capable of preventing the dietary necrosis (2) were effective also in reducing mortality of mice fed the necrogenic diet and infected with M.H.V.3 virus. In chicks a Torula yeast diet produces multiple exudates. Like necrotic degeneration in mice these lesions can be prevented by both alpha tocopherol and selenium(3). Our experiments have pro- vided more evidence that very small amounts of selenium can often replace Vit. E as an es- sential nutrient. We have also shown that mortality of mice infected with experimental viral hepatitis and receiving a casein diet supplying 15% of protein is similar to that of animals receiving a yeast diet of similar concentration and supplemented with alpha tocopherol. Although casein and yeast had a similar effect on mortality, casein was con- siderably more effective in stimulating growth than yeast supplemented with alpha tocopherol. This result confirms previous ob- servations suggesting that the growth-pro- moting and anti-infective effects of protein are independent of one another (5).
Summary. A necrogenic Torula yeast diet increased the susceptibility of mice to experi- mental viral hepatitis. Supplementation of this diet with alpha tocopherol or selenium reduced the mortality of this infection. The susceptibility of mice fed a casein diet re- sembled that of animals receiving a yeast diet of similar protein content and supple- mented with alpha tocopherol.
1. Himsworth, H. P., The Liver and its Diseases, 2nd Edit., Cambridge, Mass., 1950, p120.
2. DeWitt, W. B., Schwarz, K., Experientia, 1958, v14, 28.
3. Dam, H., Nielson, G. K., Prange, I., Sønder- gaard, E., ibid., 1957, v13, 493.
4. Ruebner, B., Bramhall, J. L., Arch. Path., 1960, v69, 190.
5. Ruebner, B., Miyai, K., J. Lab. and Clin. Med., 1961, in press.
6. Meader, R. D., Williams, W., Am. J. Anat., 1957, v100, 167.
7. Dick, G. W. A., Niven, J. S. F., Gledhill, A. W., Brit. J. Exp. Path., 1956, v37, 90.
Received April 4, 1961. P.S.E.B.M., 1961, v107.
Precursors of Etiocholanolone and Androsterone in Adrenal Carcinoma. (26650)
MORTIMER B. LIPSETT (Introduced by Roy Hertz) Endocrinology Branch, National Cancer Institute, Bethesda, Md.
Most patients with adrenal carcinoma ex- crete increased amounts of etiocholanolone (E) and variably increased amounts of an- drosterone (A) resulting in an E/A ratio well above the normal range(1,2). This has been interpreted to mean that some of the E ori- ginates from C21-steroids whose metabolism leads to 17-ketosteroids (17-KS) of the 50- series predominantly. Since the relative amounts of 5a- and 53-17-KS may be re- lated not only to the precursors but to the metabolic transformations of any particular precursor, tritium-labeled dehydroepiandros- terone (DHA) has been used to study the origin of a urinary A and E in patients with adrenal carcinoma. The secretion rate of DHA was also estimated in these patients.
Materials and methods. Five mg of DHA
acetate were added to one millicurie of 7- tritium-DHA acetate (New England Nuclear Corp.), the material chromatogrammed in phenyl Cellosolve/heptane (I), eluted, and hydrolyzed with 0.067 N NaOH. It was then successively chromatogrammed in ligroin/ propylene glycol (II), and isooctane/me- thanol: water, 10/8:2 (III). The DHA then had a specific activity (S.A.) of 51 × 106 cpm/mg.
The samples were counted in the Packard Tri-Carb liquid scintillation spectrometer with an efficiency of approximately 15%. Sufficient counts were accumulated to give a standard error of no more than 5%.
Urine was collected for 48 hours after in- travenous injection of 2.1 × 106 cpm of DHA. The urines were treated as previously
| Patient | Age | Sex | Diagnosis | Clinical syndrome | Site of metastases | Urinary excretion, mg/24 hr | ||
|---|---|---|---|---|---|---|---|---|
| 17-KS | 17-OHCS | THS | ||||||
| Wh | 27 | ₽ | Chorioca | Good health | 9 | 6.2 | .2 | |
| Cha | 23 | 오 | ", | -- | 11 | 5.5 | .2 | |
| Se | 7 | 6 | Adrenal ca | Virilism | Lung | 44 | 4.3 | .2 |
| Che | 49 | ? | ", | Cushing's & virilism | Lang, liver | 480 | 170 | 56 |
| Ma | 37 | 우 | Lung, abdomen | 58 | 15 | 4.2 | ||
| St | 55 | ¿ | .. | Jamg | 46 | 14 | 3.5 | |
described (3) and DHA separated from A and E by digitonin precipitation. The ke- tosteroids were then chromatogrammed in systems II and III, acetylated. rechromato- grammed in I, and measured by a micro-Zim- merman technic(4). The methods used in this laboratory for 17-KS, 17-hydroxycorti- coids (17-OHCS), and tetrahydro-compound S (THS) have been listed (3).
The secretory rate of DHA was calculated by the formula:
cpm DHA injected
Secretory rate = S.A. of urinary DHA
This is a maximal value since less than 100% of the isotope is excreted in 48 hours(5). Since the S.A. of A and E should equal the S.A. of DHA if they were completely derived from DHA, a decrease in S.A. could result only through the contribution of unlabelled precursor to urinary A and E. The percent- age of A derived from DHA must then be S.A.A
S.A.DHA X 100, and similarly for E. The er- rors in the methods are such that the S.A. of the urinary ketosteroids had a standard error of ± 8%.
Six patients were studied, 2 ovariectomized women and 4 patients with metastatic adre.
nal carcinoma. The 2 ovariectomized women had been treated for choriocarcinoma previ- ously. but were in good health and appar- ently free of disease at the time of study. Pertinent features of these cases are pre- sented in Table I.
Results. (Table II). In both normal sub- jects. S.A., and S.A.p. did not differ signifi- cantly from S.A.DITA. Thus the fraction of A and E derived from precursors other than DHA was negligible. Estimated secretion rates of DHA were 7 mg and 11 mg daily.
The patients with adrenal cancer excreted large amounts of E and A. In 3 of them, the E/A ratio was above normal (2.6, 4.1, 5.2). The S.A.A did not differ significantly from the S.A.DIA in 3 of the 4 patients. Only in the patient with the highest excretion of 17-KS (Che) could it be shown that as much as 19% of the androsterone was not derived from DHA. In contrast, in 3 of the 4 pa- tients with adrenal cancer, the S.A.p., was lower than S.A.DHA so that only 35%, 34%, and 76% of the E was derived from DHA.
The calculated daily secretion rates of DHA varied from 28 mg to 390 mg in the patients with carcinoma. There was a rough correspondence between these secretion rates and urinary excretion of DHA.
| Patient | Urinary excretion, mg/24 hr* | E/A | Specific activity, cpm/ug | DHA secre- tion rate, mg/24 hr | % derived from DHA | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| A | E | DHA | .1 | E | DHA | A | E | |||
| Wh | 1.7 | 2.3 | .6 | 1.5 | 93 | 95 | 98 | 11 | 95 | 97 |
| Cha | 1.9 | 1.8 | .9 | 1.0 | 161 | 139 | 150 | 7 | 107 | 93 |
| Sc | 11 | 14 | 18 | 1.3 | 35 | 34 | 38 | 28 | 92 | 90 |
| Che | 27 | 112 | 161 | 4.1 | 2.2 | .92 | 2.7 | 39 | 81 | 34 |
| Ma | 4.2 | 22 | 21 | 5.2 | 20 | 9.1 | 26 | 41 | 77 | 35 |
| St | 3.5 | 9.1 | 15 | 2.6 | 34 | 25 | 33 | 32 | 103 | 76 |
* A - Androsterone. E == Etiocholanolone. DHA = Dehydroepiandrosterone.
Discussion. The method for measuring the extent to which a steroid metabolite is derived from a precursor was described by Vande Wiele and Lieberman(5) who showed that, in the normal adult, DHA is the pre- cursor of the urinary A and E. The results in the 2 ovariectomized women are in accord with this. In 3 of the 4 cases of adrenal car- cinoma, however, urinary E must have ori- ginated from both DHA and other pre- cursors, these other precursors accounting for as much as 3/3 of the E in 2 of the patients. It is significant that the 3 patients with S.A.F < S.A.DHA were those with excess cor- ticoid production as shown by the excretion of 17-OHCS and THS.
The 17-KS produced by the metabolism of 17a-hydroxyprogesterone(6,7) and com- pound S(8) are predominantly of the 5,8- series, and it has been suggested(2) that compound S may be an important precursor of urinary E in adrenal cancer. In a series of 10 patients with adrenal carcinoma, both THS and pregnanetriol were generally found in large amounts when the cancer was pro- ducing corticoids (Lipsett and Wilson, un- published data). This implies that 17a-hy- droxyprogesterone and compound S were not being utilized normally in biosynthesis of cortisol. Thus it seems reasonable to sup- pose that the large amounts of E in adrenal cancer are due not only to over-production of DHA but to the metabolism of 17a-hy- droxyprogesterone and compound S as well, in many of the patients.
This viewpoint is supported by the study in Sc. This boy had no evidence of Cush- ing’s syndrome and excretion of THS and 17-OHCS was normal. The E/A ratio was 1.3 although both steroids were excreted in large amounts. The S.A. of A and E did not differ significantly from S.A.DHA, and thus only small quantities of A and E could have been derived from steroids other than DHA.
With respect to A, the specific activity was significantly lower than that of D in 2 of the 4 cases. The unlabelled A may have arisen from 17a-hydroxyprogesterone and compound S as discussed, and a further con- tribution of androstenedione to urinary A
and E seems possible(9). Degree of dilution of the labelled A was not of the same mag- nitude as that of E. This would not be ex- pected in view of the above considerations.
The secretion rate of DHA in the 2 nor- mal subjects and in 4 others(5) suggests that DHA is quantitatively, at least, an im- portant secretory product of the adrenal cor- tex. The accuracy of the secretion rates in adrenal cancer may be less than in the nor- mal, since it seems possible that equilibration of the injected isotope with the large pool may not be sufficiently rapid with respect to its excretion. If such an effect occurred, however, the true secretion rates would be even higher than those estimated.
Summary. The precursor of urinary an- drosterone and etiocholanolone was shown to be dehydroepiandrosterone in 2 ovariecto- mized women. In 3 women with Cushing’s syndrome due to adrenal carcinoma, there were other major precursors of etiocholano- lone and, to a lesser extent, of androsterone. It was suggested that these other precursors were 17a-hydroxyprogesterone and com- pound S. In one patient with adrenal can- cer who excreted only 11-deoxy-17-ketoster- oids, etiocholanolone was derived almost en- tirely from dehydroepiandrosterone. The se- cretion rate of DHA was 7 and 11 mg daily in 2 ovariectomized women. In 4 patients with adrenal carcinoma, it ranged from 28 mg to 390 mg daily.
I wish to thank Drs. Seymour Lieberman and Bernt Hokfelt for stimulating discussion and helpful criticism. Mrs. Barbara Riter contributed excellent technical assistance.
1. Gallagher, T. F., Cancer Res., 1957, v17, 520.
2. Vande Wiele, R., Christy, N. P., Lieberman, S., Jailer, J. W., PROC. Soc. EXP. BIOL. AND MED., 1958, v99, 520.
3. Lipsett, M. B., Damast, B., ibid., 1958, v99, 285.
4. Wilson, H., Arch. Biochem. Biophys., 1954, v52, 217.
5. Vande Wiele, R., Lieberman, S., in Biological Activities of Steroids in Relation to Cancer, G. Pin- cus and E. P. Vollmer, Eds., 1960, Academic Press, N. Y. and London, p93.
6. Jailer, J. W., Gold J. J., Vande Wiele, R., Lieb- erman, S., J. Clin. Invest., 1955, v34, 1639.
7. Axelrod, L. R., Goldzieher, J. W., J. Clin. En-
docrinol. Metab., 1960, v20, 238.
8. Birke, G., Acta Endocrinol., 1954, v15, 17.
Miller, K. L., J. Biol. Chem., 1960, v235, P.C. 48.
9. Hirschmann, H., De Courcy. C., Levy, R. P .. Received April 27, 1961. P.S.E.B.M., 1961, v107.
Effect of Certain Liquid Organopolysiloxanes on Cholesterol Atherosclerosis of the Rabbit .* (26651)
FRANK GOLLAN (Introduced by Wilson C. Grant)
Veterans Administration Hospital and University of Miami School of Medicine, Coral Gables, Fla.
It has been postulated that the stability of lipids in emulsion form depends on main- tenance of a sufficient surface charge, so that deposition of lipid from plasma at certain sites on the linings of the blood vessels might well result from a lack of surface active agents in plasma(1). Fatty acids are anionic surface active agents and their surface activ- ity is increased by presence of double bonds in the fatty acid radicle(2). Thus, the least saturated fatty acid is the most effective as a charged surface active agent. The pro- nounced lipemia and cholesterolemia produc- ing effect of a surface active detergent like Triton WR-1339 has been thoroughly ex- plored(3), whereas hydrophobic surface ac- tive agents like the silicone fluids have re- ceived little attention. Dimethylpolysilox- anes, like DC 200 and DC antifoam,t had only a suggestive effect on aortic athersclero- sis of rabbits(4), but a phenylmethylpoly- siloxane* altered cholesterol deposition pro- foundly (5). The present study compares the effect of these 2 silicone fluids in a large number of animals.
Methods and results. In experiments last- ing 2 months New Zealand albino rabbits in groups of 6 were fed a stock diet to which 2% cholesterol and .5,% 1%, 2% and 5% of the silicone fluids in weight of diet were added. Weekly serum samples were ana- lyzed for cholesterol by the method of Pear-
son(6), but the results of the terminal sam- ples only are presented since there were no specific trends in reaching the final value. Tissue cholesterol was determined by the method of Kingsley (7).
The results (Table I) show that the 2 sili- cone fluids used exert a different biological effect. Addition of phenylmethylpolysilox- ane in all concentrations prevents develop- ment of severe hypercholesterolemia. The arbitrarily chosen lowest concentration of .5% has as much effect as the 10 times larger amount. The dimethylpolysiloxane did not prevent hypercholesterolemia, except in one experiment where a 1% concentration was used. Even in this instance the effect was less pronounced than that obtained with the other silicone fluid. Cholesterol content of the liver remains esentially unchanged if di- methylpolysiloxane is added to the stock diet containing 2% cholesterol, but increases markedly if the phenylmethylpolysiloxane fluid is fed at the 2% and 5% level. The op- posite changes occur in the aorta where the dimethylpolysiloxane produces an increased cholesterol content, whereas addition of phe- nylmethylpolysiloxane in higher concentra- tions does not change the cholesterol content of the aorta. The histological lesion of the aorta in a 2% feeding experiment is shown in representative photomicrographs (Fig. 1). Addition of 2% silicone fluids to the stock diet without cholesterol does not alter choles- terol content of the blood serum or of the tissues examined and does not produce his- tological changes.
Discussion. The cause of the described biological effects of silicone fluids is un- known, therefore the discussion is specula-
* This study was partially supported by a grant from the Dow Corning Center of Aid to Medical Research.
t Supplied by R. R. McGregor, Dow Corning Center of Aid to Medical Research, Midland, Mich. # Coded as XF-10050 by Dow Corning Corp., Midland, Mich.