- Email: [email protected]
Tissue uptake and excretion of bromine-82 by rats
ARCHIVES
OF
BIOCHEMISTRY
Tissue Uptake
AND
and Excretion
B. Theodore From
the University Agricultural
BIOPHYSICS
74, 357-361
of Bromine-82
Cole’ and Homer
of Tennessee, Research Received
United Program, June
(1958)
States Atomic Oak Ridge,
by Rats
Patrick Energy Commission, Tennessee2
11, 1957
Huff el a.!. (1) have recently demonstrated a nutritional requirement for bromide in rats on a purified diet, adequate in factors known to be required for growth, containing iodinated casein with thyroid activity. Bosshardt et al. (2) gave evidence of a growth response (S-10 %) in chicks on diets supplemented with a bromide source. Winnek and Smith (3), through chemical analysis, showed that the feeding of a bromide supplement increased bromide levels in blood, liver, kidney, muscle, spleen, and brain. Abelin and Poretti (4) demonstrated an increased uptake of radioactive bromide by several tissues of hyperthyroid rats and suggested that the effect of hyperthyroidism was to induce a partial deficiency of bromide by increasing tissue requirements. Mack and Shipley (5) found the activity of gastric tissue and contents relatively high at 24 hr. after intravenous administration of 0.5-l .O millicurie (me.) BFz/kg. body weight. These workers could demonstrate no preferential uptake by thyroid tissue. A study of the tissue uptake and excretion of BF2 at various intervals of time following injection in bromide-deficient rats is pertinent to our further understanding of the role of bromide in nutrition. Evidence con1 Research Participant, Oak Ridge Institute of Nuclear Studies. On leave from Louisiana State University, Baton Rouge, Louisiana. 2 This manuscript is published with the permission of the director of the University of Tennessee, Agricultural Experiment Station, Knoxville, Tennessee. The radioactive materials used in this work were obtained from the Oak Ridge National Laboratory on allocation from the U. S. Atomic Energy Commission. This work was completed under Contract No. AT-40-l-GEN-242 between the University of Tennessee, College of Agriculture, and the U. S. Atomic Energy Commission. 357
358
COLE AN’I) PATRICK
cerning the rapidity and selectivity of tissue uptake and excretion of bromide in normal and nutritionally deficient animals is presented. MATERIALS
AND METHODS
Sixty-four Albino weanling rats (Rockland strain) were placed on an RPL diet (rats, poor quality protein, lysine) low in methionine, tryptophan, vitamin De , lysine, and completely lacking in folic acid for a period of 5 days. Taken off this diet, which served to induce a metabolic stress in the animals, these rats, weighing from 34 to 64 g., were placed on a bromide-deficient diet described in Table I. Fourteen days later, all animals received an intraperitoneal injection of 50 microcuries Gc.) of Br** as KBr. Considerable activity, was injected to provide for concentration measurement in tissues of minimal weight (i.e., adrenals 4-30 mg., ovary 9-34 mg., and pancreas 42-160 mg.) of such magnitude that they could be measured by conventional, gamma-counting procedures up to96 hr. postinjection. Animals were anesthetized with Nembutal and decapitated at the time intervals, postinjection, indicated in Table II. Four animals were sacrificed at each time period except at 4,24,72, and 96 hr. Eight animals were sacrificed at 4 and 24 hr. and six each at 72 and 96 hr. Additional animals were placed on a deficient diet, TABLE I Low Bromine Ration (grams/100 g.) cottonseed oil
Hydrogenated CereloseQ Protein “Drackett”* CaCOp Salt Mixture U. T. No. 1 K2HPO4 Vitamin Premix (G.B.I.)c
10.09 56.56 25.90 1.25 2.99 1.25 4.00 Q From General Biologicals Incorporated, Chicago, Illinois. b From Drackett Company, Cincinnati, Ohio. c From General Biologicals Incorporated. Added to diet as 5yo to give the following vitamins in micrograms/gram: thiamine 2.5, riboflavine 2.5, pyridoxine 2.5, pantothenic acid 20, nicotinic acid 10, p-aminobeneoic acid 50, inositol 100, cholesterol chloride 1009. Composition of Salt Mixture U. T. No. 1: This is a modification of the Ca phosphate-free mix used by H. G. Day et al. [J. Nutrition 16, 525 (1938)] and includes the following minerals; in per cent: 0.01 NaCl 39.84 NaF 0.03 KC1 18.26 KI KHCOa 26.29 CuSO1.5H20 0.40 (Anhyd.) MgSO* 12.00 MnSOa.4HzO 0.20 K$04.2HzO 0.02 ZnS0,.7Hz0 0.60 FeC13.6H20 2.59
g 0
1
0.5-.l l.l-.2 l.l-.2
0.5
0.4-.l 1.2-.l 1.3-.4
T-
Uptake
4
0.6-.030.7(8)-.l l.l-.l 1.4(8)-.3 l.O-.l 1.4(S)-.4
2
of Bra2 by Tissues
0.5-.04 l.l-.2 l.l-.l
8
II Per
24
0.5-.O! 0.8-.2 0.7-.l
48
(hr.)” 72
Dose/Gram
96
I
of Tissue
2
Control
8
5 0.2(6)-.03 0.1(s)-.02 0.5-.l 0.7-.03 0.3(6)-.03 0.2(6)-.04 1 .l-.25 1.4-.17 0.3(S)-.02 0.2(6)-.04 0.9-.l 1.4-.15 0.5(6)-.l 0.3(6)-.l 0.4(6)-.Ol 0.3(6)-.l 1.9-.2 2.2-.3 0.5(6)-.03 0.4(6)-.l 1.3-.4 1.7-.3 0.4(6)-.04 0.3(6)-.04 1.5-.3 2.0-.2 0.6(6)-.l 0.4(6)-.1 2.1-.3 2.1-.3 0.5(6)-.l 0.3(6)-.l 1.4-.3 2.3-.3 0.5(6)-.04 0.3(6)-.03 1.9-.4 2.0-.6 0.8(6)-.l 0.5(6)-.l 2.5(3)-1.03.4(3)-.4 0.6(6)-.l 0.4(6)-.042.4(3)-.5 2.5(3)-.4 1 .9(12)-.2 1.3(6)-.l 0.5(l) 3.2(2) 10 1.1(12)-.020.1(12)-.020.6(2) 0.4(2) -
-7
Cent of Administered
dose administration
in
L 0.5(8)-.l 0.9(8)-.2 0.8(8)-.2
.-
-
Time after
deficient
TABLE Expressed
Bromine
of Rats
2.7-.9 2.0-.6 l.O-.3 2.5(8)-.5 1.6-.2 1.4(8)-.4 1.6.5 2.5(8)-.7 1 .8(8)-.61.4(8)-.3 3.0(7)-.7 1.5(8)-.5 1.2(7)-.3 1.0(8)-.3 2.3-.4 1.7-.5 1.7-.2 2.4(8)-.4 1.6.1 1.2-.3 1.3-.5 2.0-.4 2.2-.4 2.5(8)-.6 2.0-.2 1.4-.3 1.6-.6 2.1-.3 2.1-.4 2.1-.9 1.6-.4 2.1(8)-.7 1.8-.3 1.3-.3 1.5.4 2.5-.7 2.5-.5 2.6.3 2.0(8)-.8 1.9-.2 1.3-.3 1.3-.2 4.2-.5 3.2-.4 4.0(5)-1.3 3.3-.2 2.0-.5 6.0(7)-l .: 1 2.7(7)-.9 4.3(7)-.9 2.6-.3 2.6-.l 3.1(5)-.9 2.6.2 2.4(7)-.6 2.4(2) 3.6-1.: !5 .0(20)-.4 .4(16)-.5 #.1(15)-.0: 0.2 0 .1(19)-.o: 0 Figures on blood and urine expressed as per cent of administered dose/ml. b Four animals sacrified at each time period except as indicated by figures in parentheses.
Brain Liver Heart Thyroid Pancreas Adrenals Spleen Kidney Intestine Gonads Bloodp1.O Blood ce. Urine Feces
Tissue
I
Mean
360
COLE
AND
PATRICK
dosed, and sacrificed to provide adrenals at each time period indicated in Table II. Tissues were removed, weighed immediately, and placed into Pyrex test tubes containing 8 ml. of digestion fluid made up of 0.2 M NaOH, 0.05 M NaHSOz , and 0.015 M KBr. Tubes were placed in a water bath at 85°C. until digestion was complete (3-6 hr.). Volume was maintained by addition of digestion fluid. Monitoring of cotton plugs inserted in the neck of the tubes gave no evidence of loss of Br** with water evaporation. Tubes were removed from the water bath and cooled. The volume of solution was increased to 10 ml. with digestion solution, and hard gamma emissions of the digested tissues were counted in an NaI (Tl), well type, Chicago-Nuclear model D5-3, scintillation detector. This is insensitive to volume changes in the counting test tube between 0 and 3 mm. Recording was made on a Nuclear-Chicago scaling unit, model 161A. Counting of adrenal, ovarian, and pancreatic tissues was completed within 24 hr. following sacrifice of the animal. More active tissues were counted last. Decay was calculated, and data shown in Table II are expressed as percentage of the administered dose per gram of tissue. Blood was separated, and cells were washed and hemolyzed in distilled water and counted directly. Plasma was mixed with saline and counted in similar fashion. Statistical significance is based upon calculation of standard deviation. T values were calculated where standard deviation (SD.) overlap occurred. RESULTS
AND
DISCUSSION
Results of tissue concentration measurements are shown in Table II. Brain appeared to be least active with respect to uptake of BP2. At a time when blood plasma varied from 5.9 to 3.2 % (44-2 hr.) and relatively little BP2 was being excreted (urine level 0.4 % at 2 hr.), the brain levels remained at approximately 0.5 % and remained at that level through 48 hr. Uptake of Brs2 by liver and heart tissue was significantly less than any tissue except brain, although there was no significant difference in per cent uptake between the two former organs. Levels in the pancreas and adrenal were higher than heart and liver levels throughout the 8-hr. period. The most variation in uptake of BF within tissues was noticed in the adrenals. This could be an indication of variable and high metabolic activity connected with metabolic stress in these bromide-deficient animals. If the variation was due to adsorption of activity in this organ, since injection was made intraperitoneally, a characteristic pattern of variation should likewise have appeared in the intestine, spleen, and kidney tissues. The latter observation did not appear. No appreciable difference in distribution of bromide attributable to sex of rats was found. Since no sex differences were observed, data on gonadal tissues were pooled and presented as uptake of gonads. The levels of bromide in gonad, spleen, kidney, and small intestinal tissues reached a peak concentration during the first 4 hr., did not subsequently vary with time, and were not significantly different from bromide levels
BROMINE
UPTAKE
AND EXCRETION
361
in pancreas and adrenal tissues, but remained higher than heart, liver, and brain tissues. A group of eight control animals was sacrificed in groups of four at 2 and 8 hr., respectively. Comparison between the tissue levels of radiobromide in these animals showed no significant difference from the deficient animals except at 8 hr. At this latter time bromide levels for brain, spleen, and pancreas tissues of control animals were significantly elevated above experimental animals. In no tissue was there any significant difference between uptake levels after 24 hr., and the data show that activity was almost completely gone in all tissues at 72 hr. The beginning of Br@ excretion at 4 hr. was correlated with a rapid fall in blood levels and a fall in mean values for various tissues as is evident in Fig. 1. Apparent also is the relative lack of any significant quantity of Brn lost from the animal through the feces. Blood concentration values indicate equal distribution between cells and plasma with the ratio maintained throughout the period I$&-96 hr. Initially there is a significantly higher concentration in the plasma. Quantitative comparison of these data with those of Mack and Shipley (5) cannot readily be made since their work included beta counts of dehydrated tissue and gamma counts of undigested tissue. Their data were expressed as concentration ratios rather than per cent of administered dose. Qualitative comparison of these data with appropriate tissue data reported by these workers shows agreement with their values for rats not nutritionally deficient in bromide. SUMMARY
Uptake of bromine-82 by various tissues of bromide-deficient rats has been studied. Tissues of bromide-deficient rats, with respect to per cent of original dose taken up, may be ranked as follows: brain < liver = heart < pancreas; adrenal; gonads; spleen; kidney and intestine. Excretion of Bti2 occurs principally through the kidney, with the isotope appearing in the voided urine at 4 hr. postinjection. REFERENCES 1. HUFF, J. W., BOSSHARDT, D. K., MILLER, C. P., AND BARNES, Sot. Ezptl. Biol. Med. 99, 216 (1956). 2. BOSSHARDT, K. D., HUFF, J. W., AND BARNES, R. H., Proc. Sot. Med. 92, 219 (1956). 3. WINNEX, P. S., AND SMITE, A. H., J. BioZ. Chem. 119,93 (1937). 4. ABELIN, J., AND PORETM, G., HeZv. Physiol. et Pharmacol. Acta 10, 5. MACX, J. F., AND SHIPLEY, R. A., Proc. Sot. Ezptl. BioZ. Med. 90,
R. H., Proc. Exptl. Biol. C7-9 (1952). 18 (1952).
OF
BIOCHEMISTRY
Tissue Uptake
AND
and Excretion
B. Theodore From
the University Agricultural
BIOPHYSICS
74, 357-361
of Bromine-82
Cole’ and Homer
of Tennessee, Research Received
United Program, June
(1958)
States Atomic Oak Ridge,
by Rats
Patrick Energy Commission, Tennessee2
11, 1957
Huff el a.!. (1) have recently demonstrated a nutritional requirement for bromide in rats on a purified diet, adequate in factors known to be required for growth, containing iodinated casein with thyroid activity. Bosshardt et al. (2) gave evidence of a growth response (S-10 %) in chicks on diets supplemented with a bromide source. Winnek and Smith (3), through chemical analysis, showed that the feeding of a bromide supplement increased bromide levels in blood, liver, kidney, muscle, spleen, and brain. Abelin and Poretti (4) demonstrated an increased uptake of radioactive bromide by several tissues of hyperthyroid rats and suggested that the effect of hyperthyroidism was to induce a partial deficiency of bromide by increasing tissue requirements. Mack and Shipley (5) found the activity of gastric tissue and contents relatively high at 24 hr. after intravenous administration of 0.5-l .O millicurie (me.) BFz/kg. body weight. These workers could demonstrate no preferential uptake by thyroid tissue. A study of the tissue uptake and excretion of BF2 at various intervals of time following injection in bromide-deficient rats is pertinent to our further understanding of the role of bromide in nutrition. Evidence con1 Research Participant, Oak Ridge Institute of Nuclear Studies. On leave from Louisiana State University, Baton Rouge, Louisiana. 2 This manuscript is published with the permission of the director of the University of Tennessee, Agricultural Experiment Station, Knoxville, Tennessee. The radioactive materials used in this work were obtained from the Oak Ridge National Laboratory on allocation from the U. S. Atomic Energy Commission. This work was completed under Contract No. AT-40-l-GEN-242 between the University of Tennessee, College of Agriculture, and the U. S. Atomic Energy Commission. 357
358
COLE AN’I) PATRICK
cerning the rapidity and selectivity of tissue uptake and excretion of bromide in normal and nutritionally deficient animals is presented. MATERIALS
AND METHODS
Sixty-four Albino weanling rats (Rockland strain) were placed on an RPL diet (rats, poor quality protein, lysine) low in methionine, tryptophan, vitamin De , lysine, and completely lacking in folic acid for a period of 5 days. Taken off this diet, which served to induce a metabolic stress in the animals, these rats, weighing from 34 to 64 g., were placed on a bromide-deficient diet described in Table I. Fourteen days later, all animals received an intraperitoneal injection of 50 microcuries Gc.) of Br** as KBr. Considerable activity, was injected to provide for concentration measurement in tissues of minimal weight (i.e., adrenals 4-30 mg., ovary 9-34 mg., and pancreas 42-160 mg.) of such magnitude that they could be measured by conventional, gamma-counting procedures up to96 hr. postinjection. Animals were anesthetized with Nembutal and decapitated at the time intervals, postinjection, indicated in Table II. Four animals were sacrificed at each time period except at 4,24,72, and 96 hr. Eight animals were sacrificed at 4 and 24 hr. and six each at 72 and 96 hr. Additional animals were placed on a deficient diet, TABLE I Low Bromine Ration (grams/100 g.) cottonseed oil
Hydrogenated CereloseQ Protein “Drackett”* CaCOp Salt Mixture U. T. No. 1 K2HPO4 Vitamin Premix (G.B.I.)c
10.09 56.56 25.90 1.25 2.99 1.25 4.00 Q From General Biologicals Incorporated, Chicago, Illinois. b From Drackett Company, Cincinnati, Ohio. c From General Biologicals Incorporated. Added to diet as 5yo to give the following vitamins in micrograms/gram: thiamine 2.5, riboflavine 2.5, pyridoxine 2.5, pantothenic acid 20, nicotinic acid 10, p-aminobeneoic acid 50, inositol 100, cholesterol chloride 1009. Composition of Salt Mixture U. T. No. 1: This is a modification of the Ca phosphate-free mix used by H. G. Day et al. [J. Nutrition 16, 525 (1938)] and includes the following minerals; in per cent: 0.01 NaCl 39.84 NaF 0.03 KC1 18.26 KI KHCOa 26.29 CuSO1.5H20 0.40 (Anhyd.) MgSO* 12.00 MnSOa.4HzO 0.20 K$04.2HzO 0.02 ZnS0,.7Hz0 0.60 FeC13.6H20 2.59
g 0
1
0.5-.l l.l-.2 l.l-.2
0.5
0.4-.l 1.2-.l 1.3-.4
T-
Uptake
4
0.6-.030.7(8)-.l l.l-.l 1.4(8)-.3 l.O-.l 1.4(S)-.4
2
of Bra2 by Tissues
0.5-.04 l.l-.2 l.l-.l
8
II Per
24
0.5-.O! 0.8-.2 0.7-.l
48
(hr.)” 72
Dose/Gram
96
I
of Tissue
2
Control
8
5 0.2(6)-.03 0.1(s)-.02 0.5-.l 0.7-.03 0.3(6)-.03 0.2(6)-.04 1 .l-.25 1.4-.17 0.3(S)-.02 0.2(6)-.04 0.9-.l 1.4-.15 0.5(6)-.l 0.3(6)-.l 0.4(6)-.Ol 0.3(6)-.l 1.9-.2 2.2-.3 0.5(6)-.03 0.4(6)-.l 1.3-.4 1.7-.3 0.4(6)-.04 0.3(6)-.04 1.5-.3 2.0-.2 0.6(6)-.l 0.4(6)-.1 2.1-.3 2.1-.3 0.5(6)-.l 0.3(6)-.l 1.4-.3 2.3-.3 0.5(6)-.04 0.3(6)-.03 1.9-.4 2.0-.6 0.8(6)-.l 0.5(6)-.l 2.5(3)-1.03.4(3)-.4 0.6(6)-.l 0.4(6)-.042.4(3)-.5 2.5(3)-.4 1 .9(12)-.2 1.3(6)-.l 0.5(l) 3.2(2) 10 1.1(12)-.020.1(12)-.020.6(2) 0.4(2) -
-7
Cent of Administered
dose administration
in
L 0.5(8)-.l 0.9(8)-.2 0.8(8)-.2
.-
-
Time after
deficient
TABLE Expressed
Bromine
of Rats
2.7-.9 2.0-.6 l.O-.3 2.5(8)-.5 1.6-.2 1.4(8)-.4 1.6.5 2.5(8)-.7 1 .8(8)-.61.4(8)-.3 3.0(7)-.7 1.5(8)-.5 1.2(7)-.3 1.0(8)-.3 2.3-.4 1.7-.5 1.7-.2 2.4(8)-.4 1.6.1 1.2-.3 1.3-.5 2.0-.4 2.2-.4 2.5(8)-.6 2.0-.2 1.4-.3 1.6-.6 2.1-.3 2.1-.4 2.1-.9 1.6-.4 2.1(8)-.7 1.8-.3 1.3-.3 1.5.4 2.5-.7 2.5-.5 2.6.3 2.0(8)-.8 1.9-.2 1.3-.3 1.3-.2 4.2-.5 3.2-.4 4.0(5)-1.3 3.3-.2 2.0-.5 6.0(7)-l .: 1 2.7(7)-.9 4.3(7)-.9 2.6-.3 2.6-.l 3.1(5)-.9 2.6.2 2.4(7)-.6 2.4(2) 3.6-1.: !5 .0(20)-.4 .4(16)-.5 #.1(15)-.0: 0.2 0 .1(19)-.o: 0 Figures on blood and urine expressed as per cent of administered dose/ml. b Four animals sacrified at each time period except as indicated by figures in parentheses.
Brain Liver Heart Thyroid Pancreas Adrenals Spleen Kidney Intestine Gonads Bloodp1.O Blood ce. Urine Feces
Tissue
I
Mean
360
COLE
AND
PATRICK
dosed, and sacrificed to provide adrenals at each time period indicated in Table II. Tissues were removed, weighed immediately, and placed into Pyrex test tubes containing 8 ml. of digestion fluid made up of 0.2 M NaOH, 0.05 M NaHSOz , and 0.015 M KBr. Tubes were placed in a water bath at 85°C. until digestion was complete (3-6 hr.). Volume was maintained by addition of digestion fluid. Monitoring of cotton plugs inserted in the neck of the tubes gave no evidence of loss of Br** with water evaporation. Tubes were removed from the water bath and cooled. The volume of solution was increased to 10 ml. with digestion solution, and hard gamma emissions of the digested tissues were counted in an NaI (Tl), well type, Chicago-Nuclear model D5-3, scintillation detector. This is insensitive to volume changes in the counting test tube between 0 and 3 mm. Recording was made on a Nuclear-Chicago scaling unit, model 161A. Counting of adrenal, ovarian, and pancreatic tissues was completed within 24 hr. following sacrifice of the animal. More active tissues were counted last. Decay was calculated, and data shown in Table II are expressed as percentage of the administered dose per gram of tissue. Blood was separated, and cells were washed and hemolyzed in distilled water and counted directly. Plasma was mixed with saline and counted in similar fashion. Statistical significance is based upon calculation of standard deviation. T values were calculated where standard deviation (SD.) overlap occurred. RESULTS
AND
DISCUSSION
Results of tissue concentration measurements are shown in Table II. Brain appeared to be least active with respect to uptake of BP2. At a time when blood plasma varied from 5.9 to 3.2 % (44-2 hr.) and relatively little BP2 was being excreted (urine level 0.4 % at 2 hr.), the brain levels remained at approximately 0.5 % and remained at that level through 48 hr. Uptake of Brs2 by liver and heart tissue was significantly less than any tissue except brain, although there was no significant difference in per cent uptake between the two former organs. Levels in the pancreas and adrenal were higher than heart and liver levels throughout the 8-hr. period. The most variation in uptake of BF within tissues was noticed in the adrenals. This could be an indication of variable and high metabolic activity connected with metabolic stress in these bromide-deficient animals. If the variation was due to adsorption of activity in this organ, since injection was made intraperitoneally, a characteristic pattern of variation should likewise have appeared in the intestine, spleen, and kidney tissues. The latter observation did not appear. No appreciable difference in distribution of bromide attributable to sex of rats was found. Since no sex differences were observed, data on gonadal tissues were pooled and presented as uptake of gonads. The levels of bromide in gonad, spleen, kidney, and small intestinal tissues reached a peak concentration during the first 4 hr., did not subsequently vary with time, and were not significantly different from bromide levels
BROMINE
UPTAKE
AND EXCRETION
361
in pancreas and adrenal tissues, but remained higher than heart, liver, and brain tissues. A group of eight control animals was sacrificed in groups of four at 2 and 8 hr., respectively. Comparison between the tissue levels of radiobromide in these animals showed no significant difference from the deficient animals except at 8 hr. At this latter time bromide levels for brain, spleen, and pancreas tissues of control animals were significantly elevated above experimental animals. In no tissue was there any significant difference between uptake levels after 24 hr., and the data show that activity was almost completely gone in all tissues at 72 hr. The beginning of Br@ excretion at 4 hr. was correlated with a rapid fall in blood levels and a fall in mean values for various tissues as is evident in Fig. 1. Apparent also is the relative lack of any significant quantity of Brn lost from the animal through the feces. Blood concentration values indicate equal distribution between cells and plasma with the ratio maintained throughout the period I$&-96 hr. Initially there is a significantly higher concentration in the plasma. Quantitative comparison of these data with those of Mack and Shipley (5) cannot readily be made since their work included beta counts of dehydrated tissue and gamma counts of undigested tissue. Their data were expressed as concentration ratios rather than per cent of administered dose. Qualitative comparison of these data with appropriate tissue data reported by these workers shows agreement with their values for rats not nutritionally deficient in bromide. SUMMARY
Uptake of bromine-82 by various tissues of bromide-deficient rats has been studied. Tissues of bromide-deficient rats, with respect to per cent of original dose taken up, may be ranked as follows: brain < liver = heart < pancreas; adrenal; gonads; spleen; kidney and intestine. Excretion of Bti2 occurs principally through the kidney, with the isotope appearing in the voided urine at 4 hr. postinjection. REFERENCES 1. HUFF, J. W., BOSSHARDT, D. K., MILLER, C. P., AND BARNES, Sot. Ezptl. Biol. Med. 99, 216 (1956). 2. BOSSHARDT, K. D., HUFF, J. W., AND BARNES, R. H., Proc. Sot. Med. 92, 219 (1956). 3. WINNEX, P. S., AND SMITE, A. H., J. BioZ. Chem. 119,93 (1937). 4. ABELIN, J., AND PORETM, G., HeZv. Physiol. et Pharmacol. Acta 10, 5. MACX, J. F., AND SHIPLEY, R. A., Proc. Sot. Ezptl. BioZ. Med. 90,
R. H., Proc. Exptl. Biol. C7-9 (1952). 18 (1952).