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Lithium chloride and dilithium carbamyl phosphate: Lithium distribution and toxicity in mice
TOXICOLOGY
Lithium
AND
APPLIED
PHARMACOLOGY
(1978)
Chloride and Dilithium Carbamyl Phosphate: Distribution and Toxicity in Mice’
HOWARD M. JERNIGAN, Departments
44.413-421
of Biochemistry Tennessee
JR.,
GORDON D. SCHRANK, AND LORRAINE M. KRAUS*
and Microbiology and the Comprehensive Centerfor the Health Sciences, Memphis.
Received
Lithium
Ju1.v 18, 1977: accepted
October
Sickle Cell Center, Tennessee 38163
University
of
5,1977
Lithium Chloride and Dilithium Carbamyl Phosphate: Lithium Distribution and Toxicity in Mice. JERNIGAN, H. M., JR.. SCHRANK, G. D.. AND KRAUS, L. M. (1978). Toxicol. Appl. Pharmacol. 44, 4 13-42 1. Dilithium carbamyl phosphate, an antisickling agent, dissociates into lithium cations and carbamyl phosphate dianions in solution. In order to determine which ion is primarily responsible for the toxicity observed at very high doses of dilithium carbamyl phosphate, two groups of 1.5 mice each were given similar amounts of lithium in twice-daily ip injections for 21, 23. or 30 days. Six of the fifteen mice survived in group A (lithium chloride, 12.9 mEq of Li+/kg/day), and 8 of 15 mice survived in group B (dilithium carbamyl phosphate, 13.8 mEq of Li+/kg/day). Blood l$hium concentrations measured 4-6 hr after an injection averaged 1.7 (+0.7 SD) mEq/liter for each group. Lithium concentrations were measured in 39 samples of tissue or tissue contents from mice in each group. In the surviving animals, lithium ranged from an average of 0.8-l. 1 mEq/kg in liver to 5- 15 mEq/kg in the contents of the lower intestinal tract. Lithium concentrations greater than 15 mEq/kg were often found in the tissues of the nonsurvivors. Hemoglobin carbamylation was 1.0 (i0.3 SD) mol of valine hydantoin/mol of hemoglobin tetramer in mice given dilithium carbamyl phosphate for 21-30 days. This study demonstrates that the toxic effects associated with administration of dilithium carbamyl phosphate appear to be due to the lithium cation rather than the carbamyl phosphate dianion. In all parameters measured, the toxic effects of lithium chloride paralleled those observed when an equivalent amount of lithium was administered as dilithium carbamyl phosphate.
Carbamyl phosphate carbamylates the amino-terminal valine residues of hemoglobin S in erythrocytes thereby interfering with the sickling phenomenon (Kraus and Kraus, 1971; Kraus ef al., 1972, 1977). Erythrocytes removed from patients with sickle cell anemia were incubated with carbamyl phosphate in vitro and labeled with 51Cr. When returned to the same patient, these erythrocytes had an increased survival time as compared with untreated erythrocytes (Kraus et al., 1972, 1973; Milner and Charache, 1973). Long-term studies have shown that oral or iv administration of carbamyl phosphate to dogs results in in uivo carbamylation of hemoglobin to concentrations which have been demonstrated to have an antisickling effect in erythrocytes containing deoxygenated HbS in vitro. However, a regimen of 100 mg/kg/day (8 mg or 1.2 mEq of Li+/kg/day) ’ Supported in part by USPHS Grant HL- 15 169. ’ T O whom requests for reprints should be sent at Department of Biochemistry, Center for the Health Sciences, Memphis, Tennessee 38163. 413
University of Tennessee
0041-008X/78/0442-0413$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
414
JERNIGAN,
SCHRANK,
AND
KRAUS
of dilithium salt occasionally produced toxic or near-toxic concentrations of lithium in the dogs (Kraus et al., 1975, 1977). It was necessary to distinguish between the effects of the different parts of the molecule, i.e., the lithium cation or the carbamyl phosphate dianion. The results in the mouse study described below demonstrate that, under the conditions studied, dilithium carbamyl phosphate is no more toxic than an equivalent amount of lithium administered in the form of lithium chloride, thereby implicating the lithium cation as the primary toxic ion in dilithium carbamyl phosphate.
METHODS
Thirty C57Bl mice, 168-180 days old and randomly selected as to sex, were individually identified, divided into two equal groups, and maintained on Purina Mouse Chow and water ad libitum. One group (group A) received 600 mg/kg/day of reagentgrade lithium chloride3 in a 15-mg/ml aqueous solution, while the other group (group B) received dilithium carbamyl phosphate,4 1200 mg/kg/day, in an aqueous solution of 30 mg/ml, which was frozen in aliquots and stored at -2OOC. These solutions used for injection were subsequently assayed for lithium concentration by atomic absorption spectrophotometry. The lithium content of the dilithium carbamyl phosphate solution was 345 mEq/liter (351 mEq/liter expected: compared to 322 mEq/liter in the lithium chloride solution (354 mEq/liter expected) The lower than expected lithium concentration in the lithium chloride solution was due to moisture in the granular lithium chloride. Both groups of mice were given 0.02 ml 01 solution/g body weight ip twice daily. Therefore group A received 12.9 mEq of Li+/kE body weight daily from lithium chloride, and group B received 13.8 mEq of Li+/kf body weight daily from dilithium carbamyl phosphate. Two days before the injections were started and 4-6 hr after the morning dose or Days 2, 7, 14, and 21 or until the mouse was killed or died, blood was removed from the tail after severing the tip. Blood was taken for lithium determinations (0.05 ml of blooc in 1.0 ml of water), leukocyte counts, packed cell volume, and random reticulocytc counts. Those animals which died were identified and frozen at -7O’C for late dissection. On Days 21, 23, or 30, 4-7 hr after the morning injection, final bloom samples were taken from the tail. Thereafter the mice were anesthetized with ether ant were exsanguinated by severing the major blood vessels in the neck. The mice were dissected and 39 tissues or tissue contents from each mouse wer’ sealed in weighed glass tubes. Tissue lithium was determined by the modified method a Schou (1958). A volume of concentrated nitric acid equal to 0.5- to l-fold the weight a tissue was added, and the tissues were digested for 1 hr (or more if digestion appearel incomplete) in a boiling water bath. Ten percent isopropanol was added to bring th final volume to 25 times the volume of nitric acid, the tubes were shaken, and th contents were filtered through Pyrex wool to remove insoluble material (mostly lipids Lithium analysis of nitric acid tissue digests or of water dilutions of blood was don using a Perkin-Elmer Model 290B atomic absorption spectrophotometer. White blood cell counts were done using a Coulter Counter, Model Z, 3 Mallinckrodt Chemical Works, St. Louis, 4 Sigma Chemical Co., St. Louis, Missouri;
Missouri. assayed by the supplier
to be 98% pure.
LITHIUM
DISTRIBUTION
AND
TOXICITY
415
Carbamylation of the N-terminal valines of hemoglobin was determined by the method of Manning et al. (1973) and expressed as moles of valine hydantoin per mole of hemoglobin tetramer. RESULTS The survival data for both groups of mice are reported in Fig. 1. At Day 2, when the greatest difference existed in survivors between groups A and B, the p value from a x2 determination was greater than 0.05, and, at Day 21, the p value was greater than 0.9. Therefore, there was no significant difference in survivors throughout the duration ofthe study between the mice receiving lithium chloride (group A) and those receiving dilithium carbamyl phosphate (group B). During the same time period, there were no deaths seen in the stable colony of over 75 mice, from which the experimental animals
0
10 DAYS
20
FIG. 1. Survival of mice injected ip, twice daily with (0) 12.9 mEq/kg/day of lithium chloride group A or (X) 13.8 mEq/kg/day of dilithium carbamyl phosphate designated group B.
designated
were taken. An examination of mortality, as group: survivors/total of male (A: 3/7; B: 7/10) and female (A: 3/S; B: l/5) mice did not indicate a significant difference between sexes, although the small numbers involved do not permit a definite statistical analysis. On the other hand, there were three pregnant mice (two in the lithium chloride group and one in the lithium carbamyl phosphate group) and all of these died during treatment. Subsequently, all 15 mice survived in a 17-day study using sodium chloride rather than lithium chloride administered in a similar regimen using equal amounts of the sodium cation. All mice in this group were bled, injected, and maintained in the same way as were the mice in groups A and B. Mice from groups A and B were killed on Days 21, 23, and 30, and during that time there were no spontaneous deaths. At autopsy, no infection, inflammation, or hematomas were found at the site of the injections or in the peritoneal cavity.
416
JERNIGAN,
SCHRANK,
AND
KRAUS
The mean lithium concentrations in whole blood of surviving mice of both groups are compared at intervals during the study (Table 1). The mean lithium concentrations at different time points ranged from 1 to 2 mEq/liter with a mean for each group of 1.7 & 0.7 mEq/liter averaging all values measured after lithium administration began. Because the interval of time between death and the last injection of lithium varied from mouse to mouse, there is no terminal measurement of blood lithium concentration available for comparison in the nonsurvivors. At the last routine bleeding before a nonsurvivor died, blood lithium ranged from 1.3 to 12 mEq/liter in group A and from 1.4 to 11 mEq/liter in group B. TABLE WHOLE BLOOD LITHIUM
1
CONCENTRATIONS IN MICE WHICH SURVIVED UNTIL THE END OF THE PROTOCOL”. b Days on study
Group A (lithium chloride) Group B (dilithium carbamyl phosphate)
0
2
1
14
2 l-30
0
1.9 + 1.0
1.9 f 0.5
1.6 k 0.4
1.4 t 0.5
0
1.9 + 1.1
2.0 f 0.6
1.1 i 0.3
1.9 i- 0.6
B Lithium concentrations expressed as milliequivalents per liter;‘mean 2~ SD. b Group A: six survivors; group B: eight survivors.
Table 2 showsthe concentration of lithium in 39 tissuesor tissue contents from mice of group A or group B. After normalizing the dose of lithium, there were no significant differences (p > 0.05 using Student’s t test) in survivors comparing the lithium concentration of each tissue in group A to the same tissue in group B. The only exception to this was cecum and cecal contents (p < 0.05). Lithium was present in all tissuesexamined. Liver had the lowest lithium concentration (0.8 mEq/kg, group A; 1.1 mEq/kg, group B). Blood, spleen, testis, spinal cord, and brain had intermediate concentrations of lithium (1.2-1.9 mEq/kg) which were significantly higher (p < 0.05) than concentrations in the liver. Salivary gland, muscle, kidney, epididymis, lung, and bone (cranium) had a higher lithium content than the intermediate group (p < 0.05). In the same mouse,total kidney or a portion of the cortex contained the samelithium per unit wet weight. Lithium concentration increased in the tissue or contents of the digestive tract when measureddistally from the stomach to the colon, even though the amounts of lithium found varied from mouse to mouse. In the urinary bladder and gall bladder, any residueof urine or bile could raisethe lithium content. In the mice which died during the course of the study (Table 2), lithium concentrations greater than 15 mEq/kg were often found in the tissues, with the exception of two mice in group A which died early in the study. Also, two mice in group A and one mousein group B were pregnant when they died. The lithium content of the total fetus was lower but correlated with the concentration of lithium in the tissuesof the pregnant mouse. The reaction of dilithium carbamyl phosphate with the amino-terminal valine of hemoglobin was measuredin blood from the surviving mice when they were killed. The
LITHIUM
DISTRIBUTION
AND
417
TOXICITY
TABLE 2 LITHIUM DISTRIBUTION IN TISSUES OF MICE RECEIVING LITHIUM CHLORIDE (GROUP A) OR DILITHIUM CARBAMYL PHOSPHATE (GROUP B) Surviving 3 weeks Tissueb Blood (mEq/liter) Spleen Brain Spinal cord Lumbar spinal nerve Lung Heart Skeletal muscle Parotid salivary gland Thyroid Adrenal Kidney (whole) Kidney (cortex) Kidney (remainder) Urinary bladder Urine (mEq/liter) Testes Epididymis Ovaries Oviduct Fetuses Lens Eye (lens removed) Bone (cranium) Skin and hair Liver Gall bladder Bile (gall bladder) Stomach tissue Stomach contents Duodenum tissue Duodenum contents Ileum tissue Ileum contents Cecum tissue Cecum contents Colon tissue Colon contents Feces a Lithium distribution
Group A’ (mean +_ SD) 1.4 1.2 1.2 1.3 2.4 1.9
+ 0.5 IO.3 * 0.2 & 0.4 & 0.7 k 0.6
1.8 + 0.5 2.7 2.2 1.9 2.0 2.2 2.3 2.3 2.1
i 0.9 + 0.4 2 1.3 & 1.6 + 0.5 k 0.6 + 0.5 _+ 0.7d 8.1' 1.4 & 0.4 2.2 f 1.1
1.5 i 0.8 1.0 + 0.3 1.6 & 0.4 2.5 i 0.5 2.5 k 0.3 7.1 + 2.8 0.8 & 0.3 1.9 * 1.2 10.7' 1.2 f 0.8 1.2 * 0.9 1.5 + 0.4 2.2 * 1.2
1.8 * 0.4 1.9 _+ 0.7 3.8 4.8 3.4 9.1
k 0.4 + 1.6 + 0.8 _+ 4.3
Died during 3 weeks
Group B’ (mean +_ SD)
Group AC (range)
Group Bc (range)
1.9 f 0.6
-
1.8 1.8 1.7 3.1
+ i & 2
0.5 0.6 0.4 l.od
3-13 8-12 3-12 -
7-26 6-18 6-12 -
2.6 2.4 3.4 2.9
+ & k +
0.8 0.6 0.6 0.6
11-18 4-18
5-13 6-28 6-29 6-24 9-50 5-23 6-22 6-23 6-2 8-49 -
3.1 + 2.2 1.8 f 0.6 2.7 & 0.8 2.8 & 0.8 3.0 + 0.7 4.0 _+ 1.3 19,46' 1.5 & 0.5 4.0 & 1.6 1.2' 2.3 + 0.7d 3.8 & 1.2 3.0 * 1.0 4.5 + 1.7
1.1 k 0.5 2.6 & 0.7 4.3,9.5' 2.2 _+ 2.1d 1.2 & 0.4f 1.6 k 0.8d 2.6 k 0.9 3.1 * 1.4d 5.3 _+ 4.2 7.0 k 2.5d
10.1 * 3.7 5.5 + 2.1d 15.2 _+ 7.0 25 f 10
4-20
lc-15 13-31 4-12 4-12 4-14 4-13 5-15 4-10 4-15
5-2 1 7-17 10” 4' lo-15 9-25 11-38 6-79 8-18 6-14 2-17 12e 4-11 9-27 l&32 l&42 9-3 1
8' 4-6 3-16 16-21 6-16 8-15 4-12 6-13 4-7 4-7 4-16 6-16 6-20 5-19 6-33 7-40 6-2 6-20 -
1
1
14-89 14-97 IO-27 23-80 -
is expressed as milliequivalents of Li+ per kilogram wet weight. b Because of cannibalism, not all tissues were available from all mice. Mice dying in the first 24 hr were not included in the range. c In group A, three of seven males and three of eight females survived. In group B, seven of ten males and one of five females survived. d One mouse which showed very high [LPI in this tissue was not included in the average. e Single sample. ’ Two mice which showed very high [Li+ 1 in this tissue were not included in the average.
418
JERNIGAN,
SCHRANK,
AND
KRAUS
carbamylation values expressed as moles of valine hydantoin per mole of hemoglobin tetramer at 21 Days, were 1.0 and 1.2; at 23 Days, 0.6, 0.9, and 1.0; at 30 Days, 0.8 1.1, and 1.6. No valine hydantoin was detected in the mice given lithium chloride. The initial leukocyte values were those typically found for untreated mice in this colony. The mean leukocyte counts of surviving mice in both groups decreased with time (Fig. 2). Examination of data on leukocyte counts of nonsurvivors revealed considerable variation, but in general they appeared to be comparable to those found in the survivors (Fig. 2). The hematocrits of both groups also decreased with time (Fig. 2).
OAYS FIG. 2. The leukocyte values (x 109/liter; mean + SE) are compared for group: (0) lithium chloride, group A, or (X) dilithium carbamyl phosphate, (milliliters per 100 ml; mean f SE) are compared in surviving mice from each group A, or (0) dilithium carbamyl phosphate, group B. The first injections from final blood samples (obtained on Days 21-30) were averaged and plotted
surviving group B. group; (A) were given at Day 2 1.
mice from each The hematocrits lithium chloride, on Day 0. Data
At autopsy, the reticulocyte counts were elevated in both groups (greater than 4%) in all mice examined. Reticulocyte counts were highest (up to 11%) in those individual mice which had the lowest hematocrits. The surviving mice in both groups had a small weight loss in the first week; thereafter they were gradually regaining the lost weight. DISCUSSION Jernigan and Kraus (1975) showed by enzymatic assay that the carbamyl phosphate dianion of dilithium carbamyl phosphate injected ip entered the circulation in mice, reached a peak of concentration 2 min after the injection, and gradually disappeared in 10 min. These findings show that carbamyl phosphate does enter the circulatory system and thus could be a source of toxicity after ip administration. However, dilithium carbamyl phosphate and lithium chloride were equally toxic when administered to mice in twice-daily ip injections containing similar amounts of lithium (Fig. 1). Approximately half the animals in the lithium chloride and dilithium carbamyl phosphate groups died during the study, whereas, in a similar study, identical amounts of sodium cation from sodium chloride did not cause any deaths or toxicity. Therefore, the toxicity
LITHIUM
DISTRIBUTION
AND
TOXICITY
419
of dilithium carbamyl phosphate observed in this experiment appeared to be caused primarily, if not entirely, by lithium, not by the carbamyl phosphate dianion. The concentration of lithium was determined in a wide variety of tissues from the two groups of mice (Table 2). The relative distribution between the tissues was similar in the surviving mice of both groups. There did not appear to be any sex-related difference. Liver showed the lowest lithium concentration of any tissue measured, which may be related to the secretion of lithium in the bile (Kraus et al., 1976; Table 2). This is consistent with the report by Schou (1958) who concluded that, in rat liver, lithium ions are excreted against a concentration gradient. The lithium concentrations found in other tissues were consistent with previous reports of tissue distribution in rats (Schou, 1958; Ho et al., 1970; Birch and Hullin, 1972) or mice (Messiha, 1976). Also, these studies indicated that, in rats and mice, most of the lithium disappears from the circulation within 24 hr after the last dose, a finding which we confirmed in preliminary experiments. These previous studies utilized either one injection or a single injection given daily and reported the lithium content in fewer tissues. Therefore, our results (Table 2) obtained using twice-daily injections are not exactly comparable. In general. however. the relative distribution of lithium in such tissues as liver, brain, kidney, and muscle reported in Table 2 appears to be consistent with the aforementioned reports. Lithium concentration showed a considerable degree of tissue specificity. At autopsy, the mean lithium concentration in many tissues was higher in survivors in group B than in group A. This difference was not significant (p > 0.05) when normalized for the slightly higher dose of lithium in group B. The similar survival data would indicate that similar lithium toxicity occurred in each group. Surviving mice were killed 4 to 6 hr after a morning dose. Nonsurvivors died at various intervals (usually unknown) after the previous dose. Because the lithium concentrations reported are a reflection of lithium excretion and its distribution between tissues, this may explain part of the wide range of lithium concentrations in tissues of nonsurvivors. Dehydration of nonsurvivors following freezing at -70° C appeared to be minimal. The variability in the time of death after an injection precludes the correlation of tissue lithium concentrations with duration of treatment in nonsurvivors. Very high blood lithium concentrations associated with recurring irritation of the intestine and diarrhea in dogs (Kraus et al., 1977) receiving dilithium carbamyl phosphate prompted the special attention given to lithium concentrations in the digestive tract in this study (Table 2). Higher lithium concentrations were found both in tissue and in the contents of the lower digestive tract rather than in the upper tract. To avoid the possibility of leaching lithium from tissue samples, intestinal contents were removed from tissue samples by scraping rather than rinsing. However, it is unlikely that tissue samples could have been sufficiently contaminated with contents to account for the observed differences, and, in fact, cecum tissue contained a higher lithium concentration than colon tissue, although colon contents contained a higher lithium concentration than cecum contents. The high lithium concentration found in colon contents and feces may reflect the lower water content; however, it is clear that, at the high doses of lithium used in these mice, lithium was not efficiently reabsorbed by the colon. The data do not suggest the extent to which the intraintestinal lithium originated from bile (Kraus et al., 1976) or was absorbed by the intestine from the circulation or the peritoneal cavity and was subsequently secreted into the lumen.
420
JERNIGAN,
SCHRANK,
AND
KRAUS
Intraperitoneal injection of carbamyl phosphate resulted in intraerythrocytic carbamylation of hemaglobin. This was measured as moles of valine hydantoin per mole of hemoglobin tetramer and averaged 1.O k 0.3 (mean f SD). This carbamylation occurred using a 1200-mg/kg daily dose compared to the daily dose of 100 mg/kg used in the dog studies (Kraus et al., 1977) in which carbamylation reached a plateau at 0.3-0.36 mol of valine hydantoin/mol of hemoglobin. Intraperitoneal injection of i4Clabeled carbamyl phosphate into rats has also been reported to result in carbamylation of red cell membrane proteins and hemoglobin (Crist et al., 1973). The reticulocytosis observed in the mice would indicate that many newly formed erythrocytes were present at the end of the study. These younger cells would have been exposedto carbamyl phosphate for a shorter time and would be lesscarbamylated than cells which had beenpresent for the entire 21-day period of treatment. ACKNOWLEDGMENTS
The authorswishto thank Mr. C. B. Stone,Ms. M. J. Viar, Ms. C. C. Lo, Mr. J. W. Gnadt, and Mr. M. J. Skinner for their excellenttechnical assistance. The authors alsoexpresstheir appreciationto Dr. R. A. Schreiberfor providing the miceandto Dr. E. G. Schneiderfor useof his atomic absorptionspectrophotometer.The authors thank Dr. W. H. Lawrence for his interestand advice. REFERENCES N. J., AND HULLIN, R. P. (1972). The distribution and bindingof lithium following its long-termadministration.Lif Ski. 11, 10951099. GRIST, R. D., GRISOLIA, S., BETTIS, C. J., AND GRISOLIA, J. (1973).Carbamoylationof proteins followingadministrationto rats of carbamoylphosphateand cyanate andeffectson memory. Eur. J. Biochem. 32, 109-l 16. Ho, A. K. S., GERSHON, S., AND PINCKNEY, L. (1970). The effects of acute and prolonged lithium treatment on the distribution of electrolytes, potassiumand sodium.Arch. Znt. BIRCH,
Pharmacodyn.
l&X6,54-65.
AND KRAUS, L. M. (1975). Enzymatic determinationof carbamyl phosphate in blood.J. Lab. Clin. Med. 85,694-702. KRAUS, L. M., JERNIGAN, H. M., BLATTEIS, C. M., AND KRAUS, A. P. (1975). In vivo effectsof carbamylphosphatein dogs.Progr. Clin. Biol. Res. 1, 399-4 12.
JERNIGAN,
KRAUS,
H. M.,
L. M., JERNIGAN,
H.
M.,
JR.,
HAIRE,
R.
N.,
AND
HEDLUND,
B.
E.
(1977).
Characterization of in vivo effects of dilithium carbamyl phosphateon dog hemoglobin structureandfunction. Biochim. Biophys. Acta 491,497-502. KRAUS, L. M., JERNIGAN, H. M., SCHRANK, G. D., STADLAN, E. M., AND HESSLER, J. R. (1976).Blood-bileratio of lithium andgastroenteritis.Clin. Res. 24, 566A. KRAUS, L. M., AND KRAUS,A. P. (1971). Carbamyl phosphatemediatedinhibition of the sicklingof erythrocytesin vitro. Biochem. Biophys. Res. Commun. 44, 1381-1387. KRAUS,
L. M., RASAD,
A.,
FRIEDMAN,
B. I., AVIS,
K. E., ALLEN,
C. M., AND KRAUS, A. P.
(1973). Prolongationof in vivo survival of sicklecell erythrocytes by carbamyl phosphate (CP). Clin. Res. 21, 728. KRAUS, L. M., RASAD, A., AND KUUS, A. P. (1972). Carbamyl phosphatemodificationof hemoglobinS structureand function resultingin alteredsickling.Advan. Exp. Med. Biof. 28, 279-292. J. M., LEE, C. K., CERAMI, A., AND GILLEITE, P. N. (1973). Gas chromatographic determinationof the carbamylationof hemoglobinSby cyanate.J. Lab. C/in. Med. 81, 941-
MANNING,
945.
LITHIUM
DISTRIBUTION
AND
TOXICITY
421
F. S. (1976). Distribution and retention of exogenously administered alkali metal ions in the mouse brain. Arch. Znt. Pharmacodyn. 219,87-96. MILNER,P. F., AND CHARACHE, S. (1973). Life spanof carbamylatedred cellsin sicklecell anemia.J. Clin. Invest. 52, 3 16l-3 171. SCHOU, M. (1958).Lithium studies.3. Distributionbetweenserumandtissues.Acta Pharmacol. Toxicol. 15, 115-124. MESSIHA,
Lithium
AND
APPLIED
PHARMACOLOGY
(1978)
Chloride and Dilithium Carbamyl Phosphate: Distribution and Toxicity in Mice’
HOWARD M. JERNIGAN, Departments
44.413-421
of Biochemistry Tennessee
JR.,
GORDON D. SCHRANK, AND LORRAINE M. KRAUS*
and Microbiology and the Comprehensive Centerfor the Health Sciences, Memphis.
Received
Lithium
Ju1.v 18, 1977: accepted
October
Sickle Cell Center, Tennessee 38163
University
of
5,1977
Lithium Chloride and Dilithium Carbamyl Phosphate: Lithium Distribution and Toxicity in Mice. JERNIGAN, H. M., JR.. SCHRANK, G. D.. AND KRAUS, L. M. (1978). Toxicol. Appl. Pharmacol. 44, 4 13-42 1. Dilithium carbamyl phosphate, an antisickling agent, dissociates into lithium cations and carbamyl phosphate dianions in solution. In order to determine which ion is primarily responsible for the toxicity observed at very high doses of dilithium carbamyl phosphate, two groups of 1.5 mice each were given similar amounts of lithium in twice-daily ip injections for 21, 23. or 30 days. Six of the fifteen mice survived in group A (lithium chloride, 12.9 mEq of Li+/kg/day), and 8 of 15 mice survived in group B (dilithium carbamyl phosphate, 13.8 mEq of Li+/kg/day). Blood l$hium concentrations measured 4-6 hr after an injection averaged 1.7 (+0.7 SD) mEq/liter for each group. Lithium concentrations were measured in 39 samples of tissue or tissue contents from mice in each group. In the surviving animals, lithium ranged from an average of 0.8-l. 1 mEq/kg in liver to 5- 15 mEq/kg in the contents of the lower intestinal tract. Lithium concentrations greater than 15 mEq/kg were often found in the tissues of the nonsurvivors. Hemoglobin carbamylation was 1.0 (i0.3 SD) mol of valine hydantoin/mol of hemoglobin tetramer in mice given dilithium carbamyl phosphate for 21-30 days. This study demonstrates that the toxic effects associated with administration of dilithium carbamyl phosphate appear to be due to the lithium cation rather than the carbamyl phosphate dianion. In all parameters measured, the toxic effects of lithium chloride paralleled those observed when an equivalent amount of lithium was administered as dilithium carbamyl phosphate.
Carbamyl phosphate carbamylates the amino-terminal valine residues of hemoglobin S in erythrocytes thereby interfering with the sickling phenomenon (Kraus and Kraus, 1971; Kraus ef al., 1972, 1977). Erythrocytes removed from patients with sickle cell anemia were incubated with carbamyl phosphate in vitro and labeled with 51Cr. When returned to the same patient, these erythrocytes had an increased survival time as compared with untreated erythrocytes (Kraus et al., 1972, 1973; Milner and Charache, 1973). Long-term studies have shown that oral or iv administration of carbamyl phosphate to dogs results in in uivo carbamylation of hemoglobin to concentrations which have been demonstrated to have an antisickling effect in erythrocytes containing deoxygenated HbS in vitro. However, a regimen of 100 mg/kg/day (8 mg or 1.2 mEq of Li+/kg/day) ’ Supported in part by USPHS Grant HL- 15 169. ’ T O whom requests for reprints should be sent at Department of Biochemistry, Center for the Health Sciences, Memphis, Tennessee 38163. 413
University of Tennessee
0041-008X/78/0442-0413$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
414
JERNIGAN,
SCHRANK,
AND
KRAUS
of dilithium salt occasionally produced toxic or near-toxic concentrations of lithium in the dogs (Kraus et al., 1975, 1977). It was necessary to distinguish between the effects of the different parts of the molecule, i.e., the lithium cation or the carbamyl phosphate dianion. The results in the mouse study described below demonstrate that, under the conditions studied, dilithium carbamyl phosphate is no more toxic than an equivalent amount of lithium administered in the form of lithium chloride, thereby implicating the lithium cation as the primary toxic ion in dilithium carbamyl phosphate.
METHODS
Thirty C57Bl mice, 168-180 days old and randomly selected as to sex, were individually identified, divided into two equal groups, and maintained on Purina Mouse Chow and water ad libitum. One group (group A) received 600 mg/kg/day of reagentgrade lithium chloride3 in a 15-mg/ml aqueous solution, while the other group (group B) received dilithium carbamyl phosphate,4 1200 mg/kg/day, in an aqueous solution of 30 mg/ml, which was frozen in aliquots and stored at -2OOC. These solutions used for injection were subsequently assayed for lithium concentration by atomic absorption spectrophotometry. The lithium content of the dilithium carbamyl phosphate solution was 345 mEq/liter (351 mEq/liter expected: compared to 322 mEq/liter in the lithium chloride solution (354 mEq/liter expected) The lower than expected lithium concentration in the lithium chloride solution was due to moisture in the granular lithium chloride. Both groups of mice were given 0.02 ml 01 solution/g body weight ip twice daily. Therefore group A received 12.9 mEq of Li+/kE body weight daily from lithium chloride, and group B received 13.8 mEq of Li+/kf body weight daily from dilithium carbamyl phosphate. Two days before the injections were started and 4-6 hr after the morning dose or Days 2, 7, 14, and 21 or until the mouse was killed or died, blood was removed from the tail after severing the tip. Blood was taken for lithium determinations (0.05 ml of blooc in 1.0 ml of water), leukocyte counts, packed cell volume, and random reticulocytc counts. Those animals which died were identified and frozen at -7O’C for late dissection. On Days 21, 23, or 30, 4-7 hr after the morning injection, final bloom samples were taken from the tail. Thereafter the mice were anesthetized with ether ant were exsanguinated by severing the major blood vessels in the neck. The mice were dissected and 39 tissues or tissue contents from each mouse wer’ sealed in weighed glass tubes. Tissue lithium was determined by the modified method a Schou (1958). A volume of concentrated nitric acid equal to 0.5- to l-fold the weight a tissue was added, and the tissues were digested for 1 hr (or more if digestion appearel incomplete) in a boiling water bath. Ten percent isopropanol was added to bring th final volume to 25 times the volume of nitric acid, the tubes were shaken, and th contents were filtered through Pyrex wool to remove insoluble material (mostly lipids Lithium analysis of nitric acid tissue digests or of water dilutions of blood was don using a Perkin-Elmer Model 290B atomic absorption spectrophotometer. White blood cell counts were done using a Coulter Counter, Model Z, 3 Mallinckrodt Chemical Works, St. Louis, 4 Sigma Chemical Co., St. Louis, Missouri;
Missouri. assayed by the supplier
to be 98% pure.
LITHIUM
DISTRIBUTION
AND
TOXICITY
415
Carbamylation of the N-terminal valines of hemoglobin was determined by the method of Manning et al. (1973) and expressed as moles of valine hydantoin per mole of hemoglobin tetramer. RESULTS The survival data for both groups of mice are reported in Fig. 1. At Day 2, when the greatest difference existed in survivors between groups A and B, the p value from a x2 determination was greater than 0.05, and, at Day 21, the p value was greater than 0.9. Therefore, there was no significant difference in survivors throughout the duration ofthe study between the mice receiving lithium chloride (group A) and those receiving dilithium carbamyl phosphate (group B). During the same time period, there were no deaths seen in the stable colony of over 75 mice, from which the experimental animals
0
10 DAYS
20
FIG. 1. Survival of mice injected ip, twice daily with (0) 12.9 mEq/kg/day of lithium chloride group A or (X) 13.8 mEq/kg/day of dilithium carbamyl phosphate designated group B.
designated
were taken. An examination of mortality, as group: survivors/total of male (A: 3/7; B: 7/10) and female (A: 3/S; B: l/5) mice did not indicate a significant difference between sexes, although the small numbers involved do not permit a definite statistical analysis. On the other hand, there were three pregnant mice (two in the lithium chloride group and one in the lithium carbamyl phosphate group) and all of these died during treatment. Subsequently, all 15 mice survived in a 17-day study using sodium chloride rather than lithium chloride administered in a similar regimen using equal amounts of the sodium cation. All mice in this group were bled, injected, and maintained in the same way as were the mice in groups A and B. Mice from groups A and B were killed on Days 21, 23, and 30, and during that time there were no spontaneous deaths. At autopsy, no infection, inflammation, or hematomas were found at the site of the injections or in the peritoneal cavity.
416
JERNIGAN,
SCHRANK,
AND
KRAUS
The mean lithium concentrations in whole blood of surviving mice of both groups are compared at intervals during the study (Table 1). The mean lithium concentrations at different time points ranged from 1 to 2 mEq/liter with a mean for each group of 1.7 & 0.7 mEq/liter averaging all values measured after lithium administration began. Because the interval of time between death and the last injection of lithium varied from mouse to mouse, there is no terminal measurement of blood lithium concentration available for comparison in the nonsurvivors. At the last routine bleeding before a nonsurvivor died, blood lithium ranged from 1.3 to 12 mEq/liter in group A and from 1.4 to 11 mEq/liter in group B. TABLE WHOLE BLOOD LITHIUM
1
CONCENTRATIONS IN MICE WHICH SURVIVED UNTIL THE END OF THE PROTOCOL”. b Days on study
Group A (lithium chloride) Group B (dilithium carbamyl phosphate)
0
2
1
14
2 l-30
0
1.9 + 1.0
1.9 f 0.5
1.6 k 0.4
1.4 t 0.5
0
1.9 + 1.1
2.0 f 0.6
1.1 i 0.3
1.9 i- 0.6
B Lithium concentrations expressed as milliequivalents per liter;‘mean 2~ SD. b Group A: six survivors; group B: eight survivors.
Table 2 showsthe concentration of lithium in 39 tissuesor tissue contents from mice of group A or group B. After normalizing the dose of lithium, there were no significant differences (p > 0.05 using Student’s t test) in survivors comparing the lithium concentration of each tissue in group A to the same tissue in group B. The only exception to this was cecum and cecal contents (p < 0.05). Lithium was present in all tissuesexamined. Liver had the lowest lithium concentration (0.8 mEq/kg, group A; 1.1 mEq/kg, group B). Blood, spleen, testis, spinal cord, and brain had intermediate concentrations of lithium (1.2-1.9 mEq/kg) which were significantly higher (p < 0.05) than concentrations in the liver. Salivary gland, muscle, kidney, epididymis, lung, and bone (cranium) had a higher lithium content than the intermediate group (p < 0.05). In the same mouse,total kidney or a portion of the cortex contained the samelithium per unit wet weight. Lithium concentration increased in the tissue or contents of the digestive tract when measureddistally from the stomach to the colon, even though the amounts of lithium found varied from mouse to mouse. In the urinary bladder and gall bladder, any residueof urine or bile could raisethe lithium content. In the mice which died during the course of the study (Table 2), lithium concentrations greater than 15 mEq/kg were often found in the tissues, with the exception of two mice in group A which died early in the study. Also, two mice in group A and one mousein group B were pregnant when they died. The lithium content of the total fetus was lower but correlated with the concentration of lithium in the tissuesof the pregnant mouse. The reaction of dilithium carbamyl phosphate with the amino-terminal valine of hemoglobin was measuredin blood from the surviving mice when they were killed. The
LITHIUM
DISTRIBUTION
AND
417
TOXICITY
TABLE 2 LITHIUM DISTRIBUTION IN TISSUES OF MICE RECEIVING LITHIUM CHLORIDE (GROUP A) OR DILITHIUM CARBAMYL PHOSPHATE (GROUP B) Surviving 3 weeks Tissueb Blood (mEq/liter) Spleen Brain Spinal cord Lumbar spinal nerve Lung Heart Skeletal muscle Parotid salivary gland Thyroid Adrenal Kidney (whole) Kidney (cortex) Kidney (remainder) Urinary bladder Urine (mEq/liter) Testes Epididymis Ovaries Oviduct Fetuses Lens Eye (lens removed) Bone (cranium) Skin and hair Liver Gall bladder Bile (gall bladder) Stomach tissue Stomach contents Duodenum tissue Duodenum contents Ileum tissue Ileum contents Cecum tissue Cecum contents Colon tissue Colon contents Feces a Lithium distribution
Group A’ (mean +_ SD) 1.4 1.2 1.2 1.3 2.4 1.9
+ 0.5 IO.3 * 0.2 & 0.4 & 0.7 k 0.6
1.8 + 0.5 2.7 2.2 1.9 2.0 2.2 2.3 2.3 2.1
i 0.9 + 0.4 2 1.3 & 1.6 + 0.5 k 0.6 + 0.5 _+ 0.7d 8.1' 1.4 & 0.4 2.2 f 1.1
1.5 i 0.8 1.0 + 0.3 1.6 & 0.4 2.5 i 0.5 2.5 k 0.3 7.1 + 2.8 0.8 & 0.3 1.9 * 1.2 10.7' 1.2 f 0.8 1.2 * 0.9 1.5 + 0.4 2.2 * 1.2
1.8 * 0.4 1.9 _+ 0.7 3.8 4.8 3.4 9.1
k 0.4 + 1.6 + 0.8 _+ 4.3
Died during 3 weeks
Group B’ (mean +_ SD)
Group AC (range)
Group Bc (range)
1.9 f 0.6
-
1.8 1.8 1.7 3.1
+ i & 2
0.5 0.6 0.4 l.od
3-13 8-12 3-12 -
7-26 6-18 6-12 -
2.6 2.4 3.4 2.9
+ & k +
0.8 0.6 0.6 0.6
11-18 4-18
5-13 6-28 6-29 6-24 9-50 5-23 6-22 6-23 6-2 8-49 -
3.1 + 2.2 1.8 f 0.6 2.7 & 0.8 2.8 & 0.8 3.0 + 0.7 4.0 _+ 1.3 19,46' 1.5 & 0.5 4.0 & 1.6 1.2' 2.3 + 0.7d 3.8 & 1.2 3.0 * 1.0 4.5 + 1.7
1.1 k 0.5 2.6 & 0.7 4.3,9.5' 2.2 _+ 2.1d 1.2 & 0.4f 1.6 k 0.8d 2.6 k 0.9 3.1 * 1.4d 5.3 _+ 4.2 7.0 k 2.5d
10.1 * 3.7 5.5 + 2.1d 15.2 _+ 7.0 25 f 10
4-20
lc-15 13-31 4-12 4-12 4-14 4-13 5-15 4-10 4-15
5-2 1 7-17 10” 4' lo-15 9-25 11-38 6-79 8-18 6-14 2-17 12e 4-11 9-27 l&32 l&42 9-3 1
8' 4-6 3-16 16-21 6-16 8-15 4-12 6-13 4-7 4-7 4-16 6-16 6-20 5-19 6-33 7-40 6-2 6-20 -
1
1
14-89 14-97 IO-27 23-80 -
is expressed as milliequivalents of Li+ per kilogram wet weight. b Because of cannibalism, not all tissues were available from all mice. Mice dying in the first 24 hr were not included in the range. c In group A, three of seven males and three of eight females survived. In group B, seven of ten males and one of five females survived. d One mouse which showed very high [LPI in this tissue was not included in the average. e Single sample. ’ Two mice which showed very high [Li+ 1 in this tissue were not included in the average.
418
JERNIGAN,
SCHRANK,
AND
KRAUS
carbamylation values expressed as moles of valine hydantoin per mole of hemoglobin tetramer at 21 Days, were 1.0 and 1.2; at 23 Days, 0.6, 0.9, and 1.0; at 30 Days, 0.8 1.1, and 1.6. No valine hydantoin was detected in the mice given lithium chloride. The initial leukocyte values were those typically found for untreated mice in this colony. The mean leukocyte counts of surviving mice in both groups decreased with time (Fig. 2). Examination of data on leukocyte counts of nonsurvivors revealed considerable variation, but in general they appeared to be comparable to those found in the survivors (Fig. 2). The hematocrits of both groups also decreased with time (Fig. 2).
OAYS FIG. 2. The leukocyte values (x 109/liter; mean + SE) are compared for group: (0) lithium chloride, group A, or (X) dilithium carbamyl phosphate, (milliliters per 100 ml; mean f SE) are compared in surviving mice from each group A, or (0) dilithium carbamyl phosphate, group B. The first injections from final blood samples (obtained on Days 21-30) were averaged and plotted
surviving group B. group; (A) were given at Day 2 1.
mice from each The hematocrits lithium chloride, on Day 0. Data
At autopsy, the reticulocyte counts were elevated in both groups (greater than 4%) in all mice examined. Reticulocyte counts were highest (up to 11%) in those individual mice which had the lowest hematocrits. The surviving mice in both groups had a small weight loss in the first week; thereafter they were gradually regaining the lost weight. DISCUSSION Jernigan and Kraus (1975) showed by enzymatic assay that the carbamyl phosphate dianion of dilithium carbamyl phosphate injected ip entered the circulation in mice, reached a peak of concentration 2 min after the injection, and gradually disappeared in 10 min. These findings show that carbamyl phosphate does enter the circulatory system and thus could be a source of toxicity after ip administration. However, dilithium carbamyl phosphate and lithium chloride were equally toxic when administered to mice in twice-daily ip injections containing similar amounts of lithium (Fig. 1). Approximately half the animals in the lithium chloride and dilithium carbamyl phosphate groups died during the study, whereas, in a similar study, identical amounts of sodium cation from sodium chloride did not cause any deaths or toxicity. Therefore, the toxicity
LITHIUM
DISTRIBUTION
AND
TOXICITY
419
of dilithium carbamyl phosphate observed in this experiment appeared to be caused primarily, if not entirely, by lithium, not by the carbamyl phosphate dianion. The concentration of lithium was determined in a wide variety of tissues from the two groups of mice (Table 2). The relative distribution between the tissues was similar in the surviving mice of both groups. There did not appear to be any sex-related difference. Liver showed the lowest lithium concentration of any tissue measured, which may be related to the secretion of lithium in the bile (Kraus et al., 1976; Table 2). This is consistent with the report by Schou (1958) who concluded that, in rat liver, lithium ions are excreted against a concentration gradient. The lithium concentrations found in other tissues were consistent with previous reports of tissue distribution in rats (Schou, 1958; Ho et al., 1970; Birch and Hullin, 1972) or mice (Messiha, 1976). Also, these studies indicated that, in rats and mice, most of the lithium disappears from the circulation within 24 hr after the last dose, a finding which we confirmed in preliminary experiments. These previous studies utilized either one injection or a single injection given daily and reported the lithium content in fewer tissues. Therefore, our results (Table 2) obtained using twice-daily injections are not exactly comparable. In general. however. the relative distribution of lithium in such tissues as liver, brain, kidney, and muscle reported in Table 2 appears to be consistent with the aforementioned reports. Lithium concentration showed a considerable degree of tissue specificity. At autopsy, the mean lithium concentration in many tissues was higher in survivors in group B than in group A. This difference was not significant (p > 0.05) when normalized for the slightly higher dose of lithium in group B. The similar survival data would indicate that similar lithium toxicity occurred in each group. Surviving mice were killed 4 to 6 hr after a morning dose. Nonsurvivors died at various intervals (usually unknown) after the previous dose. Because the lithium concentrations reported are a reflection of lithium excretion and its distribution between tissues, this may explain part of the wide range of lithium concentrations in tissues of nonsurvivors. Dehydration of nonsurvivors following freezing at -70° C appeared to be minimal. The variability in the time of death after an injection precludes the correlation of tissue lithium concentrations with duration of treatment in nonsurvivors. Very high blood lithium concentrations associated with recurring irritation of the intestine and diarrhea in dogs (Kraus et al., 1977) receiving dilithium carbamyl phosphate prompted the special attention given to lithium concentrations in the digestive tract in this study (Table 2). Higher lithium concentrations were found both in tissue and in the contents of the lower digestive tract rather than in the upper tract. To avoid the possibility of leaching lithium from tissue samples, intestinal contents were removed from tissue samples by scraping rather than rinsing. However, it is unlikely that tissue samples could have been sufficiently contaminated with contents to account for the observed differences, and, in fact, cecum tissue contained a higher lithium concentration than colon tissue, although colon contents contained a higher lithium concentration than cecum contents. The high lithium concentration found in colon contents and feces may reflect the lower water content; however, it is clear that, at the high doses of lithium used in these mice, lithium was not efficiently reabsorbed by the colon. The data do not suggest the extent to which the intraintestinal lithium originated from bile (Kraus et al., 1976) or was absorbed by the intestine from the circulation or the peritoneal cavity and was subsequently secreted into the lumen.
420
JERNIGAN,
SCHRANK,
AND
KRAUS
Intraperitoneal injection of carbamyl phosphate resulted in intraerythrocytic carbamylation of hemaglobin. This was measured as moles of valine hydantoin per mole of hemoglobin tetramer and averaged 1.O k 0.3 (mean f SD). This carbamylation occurred using a 1200-mg/kg daily dose compared to the daily dose of 100 mg/kg used in the dog studies (Kraus et al., 1977) in which carbamylation reached a plateau at 0.3-0.36 mol of valine hydantoin/mol of hemoglobin. Intraperitoneal injection of i4Clabeled carbamyl phosphate into rats has also been reported to result in carbamylation of red cell membrane proteins and hemoglobin (Crist et al., 1973). The reticulocytosis observed in the mice would indicate that many newly formed erythrocytes were present at the end of the study. These younger cells would have been exposedto carbamyl phosphate for a shorter time and would be lesscarbamylated than cells which had beenpresent for the entire 21-day period of treatment. ACKNOWLEDGMENTS
The authorswishto thank Mr. C. B. Stone,Ms. M. J. Viar, Ms. C. C. Lo, Mr. J. W. Gnadt, and Mr. M. J. Skinner for their excellenttechnical assistance. The authors alsoexpresstheir appreciationto Dr. R. A. Schreiberfor providing the miceandto Dr. E. G. Schneiderfor useof his atomic absorptionspectrophotometer.The authors thank Dr. W. H. Lawrence for his interestand advice. REFERENCES N. J., AND HULLIN, R. P. (1972). The distribution and bindingof lithium following its long-termadministration.Lif Ski. 11, 10951099. GRIST, R. D., GRISOLIA, S., BETTIS, C. J., AND GRISOLIA, J. (1973).Carbamoylationof proteins followingadministrationto rats of carbamoylphosphateand cyanate andeffectson memory. Eur. J. Biochem. 32, 109-l 16. Ho, A. K. S., GERSHON, S., AND PINCKNEY, L. (1970). The effects of acute and prolonged lithium treatment on the distribution of electrolytes, potassiumand sodium.Arch. Znt. BIRCH,
Pharmacodyn.
l&X6,54-65.
AND KRAUS, L. M. (1975). Enzymatic determinationof carbamyl phosphate in blood.J. Lab. Clin. Med. 85,694-702. KRAUS, L. M., JERNIGAN, H. M., BLATTEIS, C. M., AND KRAUS, A. P. (1975). In vivo effectsof carbamylphosphatein dogs.Progr. Clin. Biol. Res. 1, 399-4 12.
JERNIGAN,
KRAUS,
H. M.,
L. M., JERNIGAN,
H.
M.,
JR.,
HAIRE,
R.
N.,
AND
HEDLUND,
B.
E.
(1977).
Characterization of in vivo effects of dilithium carbamyl phosphateon dog hemoglobin structureandfunction. Biochim. Biophys. Acta 491,497-502. KRAUS, L. M., JERNIGAN, H. M., SCHRANK, G. D., STADLAN, E. M., AND HESSLER, J. R. (1976).Blood-bileratio of lithium andgastroenteritis.Clin. Res. 24, 566A. KRAUS, L. M., AND KRAUS,A. P. (1971). Carbamyl phosphatemediatedinhibition of the sicklingof erythrocytesin vitro. Biochem. Biophys. Res. Commun. 44, 1381-1387. KRAUS,
L. M., RASAD,
A.,
FRIEDMAN,
B. I., AVIS,
K. E., ALLEN,
C. M., AND KRAUS, A. P.
(1973). Prolongationof in vivo survival of sicklecell erythrocytes by carbamyl phosphate (CP). Clin. Res. 21, 728. KRAUS, L. M., RASAD, A., AND KUUS, A. P. (1972). Carbamyl phosphatemodificationof hemoglobinS structureand function resultingin alteredsickling.Advan. Exp. Med. Biof. 28, 279-292. J. M., LEE, C. K., CERAMI, A., AND GILLEITE, P. N. (1973). Gas chromatographic determinationof the carbamylationof hemoglobinSby cyanate.J. Lab. C/in. Med. 81, 941-
MANNING,
945.
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DISTRIBUTION
AND
TOXICITY
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F. S. (1976). Distribution and retention of exogenously administered alkali metal ions in the mouse brain. Arch. Znt. Pharmacodyn. 219,87-96. MILNER,P. F., AND CHARACHE, S. (1973). Life spanof carbamylatedred cellsin sicklecell anemia.J. Clin. Invest. 52, 3 16l-3 171. SCHOU, M. (1958).Lithium studies.3. Distributionbetweenserumandtissues.Acta Pharmacol. Toxicol. 15, 115-124. MESSIHA,