Einar Lystad

Reduced plasma fibrinogen, serum peroxides, lipids, and apolipoproteins after a 3-week vegetarian diet

The influence of a 3-week vegetarian diet and fasting on serum concentration of peroxides, lipids, apolipoproteins, and plasma fibrinogen was studied in ten middle-aged fibromyalgia/fibrositis patients (eight women, two men). Mean serum peroxide concentration (estimated as thiobarbituric acid reacting substances) was reduced from 3.60±0.14 to 2.82±0.15 umol/l (p=0.01) and plasma fibrinogen from 3.33±0.25 to 2.74±0.15 g/l (p=0.02). Serum total cholesterol fell from 6.61±0.50 to 4.83±0.35 mmol/l (p<0.0001), apolipoprotein B from 1.77±0.14 to 1.31±0.11 g/l (p<0.0001), and apolipoprotein A from 1.41±0.09 to 1.23±0.05 g/l (p=0.03). High density lipoprotein cholesterol concentration also decreased somewhat (from 1.26±0.09 to 1.07±0.04 mmol/l,p=0.03) An atherogenic index, reflecting the balance between low and high density lipoproteins, was reduced by 31% (from 5.74±0.79 to 3.97±0.60,p=0.02). The results suggest that vegetarian diet/fasting may have a beneficial influence on the concentration of serum peroxides and plasma fibrinogen concentration, and on the serum level of several lipoprotein-related coronary risk factors.

The effect of insulin and guanosine nucleotides on protein phosphorylations by sarcolemma membranes from skeletal muscle

In a previous report we have shown that insulin increases the phosphorylation of an endogenous protein of mol. wt. 16 000 daltons in sarcolemma membranes. In the present work we have demonstrated that phosphorylations of exogenous histones by the sarcolemma membranes are also increased by insulin. These results indicate that insulin activates a cyclic-AMP-independent protein kinase in sarcolemma membranes. The stimulatory effect of insulin on protein phosphorylations is increased by GTP and its analogue GMP-P(NH)P. The insulin effect was increased 3--4-fold by micromolar concentrations of GTP. The effect by the analogue GMP-P(NH)P was somewhat less. In the absence of insulin guanosine nucleotides had no effect on phosphorylation of the proteins. The results suggest that GTP is a modulator in the activation of a sarcolemma membrane protein kinase by insulin.

Plasma high density lipoprotein subgroup distribution in rats fed diets with varying amounts of sucrose and sunflower oil

The effect of varying the dietary sunflower oil/sucrose (SO/SU) ratio on rat plasma lipid concentration and lipoprotein distribution was studied. Four groups of 10 rats were fed for 4 weeks diets with varying SO/SU ratios. Lipoprotein components were then estimated in whole plasma and after cumulative density ultracentrifugation. Whole plasma triacylglycerol (TG), total cholesterol (TC) and free cholesterol (FC) decreased with increasing SO/SU ratio; the CE/FC ratio increased, because CE remained virtually unaltered. Plasma TG-lowering was due to a decrease in VLDL and LDL-TG. Protein, CE and FC in d=1.063–1.100 g/ml (HDL2b) and d=1.100–1.125 g/ml (HDL2a) lipoproteins decreased upon increasing the SO/SU ratio. In contrast, in d=1.125–1.200 g/ml (HDL3) lipoproteins, there was a concomitant increase in these components. Although increasing the SO/SU ratio effected more protein and CE transportation in HDL3 and less in HDL2, the total amount of these components in high density lipoproteins (d=1.063–1.200 g/ml) remained constant. Apo A-I and apo C-III decreased in HDL2 but increased in HDL3 upon increasing the SO/SU ratio. Also, HDL2 apo E, and the apo C-II/apo C-III and small apo B/large apo B ratios in VLDL and LDL were lowered by increasing the SO/SU ratio. The hepatic VLDL-TG output during isolated liver perfusion was lowest in rats fed the diet with the highest SO/SU ratio. In perfusate, like in plasma, the VLDL and LDL apo C-II/apo C-III ratio, as well as the small apo B/large apo B ratio, decreased upon increasing the dietary SO/SU ratio. The results indicate that there can be appreciable diet-dependent variations in plasma HDL subgroup distribution in spite of unchanged total HDL levels.

Influence of fatty acids and bovine serum albumin on the growth of human hepatoma and immortalized human kidney epithelial cells

SummaryThe protective influence of bovine serum albumin against growth inhibition caused by fatty acids was studied in human hepatoma (HepG2) and immortalized human kidney epithelial (IHKE) cells. In general, growth inhibition by unsaturated fatty acids (0.15 mmol/liter) increased with increasing number of double bonds. For HepG2 cells crude albumin (1g/100 ml) did not greatly modify growth inhibition by arachidonic, eicosapentaenoic, and docosahexaenoic acid. With oleic, linoleic, and linolenic acids, crude and defatted albumin stimulated cell growth. In contrast, for IHKE cells both albumins counteracted growth inhibition by unsaturated fatty acids to approximately the same extent. When HepG2 cells were cultured in the presence of saturated fatty acids (0.3 mmol/liter), C2, C6, and C8 had no or little inhibitory effect. C10 and C12 inhibited cell growth appreciably, whereas C14, and especially C16, had poor inhibitory effects. Crude albumin counteracted growth inhibition by all these fatty acids. In contrast, defatted albumin had little or no effect (except against C10 and C12), and even increased the growth inhibition by C14 and C16. With unsaturated fatty acids there seemed to be an inverse relationship between cell growth and the concentration of thiobarbituric acid reactive substances (TBARS) in media. Vitamin E abolished growth inhibition (and the increase in TBARS concentration) by unsaturated fatty acids. The complex interaction between fatty acids and albumins calls for great caution when interpreting data on growth effects.

ADP-ribosylation of sarcolemma membrane proteins in the presence of cholera toxin and its influence on insulin-stimulated membrane protein kinase activity

We have reported that insulin stimulates cyclic AMP-independent protein kinase activity in sarcolemma membranes El]. This insulin effect was enhanced by PM levels of GTP [2]. The suggestion was made that a GTP-binding protein was involved in the hormonal control of this protein kinase. To gain support for this assumption we have investigated protein kinase activity after preincubation of sarcolemma in the presence of cholera toxin and NADf. The A1 fragment of cholera toxin is known to catalyze protein ADP-ribosylation from NAD+ [3] and the wellknown stimulation of adenylated cyclase by cholera toxin has been attributed to ADP-ribosylation of a membrane protein of MI 42 000 [4,5]. It is indicated that this membrane protein is identical with the GTPbinding protein involved in control of adenylate cyclase activity [6]. Here we demonstrate ADPribosylation of a sarcolemma membrane protein of MI 56 000. This results in inhibition of cyclic AMPindependent protein kinase activity and abolishes the stimulatory effect by insulin on this enzyme.

Estimation of lipoprotein and apoprotein distribution in rat plasma by cumulative density ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Lipoprotein distribution in rat plasma determined after sequential ultracentrifugation (requiring 8 days of centrifugation to separate lipoproteins in five density classes), was compared to estimates based upon cumulative density ultracentrifugation (46 hr of ultracentrifugation). In general comparable values were obtained by the two methods with regard to protein, total cholesterol, cholesteryl ester, free cholesterol, and triacylglycerol distribution. However, the HDL3 protein concentration found by sequential ultracentrifugation was only about 50% of that found after the cumulative procedure. Apolipoproteins in lipoproteins isolated by the two methods were well separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Color of the stained bands was extracted and read photometrically. A linear standard curve was obtained with albumin. Absorbance corresponding to 1 microgram/ml was 0.057. Below d = 1.100 g/ml (HDL2b) the two ultracentrifugation methods gave comparable results for all apoproteins. In contrast to this the level of apo A-I, apo E, and apo A-IV in the more dense types of HDL was higher when estimated by cumulative than by sequential ultracentrifugation. In HDL3 isolated by sequential ultracentrifugation the apo A-IV, apo E, and apo A-I concentrations were 51, 31, and 45% respectively, of values found after cumulative ultracentrifugation. The results indicate that cumulative density ultracentrifugation, followed by colorimetric determination of apoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is a useful approach when studying lipoprotein distribution in rat plasma.

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

SummaryPeroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22∶6, n−3), arachidonic acid (C20∶4, n−6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time-and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.