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The uptake of radioactive phosphorus by rat liver following partial hepatectomy
The Uptake of Radioactive Phosphorus by Rat Liver Following Partial Hepatectomy 1, Ralph M. Johnson and S. Albert From the Richard Cohn Radiobiology Laboratory, Delroit Institute of Cancer Research, Detroit 1, Michigan Received July 13, 1951 INTRODUCTION
In rat liver following partial hepatectomy, cell hypertrophy precedes cell hyperplasia for about 1 day (2,3,4). This affords an opportunity to study the relationship of phosphorus-containing compounds to the processes of cell hypertrophy and cell division. Marshak and Byron observed an increase in the uptake of p3~ by the total P of the "regenerating" liver tissue following partial hepatectomy (5). They did not attempt any fractionation of the tissue, and their study, therefore, does not indicate whether there was a relationship between specific P-containing fractions and either the phase of cell hypertrophy or of cell division. In the experiments reported herein the incorporation of p32 into the acid-soluble organic and inorganic, phospholipide, phosphoprotein, pentose nucleic acid (PNA), and desoxypentose nucleic acid (DNA) frac -~ tions of rat liver at different intervals following partial hepatectomy has been measured. MATERIALS AND METHODS Male Holtzman rats 3-4 months old and weighing 250-350 g. were used. They were maintained on the stock diet of Purina laboratory chow and water ad libitum for at least 1 m o n t h in this laboratory. Partial hepatectomies were performed under ether anesthesia between the hours of 10:00 a. m. and noon according to the method of Higgins and Anderson (6). The 1 This investigation was supported by research grants from the National Cancer Institute of the National Institutes of Health, Public Health Service; the American Cancer Society, Inc.; the S. S. Kresge Foundation; and the Michigan Cancer Foundation. With the technical assistance of Miss R u t h Hoste. 340
UPTAKE OF RADIOACTIVE PHOSPHORUS
341
animals were given free access to the stock diet and water both before and after the operation. They were sacrificed in groups of four at 16 hr., and 1, 3, 5, 7, or 14 days following operation. Both unoperated and sham-operated rats were employed as controls. The latter were killed 24 hr. after sham-operation. Four hours prior to being sacrified, each animal received a single subcutaneous injection of 0.45 microcuries (~e.) of P~, as NaH~P3~04, per gram of body weight calculated at the time of injection.~ At the time of sacrifice the rats were anesthetized with ether, and the abdominal and thoracic cavities exposed. The livers were perfused via the thoracic inferior vena eava using ice cold 0.9% NaC1 (in water), until they appeared cleared of blood. Perfusion was employed since previous work (7) showed that the use of unperfused livers resulted in a greater variation between animals. For example, in the acidsoluble organic and inorganic, and lipide fractions of four unperfused livers the average deviations of the mean concentration coefficients were about twice those of the corresponding fractions of perfused livers. The anterior and posterior Iobules of the right lateral lobe were removed for analysis, and the eaudate lobes were discarded since they were usually found to be adherent to surrounding organs. To estimate mitotic activity, one tissue block was taken from each lobule of the right lobe remaining in the animal, and fixed in Bouin's solution. Two sections, 6 in thickness, were cut nonserially from each block, and stained with hematoxylin and eosin. A total of 12,000 nuclei were counted in each liver. A nucleus was considered in mitosis from the time definite chromatin threads were observed until membranes had formed in the daughter nuclei. The acid-soluble, lipide, phosphoprotein, DNA, and PNA fractions were isolated,. and P and protein N determinations carried out as described elsewhere (1). The acidsoluble P was further separated into organic and inorganic fractions by precipitating the latter with Mg ++ in the presence of NH4OH. Radioactivity was assayed by means of an end-window Geiger-Miiller counter and standard scaling assembly. The results were expressed as concentration coefficients, calculated in the following manner: counts per minute found in the fraction/micrograms P in fraction X 10-'. counts per minute injected/micrograms of body weight RESULTS AND DISCUSSION As e a r l y as 16 hr. a f t e r p a r t i a l h e p a t e c t o m y t h e r e was a n increase i n a c i d - s o l u b l e i n o r g a n i c P, a n d t h e a m o u n t b e g a n to decrease a f t e r 1 d a y , b e c o m i n g n o r m a l b y t h e 14th d a y ( T a b l e I). T h e r e was a n i m m e d i a t e increase i n lipide P, r e a c h i n g a m a x i m u m i n t h e 16-hr. a n d 1 - d a y groups, a n d d e c l i n i n g s t e a d i l y t h e r e a f t e r , b e c o m i n g n o r m a l a f t e r a b o u t 3 days. T h e r e were n o significant increases i n D N A or P N A P u n t i l t h e 3rd day. T h e a m o u n t s of b o t h these s u b s t a n c e s slowly decreased t h e r e after. T h e l a t t e r o b s e r v a t i o n s are e s s e n t i a l l y in a g r e e m e n t w i t h t h o s e 3Radioactive phosphorus, as H3P3~04, was furnished by the Oak Ridge National Laboratory, Oak Ridge, Tennessee. The method of preparing it for use has been described elsewhere (1).
342
RALPH
M. J O H N S O N
AND
S. A L B E R T
of Price and Laird (8). The changes seen in the concentration of acidsoluble organic and phosphoprotein P are not significant. The relative rates of uptake of p82 by the various P-containing fractions of the livers following partial hepatectomy are indicated by the concentration coefficients shown in Fig. 1. There was an immediate increase in the p32 uptake in all fractions. The highest activity in the DNA was seen at 1 day, and that in the acid-soluble organic and inorganic, phosphoprotein, and PNA at 16 hr. The rate of uptake of p32 decreased thereafter in these fractions. The maximum uptake of radioactivity in the phospholipide fraction was not seen until the 3rd day, and became normal by the 14th day. TABLE I The Phosphorus Contained in Rat Livers at Varying Periods Following Partial Hepatectomy ~ Acid-soluble
Time following partial hepatectomy
No. of animals
Controls Sham operated 16 hr. 1 day 3 days 5 days 7 days 14 days
4 4 4 3 4 4 4 4
Lipide Organic 24.2 20.8 20.9 20.5 20.5 21.9 20.8 25.9
(1.6) (1.8) (1.5) (1.8) (0.2) (1.8) (0.6) (I.0)
Phosphoprotein
DNA
PNA
Inorganic 9.9 8.0 12.0 12.6 11.1 10.6 11.3 10.2
(0.2) (0.5) (0.6) (1.2) (0.7) (0.7) (0.7) (0.6)
42.9 37.3 47.7 45.3 39.3 43.1
(1.9) (2.6) (2.9) (3.7) (2.1) (1.4)
4o.2(5.4) 41.9 (2.1)
1.2 1.4 1.4 1.2 1.5 1.4
(0.0) (0.1) (0.I) (0.2) (0.1) (0.1)
1.5 (o.1) 1.5 (0.1)
4.8 4.3 4.2 4.3 5.7 5.7 5.5 5.3
(0.8) (0.4) (0.2) (0.2) (0.3) (0.7) (0.4) (0.1)
27.2 28.1 31.8 30.8 37.9 33.9 32.7 32.0
(0.1) (0.8) (2,0) (0.9) (1.4) (1.3) (2.7) (1.4)
a Micrograms of phosphorus/mg, protein nitrogen. The average deviations from the means are shownin parentheses. Similar results were obtained in a second series of experiments performed on older male rats of the Fisher strain. The observation that the early phase of liver restoration is accompanied by significant increases in the uptake of radioactivity is in disagreement in certain respects with the conclusion of others (9,10). The differences, however, may be due to the fact that previous workers employed longer periods after ps~ injection and may, therefore, have been unable to detect the changes reported here. The percentages of liver nuclei undergoing mitosis at various intervals following partial hepatectomy are indicated in Fig. 1. The mitotic activity in the livers 16 hr. post-partial hepatectomy was not increased above that found in the controls. Mitoses were observed beginning at 24 hr., and the largest numbers were seen 3 days after operation. The
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Fro. 1. The uptake of radioactive phosphorus and mitotic activity in rat liver at different time intervals after partial hepstectomy. Each point is the average of four observations. All sham-operated controls fall on the left-hand ordinate. These values were similar to those of the unoperated controls, except in the DNA fraction, where it was slightly lower. The average deviation did not exceed 9% in the acidsoluble organic and inorganic and phosphoprotein, 16% in the PN A and phospholipide, and 25% in the DNA.
general pattern "of changes, together with the large variations seen between animals in any group, are essentially in agreement with observations previously reported (2,3,5). Our findings, together with those of others on the rate of liver restoration (2,3,6) suggest the following regarding changes which occur in
344
RALPH M. JOHNSON AND S. ALBERT
"regenerating" liver: There is a rapidly accelerating rate of uptake of P~ by the PNA, DNA, phosphoprotein, and acid-soluble fractions for about 16-24 hr. This is accompanied by an increase in the liver mass that is due to cell hypertrophy, indicating that these occurrences are related. Subsequently, the rate of uptake of p3~ by these fractions declines, accompanied by a marked increase in the rate of mitosis (Fig. 1) and a reduction in the rate of restoration of the liver mass. The exact relationship between mitotic activity and radioactivity uptake by these fractions may be obscured by the changes incident to cell hypertrophy. On the other hand, the maximum uptake of p~2 by the phospholipide fraction occurred at a time when mitoses were most frequent, suggesting a relationship between these two. The maximum uptake of p3~ did not appear to be due to the migration of phospholipide into the liver, since the occurrence of the former was not coincident with the appearance of maximum lipide P concentration. In view of the immediate response in p3~ uptake found in "regenerating" liver, it is interesting to note that other workers have found either a decrease (11,12) or no change (13) in the activity of a number of enzyme systems following partial hepatectomy. The same situation exists with regard to aerobic and anaerobic glycolysis, 02 consumption, and the respiratory quotient. No correlation was found between these functions and the amount of liver restoration or the time the tissue was taken (14). SUMMARY
1. Adult male Holtzmaa rats were given a single subcutaneous injection of radioactive phosphorus, as NaH~P3204, 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 acidsoluble 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 "r~generating" 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.
UPTAKE OF RADIOACTIVE PHOSPHORUS
345
3. In the hypertrophying liver cell following partial hepatectomy there was a marked increase in the uptake of p~2 in the acid-soluble organic and inorganic, phosphoprotein, PNA, and D N A fractions over that seen in the controls. With the shift from hypertrophy to cell hyperplasia there was a decline in the p32 uptake by these fractions. 4. The maximum uptake of p32 by the phospholipide fraction coincided with maximum mitotic activity. ~EFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
ALBERT,S., JOHNSON, R. M., ANDCOHAN,M. S., Cancer Research 10, 772 (1951). BRUES, A. M., DRURY, D. R., AND BRUES, M. C., Arch. Path: 22, 658 (1936). BRUES, A. M., AND MARBLE, B. B., J. Exptl. Med. 65, 15 (1937). BUCHER, N. L. R., AND GLINOS, A. D., Cancer Research 10, 324 (1950). MARSHAK,A., AND BrRON, R. L., JR., Proc. Soc. Exptl. Biol. Med. 59, 200 (1945). HIGGINS,(]. M., AND ANDERSON, l~. M., Arch. Path. 12, 186 (1931). JOHNSON, R. M., AND ALBERT, S., unpublished data. PRICE, J. M., AND LAIRD, A. K., Cancer Research 10, 650 (1950). CHARGAFF,]~., OLSON, K. B., AND PARTINGTON,P. F., dr. Biol. Chem. 134, 505 (1940). BRUES, A. M., TRACY, M. ~-V[.,AND COHN, W. E., Science 95, 558 (1942). NOr~RO, B., Acta Physiol. Scand. 20, 180 (1950). NOVIKOFF,A. B., AND POTTER, V. R., J. Biol. Chem. 173, 223 (1948). GREENSTEIN, J. P., J. Natl. Cancer Inst. 6, 211 (1946). NORRIS, J. L., BLANCltARD,J., AND POVOLNY, C., Arch. Path. 34, 208 (1942).
In rat liver following partial hepatectomy, cell hypertrophy precedes cell hyperplasia for about 1 day (2,3,4). This affords an opportunity to study the relationship of phosphorus-containing compounds to the processes of cell hypertrophy and cell division. Marshak and Byron observed an increase in the uptake of p3~ by the total P of the "regenerating" liver tissue following partial hepatectomy (5). They did not attempt any fractionation of the tissue, and their study, therefore, does not indicate whether there was a relationship between specific P-containing fractions and either the phase of cell hypertrophy or of cell division. In the experiments reported herein the incorporation of p32 into the acid-soluble organic and inorganic, phospholipide, phosphoprotein, pentose nucleic acid (PNA), and desoxypentose nucleic acid (DNA) frac -~ tions of rat liver at different intervals following partial hepatectomy has been measured. MATERIALS AND METHODS Male Holtzman rats 3-4 months old and weighing 250-350 g. were used. They were maintained on the stock diet of Purina laboratory chow and water ad libitum for at least 1 m o n t h in this laboratory. Partial hepatectomies were performed under ether anesthesia between the hours of 10:00 a. m. and noon according to the method of Higgins and Anderson (6). The 1 This investigation was supported by research grants from the National Cancer Institute of the National Institutes of Health, Public Health Service; the American Cancer Society, Inc.; the S. S. Kresge Foundation; and the Michigan Cancer Foundation. With the technical assistance of Miss R u t h Hoste. 340
UPTAKE OF RADIOACTIVE PHOSPHORUS
341
animals were given free access to the stock diet and water both before and after the operation. They were sacrificed in groups of four at 16 hr., and 1, 3, 5, 7, or 14 days following operation. Both unoperated and sham-operated rats were employed as controls. The latter were killed 24 hr. after sham-operation. Four hours prior to being sacrified, each animal received a single subcutaneous injection of 0.45 microcuries (~e.) of P~, as NaH~P3~04, per gram of body weight calculated at the time of injection.~ At the time of sacrifice the rats were anesthetized with ether, and the abdominal and thoracic cavities exposed. The livers were perfused via the thoracic inferior vena eava using ice cold 0.9% NaC1 (in water), until they appeared cleared of blood. Perfusion was employed since previous work (7) showed that the use of unperfused livers resulted in a greater variation between animals. For example, in the acidsoluble organic and inorganic, and lipide fractions of four unperfused livers the average deviations of the mean concentration coefficients were about twice those of the corresponding fractions of perfused livers. The anterior and posterior Iobules of the right lateral lobe were removed for analysis, and the eaudate lobes were discarded since they were usually found to be adherent to surrounding organs. To estimate mitotic activity, one tissue block was taken from each lobule of the right lobe remaining in the animal, and fixed in Bouin's solution. Two sections, 6 in thickness, were cut nonserially from each block, and stained with hematoxylin and eosin. A total of 12,000 nuclei were counted in each liver. A nucleus was considered in mitosis from the time definite chromatin threads were observed until membranes had formed in the daughter nuclei. The acid-soluble, lipide, phosphoprotein, DNA, and PNA fractions were isolated,. and P and protein N determinations carried out as described elsewhere (1). The acidsoluble P was further separated into organic and inorganic fractions by precipitating the latter with Mg ++ in the presence of NH4OH. Radioactivity was assayed by means of an end-window Geiger-Miiller counter and standard scaling assembly. The results were expressed as concentration coefficients, calculated in the following manner: counts per minute found in the fraction/micrograms P in fraction X 10-'. counts per minute injected/micrograms of body weight RESULTS AND DISCUSSION As e a r l y as 16 hr. a f t e r p a r t i a l h e p a t e c t o m y t h e r e was a n increase i n a c i d - s o l u b l e i n o r g a n i c P, a n d t h e a m o u n t b e g a n to decrease a f t e r 1 d a y , b e c o m i n g n o r m a l b y t h e 14th d a y ( T a b l e I). T h e r e was a n i m m e d i a t e increase i n lipide P, r e a c h i n g a m a x i m u m i n t h e 16-hr. a n d 1 - d a y groups, a n d d e c l i n i n g s t e a d i l y t h e r e a f t e r , b e c o m i n g n o r m a l a f t e r a b o u t 3 days. T h e r e were n o significant increases i n D N A or P N A P u n t i l t h e 3rd day. T h e a m o u n t s of b o t h these s u b s t a n c e s slowly decreased t h e r e after. T h e l a t t e r o b s e r v a t i o n s are e s s e n t i a l l y in a g r e e m e n t w i t h t h o s e 3Radioactive phosphorus, as H3P3~04, was furnished by the Oak Ridge National Laboratory, Oak Ridge, Tennessee. The method of preparing it for use has been described elsewhere (1).
342
RALPH
M. J O H N S O N
AND
S. A L B E R T
of Price and Laird (8). The changes seen in the concentration of acidsoluble organic and phosphoprotein P are not significant. The relative rates of uptake of p82 by the various P-containing fractions of the livers following partial hepatectomy are indicated by the concentration coefficients shown in Fig. 1. There was an immediate increase in the p32 uptake in all fractions. The highest activity in the DNA was seen at 1 day, and that in the acid-soluble organic and inorganic, phosphoprotein, and PNA at 16 hr. The rate of uptake of p32 decreased thereafter in these fractions. The maximum uptake of radioactivity in the phospholipide fraction was not seen until the 3rd day, and became normal by the 14th day. TABLE I The Phosphorus Contained in Rat Livers at Varying Periods Following Partial Hepatectomy ~ Acid-soluble
Time following partial hepatectomy
No. of animals
Controls Sham operated 16 hr. 1 day 3 days 5 days 7 days 14 days
4 4 4 3 4 4 4 4
Lipide Organic 24.2 20.8 20.9 20.5 20.5 21.9 20.8 25.9
(1.6) (1.8) (1.5) (1.8) (0.2) (1.8) (0.6) (I.0)
Phosphoprotein
DNA
PNA
Inorganic 9.9 8.0 12.0 12.6 11.1 10.6 11.3 10.2
(0.2) (0.5) (0.6) (1.2) (0.7) (0.7) (0.7) (0.6)
42.9 37.3 47.7 45.3 39.3 43.1
(1.9) (2.6) (2.9) (3.7) (2.1) (1.4)
4o.2(5.4) 41.9 (2.1)
1.2 1.4 1.4 1.2 1.5 1.4
(0.0) (0.1) (0.I) (0.2) (0.1) (0.1)
1.5 (o.1) 1.5 (0.1)
4.8 4.3 4.2 4.3 5.7 5.7 5.5 5.3
(0.8) (0.4) (0.2) (0.2) (0.3) (0.7) (0.4) (0.1)
27.2 28.1 31.8 30.8 37.9 33.9 32.7 32.0
(0.1) (0.8) (2,0) (0.9) (1.4) (1.3) (2.7) (1.4)
a Micrograms of phosphorus/mg, protein nitrogen. The average deviations from the means are shownin parentheses. Similar results were obtained in a second series of experiments performed on older male rats of the Fisher strain. The observation that the early phase of liver restoration is accompanied by significant increases in the uptake of radioactivity is in disagreement in certain respects with the conclusion of others (9,10). The differences, however, may be due to the fact that previous workers employed longer periods after ps~ injection and may, therefore, have been unable to detect the changes reported here. The percentages of liver nuclei undergoing mitosis at various intervals following partial hepatectomy are indicated in Fig. 1. The mitotic activity in the livers 16 hr. post-partial hepatectomy was not increased above that found in the controls. Mitoses were observed beginning at 24 hr., and the largest numbers were seen 3 days after operation. The
UPTAKE OF RADIOACTIVE PHOSPHORUS
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PARTIAL
14 HEPATECTOMu
Fro. 1. The uptake of radioactive phosphorus and mitotic activity in rat liver at different time intervals after partial hepstectomy. Each point is the average of four observations. All sham-operated controls fall on the left-hand ordinate. These values were similar to those of the unoperated controls, except in the DNA fraction, where it was slightly lower. The average deviation did not exceed 9% in the acidsoluble organic and inorganic and phosphoprotein, 16% in the PN A and phospholipide, and 25% in the DNA.
general pattern "of changes, together with the large variations seen between animals in any group, are essentially in agreement with observations previously reported (2,3,5). Our findings, together with those of others on the rate of liver restoration (2,3,6) suggest the following regarding changes which occur in
344
RALPH M. JOHNSON AND S. ALBERT
"regenerating" liver: There is a rapidly accelerating rate of uptake of P~ by the PNA, DNA, phosphoprotein, and acid-soluble fractions for about 16-24 hr. This is accompanied by an increase in the liver mass that is due to cell hypertrophy, indicating that these occurrences are related. Subsequently, the rate of uptake of p3~ by these fractions declines, accompanied by a marked increase in the rate of mitosis (Fig. 1) and a reduction in the rate of restoration of the liver mass. The exact relationship between mitotic activity and radioactivity uptake by these fractions may be obscured by the changes incident to cell hypertrophy. On the other hand, the maximum uptake of p~2 by the phospholipide fraction occurred at a time when mitoses were most frequent, suggesting a relationship between these two. The maximum uptake of p3~ did not appear to be due to the migration of phospholipide into the liver, since the occurrence of the former was not coincident with the appearance of maximum lipide P concentration. In view of the immediate response in p3~ uptake found in "regenerating" liver, it is interesting to note that other workers have found either a decrease (11,12) or no change (13) in the activity of a number of enzyme systems following partial hepatectomy. The same situation exists with regard to aerobic and anaerobic glycolysis, 02 consumption, and the respiratory quotient. No correlation was found between these functions and the amount of liver restoration or the time the tissue was taken (14). SUMMARY
1. Adult male Holtzmaa rats were given a single subcutaneous injection of radioactive phosphorus, as NaH~P3204, 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 acidsoluble 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 "r~generating" 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.
UPTAKE OF RADIOACTIVE PHOSPHORUS
345
3. In the hypertrophying liver cell following partial hepatectomy there was a marked increase in the uptake of p~2 in the acid-soluble organic and inorganic, phosphoprotein, PNA, and D N A fractions over that seen in the controls. With the shift from hypertrophy to cell hyperplasia there was a decline in the p32 uptake by these fractions. 4. The maximum uptake of p32 by the phospholipide fraction coincided with maximum mitotic activity. ~EFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
ALBERT,S., JOHNSON, R. M., ANDCOHAN,M. S., Cancer Research 10, 772 (1951). BRUES, A. M., DRURY, D. R., AND BRUES, M. C., Arch. Path: 22, 658 (1936). BRUES, A. M., AND MARBLE, B. B., J. Exptl. Med. 65, 15 (1937). BUCHER, N. L. R., AND GLINOS, A. D., Cancer Research 10, 324 (1950). MARSHAK,A., AND BrRON, R. L., JR., Proc. Soc. Exptl. Biol. Med. 59, 200 (1945). HIGGINS,(]. M., AND ANDERSON, l~. M., Arch. Path. 12, 186 (1931). JOHNSON, R. M., AND ALBERT, S., unpublished data. PRICE, J. M., AND LAIRD, A. K., Cancer Research 10, 650 (1950). CHARGAFF,]~., OLSON, K. B., AND PARTINGTON,P. F., dr. Biol. Chem. 134, 505 (1940). BRUES, A. M., TRACY, M. ~-V[.,AND COHN, W. E., Science 95, 558 (1942). NOr~RO, B., Acta Physiol. Scand. 20, 180 (1950). NOVIKOFF,A. B., AND POTTER, V. R., J. Biol. Chem. 173, 223 (1948). GREENSTEIN, J. P., J. Natl. Cancer Inst. 6, 211 (1946). NORRIS, J. L., BLANCltARD,J., AND POVOLNY, C., Arch. Path. 34, 208 (1942).