Ruth Okey

A micromethod for fractionation of lipids by silicic acid chromatography

Abstract A micromethod has been presented for separating, by silicic acid chromatography, 1–10 mg of a complex mixture of lipid of biological origin into four fractions: I, cholesterol ester; II, triglycerides; III, free cholesterol, free fatty acids, and mono- and diglycerides; and IV, phospholipids. The method was found to be rapid, convenient in handling, quantitative, and eminently suitable for further analysis of the fractions by gasliquid chromatography.

Analysis of fatty acid mixtures : comparison of two " absolute " methods of determination.

Abstract We have applied a method using an internal standard to the determination of absolute quantities of fatty acids; these quantities are more fundamental than the relative or percentage values usually obtainable by gas-liquid chromatography. The use of weight units allows direct comparison of different samples, and removes the dependence of individual values on the values of all the other components produced by the use of percentages. Use of the internal standard gives data comparable with those of older methods, but is applicable to much smaller samples. In addition to its speed and convenience it corrects automatically for small errors in manipulation during preparation of the samples for analysis; in particular, this method automatically corrects for the unknown losses which occur during injection of a sample into the gas-liquid chromatography apparatus. The internal standard method is particularly useful for samples which are too small for independent measurement of the weight of total fatty acid.

Gallstone Formation and Intake of B Vitamins in Cholesterol-Fed Guinea Pig

Thiamin, riboflavin, pantothenic acid and biotin are all reported to produce fatty livers in the rat: choline, inositol, and pyridoxine to prevent them. 1 Biotin fatty livers alone have a high cholesterol ester content. 2 The cholesterol-fed guinea pig develops a fatty liver, enlarged spleen and a severe anemia. Liver damage can to some extent be judged by red cell count. 3 Vitamin requirements of the guinea pig are not entirely known. Findings reported here are the indirect results of an attempt to improve the nutrition of our animals and so make it possible to study the lesions remaining after a period of cholesterol feeding. Diet “A” had been planned to parallel in respect to protein and fat content an egg yolk diet previously used. It supported growth in control animals slightly less well than our stock diet. Possible shortcomings seem tobe low riboflavin and pantothenic acid and too much fat. Diet “B” in which the amounts of these components were adjusted as indicated below, increased apparent well being in both control and cholesterol-fed animals. We were greatly surprised, therefore, when the cholesterol-fed animals on this diet showed an abrupt fall in red cell count without the usual preliminary loss in weight and other evidences of illness, and when the autopsies showed gallstones. Guinea pig bile contains relatively little lipid. These gallstones were rich in calcium phosphate, although they contained some cholesterol. Irritation of the membranes of the gall bladder and the biliary passages was common and some impaction of ducts was observed in cholesterol-fed animals. No stones were found in control animals. If this high incidence of gallstones is to

Bone Marrow Composition of Cholesterol-Fed Guinea Pigs.

Summary The anemia produced in guinea pigs by dietary cholesterol led to a stimulation of erythropoietic activity with a proliferation of marrow cells which displaced the fat globules normally occupying the femoral marrow cavity. The triglyceride content decreased to half of its original level while the relative proportion of PL and cholesterol, on a dry, fat-free basis, remained unchanged. The proportion of linoleic acid in the remaining portion of TG decreased. Fractionation of the PL showed changes in their composition, particularly a decrease of the less polar components. The relationship of these changes in marrow to the changes observed in the peripheral blood requires further study.

Middle and Old Age in Cholesterol-Fed Rats.

Summary Rats have been fed diets containing 1% cholesterol from weaning throughout their life span. Their growth, health and time of survival have not differed significantly from those of control animals on the same basic diet without the cholesterol.

Effect of Olive Oil and Squalene on Cholesterol Mobilization in the Rat.

Summary Much higher values for serum cholesterol were observed in 2 series of female rats fed 1% cholesterol with diets containing 10% olive oil than in the series fed like diets made up with a number of other vegetable fats. Olive oil-fed males did not have high serum cholesterols, but did have high liver lipids and cholesterol. An attempt to duplicate the effect of olive oil by adding equivalent amounts of commercial squalene to diets containing 10% cottonseed oil and 10% coconut oil is reported. The squalene-fed females on diets with added cholesterol had higher serum cholesterols than did those fed cholesterol only. Values were not nearly so high as in the rats fed olive oil. Both males and females fed squalene with cholesterol and coconut oil had significantly higher liver lipids and cholesterol than did littermates fed only coconut oil and cholesterol in the same amounts. Little or no effect attributable to squalene was observed in castrates. The possible effect of squalene as a stimulant of secretion of gonadal hormones is discussed.

Adaptation of the Bloor Oxidation Procedure to the Determination of Small Quantities of Cholesterol as Digitonid.

Micro methods for the estimation of cholesterol have hitherto been based almost entirely upon the color reaction with acetic anhydrid and concentrated sulphuric acid. The presence of interfering color producing substances in blood and tissue extracts frequently makes it highly problematical whether or not this reaction gives a true picture of the cholesterol concentration. The gravimetric estimation as the digitonid, proposed by Windaus 1 is impracticable where the amounts to be weighed are as small as those available in the average blood analysis. We have, therefore, attempted to adapt the digitonin precipitation to micro determination by oxidation of the digitonid according to the method used by Bloor 2 for fatty acids and phospholipins; i. e., with silver-chromate sulfuric acid (Nicloux reagent) and normal K2Cr2O7. Since this oxidation is not specific, the largest problem involved has been the separation of the digitonid from the excess digitonin used in precipitation, and from contaminating substances of a fatty nature. Digitonin is a saponin. Consequently, the surface tension of its water solutions is such as to make separation of the digitonid by centrifugalization impossible. Filtration of the digitonid from water solutions of digitonin has proved quantitative. However, the physical nature of the digitonid precipitate is such as to make quantitative removal of very small amounts from the filter and sides of the flask impracticable. We have, therefore, devised, with the assistance of Mr. D. J. Kooyman, a small Gooch filter tube made from a piece of Pyrex glass tubing 45 mm. long and 18 mm. in diameter, constricted somewhat at one end and fitted with a perforated porcelain disk (cut from the bottom of an ordinary Gooch crucible). This, after the filtration and washing is completed, may be placed bodily inside the flask used for the oxidation in such a way that the oxidizing solution may be made to come in contact with every part of the tube and the asbestos mat. The asbestos used for the filter must be prepared by long continued (72 to 120 hrs.) heating at 120-130° C. with bichromate-sulphuric acid which is frequently renewed. The asbestos is washed free of bichromate and suspended in distilled water in the usual way. This filter differs from that used by A. von Szent Györgyi 3 in that it makes possible complete oxidation of the digitonid by allowing control of time and temperature.

Effect of Feeding Egg Yolk on Liver Lipids of Rats

The deposition of fat and sterol-ester in the livers of animals fed diets rich in cholesterol has been reported by a number of workers. The following is a report of one of a series of experiments made to determine the influence of other dietary constituents than sterol on this lipid deposition in the livers of rats. The experimental diet fed contained dried egg yolk powder 25.3%, extracted casein 12.6%, agar 4%, Osborne Mendel salts 3%, starch 55.1% with supplements of yeast and cod liver oil. This represented egg yolk protein 8.4%, egg yolk lipids 16%, of which 1/16 was cholesterol, 1/4 lecithin. For the control diet the egg yolk powder was first extracted with alcohol-ether and the egg yolk lipid replaced by Crisco. Each experimental group consisted of 8 rats, while there were 12 in the control group, littermates of the experimental animals. The rats were placed on the diet at weaning time. Both experimental and control groups grew extremely well. They were killed after approximately 60 days on the diet. The livers of the experimental animals presented the typical whitish coloration of sterol-fed animals. The average total fatty acid content of the livers was for the males of the experimental group, 9.3%; for the females, 11.7%. For the control groups the corresponding figures were: males 7.5%; females 7.4%. Total cholesterol (digitonid precipitation) for the males of the experimental group was 2.58%; for the females 3.86%. For the control groups the corresponding figures were: males .48%; females .30%. There was very little difference in the free cholesterol in the control and experimental groups. The average for both groups was approximately 0.3%. Likewise the figures for lecithin were not very different, the average for males in both groups being 2.7% and for females in both groups 2.25%. It was noted, however, that the odor of choline, presumably from decomposition of lecithin, was very strong in the tissues of the animals of the experimental group and absent in those of the control group.

Effect of cholesterol feeding on growth of rats.

In this laboratory, during the past few years, we have fed to a total of several hundred young rats diets containing 1% cholesterol. We have found, almost without exception, that if the basic diet to which the cholesterol was added was adequate to support entirely normal growth on a long-time basis, there was no significant difference between the growth curves of the cholesterol-fed animals and those of the littermate controls which received the same diets without the added cholesterol. Moreover, this was true as well for certain vitamin-deficient diets, notably those lacking A. This finding is entirely at variance with that reported by Sperry and Stoyanoff 1 for synthetic diets. This would seem to indicate the value of analysis of the diets used in the two laboratories with this point in view. The composition of our diets has already been reported. 2 Extracted casein was used as a source of protein in both laboratories and at levels sufficiently nearly alike as to probably rule out differences in behavior due to protein intake. Sperry, et al., have used sucrose, and we have used starch as a source of carbohydrate. While the cornstarch used in our laboratory certainly did not contain enough betaine, as Best and Ridout 3 have suggested that certain samples of potato starch may, to largely prevent cholesterol ester deposition in the livers of our rats, nevertheless there is no proof that it did not contain some factor which may be necessary for the growth of cholesterol-fed rats. Again, we have used in this laboratory less highly purified sources of vitamins A, D, and, in some case, B and G, than the Columbia investigators. The behavior of vitamin-deficient animals fed cholesterol in this laboratory has been such as to indicate that lack of none of the vitamins just mentioned is in itself responsible for the retarded growth of Sperrys cholesterol-fed rats, but it does not eliminate the possibility that some other accessory factor associated with our vitamin sources may have made it possible for our cholesterol-fed rats to grow normally.