Purification and properties of two lipases from pig adipose tissue.

Two lipases were purified from pig adipose tissue after delipidation by a mild and effective procedure using mixtures of chloroform and butanol. This was followed by hydrophobic adsorption chromatography on aminohexyl-Sepharose 4B coupled with octanoic acid, gel filtration on Sephadex G-100, and isoelectric focusing. Two electrophoretically and chromatographically pure enzymes were obtained, which had the same molecular weight (60 000 +/- 3000) and specific activity, and almost identical amino acid compositions; the isoelectric points, i.e. 5.2 and 5.5, differed.

Barrier properties of lecithin/lysolecithin mixtures.

Light scattering, birefringence and X-ray studies showed that liposomes, with lipid molecules orientated in bilayers, are formed from egg licithin/lysolecithin mixtures up to 50 mol percent of lysolecithin; above this concentration much smaller mixed micelles are formed. Permeability studies demonstrated a dramatic increase in the permeability of the liposomes when the lyso concentration exceeds 22.5 mol percent. X-ray studies indicated a significant decrease in bilayer thickness with increasing lysolecithin concentration. It is suggested that decreased interaction energy between the lipid molecules in the bilayer is responsible for the inability of the thin bilayers to act as an effective permeability barrier.

Binding of unmodified low-density lipoproteins to human fibroblasts. An investigation by immunoelectron microscopy.

The bindinf of unmodified low density lipoproteins to the plasma membrane of fibroblasts was studied at the ultrastructural level. The bound low density lipoprotein was visualized by an indirect immunoperoxidase technique, with the use of an antiserum against apoprotein B. Immunoreactive regions representing bound apoprotein B were found on the plasma membrane, in indented regions with a diameter of 0.15--0.30 micrometer and a fuzzy coat on the cytoplasmic side. Fibroblasts from a patient homozygous for hyperlipoproteinaemia type IIa showed no immunoreactive material in the indented regions. The specific 125I-labelled low density lipoprotein binding to these homozygous fibroblasts was 7% compared to control fibroblasts.

The determination of lipids and proteins in suction blister fluid.

The concentration of 5 different proteins in suction blister fluid and serum was determined by immunotechniques. These proteins, varying in size and molecular weight (6,600-2,300,000) were insulin, albumin, high density lipoprotein determined as apoprotein A-I, alpha 2-macroglobulin and low density lipoprotein measured as apoprotein B. The difference in the blister fluid/serum concentration ratio of the proteins was dependent on the molecular weight and followed mainly the law of diffusion. Moreover, the amounts of insulin, albumin and apoproteins A-I and B in suction blister fluid were the same as those reported in peripheral lymph. The results indicate that the sieve function of the capillary basement membrane remains intact during the formation of the suction blisters. Suction blister fluid might therefore be regarded as representative of interstitial fluid. The concentrations of 4 different lipids (cholesterol, cholesterolesters, triglycerides and phospholipids) were also determined and their blister fluid/serum concentration ratio proved to have a fairly constant value of 0.25.

Human very low density lipoproteins: loss of electrophoretic mobility on enzymatic removal of sialic acid residues.

Incubation of very low density lipoproteins (VLDL) from three hyperlipoproteinemic patients with neuraminidase resulted in a progressive liberation of sialic acid residues. After the original sialic content had been reduced by approx. 75% a lipoprotein fraction having zero electrophoretic mobility on paper appeared. This fraction increased until, after the removal of all the sialic acid residues, the mobility of the entire VLDL fraction was reduced to zero.

The quantitative determination of apolipoprotein A-I (apo-lp-Gln I) in human serum by radial immunodiffusion assay (RID).

A radial immunodiffusion (RID) assay was developed to determine the concentration of the major apolipoprotein (apolipoprotein A-I, apo-lp-Gln I) of human high density lipoproteins (HDL): (1) In a random population sample of apparently healthy persons (104 women, 95 men) aged 40-49 years, serum levels of apo A-I were determined. For both women and men a value of 137 +/- 23 mg/dl was found. (2) Values of 139 +/- 21 mg/dl were found in a group of 31 women taking oral contraceptives, i.e. an insignificant increase in apo A-I level. (3) In two volunteers, apo A-I levels showed no significant variation in a 24-h period.

Zero electrophoretic mobility of very low density lipoproteins in type III hyperlipidemic patients during treatment.

In some cases of type III hyperlipoproteinemia reduction of lipid levels by diet and/or drugs results in a complete loss of mobility of the VLDL fraction on paper electrophoresis.

The lipoprotein composition of amniotic fluid.

The lipoprotein composition of amniotic fluid (AF) has been studied and it was found that it consists of high density lipoprotein (HDL) only. At about the 26th week of pregnancy the HDL content reaches a maximum and decreases thereafter. On crossed immunoelectrophoresis the HDL of AF was separated into two components if the agar layer contained an antiserum against apolipoprotein A-I. The component with the higher electrophoretic mobility (but not the slower moving component) could also be visualized with an antiserum against HDL. When maternal or umbilical cord serum (UCS) was analysed by crossed immunoelectrophoresis using the anti-apoA-I antiserum, we found that they only contained the faster moving lipoprotein component. If UCS was treated with phospholipase A2 and then subjected to crossed immunoelectrophoresis, the slower moving component appeared at the expense of the faster one. Likewise when UCS was incubated with AF from the 15th week of pregnancy or later, the lipoprotein component of UCS with the high mobility was partially converted to the component with the slower mobility. It is concluded that AF probably contains a phospholipase, an enzyme which may promote the onset of labour.

Studies on the surface structure of very low density lipo-proteins.

Very Low Density Lipoproteins (VLDL) were incubated with 5 different pure phospholipases. From the results the following conclusions were drawn. (1) The phospholipids ,re localized in a monomolecular layer on the outside of the VLDL particles. This supports the lipid core model proposed by several groups. (2) A minor fraction of the phospholipids (ranging from 3 to 10%) cannot be degraded by the enzymes and probably have a strong interaction with apoproteins. (3) The average surface pressure of VLDL is probably low, and comparable with a lateral surface pressure of 15 dynes/cm at the air--water interface, as concluded from experiments with two phospholipases A(2). Calculations of the thickness of the surface coat and protein coverage on the basis of this value agree very well with values reported in literature.

Efficacy of different doses of cimetidine in the treatment of reflux esophagitis. A review of three large, double-blind, controlled trials.

Four different cimetidine dosage regimens--800 mg u.i.d. HS or nocte, 800 mg u.i.d. dinnertime, 400 mg q.i.d., and 800 mg b.i.d.--were investigated for the treatment of reflux esophagitis in three independent large-scale, double-blind, controlled multicenter trials in which more than 1100 patients participated. Analysis of the data shows that the percentage of endoscopic healing after 6 and 12 weeks of treatment was fairly constant in patients with the same endoscopic grade of severity of reflux esophagitis at the start of treatment, whether they were treated with 800 mg u.i.d. (HS or dinnertime), 800 mg b.i.d., or 400 mg q.i.d. Healing percentages after 12 weeks of therapy ranged from 79%-92% for grade I, from 65%-70% for grade II, and from 41%-54% for grade III. Differences within the three grades for the various treatment regimens did not reach statistical significance. Symptomatic improvement was evaluated with the Standardized Total Heartburn Index, which is based on frequency and severity of heartburn as well as on the number of patients in the study population experiencing heartburn at a given time in relation to the total heartburn load at the start of the study. All three treatment schedules resulted in a substantial reduction of the Standardized Total Heartburn Index. Treatment with cimetidine, 800 mg u.i.d., for 6-12 weeks was efficacious in the majority of patients with reflux esophagitis grade I-III. Symptom relief was superior with dosing after dinner time compared with dosing HS. A single dose of 800 mg administered after the evening meal approached the efficacy achieved with 400 mg q.i.d. Based on these objectives and symptomatic results, a u.i.d. cimetidine regimen appears to be the treatment of choice for the initial approach of a patient with reflux esophagitis. A u.i.d. regimen may enhance patient compliance, comfort, and safety as well as ease of prescription while also being less expensive.

Immunoenzymehistochemical demonstration of the binding of low density lipoproteins to cultured human fibroblasts.

Using as indirect cytochemical immunoperoxidase technique, we were able to demonstrate the binding of low density lipoprotein to cultured human fibroblasts. With this technique, fibroblasts from a patient suffering from homozygous hyperlipoproteinaemia type IIa did not show this binding. The method described here allows study of the localization of unmodified low density lipoproteins binding to cultured fibroblasts.