolism and depletion of ATP, since concentrations of 2-dg that decreased ATP and other cell functions by more than 50% not only did not inhibit, but actually stimulated, CTX. These studies also indicate that the previously reported correlation between ATP depletion and CTX impairment observed in stored PMNs are not causally related.