Purification of porcine neurophysins I and II by high performance liquid chromatography.

Porcine neurophysins I and II have been reported to be metabolically active. This activity was suggested to be due to contaminations. Furthermore, neurophysin I has been suggested to be heterogeneous on the basis of amino acid sequence analysis. The neurophysins I and II were isolated from porcine pituitary glands. Though they seemed homogeneous in polyacrylamide gel- and sodium dodecyl sulfate electrophoresis, neurophysin I could be separated into neurophysin I1 and I2 by high performance liquid chromatography. Neurophysin I2 differs from neurophysin I1 by one additional C-terminal amino acid. The purified neurophysins had no metabolic activity.

Fast protein chromatofocusing of human very-low-density lipoproteins.

Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.

Granulocytes and three-phase bone scintigraphy for differentiation of diabetic gangrene with and without osteomyelitis.

OBJECTIVE: In diabetic gangrene, concomitant osteopathy and soft-tissue infection often render laboratory and roentgenographic signs unreliable as indicators of osteomyelitis. In this situation, scintigraphic methods can be helpful. RESEARCH DESIGN AND METHODS: Relying on the long-term clinical course as the final indicator of presence or absence of osteomyelitis, we prospectively compared in 31 patients three-phase bone scintigraphy with either indium-labeled autologous granulocytes (n = 20) or 123I-labeled antibodies against granulocytes (n = 11). RESULTS: Three-phase bone scintigraphy and imaging with indium-labeled autologous granulocytes yielded sensitivities and specificities of 95 and 70% for bone scintigraphy and 77 and 100% for granulocyte scintigraphy, respectively. One patient with severe angiopathy and proved osteomyelitis had a negative bone scintigraphy but a positive scintigraphy with labeled antibodies against granulocytes. One patient with aseptic bone necrosis presented with a formally false positive result with both methods. CONCLUSIONS: In contrast to former retrospective studies, three-phase bone scintigraphy compares very well with granulocyte scintigraphy. The care of most patients can be managed with clinical data and this widely available scintigraphic method.

In vitro lipolytic activity of beta-endorphin and its partial sequences.

beta-Endorphin stimulates glycerol release from adipose tissue in vitro in the rabbit. Thirty different amino acid sequences of this peptide were tested for lipolytic activity. Four turned out to be active: porcine and human beta-endorphin-(1-31), human beta-endorphin-(6-31), and human beta-endorphin-(1-5)-(16-31). Structure-activity investigations showed that for the lipolytic action of beta-endorphin the C-terminal part [longer than beta-endorphin-(27-31)] is relatively important. Of special importance seems to be the C-terminal amino acid residue, because none of the sequences lacking the last two amino acid residues was lipolytically active. Furthermore, a different lipolytic response to beta-endorphin was obtained in starved, ad libitum-fed, and starved-refed animals, showing that the regulation of the lipolytic potency is not only mediated by peptide concentrations in the medium.

Atorvastatin improves postprandial lipoprotein metabolism in normolipidemlic subjects.

Atorvastatin is a potent HMG-CoA reductase inhibitor that decreases low-density lipoprotein (LDL) cholesterol and fasting triglyceride concentrations. Because of the positive association between elevated postprandial lipoproteins and atherosclerosis, we investigated the effect of atorvastatin on postprandial lipoprotein metabolism. The effect of 4 weeks of atorvastatin therapy (10 mg/day) was evaluated in 10 normolipidemic men (30+/-2 yr; body mass index, 22+/-3 kg/m2; cholesterol, 4.84+/-0.54 mmol/L; triglyceride, 1.47+/-0.50 mmol/L; high-density lipoprotein cholesterol, 1.17+/-0.18 mmol/L; LDL-cholesterol, 3.00+/-0.49 mmol/L). Postprandial lipoprotein metabolism was evaluated with a standardized fat load (1300 kcal, 87% fat, 7% carbohydrates, 6% protein, 80,000 IU vitamin A) given after 12 h fast. Plasma was obtained every 2 h for 14 h. A chylomicron (CM) and a chylomicron-remnant (CR) fraction was isolated by ultracentrifugation, and triglycerides, cholesterol, apolipoprotein B, apoB-48, and retinyl-palmitate were determined in plasma and in each lipoprotein fraction. Atorvastatin therapy significantly (P < 0.001) decreased fasting cholesterol (-28%), triglycerides (-30%), LDL-cholesterol (-41%), and apolipoprotein B (-39%), whereas high-density lipoprotein cholesterol increased (4%, not significant). The area under the curve for plasma triglycerides (-27%) and CR triglycerides (-40%), cholesterol (-49%), and apoB-48 (-43%) decreased significantly (P < 0.05), whereas CR retinyl-palmitate decreased (-34%) with borderline significance (P = 0.08). However, none of the CM parameters changed with atorvastatin therapy. This indicates that, in addition to improving fasting lipoprotein concentrations, atorvastatin improves postprandial lipoprotein metabolism presumably by increasing CR clearance or by decreasing the conversion of CMs to CRs, thus increasing the direct removal of CMs from plasma.

Fat restriction alters the composition of apolipoprotein B-100 containing very low-density lipoproteins in humans.

We investigated effects of fat saturation and fat restriction on very low-density apolipoproteins (VLDL) including the isoforms. Normolipidemic women (22) were given a reference diet, a polyunsaturated diet, and a low-fat, polyunsaturated diet for 6 wk each. The polyunsaturated diet decreased cholesterol and apolipoprotein B levels in VLDL (-33.1% and -23.8%) and in LDL (-13.5% and -8.8%) without affecting HDL. The low-fat, polyunsaturated diet resulted in a reincrease of VLDL triglycerides, but not of VLDL cholesterol. Concentration of VLDL apolipoprotein B fell further (-41.6%). All VLDL apolipoprotein B was in the B-100 region. Though the apolipoprotein E phenotype (E-3/E-3) remained constant, a shift to more (nonsialated) apolipoprotein E isoforms could be confirmed, resulting in an increased apolipoprotein E-3 to apolipoprotein E-2 area ratio (+30.6%). This study indicates that restriction of dietary-fat intake alters the composition of apolipoprotein B-100 containing VLDL that may be favorable for atherogenesis.

Lipid composition of serum lipoproteins in patients with primary type IIb and type IV hyperlipoproteinemia.

Cholesterol, triglyceride and phospholipid concentrations of VLDL, LDL and HDL were studied in 20 patients with primary type IIb, 25 patients with primary type IV and in 18 controls. Both types are not only characterized by different concentrations of lipoprotein lipids, but also by their different lipid composition. Type IIb had more triglycerides in the LDL, type IV in the LDL and HDL. The HDL cholesterol content of type IV was decreased. The percent phospholipid concentration of HDL was identical in the 3 groups, demonstrating the constant role of this lipid fraction. The lipid relationships between the lipoproteins showed that the LDL/HDL lipid ratio of type IIb exceeded type IV ratio in spite of normal HDL lipid concentration in type IIb.

Effects of etiroxate on low density and high density lipoproteins in hypercholesterolemic patients.

Six female patients with a primary hyperlipoproteinemia type IIa received 40 mg/day etiroxate for 2 months. Serum lipids, low density and high density lipoprotein cholesterol, the apolipoproteins B and A-I, and cholesterol and apolipoprotein A-I in ultracentrifuged HDL2 and HDL3 were analysed before and after the treatment. Etiroxate caused a significant reduction in serum cholesterol, LDL-cholesterol, and serum apolipoprotein B. Concerning the HDL, there was a significant decrease in HDL-cholesterol. The analysis of HDL subfractions revealed a uniform fall in HDL2-cholesterol and HDL2-apolipoprotein A-I. It is concluded that detailed information about lipoproteins leads to a better understanding of the relationships between drug treatment and the possible prevention of atherosclerotic diseases.

The effect of D-thyroxine on lipoprotein lipids and apolipoproteins in primary type IIa hyperlipoproteinemia.

The effect of 3 months' treatment with D-thyroxine on the lipoprotein lipids and apolipoproteins AI and B was investigated in 12 patients with type IIa hyperlipoproteinemia. VLDL, LDL and HDL were separated by preparative ultracentrifugation. Both apolipoproteins were measured in serum by electroimmunoassay procedures with monospecific antisera. There was a significant decrease of cholesterol, phospholipids and apolipoprotein B in serum and of all lipids in the LDL class. VLDL and HDL lipids and apolipoprotein AI showed no significant alterations. The atherogenic ratios LDL/HDL lipids and apolipoprotein B/apolipoprotein AI were lowered with the most pronounced effect on the ratio between the two apoliopoproteins. It is concluded that there is an effective reduction of LDL particles by D-thyroxine. Further investigations are necessary to evaluate whether lipoprotein lipids or apolipoproteins are a better discriminator of the influence on atherosclerotic risk in type IIa hyperlipoproteinemia.

The effect of beta-pyridylcarbinol on lipoprotein lipids in primary type IIa hyperlipoproteinemia.

The effect of long-term treatment over 6 months with beta-pyridylcarbinol on the lipoprotein lipids was investigated in 12 patients with primary type IIa hyperlipoproteinemia. VLDL, LDL and HDL were separated by preparative ultracentrifugation. There was a significant decrease of serum cholesterol and phospholipids. The normal serum triglycerides were unaffected. While VLDL and HDL lipids showed no signicant alterations, the LDL lipids decreased. The atypical lipid composition of the LDL was changed towards normal. Though there was a significant decrease of the "atherogenic" LDL/HDL-lipid ratios after 6 months treatment, beta-pyridylcarbinol did not result in a normalization of this ratio.

The effect of cholestyramine on lipoprotein lipids in patients with primary type IIA hyperlipoproteinemia.

The effect of 3 months' treatment with cholestyramine on lipoprotein lipids was investigated in 12 patients. VLDL, LDL and HDL were separated by preparative ultracentrifugation. There was a significant decrease of serum cholesterol and phospholipids and an increase of serum triglycerides. All the VLDL-lipids increased by nearly 30%. The LDL-lipids decreased with a tendency for normalisation of their atypical lipid composition. The small but significant alterations of HDL triglycerides and cholesterol are correlated with the corresponding alterations of the other lipoproteins; the HDL-phospholipids were unchanged. The LDL/HDL-lipid ratios were decreased but not normalised. The 30% decrease of LDL-cholesterol is negatively correlated with an increase in all the VLDL-lipids.

High density lipoproteins unaffected by dietary fat modification.

Since diets containing a high P/S ratio have been reported to have detrimental effects on HDL, the effect of a moderately modified fat diet (P/S ratio 1.0, cholesterol content 250 mg/day) was investigated in 30 healthy male volunteers, divided into 2 groups. They were either given a modified fat diet or an isocaloric control diet (P/S ratio 0.3, cholesterol content 370 mg/day) for 3 months each in a cross-over design. After 3 months of the polyunsaturated fat diet LDL cholesterol was significantly lowered by 19 and 13%, respectively, in both groups. This effect was already apparent after 4 weeks. Apo A-I and cholesterol in serum and the subfractions HDL2 and HDL3 remained unchanged. Consequently, the ratio LDL/HDL cholesterol was decreased by this diet. Dietary adherence was good according to the typical changes of the linoleic acid content in serum cholesteryl esters, to the dietary recalls and to the constant body weight. We conclude that a moderately modified fat diet supplied from mixed general food is acceptable for longer periods, effectively lowers LDL cholesterol, even in normolipoproteinemic subjects, and has no detrimental effects on HDL.

Changes in very low and low density lipoproteins with dietary fat modification.

Since VLDL and LDL are involved in atherogenesis, their response to dietary modification was studied in 15 normal male prisoners. A 3-month reference diet (P/S ratio 0.3, daily cholesterol intake 370 mg) was compared with a modified fat diet (P/S 1.0, 250 mg) given for further 3 months. The decrement in serum cholesterol by 32 mg/dl reflected a decrease in VLDL and LDL. It was associated with a decrease in serum apolipoprotein B by 16 mg/dl and in serum apolipoprotein E by 1.2 mg/dl. The decrement in VLDL cholesterol was paralleled by a lowered VLDL apolipoprotein E content. Serum and VLDL triglycerides, HDL cholesterol and the serum apolipoproteins A-I and A-II did not change significantly. One beneficial result of a conventional dietary regimen is lowered LDL with unaffected HDL. Another effect is the apparent modification of VLDL with a decrement of cholesterol and apolipoprotein E-enriched particles.

Effect of fenofibrate on serum lipoproteins in subjects with familial hypercholesterolemia and combined hyperlipidemia.

The effects on serum lipoproteins were studied in 8 patients with familial heterozygous hypercholesterolemia and 9 patients with familial combined hyperlipidemia during an 8-week treatment with fenofibrate. VLDL, IDL, LDL and HDL were isolated by ultracentrifugation and precipitation. Lipids and apolipoproteins A-I and B were determined by enzymatic and immunonephelometric techniques, respectively. In hypercholesterolemia, administration of fenofibrate resulted in decreases of VLDL, IDL, and LDL (cholesterol -58.3%, -28.6%, and -24.4%), while, in combined hyperlipidemia, treatment with the drug lowered VLDL and IDL (-33.3% and -42.9%). HDL cholesterol and apolipoprotein A-I increased only in hypercholesterolemia (+22.9% and +6.9%).

Low density lipoprotein apolipoprotein B metabolism: comparison of two methods to establish kinetic parameters.

Elevated plasma cholesterol concentrations represent a major risk factor for coronary artery disease (CAD). Low density lipoprotein (LDL)-apolipoprotein (apo) B plays a key role in this process. Metabolic parameters of LDL-apoB such as fractional catabolic rate (FCR) and production rate help to understand the underlying pathomechanisms of elevated LDL-apoB and are usually determined with tracer studies (gold-standard). However, these parameters can also be calculated from the rebound of plasma LDL-apoB concentration following a perturbation such as apheresis, if it is assumed that the perturbation itself does not affect metabolic parameters. LDL-apoB metabolism was determined using two methods in eight hyperlipoproteinemic patients (47 +/- 15 years, body mass index (BMI) 27.5 +/- 4.1 kg/m): (a) by endogenous labeling using D3-leucine (bolus 5 mg/kg) as tracer and multicompartmental modeling; and (b) by fitting a monoexponential equation to LDL-apoB rebound concentration data following apheresis. LDL-apoB metabolic parameters determined using the two methods (mean +/- S.D.; FCR-tracer: 0.18 +/- 0.07 per day, FCR-rebound: 0.27 +/- 0.25 per day; production-tracer: 12.0 +/- 3.9 mg/kg per day; production-rebound: 15.2 +/- 8.0 mg/kg per day) were not correlated, were not concordant (intraclass correlation coefficient), and were not significantly different. Furthermore, only in five of the eight patients the rebound analysis predicted LDL-apoB steady-state concentrations that were within 20% of observed steady-state concentrations. These results indicate that parameters derived from LDL-apoB mass rebound following apheresis cannot be used as a surrogate for parameters established with tracer methodology probably because the assumption that apheresis does not affect metabolic parameters of LDL-apoB may not be true in all patients.

Cholestyramine plus pectin in treatment of patients with familial hypercholesterolemia.

The effects of a combined therapy, composed of a cholesterol-lowering diet, cholestyramine (16 g/day) and apple pectin (12 g/day), were studied on serum lipoproteins in 6 men with familial hypercholesterolemia. All subjects were treated with diet alone, diet plus cholestyramine and diet plus cholestyramine and pectin for at least 8 weeks on each regimen. In these severely hypercholesterolemic subjects the combined treatment with diet plus cholestyramine and pectin lowered serum cholesterol by 31% and LDL-cholesterol by 35% corresponding to a 19% and 22% further decrease respectively relative to diet plus cholestyramine. We conclude that the addition of the well-tolerated natural cholesterol-lowering product is valuable in the treatment of familial hypercholesterolemia.

Apolipoprotein E polymorphism has no independent effect on plasma levels of lipoprotein(a).

Previous studies show conflicting results concerning an influence of apolipoprotein E (apo E) phenotype on lipoprotein(a) (Lp(a)) plasma levels. We speculated that it is not the apo E phenotype itself but rather its effect on plasma lipid concentrations that might influence Lp(a) levels. In 1562 subjects concentrations of triglycerides, LDL-cholesterol and Lp(a) were measured by standard laboratory methods. Apo(a) and apo E isoforms were determined by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing, respectively, followed by immunoblotting. An univariate analysis revealed a significant influence of apo(a) isoforms, apo E phenotype, triglycerides and LDL-cholesterol on Lp(a) plasma levels (ANOVA: P < 0.001, P < 0.02, P < 0.001 and P < 0.001, respectively). In a multivariate analysis, however, the influence of the apo E phenotype was no longer significant (P>0.10), whereas apo(a) isoforms, LDL-cholesterol quintiles and triglyceride quintiles explained 29.2, 2.8 and 1.0% of the variation of the Lp(a) levels (for all three variables: P < 0.001). We conclude that apo E polymorphism does not exert an independent effect on Lp(a) concentrations. Any influence is mediated through the effect of apo E polymorphism on plasma lipids.

The effect of fluvastatin on cardiac events in patients with symptomatic coronary artery disease during one year of treatment.

The primary objective of the present study was to investigate the cholesterol-lowering effect of fluvastatin on the incidence of cardiac events in hyperlipidaemic patients with symptomatic, clinically-diagnosed (exercise-ECG) coronary heart disease (CHD) during 1 year of treatment. Exercise tolerance, incidence of angina pectoris episodes, use of anti-anginal medication and intimal-medial-thickness (IMT subgroup) of the A. carotis were secondary endpoints. In the double-blind trial a total of 365 male and female patients with stable symptomatic CHD and a low-density lipoprotein cholesterol (LDL-C) above 160 mg/dl on a lipid-lowering diet were randomised to fluvastatin 40 mg (o.a.d. or b.i.d.) or placebo for 1 year. Fluvastatin lowered total cholesterol by 17% and LDL-C by 27%. There was a significantly lower incidence of cardiac events (cardiac death, nonfatal myocardial infarction, unstable angina pectoris) in the fluvastatin group (3 events) as compared to the placebo group (10 events) (P < 0.05). Exercise tolerance improved and the incidence of angina pectoris episodes decreased in both groups, but more pronounced on fluvastatin (n.s.). Exercise-ECG discontinuation due to angina pectoris and ST-segment depression decreased in the fluvastatin group by 55.6 and 70.9%, respectively, and in the placebo group by 39.6 and 46.5% (n.s.). At baseline, a subgroup of 76 patients showed a mean IMT value of 0.73 mm which remained uninfluenced in the fluvastatin and the placebo groups. Fluvastatin was safe and well tolerated. In conclusion, patients with symptomatic CHD get cardiovascular benefit from lipid-lowering therapy with fluvastatin even during the first year of treatment.