Oscar W. Portman

Factors influencing the level and fatty acid specificity of the cholesterol esterification activity in human plasma

Abstract A procedure for studying the cholesterol esterification activity and fatty acid specificity of human plasma was described. The substrate was prepared by incubating heat-inactivated plasma with cholesterol-4-C 14 which had been applied to acid-washed Celite [Avigan, J. (3)]. Small quantities of homologous and heterologous unheated plasmas were incubated with this substrate. This system was characterized, and it was shown that the esterification activity of plasma from a variety of sources could be evaluated with this common substrate. The apparent esterification determined from radioactivity measurements was shown to represent net esterification and to depend chiefly on the properties of the active component. Cholesterol esters of individual fatty acids were formed in quantities proportional to the pre-existing pattern of the active plasma added, even when the active plasma was added to a substrate having a very different cholesterol ester composition.

Excretion of bile acids and β-hydroxysterols by rats

Abstract Cholic acid-24-C 14 was used to determine the quantities of cholate and degradation products of cholate in feces of rats fed different diets. Quantities of β-hydroxysterols (cholesterol and analogs) excreted by the same groups of rats were also measured. Rats fed Purina laboratory chow excreted a mean total of 36.4 mg. cholate and degradation products of cholate/day/kg. body weight. This compared with values of 10.3 mg. for rats fed completely purified diets including corn starch and a mean excretion of 7.7 mg. for rats fed the same purified diet with sucrose in lieu of starch. Inclusion of 20% Celluflour in the sucrose synthetic diet resulted in a mean excretion of 23.4 mg./kg. body weight/ day. The quantities of β-hydroxysterols were greater than the quantities of cholic acid in all dietary groups. Four hours after the intraperitoneal administration of cholate-24-C 14 , all of the radioactivity (paper chromatography) in the bile and portal blood was as taurocholate. Separation of the radioactivity in feces by chromatography indicated that those groups of animals with the most rapid turnover rates of cholate had bile acids in feces which appeared to have a different pattern of modification by intestinal bacteria.

Bile acid excretion by the rat: nutritional effects.

Abstract A modification of several published procedures for bile acid determinations has been developed, and this procedure has been applied to the 24-hr, biliary excretions of rats prepared for 15 days on a series of dietary regimens. It has been established that: 1. 1. Cholesterol supplementation of the basal synthetic diet or of Purina Chow resulted in significantly increased biliary bile acid and cholesterol excretion. 2. 2. The levels of vegetable fat did not influence the biliary excretion of bile acids and cholesterol. 3. 3. The feeding of a commercial diet (Purina Rat Chow) resulted in a much higher bile acid excretion with a higher proportion as cholic acid than did any synthetic diet tested. 4. 4. The substitution of egg albumin for casein in the synthetic diet resulted in an increased bile acid excretion; however, the addition of methionine, cystine, or taurine did not produce a significant increase. 5. 5. The addition of brewers yeast or desiccated liver to the diet or the administration of parenteral vitamin B 12 was without significant effect. 6. 6. Fasting decreased the bile acid excretion of rats previously fed Purina Chow but increased the excretion of rats fed the basal (sucrosecontaining) diet. The substitution of starch for sucrose in the basal dict resulted in a larger biliary excretion of bile acids and a larger proportion as cholic acid. Dextrose substituted for sucrose was without effect. 7. 7. Increasing the levels of Cellu-flour in the basal diet also resulted in an increased biliary bile acid excretion. The conclusion was drawn that sucrose and dextrose are inhibitors of biliary bile acid excretion in the rat.

Effect of dietary carbohydrate on experimentally induced hypercholesteremia and hyperbetalipoproteinemia in rats.

Summary Male albino rats were fed diets for 28 days in which the type of carbohydrate was varied and serum cholesterol and beta-lipoproteins were determined. 1. Rats fed diets containing sucrose as the carbohydrate and with added cholesterol and cholic acid had higher serum cholesterol and serum beta-lipoprotein concentrations than did control rats fed diets with corn starch substituted for sucrose. Liver cholesterol concentrations were not significantly different. 2. Feeding of sucrose, glucose, and fructose as the dietary carbohydrate had essentially equal effects on cholesterol-cholic acid induced hypercholesteremia. 3. Rats which were fed cholesterol containing diets without cholic acid and with corn starch as the carbohydrate had somewhat lower serum cholesterol values than did the controls which were fed sucrose. 4. When sulfasuxidine was added to sucrose containing diets, there was no change in serum cholesterol values; however, addition of sulfasuxidine to starch containing diets resulted in elevation of serum cholesterol values to the level of that in rats fed sucrose.

Swelling of liver mitochondria from rats fed diets deficient in essential fatty acids.

Summary Comparison of rates of in vitro swelling of liver mitochondria from control rats and from rats fed diets deficient in essential fatty acids were studied under a variety of conditions. Higher swelling rates of EFA deficient mitochondria were observed using 0.30 M sucrose medium. Addition of ATP or serum albumin inhibited swelling of normal but not that of EFA deficient mitochondria. Respiratory inhibitors prevented swelling of both groups of mitochondria. Cholate accentuated the swelling pattern of mitochondria from both groups of rats. The fatty acid composition of lipid from mitochondrial, microsomal and supernatant fractions from both groups of livers were also presented.

Essential fatty acid deficiency and cholesterol esterification activity of plasma and liver in vitro and in vivo

Abstract The esterification of cholesterol in vitro and in vivo was studied in rats fed diets containing and deficient in essential fatty acids (EFA). EFA deficiency resulted in increased cholesterol esterification activity in vitro , and this appeared to reflect an increased plasma cholesterol esterification activity in vitro , and this appeared to reflect an increase in enzyme concentrations. There was a decrease in plasma lipid phosphorus concentrations during the period of cholesterol esterification. Certain aspect of the fatty acid compositions of plasma phospholipids and cholesterol esters were consistent with a role of phospholipid fatty acids in the transesterification reaction which has been demonstrated by Glomset et al. (18). There was heterogeneous labeling of different subclasses of cholesterol esters when cholesterol-4-C 14 was used as the substrate, the more unsaturated fatty acid esters having the highest relative specific activities. EFA deficiency resulted in decreased cholesterol esterification activity by hepatocytic organelles. This activity was also reduced when organelles were exposed to hypotonic media, ultrasound, or taurocholate. Rats deficient in EFA had increased rates of esterification of cholesterol-4-C 14 in plasma and increased rates of elimination of radiocholesterol from the plsama after intravenous injection of labeled lipoproteins. This was associated with higher concentrations of total radioactivity in the livers of the EFA deficient rats. The specific activities of individual subclasses of cholesterol esters of plasma and liver were also heterogeneous for several hours in these in vivo experiments regardless of the dietary regimen involved. The more unsaturated fatty acid esters had the highest initial specific activities. Although liver and plasma free cholesterol specific activities rapidly equilibrated, the specific activities of the cholesterol esters and individual subclasses of cholesterol esters of plasma were greater than the activities of corresponding fractions from liver. It was proposed that the differences in plasma and liver esterification activities induced by EFA deficiency were secondary to alterations in the stability of hepatocytic organelles.

FATTY ACID SPECIFICITIES AND RATES OF CHOLESTEROL ESTERIFICATION IN VIVO AND IN VITRO.

The esterification of cholesterol and the formation of different fatty acid esters of cholesterol were studied after the administration of radiomevalonate or cholesterol-4-C14 to rats or Cebus monkeys. Cholesterol-4-C14 in the free form for intravenous injection was prepared by incubation of plasma at 0 ° C with radiocholesterol on Celite. After injection of mevalonate-2-C14 into rats or monkeys or cholesterol-4-C14 into rats, the specific activity of plasma cholesteryl arachidonate was higher and that of cholesteryl oleate lower than the specific activity of total plasma cholesterol esters for at least 6 hours. After the administration of cholesterol-4-C14 intravenously, the free cholesterol specific activity of liver was equal to that of plasma within 1 hour, but the ester cholesterol specific activity of plasma was three times greater than that of liver at 1 hour. The liver and plasma cholesterol ester specific activities did not become equal for nearly 12 hours. Cholesterol-4-C14 esterification by plasma in vitro resulted in a heterogeneous pattern similar to that seen in the early periods of the in vivo experiments. Esterification of radiocholesterol with different fatty acids by liver preparations in vitro was not similar to that seen in plasma. It was concluded from estimates of the rates of cholesterol esterification in plasma in vivo and in vitro that the plasma esterification activity was of considerable significance in maintenance of the level of plasma cholesterol esters. It was proposed that the early heterogeneity of labeling of different cholesterol esters in vivo resulted from the initial preponderant effect of the plasma esterification activity.

Alteration of bile salts by bacteria.

Summary The ability of a variety of common intestinal microrganisms to grow in bile salt-containing media and to alter the structure of those bile salts was studied. Strains of Escherichia coli, Proteus vulgaris, and Streptococcus faecalis were adapted by serial transfer in cholate-containing media to grow more rapidly in high concentrations of cholate and to alter the composition of the cholate more rapidly than unadapted cultures. E. coli was also shown to alter the structure of deoxycholate. Low concentrations of linoleic or palmitic acids when added to cholate-containing media partially overcame the inhibition of growth and cholate-altering capacity of E. coli. 7-Ketodeoxycholate was the principal metabolite of cholate in continuously aerated cultures of E. coli. The conditions used for transfer of mixed cultures and the isolation of one apparently pure culture of organisms from rat cecum which was capable on 2 first transfers, but not subsequently, of converting cholic acid to deoxycholic acid were described.

Effects of essential fatty acid deficiency on rat adrenal composition and secretory activity

The effect of feeding diets deficient in essential fatty acids on the composition of adrenal lipids and on the secretory activity of adrenocortical hormones was studied. Rats which were fed diets devoid of fat had lower levels of tetraenoic and pentaenoic acids and higher levels of trienoic acids in both the total lipid and cholesterol ester fraction than did rats receiving a supplement of corn oil. There was a greater accumulation of cholesterol esterified with saturated and monounsaturated fatty acids in the adrenals of the rats fed essential fatty acid-deficient diets. The adrenals of the essential fatty acid-deficient rats secreted smaller quantities of steroid hormones in vitro under the stimulation of ACTH.

Mitochondrial dehydrogenase activity of rat liver deficient in essential fatty acids.

Abstract Mitochondrial β-hydroxybutyric, isocitric, and malic dehydrogenase activities for normal rats and for rats fed diets deficient in essential fatty acids (EFA) were compared. Intact mitochondria from EFA deficient rats had higher activities in 0.5 M sucrose. Both groups of mitochondria had higher activities in water than in 0.5 M sucrose, but the differences between dietary groups were less marked with the hypotonic media than with 0.5 M sucrose. Sonic disruption of the mitochondria increased the dehydrogenase activities of both groups greatly, and eliminated the differences in activities related to diet.