Ralph M. Johnson

A study of the onset of unsaturated fatty acid deficiency in subcellular particles of rat livers.

Abstract 1. 1. Rats placed on a fat-free diet lose di-, tetra-, and pentaenoic acid from cytoplasmic particles at a high initial rate which declines by 6 weeks. Thereafter, the losses proceed very slowly, in some cases without change for 12–24 weeks. 2. 2. The largest losses are found in the microsomal fraction and the least in the mitochondrial, while those of the poorly sedimentable fraction are intermediate. 3. 3. The trienoic acid accumulates in all three fractions, reaching levels of four to six times those found in the fraction under normal conditions. 4. 4. Since these alterations occur approximately 6 weeks before the onset of external symptoms of fat deficiency, it is suggested that the primary lesion in fat deficiency may take place earlier than heretofore suspected and may bear only a secondary relationship to the external symptoms.

The uptake of radioactive phosphorus by rat liver following partial hepatectomy

Summary o 1. Adult male Holtzman rats were given a single subcutaneous injection of radioactive phosphorus, as NaH 2 P 32 O 4 , at 16 hr., and 1, 3, 5, 7, or 14 days after partial hepatectomy. The animals were sacrificed 4 hr. after injection, and the radioactivity and total P content of the acid-soluble organic and inorganic, lipide, phosphoprotein, pentose nucleic acid (PNA), and desoxypentose nucleic acid (DNA) fractions were determined. 2. Following partial hepatectomy there was an increase in the concentration of P per unit of protein N in the acid-soluble inorganic, lipide, PNA, and DNA fractions of the “regenerating” livers. Maximum concentrations were observed at about 1 day in the first two fractions and at 3–5 days in the nucleic acids. Thereafter the concentrations decreased slowly. There were no significant changes in the acid-soluble organic and phosphoprotein P. 3. In the hypertrophying liver cell following partial hepatectomy there was a marked increase in the uptake of P 32 in the acid-soluble organic and inorganic, phosphoprotein, PNA, and DNA fractions over that seen in the controls. With the shift from hypertrophy to cell hyperplasia there was a decline in the P 32 uptake by these fractions. 4. The maximum uptake of P 32 by the phospholipide fraction coincided with maximum mitotic activity.

Changes with age in the unsaturated fatty acids content of subcellular particles of rat livers

Abstract 1. 1. The polyunsaturated fatty acid concentration of three cytoplasmic particulate fractions of rat liver have been investigated in rats of different ages. Two groups of rats, reared on diets of different unsaturated fatty acid content, were used. 2. 2. No changes with age, or differences between the two groups were observed until 11 mouths of age. Thereafter, the group receiving the diet containing the lower fatty acid content showed increased concentrations of trienoic acid in the cytoplasmic particles, which reached levels exceeding those of either the di- or tetraenoic acids in the same particulate fractions. Concurrently there was a decline in the pentaenoic acid concentrations. No such changes occurred in the animals receiving the diet which contained the higher levels of the unsaturated fatty acids. 3. 3. The results indicate that there is no appreciable change during growth, maturation, or senescence in the levels of di-, tri-, tetra-, or pentaenoic acids in liver cytoplasmic particles studied, in rats fed adequate levels of these acids. 4. 4. The results suggest that a diet which can support good growth and reproduction in rats may not be adequate to meet the nutritive requirements of older animals, permitting some of the symptoms of a fatty acid deficiency to appear.

Phospholipide metabolism in cell fractions of regenerating liver.

Abstract The P 32 uptake in a number of phosphorus-containing fractions of the nuclei, mitochondria, microsomes, and supernatant of liver cells was investigated in normal rat liver and during the premitotic and mitotic phases of liver “regeneration” following partial hepatectomy. The cephalins of all the cell fractions of the normal livers incorporated P 32 much faster than the lecithins, while the sphingomyelins incorporated it relatively slowly. The increased P32 uptake of the phospholipides observed earlier to be associated with mitotic activity can be accounted for by an increased P 32 uptake by the cephalins of the nucleus, by the lecithins of the mitochondria, microsomes, supernatant, and possibly the nuclei, and by the sphingomyelins of the supernatant. The data, viewed in the light of observations made in earlier studies, suggest that P 32 turnover in lecithin occurs in all parts of the cell during interphase; that of nuclear cephalins and of supernatant sphingomyelins occurs following preprophase and prior to or during metaphase processes.

Metabolism of phospholipides associated with cell division

Abstract Male Holtzman rats 6–7 months old were partially hepatectomized and sacrificed 18 hr. or 3 days later. Five hours prior to sacrifice, acriflavine at a level of 2 mg./100 g. body weight was administered subcutaneously to the experimental (treated) animals, and 1 hr. prior to sacrifice both the treated and untreated rats received a subcutaneous injection containing 0.45 μc. P32/g. body weight. The uptake of P32 and total P in the acid-soluble, lecithin, cephalin, sphingomyelin, and DNA fractions, and the amounts of di-, tri-, tetra-, and pentaenoic acids were measured in the livers of all animals. The uptake of P32 in livers of unoperated control animals was much more rapid in the cephalin than in either the lecithin or sphingomyelin fractions. During “regeneration” following partial hepatectomy, the P32 uptake in the lecithin fraction increased markedly, reaching a maximum at 3 days when mitotic activity was highest. During this period, uptake in the sphingomyelin was increased to a smaller extent, while that in the cephalin fraction was not elevated prior to the onset of mitosis (18 hr.), but was elevated slightly and nonsignificantly at 3 days. Acriflavine, a preprophase inhibitor, caused a marked inhibition of P32 uptake in all of the phospholipides and in DNA in the animals 18 hr. following partial hepatectomy; 3 days afterward it markedly inhibited mitosis but had little or no effect on the incorporation of P32 into the phospholipides. This suggests that the increased phosphorylation of lecithin shown to be associated with mitosis occurs during preprophase processes. The data do not bear out a relationship between unsaturated fatty acid concentrations and mitosis.

Lipide metabolism during cell division.

Abstract 1. 1. Adult male Holtzman rats were each given a single injection of P32 as NaH2P32O4 at 1, 3, and 18 days after partial hepatectomy, and were sacrificed 1 hr. later. Six hours prior to sacrifice, unoperated controls and 3-day hepatectomized rats received 0.03 mg. colchicine/100 g. body weight by subcutaneous injection. Livers were removed and the radioactivity and P in the lecithin, cephalin, and sphingomyelin fractions, as well as di-, tri-, tetra-, and pentaenoic acids were determined. 2. 2. The high P32 uptake by the phospholipide fraction coincident with mitosis occurred in all three types of phospholipide isolated. It was not abolished by colchicine, a metaphase inhibitor, suggesting that it is associated with either interphase or prophase, but not with anaphase or telophase. 3. 3. There was an increase in the concentration of trienoic and a decrease in tetraenoic acid coincident with the period of hypertrophy (premitosis) in regenerating liver. There was an increase in dienoic acid coincident with the period of high mitotic activity. Blocking mitosis in metaphase with colchicine did not influence the level of dienoic acid.

The composition of the lipides of resting and pregnancy-stimulated mammary glands and mammary carcinomas.

Abstract 1. 1. The composition of the lipides of two growing tissues of the mouse, spontaneous mammary carcinomas and pregnancy-stimulated mammary glands, has been compared with that of resting mammary glands. About 1% of the lipide of both resting and stimulated mammary glands may be classified as “essential” (cholesterol plus phospholipide), and approximately 66% of the lipide of mammary carcinomas was found in this class. The total phospholipide contents of the three tissues were similar, but the amounts of the individual phospholipides differed. Pregnancy-stimulated glands contained more lecithin and less cephalin than resting glands. Of the three tissues, mammary carcinomas contained the least lecithin and the most cephalin. 2. 2. The ratio of free to esterified cholesterol was the same for the three tissues. The resting glands and carcinomas contained roughly similar amounts of total cholesterol and far more than that found in stimulated glands. 3. 3. It is concluded that one cannot describe altered tissue activity, defined broadly, in terms of changes in the total amount of the “constant factor” of lipides.

In vitro activation of cysteine desulfhydrase by rat liver microsomes.

Summary The cysteine desulfhvdrase of rat liver was found in the cytoplasmic supernatant but was not associated with either nuclei, mitochondria, or microsomes. There was an increase in enzyme activity when the microsome fraction was added to the cytoplasmic supernatant. This increase was greater as larger amounts of microsomes were added. No enhancement was observed when microsomes were added to a whole homogenate from which microsomes only had been removed, suggesting a complex interdependence of the various cytoplasmic elements.

Phospholipide metabolism during hypertrophy and hyperplasia in rat prostates and seminal vesicles

Abstract Control, castrated, and testosterone-treated rats were given subcutaneous injections of 0.45 μc. of radioactive phosphorus, as NaH 2 P 32 O 4 , and were sacrificed 2 hr. later. The radioactivity and total P contents of the acid-soluble, phospholipide, cephalin, lecithin, sphingomyelin, and DNA fractions of prostates and seminal vesicles, and the mitotic incidence in these tissues, were determined. Testosterone administered to normal animals caused hypertrophy of the prostates and hyperplasia and hypertrophy of the seminal vesicles. These were accompanied, in both tissues, by an increased P 32 uptake in all the P fractions. Treating the castrated animals with testosterone produced hyperplasia and hypertrophy in both tissues. This was again accompanied by increases in P 32 uptake in the P fractions. However, the increase in the phospholipide fractions that was associated with the hyperplasia in the prostates was much greater than that associated with hypertrophy and was not dependent on the P 32 uptake activity of the acid-soluble fraction. These results are interpreted as demonstrating that in the prostate, as in regenerating liver (1), mitotic activity is associated with a maximum P 32 uptake in the phospholipides.

Use of Trichloracetic Acid in Purification of Lipids.

Summary A method is described for the quantitative extraction and purification of small amounts of lipid with a minimum of manipulation. The acid-soluble material is removed from the tissue with cold 10% trichloracetic acid containing 0.4 M MgCl2. The lipid is removed from the residue using ethanol-ether. This extract is taken to near-dryness, re-extracted with petroleum ether-chloroform, and the latter solution washed with water and dried. The lipid thus prepared has been found to compare favorably in several respects with lipid purified according to McKibbin and Taylor(3).