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Lipide metabolism during cell division
Lipide Metabolism During Cell Division’ Ralph M. Johnson, Elinor Levin and S. Albert From
the Richard Cohn Radiobiology Laboratory, Detroit Institute of Cancer Research; and the Department oj Physiological Chemistry, Wayne University, College of Medicine, Detroit, Michigan Received
November
20, 1953
Previous experiments carried out in this laboratory showed that in regenerating rat liver, the maximum uptake of P32 by phospholipides occurred during cell division rather than during cell hypertrophy (1). The purpose of the present investigation was to determine whether the increase in P32 uptake by phospholipides during mitosis was shared to the same extent by the lecithins, cephalins, and sphingomyelins, and to determine whether this increase occurred before or after metaphase. The latter was accomplished through the use of colchicine, a mitotic inhibitor acting in metaphase. Di-, tri-, tetra-, and pentaenoic acids were also determined quantitatively on the tissue preparations studied, in view of the possible association between them and members of the phospholipide class. MATERIALS AND METHODS Male Holtzman rats 4-5 months old and weighing 350-400 on a stock diet of Purina laboratory chow and water ad libitum. tomies, sacrifice of the animals, and perfusion of livers were scribed previouslg (1). The animals were sacrificed in groups 18 days following partial hepatectomy, inasmuch as the earlier these intervals to be of greatest significance in investigations in liver following such procedure.
g. were maintained Partial hepateccarried out as deof 6 either I, 3, or study had shown of mitotic activity
1 Supported in part by a grant-in-aid from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service; and in part by institutional grants to the Detroit Institute of Cancer Research from The American Cancer Society, Inc., and The Kresge Foundation. Taken from a dissertation to be submitted by Elinor Levin for the Ph.D. Degree, Wayne University, February, 1955. 170
LIPIDE
METABOLISM
171
Six hours before sacrifice theexperimental animalswereinjectedsubcutaneously with an aqueous solution containing 0.03 mg. colchicin@/lOO g. body weight. Five hours later they were given a single subcutaneous injection of 0.45 microcuries (PC.) of Paz, as NaH2P320a , per gram of body weight calculated at the time of mjection.3 The control animals received only the injection of K;aII~l’W~ 1 hr. prior to sacrifice. Lipides were obtained from a portion of each liver according to the method of Johnson and Dutch (2). The petroleum ether-chloroform extracts were evaporated under NP with slight warming, to near dryness, and the residues were taken up in petroleum ether and made to convenient volumes. hliquots of these were tnkcn for total P and total radioactivity determinations (3) a,nd the remainder ~vas fractionated into cephalin, lecithin, and sphingomyelin fractions according to f hta procedure of Zilversmit, Entenman, and Chnikoff (4). The radioactivity was assayed by means of an end-window Geiger-&Killer counter and standard scaling assembly. Results were expressed as concentration coefficients (3). Di-, tri-, tetra-, and pentaenoic acids were determined4 on another sample of the liver tissue as described by Klein and Johnson (5). All quantitative detcrminntions were expressed on the basis of tissue N, determined ncacording to the method of Ma and Zuazaga (6). RESULTS
AND DISCUSSIOK
In preliminary studies, colchicine was employed in a dosage of 0.1 mg./lOO g. of body weight, since others had demonstrated this dose to be satisfactory for obtaining maximum inhibition of mitosis at metaphase (7). It was observed, however, that this amount produced a marked depression of I?* uptake in the acid-soluble organic P, when compared to that of controls (average concentration coefficients of 502 and 700, respectively). When smaller doses of colchicine were employed, it KM found that a level of 0.03 mg./lOO g. of body weight, produced the Ramp degree of mit~otic inhibition as the higher level (Table I). hut, did not cause a decreased uptake of P32in the acid-soluble organic I’ (average concentration coefficientz of 382 f 10 and 432 f 35 were found for (‘ontrols and colchicine-treated animals, respect,ively) . These results suggested that in addition to its action as a mitotic inhibitor, t,he higher dose of colchicine also influenced P metabolism. For this reason the lowel amount of colchine was used in the studies described. The changesobserved in the amounts of total lipide P following partial 2 .A gift of U.S.P. colchicine by Parke, Davis & Co. is gratefully acknowledged. 3 The radioactive phosphorus used in these experiments was furnished hi- t hrl Oak Ridge National Laboratory, Oak Ridge, Tcnn. 4 The unsaturated fatty acid determinations were math, I)>- Dr. Peter D. fileill of this Institute.
172
JOHNSON,
LEVIN
AND
TABLE Effect
of Varied
ALBERT
I
of Colchicine on Mitosis in Regenerating Following Partial Hepatectomy”
Amounts
Per cent of total Phase
mitotic
Rat Liver 3 DcqJs figures
of mitosis ~~ colchicine
(6)b
0.03 mg. colchicine/g. body wt. (6)b
29.2 50.1 8.8 11.9
Prophase Metaphase Anaphase Telophase
0.10 mg. colchicinefg. body wt. (4)b
8.2 91.5 0.2 0.2
6.3 93.2 0.2 0.2
a Five hundred mitotic figures were counted for each dose level, from tissues prepared as previously described (I). A nucleus was considered in mitosis from the time definite chromatin threads were observed until membranes had formed in the daughter nuclei. b Figures in parentheses indicate numbers of animals employed.
hepatectomy were similar to those seen in the earlier study (Table II) (1). The increases which occurred at 1 day were found to be accounted for entirely by increases in the lecithin and cephalin fractions. In both the unoperated animals and those 3 days post-partial hepatectomy, the administration of colchicine resulted in significant increases in the amounts of the lecithin fraction only. There were no changes in the other phospholipide fractions. The maximum P32 uptake activity in the phospholipide fraction, occurring 3 days following partial hepatectomy (l), was accompanied TABLE Phospholipide Total Time following partial hepatectomy
Phosphorus phospholipide
Cephalin
Rat Liver Lecithin
Sphingomyelin
_____ Pa2 uptake” __
be. ‘N/““. _-.-
Controls Controls + colcbicine 1 day 3 days 3 days + c&hi&e
II in Regenerating
37.7 40.1 50.7 36.0 43.7
(l.l)b (1.6) (1.4) (1.0) (0.6)
pg. ;[mg.
215 (13) 16.8 271 (16) 16.8 21.0 422 (27) 15.0 565 (40) 15.5
PS2 uptake Irg’ ‘N/““. ~___~___~
(0.7) 305 (18) 21.0 (0.6) 446 (28) 24.0 (0.9) 29.1 (1.0) 436 (25) 20.0 (0.4) 683 (44) 26.8
(0.7) (0.4) (1.0) (1.3) (0.9)
psz uptake 108 (7) 128 (9)
1.33 (0.08) 0.99 (0.10) 1.23 (0.08) 363 (29) 1.46 (0.25) 421 (45) 1.66 (0.10)
a Figures
for Pa2 uptake are concentration ooefficients calculated in the following counts/min. in fractionlpg. phosphorus infraction.
b Figures
within
counts/ruin. parentheses
are standard
injected/leg. errcm
of body wt. of mean values.
P&T.P/n%. N
manner:
P@ uptake 13 (3) 15 (2) 57 (16) 61 (10)
LIPIDE
METABOLISM
173
by increases in all three of the fractions examined (Table II). The administrat.ion of colchicine resulted in an increase of approximately 50 $% in the uptake of P32 in the cephalin fraction, and nonsignificant increases in the lecithin and sphingomyelin fractions. The selective effect on cephalin is not immediately apparent from these experiments-it may indicate that cephalin is associated with the detoxification or excretion of colchicine. Since this effect was observed the third day after partial hepatectomy as well as in the livers of the unoperated animals where only about 1 cell in lO,OOO-20,000 is in mitosis at any one time, it does not seem to be related to a mitotic inhibition. Therefore, it, appears that’ t’he colchicine treatment was without effect on the high P32 uptake that is seen in the phospholipide P coincident with a high mitotic activit,y. The results indicate that the increased phospholipide P uptake associated with mitosis is associated with a phase of cell division that occurs prior to metaphase, i.e., interphase or prophase, but not with either of the phases occurring subsequent to it, i.e., anaphase or telophase. This is of interest in view of the belief that the nuclear envelope contains phospholipides and is formed during telophase (8). It would seem reasonable t>o expect neoformation of phospholipide to oc(‘ur in anaphase or telophase, and that arrest of cell division in metaphase by colchicine would depress the P32 uptake by phospholipide. The lat#ter did not occur, suggesting that neoformation of phospholipide to be used in nuclear membranes occurred earlier in interphase or prophase. The failure to find an increase in botal phospholipide at this time does not support, the latter view, unless subtle changes occurred in the various components of the cell that were masked in this study. The latter is being investigated. There was a decrease in the amount of tetraenoic and an increase in the trienoic acid, but no change in the amount’s of either di- or pentaenoic acids at one day following partial hepatectomy (Table III). At, this time there is an increa,se in liver cell mass with a rapid uptake of P3? in acidsoluble, deoxypentose and pentose nucleic acids, and phosphoprotein I’, but very little mitotic activity (1). At 3 days following partia,l hepat’ectomy, coincident with maximum mitotic activity, there is a large increase in the concentration of dienoic acid and no change in tetra- or pentaenoic: acids over the values found in the unoperated cont’rols. The trienoic acid values remained at the same level as those found in animals 24 hr. poshpart.ial hepatectomy. Colchicine had no effect on t)he concentrations of these acids in either the unoperated controls or the 3-da,y hcpat,ectomized animals, when compared with similar animals not rcc4ving colchic*ine.
174
JOHNSON, LEVIN AND ALBERT TABLE Unsaturated
Patty
Acids
Micrograms
-
III Regenerating X 10-l/mg. N in
Fattyacids
Timefollowing partialhepatectomy Dienoic
Controls Controls + colchicine 1 day 3 days 3 days + colchicine 18 days a Figures in parentheses
Rat Liver
18.6 21.4 19.7 27.6 30.9 16.5
(1.1)5 (1.3) (1.6) (2.3) (3.3) (1.2)
are standard
Trienoic 0.3 1.8 3.8 4.2 3.4 3.0
(0.1) (0.4) (0.6) (0.7) (0.4) (0.5)
Tetraenoic Pentaenoic _
20.7 19.9 14.3 19.0 17.7 13.4
(1.0) (0.8) (0.8) (0.6) (0.6) (0.8)
-
6.0 6.5 7.2 7.7 8.4 7.2
(0.8) (0.5) (0.5) (0.5) (0.6) (0.6)
errors of the means.
(The slight increase in trienoic acid seen in the colchicine-treated control animals is not considered significant.) It is inferred from these data that if the increase in dienoic acid observed at 3 days following partial hepatectomy is associated with the mitotic process, it is concerned with that part of the process occurring prior to metaphase. The increase in trienoic acid following partial hepatectomy was still evident at least 18 days following the operation, and it is believed not to be related to the process of cell division. SUMMARY
1. Adult male Holtzman rats were each given a single injection of P32 as NaH2P3*04 at 1, 3, and 18 days after partial hepatectomy, and were sacrificed 1 hr. later. Six hours prior to sacrifice, unoperated controls and S-day hepatectomized rats received 0.03 mg. colchicine/lOO 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. 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. 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
LIPIDE
METABOLISM
coincident with the period of high mitotic activity. Blocking mitosis metaphase with colchicine did not influence the level of dienoic acid.
175 in
REFERENCES
1. JOHNSON, R. M., AND ALBERT, S., Arch. Bioch.em. and Biophys. 36, 340 (1952). 2. JOHNSON, R. M., AND DUTCH, 1’. H., Proc. Sm. Exptl. Biol. Med. 78, 662 (1951). 3. ALBERT, S., JOHNSON, R. M., AXD COHAS, Al. S., ‘Cancer Resewch 11, 772 (1951).
4. ZILVERSXIT, D. B., ENTENMAX,
C., AND CHAIKOBF, I. L., J. Biol. Cken~. 176, 193 (1948). 5. KLEIN, P. D., AND Joaxsox, R. bi., Arch. Biochem. and Biophys. 48, Ii2 (1934). 6. T&r, T. S., AND ZUAZAGA, G., Ind. Eng. Chem., Anal. Ed. 14, 280 (19W. 7. LEBLOSD, C. P., AND STEVENS, C. E., Bnat. Record 100, 357 (l!M). 8. ANDERSON, N. G., Science 117, 517 (1953).
the Richard Cohn Radiobiology Laboratory, Detroit Institute of Cancer Research; and the Department oj Physiological Chemistry, Wayne University, College of Medicine, Detroit, Michigan Received
November
20, 1953
Previous experiments carried out in this laboratory showed that in regenerating rat liver, the maximum uptake of P32 by phospholipides occurred during cell division rather than during cell hypertrophy (1). The purpose of the present investigation was to determine whether the increase in P32 uptake by phospholipides during mitosis was shared to the same extent by the lecithins, cephalins, and sphingomyelins, and to determine whether this increase occurred before or after metaphase. The latter was accomplished through the use of colchicine, a mitotic inhibitor acting in metaphase. Di-, tri-, tetra-, and pentaenoic acids were also determined quantitatively on the tissue preparations studied, in view of the possible association between them and members of the phospholipide class. MATERIALS AND METHODS Male Holtzman rats 4-5 months old and weighing 350-400 on a stock diet of Purina laboratory chow and water ad libitum. tomies, sacrifice of the animals, and perfusion of livers were scribed previouslg (1). The animals were sacrificed in groups 18 days following partial hepatectomy, inasmuch as the earlier these intervals to be of greatest significance in investigations in liver following such procedure.
g. were maintained Partial hepateccarried out as deof 6 either I, 3, or study had shown of mitotic activity
1 Supported in part by a grant-in-aid from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service; and in part by institutional grants to the Detroit Institute of Cancer Research from The American Cancer Society, Inc., and The Kresge Foundation. Taken from a dissertation to be submitted by Elinor Levin for the Ph.D. Degree, Wayne University, February, 1955. 170
LIPIDE
METABOLISM
171
Six hours before sacrifice theexperimental animalswereinjectedsubcutaneously with an aqueous solution containing 0.03 mg. colchicin@/lOO g. body weight. Five hours later they were given a single subcutaneous injection of 0.45 microcuries (PC.) of Paz, as NaH2P320a , per gram of body weight calculated at the time of mjection.3 The control animals received only the injection of K;aII~l’W~ 1 hr. prior to sacrifice. Lipides were obtained from a portion of each liver according to the method of Johnson and Dutch (2). The petroleum ether-chloroform extracts were evaporated under NP with slight warming, to near dryness, and the residues were taken up in petroleum ether and made to convenient volumes. hliquots of these were tnkcn for total P and total radioactivity determinations (3) a,nd the remainder ~vas fractionated into cephalin, lecithin, and sphingomyelin fractions according to f hta procedure of Zilversmit, Entenman, and Chnikoff (4). The radioactivity was assayed by means of an end-window Geiger-&Killer counter and standard scaling assembly. Results were expressed as concentration coefficients (3). Di-, tri-, tetra-, and pentaenoic acids were determined4 on another sample of the liver tissue as described by Klein and Johnson (5). All quantitative detcrminntions were expressed on the basis of tissue N, determined ncacording to the method of Ma and Zuazaga (6). RESULTS
AND DISCUSSIOK
In preliminary studies, colchicine was employed in a dosage of 0.1 mg./lOO g. of body weight, since others had demonstrated this dose to be satisfactory for obtaining maximum inhibition of mitosis at metaphase (7). It was observed, however, that this amount produced a marked depression of I?* uptake in the acid-soluble organic P, when compared to that of controls (average concentration coefficients of 502 and 700, respectively). When smaller doses of colchicine were employed, it KM found that a level of 0.03 mg./lOO g. of body weight, produced the Ramp degree of mit~otic inhibition as the higher level (Table I). hut, did not cause a decreased uptake of P32in the acid-soluble organic I’ (average concentration coefficientz of 382 f 10 and 432 f 35 were found for (‘ontrols and colchicine-treated animals, respect,ively) . These results suggested that in addition to its action as a mitotic inhibitor, t,he higher dose of colchicine also influenced P metabolism. For this reason the lowel amount of colchine was used in the studies described. The changesobserved in the amounts of total lipide P following partial 2 .A gift of U.S.P. colchicine by Parke, Davis & Co. is gratefully acknowledged. 3 The radioactive phosphorus used in these experiments was furnished hi- t hrl Oak Ridge National Laboratory, Oak Ridge, Tcnn. 4 The unsaturated fatty acid determinations were math, I)>- Dr. Peter D. fileill of this Institute.
172
JOHNSON,
LEVIN
AND
TABLE Effect
of Varied
ALBERT
I
of Colchicine on Mitosis in Regenerating Following Partial Hepatectomy”
Amounts
Per cent of total Phase
mitotic
Rat Liver 3 DcqJs figures
of mitosis ~~ colchicine
(6)b
0.03 mg. colchicine/g. body wt. (6)b
29.2 50.1 8.8 11.9
Prophase Metaphase Anaphase Telophase
0.10 mg. colchicinefg. body wt. (4)b
8.2 91.5 0.2 0.2
6.3 93.2 0.2 0.2
a Five hundred mitotic figures were counted for each dose level, from tissues prepared as previously described (I). A nucleus was considered in mitosis from the time definite chromatin threads were observed until membranes had formed in the daughter nuclei. b Figures in parentheses indicate numbers of animals employed.
hepatectomy were similar to those seen in the earlier study (Table II) (1). The increases which occurred at 1 day were found to be accounted for entirely by increases in the lecithin and cephalin fractions. In both the unoperated animals and those 3 days post-partial hepatectomy, the administration of colchicine resulted in significant increases in the amounts of the lecithin fraction only. There were no changes in the other phospholipide fractions. The maximum P32 uptake activity in the phospholipide fraction, occurring 3 days following partial hepatectomy (l), was accompanied TABLE Phospholipide Total Time following partial hepatectomy
Phosphorus phospholipide
Cephalin
Rat Liver Lecithin
Sphingomyelin
_____ Pa2 uptake” __
be. ‘N/““. _-.-
Controls Controls + colcbicine 1 day 3 days 3 days + c&hi&e
II in Regenerating
37.7 40.1 50.7 36.0 43.7
(l.l)b (1.6) (1.4) (1.0) (0.6)
pg. ;[mg.
215 (13) 16.8 271 (16) 16.8 21.0 422 (27) 15.0 565 (40) 15.5
PS2 uptake Irg’ ‘N/““. ~___~___~
(0.7) 305 (18) 21.0 (0.6) 446 (28) 24.0 (0.9) 29.1 (1.0) 436 (25) 20.0 (0.4) 683 (44) 26.8
(0.7) (0.4) (1.0) (1.3) (0.9)
psz uptake 108 (7) 128 (9)
1.33 (0.08) 0.99 (0.10) 1.23 (0.08) 363 (29) 1.46 (0.25) 421 (45) 1.66 (0.10)
a Figures
for Pa2 uptake are concentration ooefficients calculated in the following counts/min. in fractionlpg. phosphorus infraction.
b Figures
within
counts/ruin. parentheses
are standard
injected/leg. errcm
of body wt. of mean values.
P&T.P/n%. N
manner:
P@ uptake 13 (3) 15 (2) 57 (16) 61 (10)
LIPIDE
METABOLISM
173
by increases in all three of the fractions examined (Table II). The administrat.ion of colchicine resulted in an increase of approximately 50 $% in the uptake of P32 in the cephalin fraction, and nonsignificant increases in the lecithin and sphingomyelin fractions. The selective effect on cephalin is not immediately apparent from these experiments-it may indicate that cephalin is associated with the detoxification or excretion of colchicine. Since this effect was observed the third day after partial hepatectomy as well as in the livers of the unoperated animals where only about 1 cell in lO,OOO-20,000 is in mitosis at any one time, it does not seem to be related to a mitotic inhibition. Therefore, it, appears that’ t’he colchicine treatment was without effect on the high P32 uptake that is seen in the phospholipide P coincident with a high mitotic activit,y. The results indicate that the increased phospholipide P uptake associated with mitosis is associated with a phase of cell division that occurs prior to metaphase, i.e., interphase or prophase, but not with either of the phases occurring subsequent to it, i.e., anaphase or telophase. This is of interest in view of the belief that the nuclear envelope contains phospholipides and is formed during telophase (8). It would seem reasonable t>o expect neoformation of phospholipide to oc(‘ur in anaphase or telophase, and that arrest of cell division in metaphase by colchicine would depress the P32 uptake by phospholipide. The lat#ter did not occur, suggesting that neoformation of phospholipide to be used in nuclear membranes occurred earlier in interphase or prophase. The failure to find an increase in botal phospholipide at this time does not support, the latter view, unless subtle changes occurred in the various components of the cell that were masked in this study. The latter is being investigated. There was a decrease in the amount of tetraenoic and an increase in the trienoic acid, but no change in the amount’s of either di- or pentaenoic acids at one day following partial hepatectomy (Table III). At, this time there is an increa,se in liver cell mass with a rapid uptake of P3? in acidsoluble, deoxypentose and pentose nucleic acids, and phosphoprotein I’, but very little mitotic activity (1). At 3 days following partia,l hepat’ectomy, coincident with maximum mitotic activity, there is a large increase in the concentration of dienoic acid and no change in tetra- or pentaenoic: acids over the values found in the unoperated cont’rols. The trienoic acid values remained at the same level as those found in animals 24 hr. poshpart.ial hepatectomy. Colchicine had no effect on t)he concentrations of these acids in either the unoperated controls or the 3-da,y hcpat,ectomized animals, when compared with similar animals not rcc4ving colchic*ine.
174
JOHNSON, LEVIN AND ALBERT TABLE Unsaturated
Patty
Acids
Micrograms
-
III Regenerating X 10-l/mg. N in
Fattyacids
Timefollowing partialhepatectomy Dienoic
Controls Controls + colchicine 1 day 3 days 3 days + colchicine 18 days a Figures in parentheses
Rat Liver
18.6 21.4 19.7 27.6 30.9 16.5
(1.1)5 (1.3) (1.6) (2.3) (3.3) (1.2)
are standard
Trienoic 0.3 1.8 3.8 4.2 3.4 3.0
(0.1) (0.4) (0.6) (0.7) (0.4) (0.5)
Tetraenoic Pentaenoic _
20.7 19.9 14.3 19.0 17.7 13.4
(1.0) (0.8) (0.8) (0.6) (0.6) (0.8)
-
6.0 6.5 7.2 7.7 8.4 7.2
(0.8) (0.5) (0.5) (0.5) (0.6) (0.6)
errors of the means.
(The slight increase in trienoic acid seen in the colchicine-treated control animals is not considered significant.) It is inferred from these data that if the increase in dienoic acid observed at 3 days following partial hepatectomy is associated with the mitotic process, it is concerned with that part of the process occurring prior to metaphase. The increase in trienoic acid following partial hepatectomy was still evident at least 18 days following the operation, and it is believed not to be related to the process of cell division. SUMMARY
1. Adult male Holtzman rats were each given a single injection of P32 as NaH2P3*04 at 1, 3, and 18 days after partial hepatectomy, and were sacrificed 1 hr. later. Six hours prior to sacrifice, unoperated controls and S-day hepatectomized rats received 0.03 mg. colchicine/lOO 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. 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. 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
LIPIDE
METABOLISM
coincident with the period of high mitotic activity. Blocking mitosis metaphase with colchicine did not influence the level of dienoic acid.
175 in
REFERENCES
1. JOHNSON, R. M., AND ALBERT, S., Arch. Bioch.em. and Biophys. 36, 340 (1952). 2. JOHNSON, R. M., AND DUTCH, 1’. H., Proc. Sm. Exptl. Biol. Med. 78, 662 (1951). 3. ALBERT, S., JOHNSON, R. M., AXD COHAS, Al. S., ‘Cancer Resewch 11, 772 (1951).
4. ZILVERSXIT, D. B., ENTENMAX,
C., AND CHAIKOBF, I. L., J. Biol. Cken~. 176, 193 (1948). 5. KLEIN, P. D., AND Joaxsox, R. bi., Arch. Biochem. and Biophys. 48, Ii2 (1934). 6. T&r, T. S., AND ZUAZAGA, G., Ind. Eng. Chem., Anal. Ed. 14, 280 (19W. 7. LEBLOSD, C. P., AND STEVENS, C. E., Bnat. Record 100, 357 (l!M). 8. ANDERSON, N. G., Science 117, 517 (1953).