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Effects of DMSO on SGOT during hypothermia in adrenalectomized rats
Life Sciences Vol . 22, pp . 329-336 Printed in the II .S .A .
Pergamon Prese
EFFECTS OF DMSO ON SGOT DURING HYPOTHERMIA IN ADRENALECTOMIZED RATS Louis J . Ramazzotto, Ph .D . and Ronald Carlin, Ph .D . Fairleigh Dickinson University School of Dentistry Department of Human Physiology 110 Fuller Place Hackensack, New Jersey 07601 (Received in final form December 1, 1977)
SUMMARY
A study of changes in SGOT levels was made in by othermic Sprague-Dawley rats (rectal temperatures 23 ° C~ which were adrenalectomized and/or treated with DMSO (41~ aqueous solution injected IP) prior to hypothermia . Adrenalectomized animals exhibited slight initial increases in SGOT, but returned to control levels within 24 hours after exposure . Animals treated with DMSO alone or adrenalectomized and treated with DMSO showed significant increases in SGOT, which persisted 24 hours after exposure . Data suggests that DMSO exerts its action directly upon the cell membrane, causing permeability changes rather than indirectly by influencing the release of adrenal hormones which in turn affect membrane permeability . The effects of environmental stress on serum enzyme levels is well documented . It has been reported that serum levels of glutamic oxalacetic transaminase (SGOT) are altered after administration of catecholamines (1 , exposure to high altitude (2), prolonged exercise (3), cold exposure (4), and hypothermia (5,6) . In many of these studies, histological examinations failed to demonstrate substantial necrosis or other morphological changes which mi ht account for the significant rise in SGOT levels (3,6,7,8 . It has been shown that treatment with dimethyl sulfoxide (DMSO) prior to the induction of stress may potentlate the elevation of SGOT levels (9,4) . Histological examination following treatment wlth DMSO indicates that increased titers of SGOT are not a result of cell necrosis (2,3,10) . Since cellular damage is not visibly evident following stress alone or stress subsequent to treatment with DMSO, it is suggested that these factors may act singly or in combination to alter membrane permeability and therefore, bring about the observed increase in levels of SGOT (10,11,12) . The role of the adrenal gland in relation to stress induced elevation of SGOT has been investigated using restraint as the 329
33 0
Effect of DMSO and Hypothermia oa SGOT
Vol . 22, No . 4, 1978
stressing factor (13) . It was reported that adrenalectomy reduced the level of SGOT which had been elevated due to the restraint . Injection of epinephrine into adrenalectomized animals again increased levels of SGOT ; while administration of an alpha blocker plus epinephrine did not . It has been proposed that DMSO acts to raise SGOT levels by influencing the release of adrenal hormones, which in turn, affects membrane permeability (4) . Epinephrine acting alone, in combination with, or potentiated by adrenocortical secretions may bring about this alteration of membrane permeability . The present set of experiments were designed to test this hypothesis employing hypothermia (23°C .) as a stress . MATERIALS AND METHODS Experimental Animal Eighty-four male Sprague-Dawley rats (430±70 grams) were divided into seven main groups, each containing 12 rats . Experimental groups were sub-divided into two categories, each containing six rats . (Table 1) . Animals to be adrenalectomized were anesthesized by intraperitoneal injection of sodium pentobarbital (33 mg/Kg) . A 3 cm lateral incision was made beneath the rib cage, the posterior portion of the kidney exposed, and the adrenal gland lying within the fatty tissue identified and carefully excised . A three day interval was alloted between adrenalectomy and beginning any experiment . During this interval animals were provided with a one percent sodium chloride solution instead of drinking water . DMSO Administration Animals treated with DMSO received an intraperitoneal injection of 1 ml/100 gm body weight of a 41~ aqueous solution of DMSO kept at 37°C . DMSO was administered subsequent to anesthesia, but 5 minutes prior to initiating hypothermia . Induçtion of Hypothermia Anesthesized animals were rendered hypothermic by placing them on ice, A thin plastic shield separated the animal from the ice . The animals rectal temperature was measured by means of a clinical thermometer, heart rate was monitored with a stethoscope and respiratory rate determined visually . All parameters were recorded every ten minutes during hypothermia . Upon reaching a rectal temperature of 23°C the animals were removed from the ice 'and placed 24 to 36 inches from a 500 watt lamp for one hour to re-establish normal body temperature . Determination of Enzyme Activity Blood samples for enzyme assay were drawn by cardiac puncture utilizing a peristaltic pump . Blood samples (5 .0 ml) were taken under ether anesthesia at 5 and 24 hours following treatment with DMSO and/or exposure to cold, Only one sample per rat was obtained . Samples were centrifuged (6000 RPM for 13 minutes) and the serum withdrawn to be assayed for SGOT activity using the method developed by Karmen (14) . Employing a Beckman DB-G spectrophoto-
Vol . 22, No . 4, 1978'
Effect of DMSO and Hypothermia on SGOT
331
meter, the reduction of optical density (OD) at 340 nm due to the formation of beta-NAD was measured in terms of Karmen units, where one Karmen unit causes the formation of .000482 uM of glutamic acid per minute (15) . Mean enzyme activities with standard error were calculated and statistically significant differences determined using the Student's "t" test . TABLE 1 Animal
Distribution in Experimental and Control Groups
Experimental Each group contained 12 animals . groups had SGOT activity determined (in six animals) 5 hours and (in remaining six animals) 24 hours after treatment . 1 . Controls A . Normal controls B . Adrenalectomized controls 2 . Hypothermia 3 . DMSO 4 . DMSO + Hypothermia 5 . Adrenalectomy + Hypothermia 6 . Adrenalectomy + DMSO 7 . Adrenalectomy + Hypothermia + DMSO RES ULTS Effeçt of Adrenaleçtomy Serum GOT activity in adrenalectomized control animals was not significantly different from the activity measured in nonadrenalectomized control rats . Effect o f Hypothermia Adrenalectomized animals exposed to cold did exhibit .a slight (p< .05) initial increase in SGOT activity five hours after exposure . SGOT activity did return to control levels within 24 hours following exposure . Nan-adrenalectomized animals exposed to cold did exhibit a significantly higher SGOT activity (p<,001) at both 5 and 24 hours after hypothermia .
332
Effect
of DMSO and Hypothermia on
SCOT
Vol . 22, No . 4, 1978
ve
é , s~
FIG . 1 Effect of hypothermia on serum glutamic oxalacetic transaminase levels in adrenalectomized and nonadrenalectomized rats . Values shown are means ; vertical bars denote standard error of the means . Effect of DMSO Treatment with DMSO caused significant increases (p<.001) in the SGOT activity of both non-adrenalectomized and adrenalectomized rats . These increases persisted even 24 hours following treat ment . There was no statistically significant difference in enzyme activity between these two groups in both the 5 and 24 hour sample.
Vol . 22, No . 4 , 1978
Effect of DMSO aad Hypothermia on SCOT
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FI G . 2 Effect of dimethyl sulfoxide on serum glutamic oxalacetic transamlnase levels in adrenalectomized and non-adrenalectomized rats . Values shown are means ; vertical bars denote standard error of the meansw The combination of DMSO + Hypothermia resulted in a significant increase (p< .001) in SGOT activity, whether or not the animal had been adrenalectomized . These increases persisted even 24 hours following treatment and exposure . Non-adrenalectomized DMSO treated animals showed SGOT activity that was significantly greater (p< .O1) than that of the adrenalectomized DMSO treated rats .
333
334
Vol . 22, No . 4, 1978
Effect of DMSO aad Hypothermia on SGOT
sao
iso
vs
T
50
rs
0
FIG . 3 Combined effect of dimethyl sulfoxide and hypothermia on serum glutamic oxalacetic transaminase levels in adrenalectomized and non-adrenalectomized rats . Values shown are means ; vertical bars denote standard error of the means . DISCUSSION It has been demonstrated that exposure to cold may induce inAltland reported an creased adrenocortical secretion (16) . elevation of serum enzyme levels in proportion to the degree of hypothermia, and suggested that adrenal hormones may stimulate the release of GOT (3) . Results of the experiments described here appear to correlate with these observations in that adrenalectomized hypothermic animals exhibited significantly lower SGOT activity as com ared to the non-adrenalectomized hypothermic animals . Pearl (13~ demonstrated that adrenalectomy suppressed the rise of SGOT when stress was due to restraint . Our studies, using hypothermia as the stressing factor, lmplicate the adrenal glands as the mediators of the increase in SGOT activity . The slight increase in SGOT activity measured in adrenalectomized rats 5 hours
vol . 22, No . 4, 197$
Effect of DMSO and Hypothermia on SCOT
335
after hypothermia may be attributed to the release of catecholamines from some source other than the adrenals (17) . Hy othermia causes a prolonged duration of action of epinephrine (18~ . This might be a contributing factor in bringing about the continued rise of SGOT levels in the non-adrenalectomized rats hours after exposure to cold . Similarly, in adrenalectomized animals which possess small amounts of non-adrenal catecholamines, there should be a smaller rise in SGOT levels and therefore a faster return to control levels . The mechanism by which adrenal hormones cause the increased However, it has been established levels of SGOT is not known . that epinephrine does not increase the de novo synthesis of GOT
(13) .
Changes in SGOT levels after treatment with DMSO closely paralleled the response due to hypothermia alone, initially suggesting that both stimuli were operating through a common mechanism . However, adrenalectomy did not prevent a rise in SGOT activity in animals receiving DMSO treatment, thus indicating the existence of two different mechanisms . While the ability of DMSO to cross various types of membranes is well documented, the precise mechanism by which this is accomplished has yet to be elucidated . This ability may be related to a reversible substitution of DMSO into the membrane structure such that the membranes are only temporarily altered, and resume normal function on removal of the DMSO . Such a temporary alteration could cause a change in the permeability of the membrane and this in turn could affect serum enzyme levels . The oxidizing ability of DMSO has also been shown to produce changes in isomeric conformation of cellular fatty acids which may be a factor in creating increased membrane permeability (19,20,21,22,23,24) . The observation that adrenalectomy significantly reduces the serum levels of GOT in those animals which had been treated with DMSO and then exposed to hypothermia indicates that the adrenal hormones either directly or indirectly increased SGOT levels . Animals which were adrenalectomized and then treated with DMSO prior to cold exposure demonstrated an increase in the level of SGOT as compared to those animals which were adrenalectomized but did not receive DMSO prior to inducing hypothermia, hence, there is an lmplication that DMSO itself initiates an increase in SGOT . It is equally important to note that SGOT levels remain high in those animals which were adrenalectomized and treated with DMSO, even 24 hours after the hypothermic period . It is suggested that DMSO may act directly to increase and maintain SGOT levels . On the basis of the present experiments it appears that hypothermia indirectly affects cell membranes by way of adrenal hormones ; apparently DMSO affects the membranes in a more direct manner through some unknown mechanism .
336
Effect of DMSO and Hypothermia on SCOT
Vol . 22, No . 4, 1978
REFERENCES : 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 13 14 15
. . . .
16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 .
B . HIGHMAN, H . MALING 8 E . THOMPSON, Am . J . Physiol . , 196 436-440 (1959) P . ALTLAND, B . HIGHMAN, 8 J . GARBUS, Aerospace Med . , 35 10341039 (1964) P . ALTLAND, 8 B . HIGHMAN, Am . J . ~Ph~siol ., 201 393-395 (1961) P . ALTLAND, B . HIGHMAN * N~AkZIER, Proc . Soc . Exp . Biol . Med . 12 3 853-859 (1966) E-~LAIR, R . HOOK, H . TOLLEY 8 G . BRUCE, Sci . , _133 105-106 (1961) 0 . WENDELL, Anaes . 16 24-31 (1961) W . BIGELOW, Am .~. ~ ~Ph ys~io~l ., 160 125-137 (1950) E . FISHER, E~Eb6R~ B . FISHER,~ C.M .A . Arch . Surg . 7 5 817-827 (1957) P . ALTLAND, B . HIGHMAN 8 J . GARBUS, Life Sci . _5 375-383 (1966) B . HIGHMAN 8 P . ALTLAND, Proc . Soc . ~ ~ôT . Med . ,109 523526 (1962) . M . BERCOT, M . DE MENDONCA, K . SCHWARTZ, J . LEANDRI 8 J . CACHERA, Cr obiolo , 9 479-487 H . ALBAUM, , U.S .A .F . Re ort No . 57-108 1-4 1957 We . PEARL, T .~BALAZE 8 . c 7-74 1966 ; A . KARMEN, J . Clin . Invest . 34 13T-T~3~TS55T SIGMA CHEMI~AL~6f~1FAAY,Tech . Bulletin No . 155-UV , (Rev . Feb ., 1973) R. BOULOVARD, Fed . Proc . 22 750-753 (1962) C . TURNER 8 J .~~`N1~R1~,General Endocrinolo , 5th Ed . W . B . Saunders, Phila ., ~TTS7 E . BLAIR, Fed . Proc . 28 1456-1462 (1969) A . ALLISON,T~atu~0~141-143 (1965) A . BANGHAM, K~FE1'S~V . SHATLANDER, Nature 193 754-756 (1962) A . BUCKLEY, J . Chem . Educ ., 42 674 (1~E . FRANKEL, T~e nn . ~u6T- (TS62) P . PUIG MUSET,BT~IA1~TiFf-ESTEVE, Experentia . _XX 649-651 (1965) K . SHILKIN, J . PAPADEMITRIOU, M . WALTERS, Aust . J . Exp . Med . Sci . 44 581-584 (1966)
Pergamon Prese
EFFECTS OF DMSO ON SGOT DURING HYPOTHERMIA IN ADRENALECTOMIZED RATS Louis J . Ramazzotto, Ph .D . and Ronald Carlin, Ph .D . Fairleigh Dickinson University School of Dentistry Department of Human Physiology 110 Fuller Place Hackensack, New Jersey 07601 (Received in final form December 1, 1977)
SUMMARY
A study of changes in SGOT levels was made in by othermic Sprague-Dawley rats (rectal temperatures 23 ° C~ which were adrenalectomized and/or treated with DMSO (41~ aqueous solution injected IP) prior to hypothermia . Adrenalectomized animals exhibited slight initial increases in SGOT, but returned to control levels within 24 hours after exposure . Animals treated with DMSO alone or adrenalectomized and treated with DMSO showed significant increases in SGOT, which persisted 24 hours after exposure . Data suggests that DMSO exerts its action directly upon the cell membrane, causing permeability changes rather than indirectly by influencing the release of adrenal hormones which in turn affect membrane permeability . The effects of environmental stress on serum enzyme levels is well documented . It has been reported that serum levels of glutamic oxalacetic transaminase (SGOT) are altered after administration of catecholamines (1 , exposure to high altitude (2), prolonged exercise (3), cold exposure (4), and hypothermia (5,6) . In many of these studies, histological examinations failed to demonstrate substantial necrosis or other morphological changes which mi ht account for the significant rise in SGOT levels (3,6,7,8 . It has been shown that treatment with dimethyl sulfoxide (DMSO) prior to the induction of stress may potentlate the elevation of SGOT levels (9,4) . Histological examination following treatment wlth DMSO indicates that increased titers of SGOT are not a result of cell necrosis (2,3,10) . Since cellular damage is not visibly evident following stress alone or stress subsequent to treatment with DMSO, it is suggested that these factors may act singly or in combination to alter membrane permeability and therefore, bring about the observed increase in levels of SGOT (10,11,12) . The role of the adrenal gland in relation to stress induced elevation of SGOT has been investigated using restraint as the 329
33 0
Effect of DMSO and Hypothermia oa SGOT
Vol . 22, No . 4, 1978
stressing factor (13) . It was reported that adrenalectomy reduced the level of SGOT which had been elevated due to the restraint . Injection of epinephrine into adrenalectomized animals again increased levels of SGOT ; while administration of an alpha blocker plus epinephrine did not . It has been proposed that DMSO acts to raise SGOT levels by influencing the release of adrenal hormones, which in turn, affects membrane permeability (4) . Epinephrine acting alone, in combination with, or potentiated by adrenocortical secretions may bring about this alteration of membrane permeability . The present set of experiments were designed to test this hypothesis employing hypothermia (23°C .) as a stress . MATERIALS AND METHODS Experimental Animal Eighty-four male Sprague-Dawley rats (430±70 grams) were divided into seven main groups, each containing 12 rats . Experimental groups were sub-divided into two categories, each containing six rats . (Table 1) . Animals to be adrenalectomized were anesthesized by intraperitoneal injection of sodium pentobarbital (33 mg/Kg) . A 3 cm lateral incision was made beneath the rib cage, the posterior portion of the kidney exposed, and the adrenal gland lying within the fatty tissue identified and carefully excised . A three day interval was alloted between adrenalectomy and beginning any experiment . During this interval animals were provided with a one percent sodium chloride solution instead of drinking water . DMSO Administration Animals treated with DMSO received an intraperitoneal injection of 1 ml/100 gm body weight of a 41~ aqueous solution of DMSO kept at 37°C . DMSO was administered subsequent to anesthesia, but 5 minutes prior to initiating hypothermia . Induçtion of Hypothermia Anesthesized animals were rendered hypothermic by placing them on ice, A thin plastic shield separated the animal from the ice . The animals rectal temperature was measured by means of a clinical thermometer, heart rate was monitored with a stethoscope and respiratory rate determined visually . All parameters were recorded every ten minutes during hypothermia . Upon reaching a rectal temperature of 23°C the animals were removed from the ice 'and placed 24 to 36 inches from a 500 watt lamp for one hour to re-establish normal body temperature . Determination of Enzyme Activity Blood samples for enzyme assay were drawn by cardiac puncture utilizing a peristaltic pump . Blood samples (5 .0 ml) were taken under ether anesthesia at 5 and 24 hours following treatment with DMSO and/or exposure to cold, Only one sample per rat was obtained . Samples were centrifuged (6000 RPM for 13 minutes) and the serum withdrawn to be assayed for SGOT activity using the method developed by Karmen (14) . Employing a Beckman DB-G spectrophoto-
Vol . 22, No . 4, 1978'
Effect of DMSO and Hypothermia on SGOT
331
meter, the reduction of optical density (OD) at 340 nm due to the formation of beta-NAD was measured in terms of Karmen units, where one Karmen unit causes the formation of .000482 uM of glutamic acid per minute (15) . Mean enzyme activities with standard error were calculated and statistically significant differences determined using the Student's "t" test . TABLE 1 Animal
Distribution in Experimental and Control Groups
Experimental Each group contained 12 animals . groups had SGOT activity determined (in six animals) 5 hours and (in remaining six animals) 24 hours after treatment . 1 . Controls A . Normal controls B . Adrenalectomized controls 2 . Hypothermia 3 . DMSO 4 . DMSO + Hypothermia 5 . Adrenalectomy + Hypothermia 6 . Adrenalectomy + DMSO 7 . Adrenalectomy + Hypothermia + DMSO RES ULTS Effeçt of Adrenaleçtomy Serum GOT activity in adrenalectomized control animals was not significantly different from the activity measured in nonadrenalectomized control rats . Effect o f Hypothermia Adrenalectomized animals exposed to cold did exhibit .a slight (p< .05) initial increase in SGOT activity five hours after exposure . SGOT activity did return to control levels within 24 hours following exposure . Nan-adrenalectomized animals exposed to cold did exhibit a significantly higher SGOT activity (p<,001) at both 5 and 24 hours after hypothermia .
332
Effect
of DMSO and Hypothermia on
SCOT
Vol . 22, No . 4, 1978
ve
é , s~
FIG . 1 Effect of hypothermia on serum glutamic oxalacetic transaminase levels in adrenalectomized and nonadrenalectomized rats . Values shown are means ; vertical bars denote standard error of the means . Effect of DMSO Treatment with DMSO caused significant increases (p<.001) in the SGOT activity of both non-adrenalectomized and adrenalectomized rats . These increases persisted even 24 hours following treat ment . There was no statistically significant difference in enzyme activity between these two groups in both the 5 and 24 hour sample.
Vol . 22, No . 4 , 1978
Effect of DMSO aad Hypothermia on SCOT
>o
vs i iao
~s
I
a E e
x
Ë
I
i
i
e
~6
x i
i
FI G . 2 Effect of dimethyl sulfoxide on serum glutamic oxalacetic transamlnase levels in adrenalectomized and non-adrenalectomized rats . Values shown are means ; vertical bars denote standard error of the meansw The combination of DMSO + Hypothermia resulted in a significant increase (p< .001) in SGOT activity, whether or not the animal had been adrenalectomized . These increases persisted even 24 hours following treatment and exposure . Non-adrenalectomized DMSO treated animals showed SGOT activity that was significantly greater (p< .O1) than that of the adrenalectomized DMSO treated rats .
333
334
Vol . 22, No . 4, 1978
Effect of DMSO aad Hypothermia on SGOT
sao
iso
vs
T
50
rs
0
FIG . 3 Combined effect of dimethyl sulfoxide and hypothermia on serum glutamic oxalacetic transaminase levels in adrenalectomized and non-adrenalectomized rats . Values shown are means ; vertical bars denote standard error of the means . DISCUSSION It has been demonstrated that exposure to cold may induce inAltland reported an creased adrenocortical secretion (16) . elevation of serum enzyme levels in proportion to the degree of hypothermia, and suggested that adrenal hormones may stimulate the release of GOT (3) . Results of the experiments described here appear to correlate with these observations in that adrenalectomized hypothermic animals exhibited significantly lower SGOT activity as com ared to the non-adrenalectomized hypothermic animals . Pearl (13~ demonstrated that adrenalectomy suppressed the rise of SGOT when stress was due to restraint . Our studies, using hypothermia as the stressing factor, lmplicate the adrenal glands as the mediators of the increase in SGOT activity . The slight increase in SGOT activity measured in adrenalectomized rats 5 hours
vol . 22, No . 4, 197$
Effect of DMSO and Hypothermia on SCOT
335
after hypothermia may be attributed to the release of catecholamines from some source other than the adrenals (17) . Hy othermia causes a prolonged duration of action of epinephrine (18~ . This might be a contributing factor in bringing about the continued rise of SGOT levels in the non-adrenalectomized rats hours after exposure to cold . Similarly, in adrenalectomized animals which possess small amounts of non-adrenal catecholamines, there should be a smaller rise in SGOT levels and therefore a faster return to control levels . The mechanism by which adrenal hormones cause the increased However, it has been established levels of SGOT is not known . that epinephrine does not increase the de novo synthesis of GOT
(13) .
Changes in SGOT levels after treatment with DMSO closely paralleled the response due to hypothermia alone, initially suggesting that both stimuli were operating through a common mechanism . However, adrenalectomy did not prevent a rise in SGOT activity in animals receiving DMSO treatment, thus indicating the existence of two different mechanisms . While the ability of DMSO to cross various types of membranes is well documented, the precise mechanism by which this is accomplished has yet to be elucidated . This ability may be related to a reversible substitution of DMSO into the membrane structure such that the membranes are only temporarily altered, and resume normal function on removal of the DMSO . Such a temporary alteration could cause a change in the permeability of the membrane and this in turn could affect serum enzyme levels . The oxidizing ability of DMSO has also been shown to produce changes in isomeric conformation of cellular fatty acids which may be a factor in creating increased membrane permeability (19,20,21,22,23,24) . The observation that adrenalectomy significantly reduces the serum levels of GOT in those animals which had been treated with DMSO and then exposed to hypothermia indicates that the adrenal hormones either directly or indirectly increased SGOT levels . Animals which were adrenalectomized and then treated with DMSO prior to cold exposure demonstrated an increase in the level of SGOT as compared to those animals which were adrenalectomized but did not receive DMSO prior to inducing hypothermia, hence, there is an lmplication that DMSO itself initiates an increase in SGOT . It is equally important to note that SGOT levels remain high in those animals which were adrenalectomized and treated with DMSO, even 24 hours after the hypothermic period . It is suggested that DMSO may act directly to increase and maintain SGOT levels . On the basis of the present experiments it appears that hypothermia indirectly affects cell membranes by way of adrenal hormones ; apparently DMSO affects the membranes in a more direct manner through some unknown mechanism .
336
Effect of DMSO and Hypothermia on SCOT
Vol . 22, No . 4, 1978
REFERENCES : 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 13 14 15
. . . .
16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 .
B . HIGHMAN, H . MALING 8 E . THOMPSON, Am . J . Physiol . , 196 436-440 (1959) P . ALTLAND, B . HIGHMAN, 8 J . GARBUS, Aerospace Med . , 35 10341039 (1964) P . ALTLAND, 8 B . HIGHMAN, Am . J . ~Ph~siol ., 201 393-395 (1961) P . ALTLAND, B . HIGHMAN * N~AkZIER, Proc . Soc . Exp . Biol . Med . 12 3 853-859 (1966) E-~LAIR, R . HOOK, H . TOLLEY 8 G . BRUCE, Sci . , _133 105-106 (1961) 0 . WENDELL, Anaes . 16 24-31 (1961) W . BIGELOW, Am .~. ~ ~Ph ys~io~l ., 160 125-137 (1950) E . FISHER, E~Eb6R~ B . FISHER,~ C.M .A . Arch . Surg . 7 5 817-827 (1957) P . ALTLAND, B . HIGHMAN 8 J . GARBUS, Life Sci . _5 375-383 (1966) B . HIGHMAN 8 P . ALTLAND, Proc . Soc . ~ ~ôT . Med . ,109 523526 (1962) . M . BERCOT, M . DE MENDONCA, K . SCHWARTZ, J . LEANDRI 8 J . CACHERA, Cr obiolo , 9 479-487 H . ALBAUM, , U.S .A .F . Re ort No . 57-108 1-4 1957 We . PEARL, T .~BALAZE 8 . c 7-74 1966 ; A . KARMEN, J . Clin . Invest . 34 13T-T~3~TS55T SIGMA CHEMI~AL~6f~1FAAY,Tech . Bulletin No . 155-UV , (Rev . Feb ., 1973) R. BOULOVARD, Fed . Proc . 22 750-753 (1962) C . TURNER 8 J .~~`N1~R1~,General Endocrinolo , 5th Ed . W . B . Saunders, Phila ., ~TTS7 E . BLAIR, Fed . Proc . 28 1456-1462 (1969) A . ALLISON,T~atu~0~141-143 (1965) A . BANGHAM, K~FE1'S~V . SHATLANDER, Nature 193 754-756 (1962) A . BUCKLEY, J . Chem . Educ ., 42 674 (1~E . FRANKEL, T~e nn . ~u6T- (TS62) P . PUIG MUSET,BT~IA1~TiFf-ESTEVE, Experentia . _XX 649-651 (1965) K . SHILKIN, J . PAPADEMITRIOU, M . WALTERS, Aust . J . Exp . Med . Sci . 44 581-584 (1966)