High correlation but lack of agreement between direct high-performance gel chromatography analysis and conventional indirect methods for determining lipoprotein cholesterol.

Low-density lipoprotein-cholesterol (LDL-C) is currently estimated clinically by using the Friedewald formula, when plasma triglycerides are < 4000 mg/L, or as the difference between infranatant and high-density lipoprotein-cholesterol (HDL-C) values after ultracentrifugation of plasma at native density, when plasma triglycerides are > or = 4000 mg/L (beta quantification). HDL-C is measured by selective precipitation of apolipoprotein B-containing lipoproteins from whole plasma or from the density > 1.006 kg/L infranatant. We compared these conventional methods for LDL-C and HDL-C with "high-performance" gel chromatography (HPGC), a method that directly and simultaneously measures both LDL-C and HDL-C in a single, microliter volume of plasma. Not surprisingly, we found that the results by all these methods were highly correlated. However, LDL-C values were significantly higher and HDL-C values significantly lower by the direct HPGC method than by the conventional methods (paired t-test). In addition, both Bland-Altman plots and concordance correlation analyses indicated lack of agreement between the methods' results in the majority of patients' subgroups.

Psyllium for the reduction of cholestyramine-associated gastrointestinal symptoms in the treatment of primary hypercholesterolemia.

OBJECTIVE: To determine if the bulk-forming laxative, psyllium hydrophilic mucilloid (PHM), reduces the gastrointestinal side effects and enhances the cholesterol-lowering efficacy of cholestyramine resin in patients with primary hypercholesterolemia. DESIGN: After a dietary lead-in period and 6 weeks of treatment with cholestyramine, the study followed a double-blinded, placebo-controlled, crossover format. SETTING: Lipid clinic affiliated with a large metropolitan community hospital. PARTICIPANTS: Twenty-seven randomly selected male and female patients with a diagnosis of primary hypercholesterolemia. Entry criteria required a fasting low-density lipoprotein cholesterol (LDL-C) concentration of 4.91 mmol/L (190 mg/dL) or greater and a triglyceride concentration of less than 2.26 mmol/L. Patients using steroids, beta-blockers, thiazide diuretics, and lipid-lowering agents, or having a history of allergy to psyllium or aspartame were excluded. INTERVENTION: The study consisted of four interventional phases of 6 weeks' duration that included (1) dietary stabilization (National Cholesterol Education Program Step I Diet); (2) cholestyramine therapy (4 g twice daily); (3) cholestyramine with study medication (PHM [5.1 g twice daily] or placebo); and (4) cholestyramine with crossover to alternate study medication. MAIN RESULTS: Following the 6-week dietary lead-in phase, four patients were eliminated from the study because their fasting LDL-C concentrations fell below 4.14 mmol/L (160 mg/dL), and one patient was eliminated because testosterone therapy was initiated by his internist. The remaining 22 patients entered the cholestyramine treatment phase. Four left the study within 2 weeks because of intolerable gastrointestinal tract symptoms. The 18 patients who completed this phase demonstrated significant reductions in their plasma total cholesterol (7.27 vs 6.67 mmol/L [281 vs 258 mg/dL]) and LDL-C (5.38 vs 4.63 mmol/L [208 vs 179 mg/dL]) concentrations compared with baseline levels. The addition of PHM to the cholestyramine regimen provided a tendency toward further reductions in total cholesterol and LDL-C levels (6.67 vs 6.46 mmol/L [258 vs 250 mg/dL] and 4.63 vs 4.29 mmol/L [179 vs 166 mg/dL], respectively), although statistical significance was not achieved. Psyllium hydrophilic mucilloid significantly reduced the frequency and severity of constipation, abdominal discomfort, and heartburn. No reports of new gastrointestinal tract symptoms or untoward effects were noted with the addition of PHM. CONCLUSION: These data suggest that the addition of PHM to cholestyramine therapy may improve a patient's compliance by reducing the associated gastrointestinal tract side effects.

Efficacy and safety of pravastatin in the long-term treatment of elderly patients with hypercholesterolemia.

PURPOSE: Elevated cholesterol levels are a major risk factor for coronary heart disease, which remains a significant problem in patients beyond age 65 years. Because drug therapy for the control of hypercholesterolemia in elderly patients is frequently considered to be indicated, we investigated the efficacy and safety of pravastatin in the treatment of elderly subjects with primary hypercholesterolemia. PATIENTS AND METHODS: In this 96-week, multicenter, double-blind, placebo-controlled study, 142 subjects (95 women, 47 men) 64 to 90 years of age with elevated cholesterol levels despite dietary intervention were randomized to receive pravastatin 20 mg at bedtime or matching placebo (2:1). Dosage could be doubled after 8 weeks, a bile acid-binding resin could be added after 16 weeks, and nicotinic acid or probucol could be added after 32 weeks, as needed, to adequately lower the low-density lipoprotein cholesterol (LDL-C) levels. RESULTS: Significant reductions in the levels of LDL-C (-30.9%), total cholesterol (Total-C; -21.9%), and triglycerides (TG; -16.7%) and significant increases in the levels of high-density lipoprotein cholesterol (HDL-C; 11.3%) were noted in the group receiving pravastatin treatment at 16 weeks (P < or = 0.001 compared with baseline, P < or = 0.01 compared with placebo). The cholesterol-lowering effects of pravastatin were sustained throughout the 96 weeks of the trial. Pravastatin was well tolerated, with an overall incidence of adverse events nearly identical to that of placebo. CONCLUSIONS: In this study, pravastatin was well tolerated and effective in lowering LDL-C, Total-C, and TG and in raising HDL-C during long-term treatment of elderly patients with primary hypercholesterolemia.

CP-66,948: an antisecretory histamine H2-receptor antagonist with mucosal protective properties.

CP-66,948 is a histamine H2-receptor antagonist with gastric antisecretory activity and mucosal protective properties. The affinity of CP-66,948 for the guinea pig atria histamine H2-receptor is 15 times greater than that of cimetidine and seven times greater than that of ranitidine. In vivo, the ED50 value for inhibition of gastric acid secretion in pylorus-ligated rats is 2 mg/kg intraduodenally, and in histamine or pentagastrin-stimulated Heidenhain pouch dogs the antisecretory ED50 values are 0.3 mg/kg per os and 1.0 mg/kg per os, respectively. CP-66,948 also inhibits ethanol-induced gastric hemorrhagic lesions in rats following either oral or systemic administration (ED50 values of 12 mg/kg per os and 6 mg/kg subcutaneously). In addition, the mucosal protective activity is independent of prostaglandin synthesis. CP-66,948 inhibits gastric acid secretion in man, and its mucosal protective activity may provide additional benefits in peptic ulcer therapy.

The effect of pravastatin on plasma lipoprotein and apolipoprotein levels in primary hypercholesterolemia. The Southeastern Michigan Collaborative Group.

Pravastatin is a metabolic product of mevastatin and a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. It was investigated for its cholesterol-lowering properties in a double-blind, placebo-controlled, multicenter study of 82 patients with primary hypercholesterolemia. Following a 6- to 8-week dietary lead-in period, patients were randomized to twice-daily placebo or active drug for 16 weeks. Patients receiving 10 mg of pravastatin twice a day for 8 weeks experienced mean total cholesterol and low-density lipoprotein cholesterol (LDL-C) level reductions of 20% (6.85 vs 5.48 mmol/L [265 vs 212 mg/dL]) and 28% (5.17 vs 3.75 mmol/L [200 vs 145 mg/dL]), respectively. At 20 mg twice a day for an additional 8 weeks, pravastatin reduced plasma total cholesterol, LDL-C, and apolipoprotein B-100 levels by 23% (6.85 vs 5.30 mmol/L [265 vs 205 mg/dL]), 31% (5.17 vs 3.59 mmol/L [200 vs 139 mg/dL]), and 23% (118 vs 91 mg/dL), respectively. High-density lipoprotein cholesterol (HDL-C), HDLb-C, HDLb-C, and apolipoprotein A-I plasma concentrations increased by 11%, 60%, 7%, and 10%. Plasma triglyceride concentrations decreased in both the pravastatin- and placebo-treated patients. Pravastatin was generally well tolerated and an effective agent for the treatment of primary hypercholesterolemia.

The relationship between apolipoprotein B-100 and low-density lipoprotein cholesterol after treatment with an HMG CoA reductase inhibitor.

Measurement of apolipoproteins AI and B-100 has been shown to provide at least equivalent information to measurement of lipoprotein cholesterol levels for evaluating risk of cardiovascular disease. In the present study, the authors examined the relationship between APO B-100 and low-density lipoprotein (LDL) cholesterol in a hypercholesterolemic population that was treated with 16 weeks of hydroxymethylglutaryl CoA reductase inhibitor therapy to lower plasma cholesterol levels. The average decrease in APO B-100 was -0.27 g/L (23% of baseline), which was similar to the decrease in LDL cholesterol, -1.6 mmol/L (-0.61 g/L) (30% of baseline). Correlation data (r = 0.902) indicated that the information provided by the two parameters corresponded in individual cases. Apolipoproteins assayed on automated equipment by kit methods are simpler, more straightforward, and provide more reproducible results than measurement of lipoprotein cholesterols. The authors conclude that, in addition to being more reliable for appraising risk of coronary artery disease, measurement of apolipoproteins may be equally useful for monitoring lipoprotein-lowering therapy.

Dopamine and dobutamine induce hypokalemia in anesthetized dogs.

Epinephrine-induced hypokalemia appears to be mediated by beta 2-agonist activation of Na+/K+ ATPase. To determine whether dopamine and dobutamine induce hypokalemia, eight adult mongrel dogs were anesthetized and studied in random crossover fashion. Potassium [K+] was measured with an ion-selective microelectrode, and central hemodynamics were measured continuously. After stabilization, dopamine and dobutamine were infused at doses of 2, 4, 8, and 20 micrograms/kg/min (15-min increments/dose), and 0.9% NaCl was infused at equivalent volumes, with a 1-h washout between treatments. The mean change in [K+] at each infusion rate was compared between treatments among dogs with an adequate hemodynamic response. Among dopamine responders (n = 5), [K+] decreased from 3.74 +/- 0.42 mEq/L at baseline to 3.63 +/- 0.51 at 2 micrograms/kg/min (p less than 0.02) and was not significantly different at higher doses. Among dobutamine responders (n = 7), [K+] decreased from 3.52 +/- 0.74 at baseline to 3.31 +/- 0.87 at 8 micrograms/kg/min (p less than 0.02) and 3.25 +/- 0.86 at 20 micrograms/kg/min (p less than 0.02), and was not significantly different at lower doses. We conclude that dopamine and dobutamine induce significant hypokalemia, consistent with their adrenergic agonist activity, and this may be related to the known arrhythmogenicity of these agents.

New assay of apolipoproteins A-I and B by rate nephelometry evaluated.

We evaluated new, commercially available reagents for assaying apolipoproteins (apo) A-I and B by rate nephelometry (INA). Our initial linearity studies for apoA-I indicated that use of the commercial diluent resulted in incomplete immunoreactivity. Subsequent revision of the calibration line by the manufacturer compensated for this and improved the linearity for the apoA-I assay. We observed good linearity for the apoB assay. The within-run CVs were less than 4.0% and the between-run CVs were less than 5.5% for both assays. Results were 109% for apoA-I and 101% for apoB as compared with those measured for IUIS-WHO reference materials from the Centers for Disease Control. Recovery averaged 103% for apoA-I and 105% for apoB, for duplicate assays of three concentrations of purified apoA-I and low-density lipoprotein (LDL). Assaying sera from 45 patients, we demonstrated a good correlation between INA and radial immunodiffusion for both apoA-I (r = 0.92) and apoB (r = 0.95). Correlations between apoA-I and high-density lipoprotein cholesterol, and between apoB and LDL cholesterol compared favorably with previous reports. We conclude that these assays are accurate, precise, and easily automated for clinical application.

The effect of lipoproteins on the synthesis of prostacyclin, von Willebrand factor and apolipoproteins A-I and A-II in cultured human endothelial cells.

Primary cultures of confluent human endothelial cells (ECM) were grown in media containing the major lipoproteins (LP) and lipoprotein deficient serum (LDS). The release of 6-keto-PGF1 alpha, von Willebrand factor (VIII RAg) and apolipoproteins (apo) A-I and A-II were investigated by radioimmunoassay. The cell-associated VIII RAg, apo A-I and apo A-II were also confirmed by fluorescein antibodies, and the synthesis of the apolipoproteins was examined by incorporation of [3H]leucine. Apo A-I and apo A-II were located and synthesized in ECM, yet only apo A-I was released into the medium. Very low density (VLDL) and low density lipoproteins (LDL) in concentrations of 50-600 micrograms/ml stimulated release of apo A-I. Stimulation of ECM for 5 min with thrombin (T) or arachidonic acid (A) did not induce apo A-I release. VIII RAg was always released into the media from ECM. The release was not affected by the lipoproteins. VIII RAg was also localized on the cell surface (VIII RAgC) and approximately 80% was released by trypsin. LDL stimulated the occurrence of factor VIII RAg on the cell surface. 6-Keto PGF1 alpha was always released into the medium and the production was stimulated by T and AA. The main lipoproteins (50-600 micrograms/ml) and apo A-I and A-II did not affect the release of 6-keto-PGF1 alpha. This study shows that endothelial cells synthesize and release proteins important for thrombogenesis and atherosclerosis. The release of apolipoproteins A-I was stimulated by VLDL and LDL, and the concentration of cell-related factor VIII RAg was stimulated by LDL.

Apolipoprotein A-I as a marker of angiographically assessed coronary-artery disease.

This study was designed to determine whether the plasma level of apolipoprotein A-I is a better discriminator of angiographically documented coronary-artery disease than the level of high-density-lipoprotein (HDL) cholesterol in male subjects. The level of plasma apolipoprotein A-I in 83 patients with coronary-artery disease was 96.7 +/- 4.2 mg per deciliter (mean +/- S.E.M.), which was significantly lower (P less than 0.0001) than the level in 25 patients without coronary-artery disease (146.9 +/- 2.1 mg per deciliter). The levels of HDL cholesterol were also lower (P less than 0.0001) in patients with coronary-artery disease (31.9 +/- 1.5 mg per deciliter) than in those without it (45.9 +/- 2.3 mg per deciliter). A stepwise discriminant analysis, however, indicated the superiority of apolipoprotein A-I over HDL cholesterol in detecting coronary-artery disease. Furthermore, a linear discriminant analysis suggested that although HDL cholesterol by itself was a discriminator of coronary-artery disease, it did not provide a substantial increase in discriminatory value over that provided by apolipoprotein A-I; in contrast, apolipoprotein A-I levels added discriminatory value to the information obtained by measuring HDL cholesterol alone. We conclude that apolipoprotein A-I by itself is more useful than HDL cholesterol for identifying patients with coronary-artery disease.

Radioimmunoassay of apolipoprotein A-i. application of a non-ionic detergent (Tween-20) and solid-phase staphylococcus.

We describe two techniques for radioimmunoassay of apolipoprotein A-I (apoA-I) in human plasma, each involving use of a non-ionic detergent, Tween-20, to expose antigenic sites, and one involving "IgG SORB" (a suspension of killed staphylococci) as a solid-phase separator. Tween-20 (3.75 g/L) decreased nonspecific binding and unmasked the antigenic sites on the apoA-I molecule in plasma to the same extent as did a tedious delipidation procedure, without altering the binding affinity between apoA-I and apoA-I antibodies as determined by Scatchard analysis (Ka congruent to 2.83 X 10(8) L/mol). The widely accepted double-antibody immunoprecipitation technique for separating bound and unbound 125I-labeled apoA-I is time-consuming, owing to extended periods of incubation and centrifugation IgG SORB effectively separates bound from unbound 125I-labeled apoA-I and the reaction is complete within 10 min. On comparing concentrations of apoA-I in human plasma by the conventional second-antibody (y) and solid-phase IgG SORB methods (x), we found results by the two techniques to be reasonably identical (r = 0.98, y = 1.2x -- 0.17). The mean concentrations of apoA-I in plasma from 65 normal and five hyperlipidemic patients were 1.33 (SD 0.32) and 0.78 (SD 0.35) g/L, respectively, and apoA-I and high-density lipoprotein cholesterol were significantly correlated (r = 0.72, p less than 0.001).

Effects of locally and systemically infused bradykinin on transvascular fluid and protein transfer in the canine forelimb.

Local intra-arterial infusions of bradykinin into canine forelimbs perfused either naturally or at constant inflow markedly increase skin lymph protein concentration promoting edema formation. However, prolonged systemic infusions of this agent either intravenously or into the left ventricular chamber, in blood concentrations calculated to exceed those that produce massive protein and fluid efflux on local administration, causes only minimal increases in lymph protein concentration, and in naturally perfused forelimbs promotes extravascular fluid reabsorption rather than net fluid filtration. Local infusions of bradykinin fail to alter aortic pressure whereas systemic infusions produce a profound but transient decrease in this variable. Moreover, the local intra-arterial infusion of bradykinin into forelimbs perfused at constant inflow fails to increase skin lymph protein concentration after 60 minutes of systemic hypotension. In contrast to bradykinin alone, the simultaneous infusion of bradykinin and norepinephrine or bradykinin and isoproterenol fails to increase skin lymph protein concentration. The antagonism of the bradykinin protein efflux by both norepinephrine and isoproterenol can be prevented by prior treatment with propranolol. These data suggest that the liberation of catecholamines may account, in part, for the minimal increases in forelimb protein efflux during systemic infusions of bradykinin relative to that produced by local intra-arterial infusions of this agent.

Antagonism of histamine edema formation by catecholamines.

Histamine (4 microgram base/min) infused into the brachial artery for 60 min greatly increases skin lymph flow and lymph protein concentration in forelimbs perfused either naturally or at constant inflow. In contrast, the simultaneous intrabrachial infusion of histamine and norepinephrine (4 microgram base/min of each) or histamine and isoproterenol (4 microgram base/min and 3 microgram/min, respectively) for 60 min failed to alter lymph protein concentration in forelimbs perfused either naturally or at constant inflow. The edema in forelimbs naturally perfused by histamine (4 microgram base/min) infused into the brachial artery for 60 min was also greatly reduced by the simultaneous histamine-catecholamine infusions. Phentolamine (in concentrations that antagonize the vascular actions of norepinephrine) failed to prevent the antagonism of histamine protein efflux by norepinephrine. Hence, it must be concluded that catecholamines antagonize the protein efflux by locally infused histamine, independent of alpha-adrenergic receptor activity.

Evidence for a centrally mediated hypotensive effect of Escherichia coli endotoxin in the anesthetized dog.

The centrally mediated cardiovascular effects of Escherichia coli endotoxin were studied utilizing a neurally intact vascularly isolated head-trunk preparation in the anesthetized dog. The vascularly isolated head was perfused at constant flow with arterial blood supplied by a donor animal. Spectrophotometric examination of the donor and recipient trunk blood after administration of Evan's blue dye indicated that there was no blood exchanged between the head and trunk of the recipient dog. The responses to various physiological maneuvers and denervations indicated that the central nervous system and all afferents and efferents involved in the control of the cardiovascular system were functioning normally. The infusion of purified E. coli endotoxin into the arterial perfusion circuit to the head, either before or after bilateral denervation of the carotid sinus-body complexes, resulted in marked hypotension within 30 minutes in the trunk of the recipient dog. These findings indicate that purified E. coli endotoxin is capable of eliciting marked centrally mediated hypotensive responses. The time course of these responses suggests that the centrally mediated hypotensive effects of endotoxin do not participate in the initial precipitous fall in blood pressure seen after systemic administration of endotoxin, but rather that they may contribute significantly to the maintenance of the hypotension.

Effects of systemically infused histamine on transvascular fluid and protein transfer.

Histamine (4-64 microgram base/min) infused into the brachial artery clearly promotes edema formation in forelimbs perfused at natural flow. In contrast, intravenously administered histamine, even in blood concentrations exceeding those achieved by local infusion, not only fails to promote edema formation, but rather causes net extravascular fluid reabsorption. In this study, high concentrations of histamine were infused into the left ventricular chamber to bypass the pulmonary circuit. Histamine (400-800 microgram base/min) infused into the left ventricle of the heart for 90 min produced marked hypotension and only very slight increases in forelimb skin lymph flow and lymph protein concentration and failed to produce visible signs of edema. Thus the differential actions of local and intravenous histamine on lymph flow, protein efflux, and fluid fluxes cannot be explained by uptake or destruction of histamine in the lung during intravenous infusions of this agent. It seems more likely that they result from different actions on microvascular pressure, surface area, and/or permeability to plasma proteins. Prior hypotension produced either by acetylcholine, systemically infused histamine, or arterial hemorrhage almost completely prevents the increase in skin lymph flow and lymph protein concentration by histamine infused locally into the brachial artery, even in forelimbs perfused at constant flow.