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Solubilities of 17-ketosteroids in water
849
SOLUBILITIES OF 17-KETOSTEROIDS IN WATER
Gert M . Jacobsohn and David Levenberg Department of Biological Chemistry Hahnemann Medical College, Philadelphia 2, Pa°
Received
October 13, 1964
Steroids, in general, are classified as water insoluble compounds. Though the extent of their insolubillty may bear important physiological significance, it does not appear to have been investigated systematically. We undertook an examination of neutral 17-ketosterolds in connection with metabolism studies and found, to our surprise, that solubility is quite high when related to biological functions, such as rates of excretion or concentrations in urine.
In terms of what a living organism requires,
solubility must be interpreted on different grounds from purely chemical considerations involved in problems of laboratory handlingo METHOD Individual steroids were shaken overnight with 50 ml.-quantities of distilled water. Only a portion of the added substances dissolved. The saturated supernatants were filtered twice through the same filter paper. Suitable allquots, which varied between I and 3 ml., were shaken three times with 3 ml. of ether each time. Zimmermann tests, modified according to Holtorff and Koch I , were performed on the residues after evaporation of ether from the pooled extracts. Colorimetric standards of 20, 40, and 60 I~g. of each steroid were prepared and assayed simultaneously. Four or 5 replicates were performed in each experiment. For a temperature of 37o , the solutions were equilibrated in a water bath and filtered with a jacketed funnel with circulating water from the bath. For steroids with high solubil ities, 0.1 or 0.2 ml ° portions were removed from the filtered solutions and assayed directly after evaporation of water by a nitrogen jet.
850
S T E R O I D S
4:6
TABLE 1. Solubility in water, with standard deviations. Number of determinations in parentheses. Compound a
Concentration at Saturation (rag. per 100 ml.) 23-24 °
Androsterone Isoandrosterone Dehydroisoandrosterone Etiocholanolone Androstanedione 11-Hydroxyetioc holanol one 11-Hydroxyisoandrosterone
1.15 (2) 2.02 (5) 2.18 (6) 2.91 (2) 3.29 (2) 4.29 (2) 7.82
0.06 0.30 0.16 0.02 0.12 0.07
37 ° 1.79 (2) 2°37 (3) 3,30 (4) 3.92 (2) 4.86 (2) 5.30 (2)
0.05 0.05 0.17 0.09 0.04 0.10
0.28
(2) Adrenosterone 11-Ketoetiocholanolone
9.85 (2) 22.7 (4)
0.22
15.2
(I) 3.7
28.8 (2)
1.1
(a) The correct chemical names for the compounds, in proper sequence, are: 5a-a ndrostan-3a-ol- 17-one, 5a-a ndrosta n - 3 ~ o l - 17-one, 5-androsten-3~ o1-17-one, 5j3-androstan-3a-ol- 17-one, 5a-androstan-3, 17-dione, 5~androstan-3a, 11~diol-17-one, 5a-androstan-3~, 11J~diol- 17-one, 4-androsten-3,11,17-triene, 5J3-androstan-3a-ol- 11,17-dionep RESULTS AND DISCUSSION Table 1 has been arranged in ascending order of solubility of steroids. Among the substances included in the table are those which occur as main urinary excretory products of the 19-carbon steroids. The first membe r of the series, androsterone, dissolves to the extent of 1.8 mg. per 100 ml. of water at normal body temperature. A normal adult excretes, on the average, 1200 ml. of water by the urinary route per day, and rarely less than 600 ml.
Dec. 1964
S T E R 0 I D S
851
Excretion of androsterone fluctuates normally around 2 or 3 mg. per day, and almost never exceeds 6 rag.
Of the other steroids assayed, only
dehydroisoandrosterone and etiocholanolone may be excreted to a larger extent than androsterone, though, again the values do not usually rise above 6 mg. per day of either compound. Water solubility of the latter two steroids is about twice that of androsterone. The quantity of urine produced, therefore, is amply sufficient to eliminate all 17-ketosteroids if these were present in the free form. Salts occur in urine but at a relatively low concentration.
They are not likely to interfere because the solu-
bility of steroids in water is much in excess over their actual concentration in urine. On a teleological basis, the reason commonly offered for biological conjugation is the ease with which the conjugates are excreted.
17-
Ketosteroids occur in urine as hemiacetals of ~-D-glucopyranosiduronic acid (glucuronosides), as monoesters of sulfuric acid (sulfates), and, in trace amounts, in free form. Conjugation is said to facilitate excretion since conjugates are water-soluble substances, whereas the unconjugated, or free, compounds are "insoluble".
Data presented here indicates that
it may not be necessary to postulate the formation of conjugates as part of the excretory mechanism on the basis of increased salubility.
Free
17-ketosterolds are sufficiently soluble by themselves to account entirely for the sum of glucuronosldes, sulfates, and free compounds in urine. The question remains as to the biological purpose of the conjugating mechanism. Conjugates may be involved in the excretory process
852
S T E R 0 I D S
4:6
not because of solubility properties but because of some special ized functions such as may be necessary for the process of transfer across a biological membrane. On the other hand, steroid conjugates have been shown recently to enter direct pathways of steroid transformations which until then were thought to occur only with the Free compounds. Dehydroisoandrosterone sulfate is secreted directly by the adrenal gland 2 Pregnenolone sul fate can be converted to dehydroisoandrosterone sulfate without preliminary splitting of the ester bond 3 and the latter can serve as precursor of estrogens4° Androstenediol sulfate may be oxidized directly to dehydroisoandrosterone sulfate 5 Data of Table 1 can be used to assess the relative contribution of various functional groups to solubility. In general, the higher the number of oxygen functions on a given molecule, the greater the solubility in a polar solvent; i . e . , the polarity. Ketone groups confer a greater polarity than hydroxyl functions; compare androsterone with androstanedlone and 11-hydroxyetiocholanolone with 11-ketoetiocholanolone. A double bond increases polarity; dehydroisoandrosterone is more soluble than isoandrosterone. Hydroxyl groups in the 36 (equatorial) position are more polar than those in the 3a (axial) position, as in the example androsterone and isoandrosterone. Finally, rings A and B in trans configuration have greater solubility than those in the cls form, indicated by the pair androsterone-etiocholanoloneo Similar conclusions can be drawn from chromatographic mobility data~ although numerous exceptions appear due to interactions between solid phases employed and substances studied.
Dec. 1 9 6 4
S T E R 0 I D S
The data suggest that determinations of steroid concentrations in saturated solutions of low solubilities may be used to advantage for identification or structure determinations.
Saturation solubilities of the
stereoisomers tested seem sufficiently wide apart to permit unequivocal identification. ACKNOWLEDGMENT Supported, in part, by NIH Grant No. AM-06746-03. D . L . was recipient of a Student Summer Fellowship from funds provided by Grant FR 05413-01 of the NIH. REFERENCES °
2o 3° 4. 5°
Holtorff, A. Fo, Koch, Fo C., J° BIOL. CHEM. 135, 377 (1940). Vande Wiele, R. L°, MacDonald, Po Co, Gurpide, Eo, Lieberman, S., RECENTPROGR. HORMONE RES° 19, 275 (1963}. Calvin, H° I°, Vande Wiele, R° L., Lieberman, So, BIOCHEM° 2, 648 (1963}. Siiteri, Po K., MacDonald, P. C., STEROIDS 2, 713 (1963). Baulieu, E. E°, Corpechot, C., Emiliozzi, R., STEROIDS 2, 429 (1963).
853
SOLUBILITIES OF 17-KETOSTEROIDS IN WATER
Gert M . Jacobsohn and David Levenberg Department of Biological Chemistry Hahnemann Medical College, Philadelphia 2, Pa°
Received
October 13, 1964
Steroids, in general, are classified as water insoluble compounds. Though the extent of their insolubillty may bear important physiological significance, it does not appear to have been investigated systematically. We undertook an examination of neutral 17-ketosterolds in connection with metabolism studies and found, to our surprise, that solubility is quite high when related to biological functions, such as rates of excretion or concentrations in urine.
In terms of what a living organism requires,
solubility must be interpreted on different grounds from purely chemical considerations involved in problems of laboratory handlingo METHOD Individual steroids were shaken overnight with 50 ml.-quantities of distilled water. Only a portion of the added substances dissolved. The saturated supernatants were filtered twice through the same filter paper. Suitable allquots, which varied between I and 3 ml., were shaken three times with 3 ml. of ether each time. Zimmermann tests, modified according to Holtorff and Koch I , were performed on the residues after evaporation of ether from the pooled extracts. Colorimetric standards of 20, 40, and 60 I~g. of each steroid were prepared and assayed simultaneously. Four or 5 replicates were performed in each experiment. For a temperature of 37o , the solutions were equilibrated in a water bath and filtered with a jacketed funnel with circulating water from the bath. For steroids with high solubil ities, 0.1 or 0.2 ml ° portions were removed from the filtered solutions and assayed directly after evaporation of water by a nitrogen jet.
850
S T E R O I D S
4:6
TABLE 1. Solubility in water, with standard deviations. Number of determinations in parentheses. Compound a
Concentration at Saturation (rag. per 100 ml.) 23-24 °
Androsterone Isoandrosterone Dehydroisoandrosterone Etiocholanolone Androstanedione 11-Hydroxyetioc holanol one 11-Hydroxyisoandrosterone
1.15 (2) 2.02 (5) 2.18 (6) 2.91 (2) 3.29 (2) 4.29 (2) 7.82
0.06 0.30 0.16 0.02 0.12 0.07
37 ° 1.79 (2) 2°37 (3) 3,30 (4) 3.92 (2) 4.86 (2) 5.30 (2)
0.05 0.05 0.17 0.09 0.04 0.10
0.28
(2) Adrenosterone 11-Ketoetiocholanolone
9.85 (2) 22.7 (4)
0.22
15.2
(I) 3.7
28.8 (2)
1.1
(a) The correct chemical names for the compounds, in proper sequence, are: 5a-a ndrostan-3a-ol- 17-one, 5a-a ndrosta n - 3 ~ o l - 17-one, 5-androsten-3~ o1-17-one, 5j3-androstan-3a-ol- 17-one, 5a-androstan-3, 17-dione, 5~androstan-3a, 11~diol-17-one, 5a-androstan-3~, 11J~diol- 17-one, 4-androsten-3,11,17-triene, 5J3-androstan-3a-ol- 11,17-dionep RESULTS AND DISCUSSION Table 1 has been arranged in ascending order of solubility of steroids. Among the substances included in the table are those which occur as main urinary excretory products of the 19-carbon steroids. The first membe r of the series, androsterone, dissolves to the extent of 1.8 mg. per 100 ml. of water at normal body temperature. A normal adult excretes, on the average, 1200 ml. of water by the urinary route per day, and rarely less than 600 ml.
Dec. 1964
S T E R 0 I D S
851
Excretion of androsterone fluctuates normally around 2 or 3 mg. per day, and almost never exceeds 6 rag.
Of the other steroids assayed, only
dehydroisoandrosterone and etiocholanolone may be excreted to a larger extent than androsterone, though, again the values do not usually rise above 6 mg. per day of either compound. Water solubility of the latter two steroids is about twice that of androsterone. The quantity of urine produced, therefore, is amply sufficient to eliminate all 17-ketosteroids if these were present in the free form. Salts occur in urine but at a relatively low concentration.
They are not likely to interfere because the solu-
bility of steroids in water is much in excess over their actual concentration in urine. On a teleological basis, the reason commonly offered for biological conjugation is the ease with which the conjugates are excreted.
17-
Ketosteroids occur in urine as hemiacetals of ~-D-glucopyranosiduronic acid (glucuronosides), as monoesters of sulfuric acid (sulfates), and, in trace amounts, in free form. Conjugation is said to facilitate excretion since conjugates are water-soluble substances, whereas the unconjugated, or free, compounds are "insoluble".
Data presented here indicates that
it may not be necessary to postulate the formation of conjugates as part of the excretory mechanism on the basis of increased salubility.
Free
17-ketosterolds are sufficiently soluble by themselves to account entirely for the sum of glucuronosldes, sulfates, and free compounds in urine. The question remains as to the biological purpose of the conjugating mechanism. Conjugates may be involved in the excretory process
852
S T E R 0 I D S
4:6
not because of solubility properties but because of some special ized functions such as may be necessary for the process of transfer across a biological membrane. On the other hand, steroid conjugates have been shown recently to enter direct pathways of steroid transformations which until then were thought to occur only with the Free compounds. Dehydroisoandrosterone sulfate is secreted directly by the adrenal gland 2 Pregnenolone sul fate can be converted to dehydroisoandrosterone sulfate without preliminary splitting of the ester bond 3 and the latter can serve as precursor of estrogens4° Androstenediol sulfate may be oxidized directly to dehydroisoandrosterone sulfate 5 Data of Table 1 can be used to assess the relative contribution of various functional groups to solubility. In general, the higher the number of oxygen functions on a given molecule, the greater the solubility in a polar solvent; i . e . , the polarity. Ketone groups confer a greater polarity than hydroxyl functions; compare androsterone with androstanedlone and 11-hydroxyetiocholanolone with 11-ketoetiocholanolone. A double bond increases polarity; dehydroisoandrosterone is more soluble than isoandrosterone. Hydroxyl groups in the 36 (equatorial) position are more polar than those in the 3a (axial) position, as in the example androsterone and isoandrosterone. Finally, rings A and B in trans configuration have greater solubility than those in the cls form, indicated by the pair androsterone-etiocholanoloneo Similar conclusions can be drawn from chromatographic mobility data~ although numerous exceptions appear due to interactions between solid phases employed and substances studied.
Dec. 1 9 6 4
S T E R 0 I D S
The data suggest that determinations of steroid concentrations in saturated solutions of low solubilities may be used to advantage for identification or structure determinations.
Saturation solubilities of the
stereoisomers tested seem sufficiently wide apart to permit unequivocal identification. ACKNOWLEDGMENT Supported, in part, by NIH Grant No. AM-06746-03. D . L . was recipient of a Student Summer Fellowship from funds provided by Grant FR 05413-01 of the NIH. REFERENCES °
2o 3° 4. 5°
Holtorff, A. Fo, Koch, Fo C., J° BIOL. CHEM. 135, 377 (1940). Vande Wiele, R. L°, MacDonald, Po Co, Gurpide, Eo, Lieberman, S., RECENTPROGR. HORMONE RES° 19, 275 (1963}. Calvin, H° I°, Vande Wiele, R° L., Lieberman, So, BIOCHEM° 2, 648 (1963}. Siiteri, Po K., MacDonald, P. C., STEROIDS 2, 713 (1963). Baulieu, E. E°, Corpechot, C., Emiliozzi, R., STEROIDS 2, 429 (1963).
853