Virgil S. LeQuire

LIPASE ACTIVITY IN BLOOD FROM THE HEPATIC AND PERIPHERAL VASCULAR BEDS FOLLOWING HEPARIN.

Summary Clearing factor lipase, an enzyme with the ability to clear a lipemic substrate and to cause hydrolysis of triglyceride, is released during a single passage of heparin through the vascular beds of the dog liver, dog hindlimb and human forearm.

Isolation and characterization of hepatic Golgi lipoproteins from hypercholesterolemic rats.

The feeding of cholesterol-rich diets alters the serum lipoproteins of a number of mammalian species. These lipoproteins are characterized by the presence of several classes of particles enriched in cholesteryl esters and apolipoprotein E (apo E). It was the aim of this study to determine whether one or more of these particles arises by de novo hepatic synthesis by characterizing nascent lipoproteins isolated from the hepatic Golgi apparatus of hypercholesterolemic rats. Characterization of these lipoproteins afforded the opportunity to assess morphologic, biochemical, and biophysical properties of newly synthesized lipoproteins before enzymatic alterations and apoprotein transfer known to occur after secretion into the plasma compartment. Golgi very low density lipoproteins (VLDL, d < 1.006 g/ml) from hypercholesterolemic rats contained nearly four times the total cholesterol mass found in control Golgi VLDL. They exhibited electrophoretic mobility intermediate between beta and pre-beta and were devoid of apo C. A second population of hepatic Golgi lipoproteins was isolated from hypercholesterolemic rats at 1.006--1.040 g/ml d. These low density lipoproteins were smaller than VLDL, displayed beta electrophoretic mobility, were enriched in cholesteryl esters, and contained apo E as well as apo B. The fatty acid composition of the core lipids of the nascent lipoproteins was found to reflect that of dietary triglyceride. The liver of the hypercholesterolemic rat thus plays an active role in dietary-induced hypercholesterolemia by synthesizing a modified VLDL and a low density lipoprotein resembling serum low density lipoprotein.

Beneficial effects of penicillamine treatment on hereditary avian muscular dystrophy.

Hereditary muscular dystrophy in chickens of the New Hampshire strain was treated with penicillamine from the 9th day after hatching to the 425th day. The adult maintenance dose for males was 50 mg/kg per day and for females, 13-65 mg/kg per day. In avian dystrophy, deterioration of the muscle fibers is evidenced in the 2nd mo by an inability of the birds to rise after falling on their backs and by a progressive rigidity of the wings. The drug delayed the onset of symptoms and partially alleviated the debilitating aspects of the disease. Penicillamine produced three major improvements: (a) better righting ability when birds were placed on their backs; (b) greater wing flexibility; (c) and suppression of plasma creatine phosphokinase activity. The results are statistically analyzed and discussed in relationship to Duchenne dystrophy. Normal birds were not affected by penicillamine as judged by these parameters. The rationale for using penicillamine, a sulfhydryl compound with reducing properties, was (a) to attempt to protect essential thiol enzymes in the anabolic and glycolytic pathways against inactivation and (b) to prevent collagen cross-linking and deposition in muscle. Although the precise mechanism of drug action has not been determined. the possible role of penicillamine in mitigating the symptoms of genetic dystrophy in man is under consideration. Further, penicillamine may have a more generalized application i the prevention of contractures in a variety of neuromuscular disorders.

Pyrogenicity of Influenza Virus in Rabbits.

During an investigation of the toxicity of influenza virus in rabbits, it was noted that the intravenous injection of infected chorio-allantoic fluid was followed by fever. To study this phenomenon, animals were placed in individual stalls, 3 preliminary rectal temperatures were taken at 30-minute intervals to establish a baseline, and the virus preparation to be tested was then injected into the marginal ear-vein. Rectal temperatures were recorded every 30 minutes throughout an observation period of 6 hours after injection. All glassware and saline solutions used were uncontaminated with bacterial pyrogens. A single pool of chorio-allantoic fluid from embryos infected with the PR-8 strain, having a hemagglutinin titer of 1:1024, 1 was used. One ml of this material consistently caused a rise in temperature beginning l 1/2 to 2 hours after injection, reaching a peak of 3-4°F above the baseline in the next 4 hours, and gradually falling to normal. Although doses as small as 0.025 ml produced fever, the increases in temperature were of a lower order. Normal chorio-allantoic fluid and infected fluid from which the virus particles had been removed by centrifugation at 30,000 R.P.M. or by adsorption on chicken erythrocytes gave no fever. The virus resuspended in normal saline solution produced typical temperature elevations. No fever followed the injection of PR-8 virus neutralized with homologous immune serum. The febrile response in rabbits is independent of the infectivity of the virus but seems to be related to its adsorptive capacity. Heating at 56°C for 30 minutes destroyed the infectivity of PR-8 for chick embryos but a portion of the hemagglutinin was retained (1024 to 128); this material produced definite fever in rabbits. Heating at 62°C for 30 minutes destroyed the hemagglutinin titer and these preparations were then non-pyrogenic.

Mechanism of action of penicillamine in the treatment of avian muscular dystrophy.

Penicillamine, a cysteine analog with a reduced sulfhydryl group, has been used in this laboratory for the treatment of hereditary avian dystrophy. The drug delays the onset of symptoms and alleviates the debilitating aspects of the disease. To study the mechanism of drug action, the effects of penicillamine on white and red muscles of dystrophic chickens were examined with regard to the specific activities of the soluble enzymes glyceraldehyde-3-phosphate dehydrogenase, acetylphosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, glutathione preoxidase, superoxide dismutase, and catalase. The sulfhydryl contents of the soluble proteins and the concentration of myoglobin were also determined. In white dystrophic muscle (pectoral), there were large alterations in the various enzymatic activities compared to normal levels. In the DISCUSSION, these changes are related to the pathogenesis of the disease and to the adaptive response for protection of the severely affected fast fibers. Red dystrophic muscles (thigh) were minimally involved, in accordance with the known sparing action of the slow fiber type. The results suggested that the disease process in dystrophic muscle may be due to oxidation of the essential sulfhydryl groups of proteins. Penicillamine may produce therapeutic effects by altering the intracellular redox status, thereby promoting better regulation of enzymatic activity, membrane stability, and improved muscle function.

Leukocytic and pyrogenic effects of typhoid vaccine and augmentation by homologous plasma.

Summary and Conclusions The effects of intravenously administered typhoid vaccine upon the leukocyte population and the temperature of the rabbit were studied. The response of the temperature and of the leukocyte count was dependent upon the amount of typhoid vaccine given. Small amounts of the vaccine caused little or no fever, a heterophile leukocytosis, and a mononuclear leukopenia. When larger doses of vaccine were given, the rabbits responded with high fever, an initial heterophile leukopenia followed by a secondary leukocytosis, and a sustained mononuclear leukopenia. Ten times the number of organisms were required in saline suspensions to produce a thermogenic and leukocytic response comparable to that produced when the vaccine was given in homologous plasma. The plasma-typhoid and saline-typhoid reaction differed only in degree except for the earlier onset of fever when the organisms were given in plasma. These experiments on rabbits with typhoid vaccine indicate that when leukocyte changes are used as a test for pyrogens, it is necessary to employ a series of total and differential counts on each animal.

Transfer of esterified cholesterol from serum lipoproteins to the liver

The fate of cholesteryl esters of the serum lipoproteins was studied in intact rats and in isolated perfused rat livers. The lipoproteins of fasting rat serum were labeled in vitro with [3H]cholesteryl oleate. Following intravenous injection, it was found that the majority of the radioactive ester was rapidly taken up by the liver where hydrolysis of the ester bond occurred. At 5 min, 58% of the injected material was recovered in the liver, 85% of which was still in the ester form, while at 30 min only 22% of the liver radioactivity was in cholesteryl esters. There was very little difference in the rate at which radioactivity was taken up from the different lipoprotein classes. Similar phenomena were observed in the perfused liver, but it was found that although the radioactive esters were being taken up, there was no change in the concentrations of free or esterified cholesterol in the perfusing medium, indicating that the lipoprotein cholesteryl ester was gaining access to the liver through an exchange of molecules. After uptake, cell fractionation experiments showed that the plasma membranes had the greatest relative amounts of radioactivity, suggesting that this is the site of exchange. Small amounts of radioactivity were recovered in the bile, demonstrating that serum lipoproteins can serve as precursors of at least some of the bile steroids.

Electron paramagnetic resonance and saturation transfer electron paramagnetic resonance studies on erythrocytes from goats with and without heritable myotonia

SummaryErythrocytes from myotonic goats, an animal model of heritable myotonia, and normal goats were studied using electron paramagnetic resonance (EPR) and saturation transfer electron paramagnetic resonance (ST-EPR) spin labeling techniques. Three fatty acid spin labels with the nitroxide moiety at progressively greater distances from the carboxyl group were used to monitor different regions within the erythrocyte membrane. Since spin labels have been shown to induce hemolytic and morphologic alterations in erythrocytes, conditions for minimizing these alterations were first defined by hemolysis studies and scanning electron microscopy. Using these defined conditions for our studies we observed no significant differences in any of the EPR or ST-EPR parameters for normal and myotomic goat erythrocytes with any of the fatty acid spin labels used. Our results do not support the theory that myotonia is the result of a generalized membrane defect characterized by increased membrane fluidity as determined by fatty acid spin labels.

Alterations in plasma lipid concentrations in normal and hyperlipidemic patients with morbid obesity before and after jejunoileal bypass

In a study of 193 massively obese patients plasma lipoprotein concentrations were elevated in 94 patients and were within normal limits in 99 patients. Jejunoileal bypass, in addition to causing weight loss, lowered plasma lipids in both groups to normal or below normal levels.

A Generalized Membrane Defect in Heritable Myotonia: Studies of Erythrocytes in an Animal Model and Patients

Summary Studies were conducted on erythrocytes from myotonic goats, an animal model of heritable myotonia, and from patients with myotonic muscular dystrophy. When compared to those of normal goats, erythrocyte membranes from myotonic goats had (i) increased levels of calcium; (ii) an increased sialic acid content, and (iii) an altered protein composition with reduction of a 140,000 molecular weight protein. Erythrocytes from myotonic goats were also less susceptible to hemolysis than those from normal. Membranes of erythrocytes from patients with myotonic dystrophy had an increased content of sialic acid as compared to controls. The differences in calcium and sialic acid are consistent with previously reported abnormalities in the sarcoplasmic reticulum from the myotonic goat, and these findings support the concept of a generalized membrane defect in myotonia. This work was supported by grants from the Muscular Dystrophy Association. The authors acknowledge the excellent technical assistance of Patty Soule and Rita Bennett.