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Solubility of benzene hexachloride isomers in rat fat
TOXICOLOGY
AND
APPLIED
Solubility
PHARMACOLOGY
of
Benzene
7: 79-83
Hexachloride VINCENT
Toxicology
Sectiott,
(1965)
A.
Received
March
in Rat
Fat
SEDLAK
Technology Branch, Communicable of Health, Education, and
Department
Isomers
Disease Welfare,
Center, Atlanta,
Public Health Georgia
Service,
26, 1964
Davidow and Frawley (1951) found that the four common isomers of benzene hexachloride (BHC, or hexachlorocyclohexane) are preferentially stored in adipose tissue of both rats and dogs, but that some isomers are stored in all tissues much more avidly than others. Because the storage levels of the various isomers of BHC in adipose tissue differ by factors as great as 19, these isomers were considered good material for testing the relationship between storage and solubility in fat. The work of Davidow and Frawley makes it possible to compare solubility with the concentration of each isomer in the fat tissue of rats after it has reached a steady state following daily feeding at a uniform dietary rate (100 ppm). (Their work gave evidence that the alpha, gamma, and delta isomers reach a state of equilibrium of storage between the fourth and sixth week of feeding, whereas the beta isomer reaches a steady state by the twelfth week in females and slightly earlier in males.) METHODS
Abdominal fat from several rats was pooled and refrigerated for use as needed. Rat fat for the solubility determination was separated from connective tissue by taking 10 or 12 g of adipose tissue and grindin, v it with mortar and pestle and a small quantity of sodium sulfate. Only enough sodium sulfate was added to absorb moisture and aid in abrading the connective tissue supporting the fat. Then the mixture was transferred mechanically to plastic centrifuge tubes and centrifuged for 1 hour or more at about 2500 rpm. The fat that accumulated at the surface was an oily liquid. This liquid was pipetted off and used immediately for preparation of mixtures with individual BHC isomers. Since comparison between solubility and storage in living adipose tissue was the point of interest, the solubility tests were made at 37.0”. The body temperature of the white rat (Rattus norvegicus) is 37.3” (range 34.5-40.0”) (Spector, 1956). The starting mixture for each determination of saturation concentration was two or more grams of isolated rat fat and a weight of crystalline BHC isomer equal to about 15%’ of the weight of fat. The mixtures were stirred by glass paddles in test tubes at 37.0’ for 14-88 hours. At 14-17 hours, the stirring was stopped and the mixtures were allowed to settle for half an hour. An aliquot was taken from the upper half of the liquid and transferred to a centrifuge tube. Then the test tubes and their remaining contents were returned to the constant temperature water bath and stirred until the total stirring time of 86-88 hours was reached. Again, each mixture was allowed to settle for half an hour, and an aliquot was transferred to a centrifuge tube. 79
80
VINCENT
A. SEDLAK
The aliquot from each settled mixture was immediately centrifuged at 2500 rpm for half an hour at room temperature to separate any suspended crystalline material. Since the temperature of the aliquots during centrifugation dropped to only 34.5’, thus remaining within the body temperature range reported for rats (Spector, 1956), it was not considered critical. Two replicate aliquots were then removed from the centrifuged solutions and weighed before analysis for their respective isomer content. When necessary, the procedure for preparing solutions of individual isomers of BHC in rat fat was repeated with increasing quantities of the crystalline material in the starting mixture until the analyses indicated that at least 30% of the original quantity of isomer remained in the solid state after 86 hours of incubation. Replicate aliquots (0.12-0.60 g) of the centrifuged solutions of the isomers of BHC in rat fat were transferred to volumetric flasks and diluted in order to permit direct analysis without any cleanup being required. Aliquots containing the gamma or delta BHC isomer were transferred and diluted with n-hexane to either 50 or 100 ml, because n-hexane is an ideal solvent in connection with the instrument used for analysis. However, the beta isomer is essentially insoluble in n-hexane, and the alpha isomer is only slowly soluble. Therefore, aliquots containing these isomers were transferred and diluted with xylene to either 10 or 25 ml. Independent tests showed that the use of xylene did not limit recovery of the alpha and beta isomers. The concentration of isomer in each diluted aliquot was determined by microcoulometric gas chromatography (Coulson and Cavanagh, 1960; Co&on et al., 1960). The Dohrmann gas chromatograph equipped with a 6 foot X & inch aluminum column packed with 205% silicone grease on acid-washed 30/60 Chromosorb P (Coulson et al., 1959) was used. The chromatographed material was fed directly into a combustion furnace, and the products containin g chlorine were detected and determined quantitatively by the Dohrmann TC 100 microcoulometric cell (Coulson and Cavanagh, 1960). The results were recorded on a Sargent model SR recorder equipped with a disk integrator. This apparatus was calibrated with 6- to lO+g aliquots of standard BHC isomersin either hexane or xylene solutions, and 97.0% recovery was obtained. The electrometer was operated at 16 ohms attenuation in order to utilize the maximal capacity range of the titration cell, and was thus capable of detecting 0.4 to 16.6 yg of BHC isomer. Aliquots of 6-80 yl from the n-hexane or xylene diluted fat solutions were then analyzed in replicate. The aliquots contained 4.8-16.2 pg. RESULTS
The results of experiments to determine the maximal solubility of isomers of BHC in rat fat at 37” are as follows: alpha, 4.05%; beta, 0.6670; gamma, 12.55%; and delta, 19.10% (Table 1). The solubilities of the alpha and beta isomers were quickly determined because the physical mixtures prepared at the start contained a great excess of BHC. The gamma and delta isomers respectively have solubilities close to or in excess of the concentration which was available in the first test mixtures. However, the amount of gamma and delta isomer that was finally added permitted an excessof 40% or more of the solid crystalline material to remain in equilibrium after saturation of the solution had been achieved. The solubilities of the BHC isomersin rat fat were compared with the solubilities reported in benzene, decahydronaphthalene, toluene, and acetic acid (Slade, 1945). The comparison is shown for benzene and decahydronaphthalene in Table 2. The
SOLUBILITY
OF
TABLE DETERMINATION
BHC isomer Alpha
Beta
OF THE SOLUBILITY
Isomer in mixture” (‘/c) 16.7
16.8
OF BHC
ISOMERS
Stirring time (hours)
BHC
ISOMERS
81
1 IN MKCHANICALLY
ISOLATED
BHC Aliquot number
RAT
FAT AT 37.O"C
concentration
Replicates (%I
-4verape (70) 4.18
14
1 2
4.06 4.31
86
3
3.91
4
4.20
4.05
14
1
0.706 0.736
0.721
86
2 3
0.697 0.672
4
Gamma
13.0
48
1
0.620 0.666
-
0.664
11.21 11.90 11.38
16.5
25.0
13.27
11.94 11.72
3
11.61 11.84 11.86
4
12.87 12.76 12.65
12.33
1
12.51 12.72
12.58
14
1 2
86
17
2 88
Delta
15.2
3 4
12.37
-
12.55
17
1
12.25 12.35 12.51
12.30
88
2 3
12.55
17
4 1
12.60
23.4
88
2 3
18.99 19.19
19.00
27.4O
4
19.02
19.10
u Based on weight. b Increased from 23.4 to 27.4%
19.00
at 17 hours.
relationship of the solubility of the isomers in rat fat to their solubility in any one of the four organic solvents was close. This parallel behavior shows that the complex fatty acid glyceride mixture constituting rat fat behaves as a simple solvent in vitro.
A comparison of the rat fat solubility results with storage data is given in Table 3. At a dietary level of lOOppm, rats store only about 0.08% of the gamma isomer potentially soluble in their fat, while at the same dosage they store about 28.6% of the beta isomer that the fat is chemically able to dissolve. Expressed in another manner, these studies indicated that, at the dosageused for comparison, from about 3.5 to nearly 2000 times the storage concentrations of the various isomers are soluble in rat fat. The storage levels of BHC isomers in adipose tissue are not related to the solubility of the isomer in rat fat except, of course, that physical
82
VINCENT
A. SEDLAK
TABLE CO&IPARISON
OF BHC
OF SOLUBILITY
Decahydronaphthalene
Solubilityt (g/100 g benzene)
Ratio (rat fat/ benzene)
Solubilityf (g/100 g solvent)
9.9 1.9 28.9 41.1 -
0.433 0.352 0.497 0.574 0.464
2.5 0.4 8.7 10.4 -
4.29 0.668 14.35 23.61
Alpha Beta Gamma Delta Average
AND DECAHYDRONAPHTHALENE
Benzene
Rat fat Solubility” (&vl@J a rat fat)
BHC isomer”
2
IN RAT FAT, BENZENE,
a A fifth isomer, epsilon, was isolated by Kauer et al. (1947) than the other isomers. ti The corresponding values for the different isomers expressed 0.664, 12.55, and 19.10. C Slade (1945). TABLE COMPARISON AND
OF THE
OF BHC
SOLUBILITY
STORAGE IN THE FAT TISSUE CONCENTRATION OF 100
ISOMERS
and
is reported
as per cent by
Ratio (rat fat/ solvent) 1.716 1.670 1.649 2.270 1.826
to be less soluble weight
are:
4.11,
3 IN MECHANICALLY
LIBERATED
RAT
FAT
AT 37°C
OF RATS FED THE SEPARATE ISOMERS AT A DIETARY PPM UNTIL STORAGE WAS AT EQUILIBRIUM
BHC isomer
Solubility in liberated rat fat (wt.%)
Storagea (%I
Proportion potential storage achieved* (%)
Alpha Beta Gamma Delta
4.05 0.66 12.55 19.10
0.04 0.19 0.01 0.01
0.99 28.6 0.08 0.05
a Storage in fresh adipose tissue, based on Fig. 1 of Davidow and Frawley (1951) 0 Calculation of the exact proportion would require knowledge of the storage in extractable lipid or knowledge of the exact proportion this lipid constituted of the fresh adipose tissue.
solubility is eventually a limiting factor. The reported differences in storage must be explained by differences in the metabolism of the isomers and not by differences in their solubility. SUMMARY The solubilities of the isomers of benzene hexachloride in rat fat mechanically removed from adipose tissue were determined at 37” and found to be: alpha, 4.05’$; beta, 0.66%; gamma, 12.55%; and delta, 19.10%. A parallelism between the solubility of the isomers in rat fat and their solubility in benzene, decahydronaphthalene, toluene, and acetic acid shows that rat fat behaves as a simple solvent in vitro. The equilibrium storage levels of the BHC isomers following dietary intake are unrelated to the solubility of the isomers in rat fat, with the exception that solubility is a limiting factor. The reported differences in storage must be explained by differences in the metabolism of the isomers and not by differences in their solubility. REFERENCES COULSON,
D. M.,
and
L.
CAVANAGH,
A.
(1960).
Automatic
chloride
analyzer.
Bnol.
C&m.
32,
124.5-1247. COWLSON,
tography
D.
M.,
CAVANAGH,
of pesticides.
/. Agr.
L.
A., Food
and STUART, J. Chem. 7, 250-251.
(1959).
Pesticide
analysis.
Gas
chroma-
SOLUBILITY
OF
BHC
ISOMERS
83
COULSON, D. M., CAVANACH, L. A., DEVRIES, J. E., and WALTHER, B. (1960). Microcoulometric gas chromatography of pesticides. J. Agr. Food Chem. 8, 399-402. DAVIDOW, B., and FRAWLEY, J. P. (1951). Tissue distribution, accumulation and elimination of the isomers of benzene hexachloride. Proc. Sot. Exptl. Biol. Med. 76, 780-783. K?.uER, I<. C., DUVALL, R. B., and ALQUIST, F. N. (1947). e-Isomer of 1,2,3,4.5.6-hexachlorocyclohexane. Ind. Eng. Chem. 39, 1335-1338. SLADE, R. E. (1945). The y-isomer of hexachlorocyclohexane (Gammexane). An insecticide with outstanding properties. Chem. Znd. 64, 314-319. SPECTOR, W. S. (1956). Handbook of Biological Data, p. 343. Saunders, Philadelphia,
AND
APPLIED
Solubility
PHARMACOLOGY
of
Benzene
7: 79-83
Hexachloride VINCENT
Toxicology
Sectiott,
(1965)
A.
Received
March
in Rat
Fat
SEDLAK
Technology Branch, Communicable of Health, Education, and
Department
Isomers
Disease Welfare,
Center, Atlanta,
Public Health Georgia
Service,
26, 1964
Davidow and Frawley (1951) found that the four common isomers of benzene hexachloride (BHC, or hexachlorocyclohexane) are preferentially stored in adipose tissue of both rats and dogs, but that some isomers are stored in all tissues much more avidly than others. Because the storage levels of the various isomers of BHC in adipose tissue differ by factors as great as 19, these isomers were considered good material for testing the relationship between storage and solubility in fat. The work of Davidow and Frawley makes it possible to compare solubility with the concentration of each isomer in the fat tissue of rats after it has reached a steady state following daily feeding at a uniform dietary rate (100 ppm). (Their work gave evidence that the alpha, gamma, and delta isomers reach a state of equilibrium of storage between the fourth and sixth week of feeding, whereas the beta isomer reaches a steady state by the twelfth week in females and slightly earlier in males.) METHODS
Abdominal fat from several rats was pooled and refrigerated for use as needed. Rat fat for the solubility determination was separated from connective tissue by taking 10 or 12 g of adipose tissue and grindin, v it with mortar and pestle and a small quantity of sodium sulfate. Only enough sodium sulfate was added to absorb moisture and aid in abrading the connective tissue supporting the fat. Then the mixture was transferred mechanically to plastic centrifuge tubes and centrifuged for 1 hour or more at about 2500 rpm. The fat that accumulated at the surface was an oily liquid. This liquid was pipetted off and used immediately for preparation of mixtures with individual BHC isomers. Since comparison between solubility and storage in living adipose tissue was the point of interest, the solubility tests were made at 37.0”. The body temperature of the white rat (Rattus norvegicus) is 37.3” (range 34.5-40.0”) (Spector, 1956). The starting mixture for each determination of saturation concentration was two or more grams of isolated rat fat and a weight of crystalline BHC isomer equal to about 15%’ of the weight of fat. The mixtures were stirred by glass paddles in test tubes at 37.0’ for 14-88 hours. At 14-17 hours, the stirring was stopped and the mixtures were allowed to settle for half an hour. An aliquot was taken from the upper half of the liquid and transferred to a centrifuge tube. Then the test tubes and their remaining contents were returned to the constant temperature water bath and stirred until the total stirring time of 86-88 hours was reached. Again, each mixture was allowed to settle for half an hour, and an aliquot was transferred to a centrifuge tube. 79
80
VINCENT
A. SEDLAK
The aliquot from each settled mixture was immediately centrifuged at 2500 rpm for half an hour at room temperature to separate any suspended crystalline material. Since the temperature of the aliquots during centrifugation dropped to only 34.5’, thus remaining within the body temperature range reported for rats (Spector, 1956), it was not considered critical. Two replicate aliquots were then removed from the centrifuged solutions and weighed before analysis for their respective isomer content. When necessary, the procedure for preparing solutions of individual isomers of BHC in rat fat was repeated with increasing quantities of the crystalline material in the starting mixture until the analyses indicated that at least 30% of the original quantity of isomer remained in the solid state after 86 hours of incubation. Replicate aliquots (0.12-0.60 g) of the centrifuged solutions of the isomers of BHC in rat fat were transferred to volumetric flasks and diluted in order to permit direct analysis without any cleanup being required. Aliquots containing the gamma or delta BHC isomer were transferred and diluted with n-hexane to either 50 or 100 ml, because n-hexane is an ideal solvent in connection with the instrument used for analysis. However, the beta isomer is essentially insoluble in n-hexane, and the alpha isomer is only slowly soluble. Therefore, aliquots containing these isomers were transferred and diluted with xylene to either 10 or 25 ml. Independent tests showed that the use of xylene did not limit recovery of the alpha and beta isomers. The concentration of isomer in each diluted aliquot was determined by microcoulometric gas chromatography (Coulson and Cavanagh, 1960; Co&on et al., 1960). The Dohrmann gas chromatograph equipped with a 6 foot X & inch aluminum column packed with 205% silicone grease on acid-washed 30/60 Chromosorb P (Coulson et al., 1959) was used. The chromatographed material was fed directly into a combustion furnace, and the products containin g chlorine were detected and determined quantitatively by the Dohrmann TC 100 microcoulometric cell (Coulson and Cavanagh, 1960). The results were recorded on a Sargent model SR recorder equipped with a disk integrator. This apparatus was calibrated with 6- to lO+g aliquots of standard BHC isomersin either hexane or xylene solutions, and 97.0% recovery was obtained. The electrometer was operated at 16 ohms attenuation in order to utilize the maximal capacity range of the titration cell, and was thus capable of detecting 0.4 to 16.6 yg of BHC isomer. Aliquots of 6-80 yl from the n-hexane or xylene diluted fat solutions were then analyzed in replicate. The aliquots contained 4.8-16.2 pg. RESULTS
The results of experiments to determine the maximal solubility of isomers of BHC in rat fat at 37” are as follows: alpha, 4.05%; beta, 0.6670; gamma, 12.55%; and delta, 19.10% (Table 1). The solubilities of the alpha and beta isomers were quickly determined because the physical mixtures prepared at the start contained a great excess of BHC. The gamma and delta isomers respectively have solubilities close to or in excess of the concentration which was available in the first test mixtures. However, the amount of gamma and delta isomer that was finally added permitted an excessof 40% or more of the solid crystalline material to remain in equilibrium after saturation of the solution had been achieved. The solubilities of the BHC isomersin rat fat were compared with the solubilities reported in benzene, decahydronaphthalene, toluene, and acetic acid (Slade, 1945). The comparison is shown for benzene and decahydronaphthalene in Table 2. The
SOLUBILITY
OF
TABLE DETERMINATION
BHC isomer Alpha
Beta
OF THE SOLUBILITY
Isomer in mixture” (‘/c) 16.7
16.8
OF BHC
ISOMERS
Stirring time (hours)
BHC
ISOMERS
81
1 IN MKCHANICALLY
ISOLATED
BHC Aliquot number
RAT
FAT AT 37.O"C
concentration
Replicates (%I
-4verape (70) 4.18
14
1 2
4.06 4.31
86
3
3.91
4
4.20
4.05
14
1
0.706 0.736
0.721
86
2 3
0.697 0.672
4
Gamma
13.0
48
1
0.620 0.666
-
0.664
11.21 11.90 11.38
16.5
25.0
13.27
11.94 11.72
3
11.61 11.84 11.86
4
12.87 12.76 12.65
12.33
1
12.51 12.72
12.58
14
1 2
86
17
2 88
Delta
15.2
3 4
12.37
-
12.55
17
1
12.25 12.35 12.51
12.30
88
2 3
12.55
17
4 1
12.60
23.4
88
2 3
18.99 19.19
19.00
27.4O
4
19.02
19.10
u Based on weight. b Increased from 23.4 to 27.4%
19.00
at 17 hours.
relationship of the solubility of the isomers in rat fat to their solubility in any one of the four organic solvents was close. This parallel behavior shows that the complex fatty acid glyceride mixture constituting rat fat behaves as a simple solvent in vitro.
A comparison of the rat fat solubility results with storage data is given in Table 3. At a dietary level of lOOppm, rats store only about 0.08% of the gamma isomer potentially soluble in their fat, while at the same dosage they store about 28.6% of the beta isomer that the fat is chemically able to dissolve. Expressed in another manner, these studies indicated that, at the dosageused for comparison, from about 3.5 to nearly 2000 times the storage concentrations of the various isomers are soluble in rat fat. The storage levels of BHC isomers in adipose tissue are not related to the solubility of the isomer in rat fat except, of course, that physical
82
VINCENT
A. SEDLAK
TABLE CO&IPARISON
OF BHC
OF SOLUBILITY
Decahydronaphthalene
Solubilityt (g/100 g benzene)
Ratio (rat fat/ benzene)
Solubilityf (g/100 g solvent)
9.9 1.9 28.9 41.1 -
0.433 0.352 0.497 0.574 0.464
2.5 0.4 8.7 10.4 -
4.29 0.668 14.35 23.61
Alpha Beta Gamma Delta Average
AND DECAHYDRONAPHTHALENE
Benzene
Rat fat Solubility” (&vl@J a rat fat)
BHC isomer”
2
IN RAT FAT, BENZENE,
a A fifth isomer, epsilon, was isolated by Kauer et al. (1947) than the other isomers. ti The corresponding values for the different isomers expressed 0.664, 12.55, and 19.10. C Slade (1945). TABLE COMPARISON AND
OF THE
OF BHC
SOLUBILITY
STORAGE IN THE FAT TISSUE CONCENTRATION OF 100
ISOMERS
and
is reported
as per cent by
Ratio (rat fat/ solvent) 1.716 1.670 1.649 2.270 1.826
to be less soluble weight
are:
4.11,
3 IN MECHANICALLY
LIBERATED
RAT
FAT
AT 37°C
OF RATS FED THE SEPARATE ISOMERS AT A DIETARY PPM UNTIL STORAGE WAS AT EQUILIBRIUM
BHC isomer
Solubility in liberated rat fat (wt.%)
Storagea (%I
Proportion potential storage achieved* (%)
Alpha Beta Gamma Delta
4.05 0.66 12.55 19.10
0.04 0.19 0.01 0.01
0.99 28.6 0.08 0.05
a Storage in fresh adipose tissue, based on Fig. 1 of Davidow and Frawley (1951) 0 Calculation of the exact proportion would require knowledge of the storage in extractable lipid or knowledge of the exact proportion this lipid constituted of the fresh adipose tissue.
solubility is eventually a limiting factor. The reported differences in storage must be explained by differences in the metabolism of the isomers and not by differences in their solubility. SUMMARY The solubilities of the isomers of benzene hexachloride in rat fat mechanically removed from adipose tissue were determined at 37” and found to be: alpha, 4.05’$; beta, 0.66%; gamma, 12.55%; and delta, 19.10%. A parallelism between the solubility of the isomers in rat fat and their solubility in benzene, decahydronaphthalene, toluene, and acetic acid shows that rat fat behaves as a simple solvent in vitro. The equilibrium storage levels of the BHC isomers following dietary intake are unrelated to the solubility of the isomers in rat fat, with the exception that solubility is a limiting factor. The reported differences in storage must be explained by differences in the metabolism of the isomers and not by differences in their solubility. REFERENCES COULSON,
D. M.,
and
L.
CAVANAGH,
A.
(1960).
Automatic
chloride
analyzer.
Bnol.
C&m.
32,
124.5-1247. COWLSON,
tography
D.
M.,
CAVANAGH,
of pesticides.
/. Agr.
L.
A., Food
and STUART, J. Chem. 7, 250-251.
(1959).
Pesticide
analysis.
Gas
chroma-
SOLUBILITY
OF
BHC
ISOMERS
83
COULSON, D. M., CAVANACH, L. A., DEVRIES, J. E., and WALTHER, B. (1960). Microcoulometric gas chromatography of pesticides. J. Agr. Food Chem. 8, 399-402. DAVIDOW, B., and FRAWLEY, J. P. (1951). Tissue distribution, accumulation and elimination of the isomers of benzene hexachloride. Proc. Sot. Exptl. Biol. Med. 76, 780-783. K?.uER, I<. C., DUVALL, R. B., and ALQUIST, F. N. (1947). e-Isomer of 1,2,3,4.5.6-hexachlorocyclohexane. Ind. Eng. Chem. 39, 1335-1338. SLADE, R. E. (1945). The y-isomer of hexachlorocyclohexane (Gammexane). An insecticide with outstanding properties. Chem. Znd. 64, 314-319. SPECTOR, W. S. (1956). Handbook of Biological Data, p. 343. Saunders, Philadelphia,