THE IN VIVO GLUCURONIDE CONJUGATION OF RADIOACTIVE AND BY THE DOG KIDNEY

George L. Cohn, Michael Hume

J Clin Invest. 1960;39(10):1584-1589. https://doi.org/10.1172/JCI104180.

Research Article

Find the latest version: https://jci.me/104180/pdf THE IN VIVO GLUCURONIDE CONJUGATION OF RADIOACTIVE ETIOCHOLANOLONE AND ANDROSTERONE BY THE DOG KIDNEY * By GEORGE L. COHN t AND MICHAEL HlUIE t (Fromi tfe Departmnentts of Internial Medicine and Sutrgery, Yale tUninversity School of Medicinie, New Haven, Conn.) (Submitted for publicationi February 19, 1960; accepted Junie 22, 1960)

It is recognized that the liver readily conjugates ent in the urine during the same symptomatic epi- reduced (1) so rapidly that, under nor- sode. When the patients were afebrile and with- mal conditions, reduced steroids are not recovered out symptoms, the 24-hour urinary 17- in an unconjugated form in plasma but rather oc- patterns were normal, and unconjugated etiocho- cur as sulfate (2-4), glucuronide (5), and prob- lanolone was not detected in the plasma. It was ably phosphate (6) conjugates. Although postulated that an attack of this type of fever might Weichselbaum and Margraf reported the presence be associated vith 1) transient overproduction of of small quantities of unconj ugated tetrahydro- etiocholanolone and/or 2) an intermittent inability 1 in 10 L of normal pooled plasma (7), to conjugate. The presence of conjugated etio- confirming a previous observation in plasma from cholanolone in urine and its absence in plasmiia dur- patients with advanced cirrhosis (8), this has not ing an attack of periodic fever is difficult to recon- been the experience of others analyzing 50 to 100 cile unless another organ in addition to the liver ml of normal plasma. Moreover, most investiga- is involved in conjugation. tors have been unable to isolate significant amounts The rapid clearance of etiocholanolone and of unconjugated reduced metabolites, androsterone, after intravenous injection (12) and viz., androsterone and etiocholanolone, in normal oral and intravenous administration of plasma 2 (2, 3, 9). (13-15), suggests the possibility that these com- Recently, however, Bondy, Cohn, Herrmann pounds may be conjugated by another organ be- and Crispell (10) demonstrated unconjugated sides the liver. The reports of Dutton (16), and etiocholanolone in the plasma of two patients with Lipschitz and Bueding (17), that kidney slices symptomatic etiocholanolone fever.3 Conjugated synthesize glucuronide conjugates in vitro stimtu- etiocholanolone (12 to 15 mg per day) was pres- lated an investigation to determine whether dog kidney could conjugate radioactive etiocholanolone * Supported by Grant 254- (C-7) from the National Institutes of Health, Bethesda, Md. and androsterone in 'vivo. t Advanced Research Fellow of The American Heart Association. MATERIALS AND METHODS t Markle Scholar in Medical Science. Six apparently healthy mongrel dogs (4 males and 2 1 Compounds referred to are etiocholanolone (3a-hy- females), weighing 8 to 17 kg, were used in these ex- droxy-etiocholane-1 7-one); androsterone (3a-hydroxy- periments. The dogs were anesthetized with N\embutal, -17-one); tetrahydrocortisol (3a, 11t8, 17a, 21- 30 mg per kg. In order to insure an adequate diuresis, tetrahydroxy--20-one); (3a, 5 per cent mannitol in hypertonic saline (166 mEq per L) 17a, 21-trihydroxy-pregnane-11, 20-dione); allo-tetrahy- was infused at a rate of 1.5 ml per minute through a drocortisol (3a, 11,8, 17a, 21-tetrahydroxy-- cannula placed into the superior vena cava via the in- 20-one); allo-tetrahydrocortisone (3a, 17a, 21-trihydroxy- ternal jugular vein. Through a mid-line incision the alldpregnane-11, 20-dione). left kidney pedicle was exposed, the left ureter cannu- 2 Dubrulle and Claeys (9) measured unconjugated lated with polyethylene tubing (PE 205, Clay-Adams, plasma 17- in 16 normal adolescents and New York, N. Y.) and the urine collected. After the found 7 to 28 Atg per 100 ml. renal vein was dissected free from the renal pedicle to 3 Recently, this clinical entity was named etiocholano- the inferior vena cava, a polyethylene tubing (PE 160) lone fever in order to differentiate it from the familial with side holes cut near the tip was inserted into the re- Mediterranean fever on one hand, and the many periodic nal vein at its junction with the spermatic (or ovarian) fevers on the other (11). vein. At intervals this cannula was flushed with 10 ml 1584 IN 1VIVO GLUCURONIDE CONJUGATION BY THE DOG KIDNEY 1585 volumes of heparin-isotonic saline solution (0.2 mig per After injection of the isotope(s), full perfusion of the ml). The renal artery was dissected free and a liga- kidney was re-established by removal of the sling or the ture passed beneath it. At zero time the renal vein was clamp. Up to 225 to 260 ml of renal vein blood was col- cross-clamped at its junction with the inferior vena cava lected in 6 to 8 minutes. Hypotension occurred in only with a Potts bulldog (serrefine) clamp. Renal vein one dog. At the end of this collection time the pedicle blood collection was begun and was manually controlled was clamped and the renal pelvis washed three times with to prevent collapse or over-distentioni of the renal vein. isotonic saline. These washings were added to the 4 to Heparin-rinsed syringes were used for the blood col- 6 ml of urine formed during the experimental period. lection. Systemic venous blood was collected via the Nephrectomy was performed at the conclusion of the ex- jugular catheter during the same period and urine flow periment. was also collected as a separate fraction. WVith the re- The plasma was separated from the cells by centrifu- nal artery delivered up into the wound by traction on the gation within 15 minutes of collection (18) and frozen ligature passed around it, thus serving to partially oc- until analyzed. The urine sample and kidney were clude the artery during the injection period, isotopically processed on the day of the experiment. The plastic bag labeled (s) (Table I), dissolved in 0.2 ml absolute and lymph contained in it, as well as the catheters, were ethanol and diluted to 2.0 ml with heparin-isotonic sa- analyzed for radioactivity. line, were injected by direct needle puncture of the re- nal artery. The injection time varied from 15 to 30 sec- Separation of plasma steroids onds. It should be pointed out that the tip of the needle Free steroids. The plasma was thawed at room tem- was positioned as closely as possible to the entrance of perature. Two ml of 1 N NaOH per 40 ml of plasma the renal artery into the kidney (Figure 1). In one was added, mixed, and the plasma extracted three times experiment, a serrefine clamp instead of the sling liga- with 2 vol of freshly distilled chloroform. Emulsions ture was used to constrict the renal artery at its origin. were broken by centrifugation. The chloroform extract In order to eliminate any return of injected isotope by was dried in a rotating vacuum evaporator at 370 C and venous radicles in the renal capsule or by the lymphatics, the residue dissolved in 10 to 15 ml of chloroform. Ra- the entire kidlney as well as its pedicle was dissected from dioactivity (cpm) was determined with corrections for its bed and surrounded by a plastic envelope. quenching. This extract (i.e., that portion of steroids extractable with chloroform without hydrolytic proce- dures) has been designated as the "free" (unconjugated) fraction. The redissolved residue was concentrated to dryness in vacuo, carefully dissolved in 1.0 ml of chloro- form and applied as a 2.0 cm streak, along with appropri- ate reference standards, to washed (19) Whatman no. 1 filter paper in the heptane-propylene glycol system 4 (20). The chromatogram was scanned for radioactivity and the data from a count rate meter traced on an Ester- line-Angus recorder. The pilot strip containing the ref- erence standards and a 0.25 cm portion from the center of the "extract strip" were cut out and stained with Zimmermann reagent. There was no Zimmermann posi- tive material detected on the "extract strip" except in the carbon" experiment. However, there was very close agreement of Rf between the Zimmermann positive refer- ence compounds and the location of the radioactive com- 6 pound(s) by isotopic scanning. The appropriate area was FIG. 1. A DIAGRAMMATIC REPRESENTATION OF THE cut out and eluted with 4.0 ml of absolute ethanol. An DOG KIDNEY PREPARATION. The left ureter (U) is shown aliquot of this eluate was counted for radioactivity. The with the catheter in place. The spermatic vein (Sp V) remainder was dried in vacuo, dissolved in chloroform is cannulated and a polyethylene catheter, with a side and chromatographed in the 96 per cent methanol: hep- hole at its tip, is passed into the renal vein (RV). The tane (1: 1) system of Bush and Willoughby (21). The renal vein is occluded with a serrefine bulldog clamp at "free" compound(s) was located and counted as its junction with the inferior vena cava (IVC) just as described. blood is withdrawn from the renal vein catheter. The Radioactivity was counted to a standard error of rate of withdrawal is adjusted to prevent collapse or over- ± 2 per cent in a liquid-phosphor scintillation counter distention of the vein. As soon as the renal vein is oc- (Technical Measurements Co., model LP-2, New Haven, cluded, isotopically labeled steroid(s) is injected rapidly Conn.) which had a counting efficiency of 15 per cent by direct puncture of the renal artery (RA). A single for tritium and 65 per cent for carbon"4. When the ligature around the renal artery near its origin from the aorta (Ao) delivers the vessel into the wound and par- 4 All chromatograms were run in a room with constant tially occludes it. temperature and humidity. 1586 GEORGE L. COHN AND MICHAEL HUME carbon scintillation counting eluates were low, they was located, eluted, anid radioactivity was determined as were quantitatively transferred to 2.0 sq cm areas on described above. aluminum planchets at infinite thinness and counted in a specially constructed windowless gas-flow counter 5 Separationi of urbiarv steroids and stcroids in nPlymp and which had a background of 1.5 to 2.0 cpm. plastic lbag Conjugated steroids. After the chloroform extraction The urine, lymph ani(l )lastic bag were each extracted 2 volumes of absolute ethanol was added to the plasma. twice witlh 100 mil of chloroform for "free" steroids. The precipitated- protein was removed by centrifugation This was followed by extraction twice with 100 ml of and further extracted three times with 1 volume of re- n-butanol for conjugated steroids. The extracts were distilled n-butanol saturated witlh water. The precipitate counted with allowances for quenching b)ut were iiot was discarded and the ethanol and butanol extracts were processed further. pooled and dried in vacuo. The residue was dissolved in 25 ml of 70 per cent methaniol and wxashed three times Separationt of ster-oids froni the kidntey with equal volumes of redistilled hexane. The hexane The kidney was cut inito small pieces, homogeniized layers, which contained no signiificant radioactivity, were with 2 vol of 0.25 MI sucrose anid extracted three times discarded and the washed methanol layer was taken to with 100 ml chloroform washes. The extract was filtered dryness in vacuo. The resi(lue was redissolved in 1.0 ml through washedl glass wNool, concenitrated to about 2.0 of 70 per cent methanol. An aliquot was counted and ml in zvacito anid ra(lioactivity measured. the remainder streaked oln Whatman no. 3 MM filter The homogenate was extracted twice with 200 ml of paper for electrophoresis. Referenice standards of an- n-butanol for conijugated steroids. This extract was fil- drosterone and etiocholanolone glucuronides alnd sulfates tered, conicenitrated and counted as descril)ed. The "free" were applied adjacent to the streak. Paper electrophore- and conijugated fractionis were niot characterized furtlher. sis was carried out as previously described (5). The conjugated compounds were located oni the electrophero- 1RESULTS gram by radioactive scanning anid staining of the refer- ence compound with Zimmermann reagent. The mo- Renal vein plasmna. The total radioactivities of bility of the Zimmermann positive standard coincided the "free" and conjugated fractions are listed in with the location of the radioactive peak.6 No "free" Table I. Percentages are corrected to 100 ml of androsterone or etiocholanolone was detected at the ori- gin of the electropherogram by these techniques or after plasma. These values are lower than were the elution and counting (as described for tritium or car- actual radioactive recoveries (except experiment bon"4). The sulfate area and remainder of the paper VI) since miiore than 100 ml of 1)lasma was ob- contained no measurable radioactivity after elution with tained. The uncorrected total "free" and con- 70 per cent methanol. jugated radioactive steroid recoveries in the re- The radioactive conjugate area was cut out and eluted nal vein plasma represent 30 to 56 cent of the with 70 per cent methanol. An aliquot was counted and per the remainder, along with adjacent reference standards, original amouint of radioactivty (cpm) injected. was chromatographed in the ethyl acetate: hexane: glacial When tracer qtuantities of etiocholanolone-H3 acetic acid: water (120: 80: 60: 140) system of Schneider were injected, almost twice as much conjugated and Lewbart (22). The steroid conjugate was located tritium-labeled etiocholanolone as "free" was by radioactive scanning and staining with Zimmermann foundl. There was twice as much "free" steroid as reagent, eluted, counted, and then rechromatographed in the n-butanol: butyl acetate: 10 per cent acetic acid (20: the conjugated form when tracer amounts of an- 80: 100) system (23). After radioactivity was measured, drosterone-H3 were injected. After tracer amounts the eluate from the second chromatogram was hydrolyzed of androsterone-H3 and etiocholanolone-H3 were with ,-glucuronidase7 as previously described (5). The injected simultaneously, a greater amount of etio- released "free" radioactive steroid was extracted witlh cholanolone glucuronide (16 per cent) than an- chloroform, counted, and chromatographed in the 96 per drosterone glucuronide was found (12 per cent of cent methanol: heptane system. The "free" compound injected dose per 100 ml). 5 Dr. Melvin V. Simpson of the Department of Bio- Jugular vein plasma, urine, lymph, plastic bag chemistry, Yale University, generously allowed us to use and kidney. No significant radioactivity was this specially constructed counter. found in either the "free" or conjugated urinary 6 In experiment VI (Table I) the androsterone glu- fractions, tubings, lymph or plastic bag. There curonide and sulfate areas were also located directly on was 0.08 to 0.4 per cent of the dose "free," and 0 0.25 cm of the extract strips. cent of the dose radioactive 7 Beta-glucuronidase was obtained from Warner-Chil- to 0.1 per conjugated cott Laboratories, Warner-Lambert Pharmaceutical Co., fractions per 100 ml in the internal jugular vein New York, N. Y. l)lasmla. After homiogenization and extraction of IN VIVO GLUCURONIDE CONJUGATION BY THE DOG KIDNEY 1587

TABLE I "Free" and conjugated steroids in renal vein plasma

Dose recovered Volume Specific Amount "Free" Coni. renal vein Experiment Steroid* activity administeredt steroid steroid plasma 1JX;,ug cpm %/100 ml renal ml vein plasma I Androsterone-4-C'4 0.0035 2.22 X 104 15.5 10.5 142 II Etiocholanolone-H3 5.2 4 X 105 8 16 125 III Androsterone-H3 2.6 5.7 X 105 28 14 115 IV Etiocholanolone-H3 5.2 6.2 X 105 15 3$ 120 Androsterone-H3 2.6 6.1 X 105 20 V Etiocholanolone-H3 5.2 6.4 X 105 8 16.5 122 Androsterone-H3 2.6 6.15 X 105 34 12 VI§ Androsterone-H3 2.6 2.75 X 105 18.2 13.3 90 * Androsterone-4-C'4 was prepared biosynthetically from testosterone-4-C'4 (New England Nuclear Corp., Boston, Mass.). The compound was isolated by gradient elution chromatography (24) after enzymatic hydrolysis and extraction of urine. Its identity was verified by infrared analysis. Androsterone (mp 181 to 1830 C) and etiocholanolone (mp 149 to 1510 C; donated by Dr. Seymour Lieberman, Columbia University, New York, N. Y.), were exposed to tritium gas (Wilzbach procedure) by the New England Nuclear Corp. The tritium-labeled steroids were thoroughly washed, puri- fied, identified and characterized to constant specific activity by paper and column chromatography. The details of the method will be reported in a separate publication. t The dosage of steroid represents the difference between the amount administered and the quantity extractable from the syringe and needle after injection. $ This animal was profoundly hypotensive 4 minutes after the injection of steroid. Because of the low percentage, conjugated steroids were not further separated. § Carrier androsterone glucuronide and sulfate (500 jAg of each) were added to the renal vein blood at the conclusion of this experiment. the renal tissue, 36 per cent "free" and 0 to 1 per jugate was not identified, it is unlikely that any cent conjugated, of the injected isotopic dose, were other conjugate would behave in the manner de- detected. A typical isotope recovery analysis is scribed. Because of their negative charge, glu- summarized in Table II. curonides of either the C21 or C19 steroids migrate Identification of androsterone and etiocholano- as a class and at a slower rate than do the sulfates lone glucuronides. The isolated radioactive ster- toward the anode on paper electrophoresis (5). oid conjugates were identified as glucuronides of Thus it is impossible to separate the C21 and C19 androsterone and etiocholanolone by virtue of glucuronides by this technique. However, using their mobilities on paper by electrophoresis fol- Schneider and Lewbart paper chromatographic lowed by chromatography in two systems. After systems (22, 23), labeled etiocholanolone and an- separation of the compounds by these techniques, drosterone glucuronides were easily isolated. ,f-glucuronidase hydrolysis resulted in the release An insignificant amount of radioactivity was of the "free" steroid which was identified by pa- found in the peripheral plasma, indicating that the per chromatography.8 Significant radioactivity in vivo technique excluded the liver (1, 25) and was detected throughout the isolation and identi- duodenum (26) from this conjugation. The effi- fication procedures. TABLE II DISCUSSION Percent isotope recoveries after the administraion of andros- These studies demonstrate that the dog kidney terone-H3 into the renal artery formed carbon14 and tritium-labeled androsterone Per cent of and etiocholanolone glucuronide in vivo. Al- total dose though the glucuronide portion of the steroid con- Total renal vein plasma (per 100 ml) 31.5 8 In experiment VI carrier androsterone glucuronide Peripheral venous plasma (per 100 ml) 0.08 Kidney 37.0 and sulfate (Table I) were added to the renal vein plasma. Plastic bag and lymph 1.0 After processing and purification by the methods de- Polyethylene tubings 0 scribed, the androsterone glucuronide fraction maintained Urine 3.0 constant specific activity. There was no detectable ra- Total 72.58 dioactivity in the fraction. 1588 GEORGE L. COHN AND MICHAEL HUME ciency of the dog kidney glucuronide conjugation ney by tubular secretion (29). Kellie and Smith system was 3 to 16.5 per cent of the injected iso- (30), on the other hand, reported clearance values topic dose per 100 ml per 6 to 8 minutes (Table for androsterone and etiocholanolone glucuronides I) . comparable with those of inulin and suggested that Because there are conformational differences these compotun(ls muay be excreted by glomerular between androsterone and etiocholanolone at the filtration alone. I n otur experiments we were third carbon atom, speculation arises concerning able to detect no miiore thain 3 per cent of the in- the relative ease of conjugation of these com- jected radioactivity in the urine despite the large pounds. The a-hydroxyl group at the third carbon amounit of free isotope "trapped" in the kidney. atom in the 5,8 compound, etiocholanolone, is The significance of these findings is not uinider- equatorial to the A ring. On the other hand, the stood and warrants further investigation. hydroxyl group on this carbon atom is axial to the Although it was demonstrated that the dog kid- A ring and more sterically hindered in the 5a com- ney synthesized steroid glucuronides in vivo, the pound, androsterone. Since it is known that an site of renal conjugationi and the eluicidation of esterifying reaction proceeds more rapidly when this process in man remain to be (letermined. the acceptor group is equatorial rather than axial (27, 28), it is reasonable to predict that under SUMMARY similar conditions mnore etiocholaiiolone than an- drosterone will be conjtugated. Of course, this 1. The conjugation of radioactive etiocholano- will be true only if the enzyme systems, cofactors, lone and androsterone to their respective glucu- substrates and so forth are available to effect glu- ronides was demonistrated (in vivo) in a dog kid- curonide synthesis. ney preparation. These glucuronides, isolated The data in Table I favor this hypothesis since, froml the renal vein plasma, were characterized by under similar experimental conditions, a higher their imobilities on paper electrophoresis, paper percentage of etiocholanolone glucuronide than chromiiatography, hydrolysis with /3-glucuronidase, of androsterone glucuironide was found. Slaun- and paper chromatography of the resultant "free" white and Sandberg (12) injected carbon14-la- compound. Significant radioactivity persisted beled androsterone and etiocholanolone into three throughout the isolation and identification pro- women and found that the etiocholanolone glu- cedures. In one experiment androsterone glu- curonides were cleared more rapidly than were the curonide was characterized by purification to con- glucuronides of androsterone. Two of the three stant specific activity. No significant amounts of women had slightly higher levels of etiocholano- radioactive glucuronide conjugates were found in lone glucuronides than of androsterone glucuro- the urine or internal jugular vein plasma. Highly nides, 15 minutes after injection. Thus, the more significant amounts of free isotope were "trapped" rapid conjugation of etiocholanolone as compared in the kidney parenchyma. with androsterone may be explained by the con- 2. Three to 16.5 per cent of the injected dose formational differences between the 3a-hydroxy of the labeled steroid per 100 ml of plasma was groups of the two compounds. It follows, if this converted to the steroid glucuronide during the hypothesis is correct, that under similar condi- 6- to 8-minute perfusion of the kidney. tions tetrahydrocortisol and tetrahydrocortisone 3. A hypothesis is presented which may ex- may be conjugated more rapidly than are their allo plain the higher yields of etiocholanolone glu- derivatives, since the 3a-hydroxy group is eqtta- curonide than of anidrosterone glucuronide formed torial to the A ring in the former, and axial in the by this technique. The lesser steric hindrance of latter compounds. the equatorial a-hydroxyl group on the third car- After conjugation, C1, glucuronides an(d sul- bon atom of etiocholanolone may facilitate rapid fates are rapidly excreted by the kidneys (12-15). glucturonide conijugation. Although androsterone However, the actual physiological mechanism is is conijugated readily, the rate of the reaction may not completely resolved. Bongiovanni and EIber- be slightly less because of steric hindrance of the lein demonstrated in a hluman volunteer that an- axiallv placed a-hydroxyl group oni the third drosterone glucuroni(le was cleared from the kid- carbon atolmi. IN VIVO GLUCURONIDE CONJUGATION BY THE DOG KIDNEY 1589

ACK NOWLEDGM ENTS fused intravenously in man. J. biol. Chem. 1954, 206, 863. The authors warmly appreciate the genierous gifts of 14. Sandberg, A. A., and Slaunwhite, W. R., Jr. Metabo- steroid glucuronides and sulfates from Dr. John J. lism of 4-C14-testosterone in human subjects. I. Schneider, Jefferson Medical College, Philadelphia. Pa.; Distribution in bile, blood, feces and urine. J. clin. and etiocholanolone and androsterone from Dr. H. Leon Invest. 1956, 35, 1331. Bradlow and Dr. Seymour Lieberman. 15. West, C. D., Reich, H., and Samuels, L. T. Urinary metabolites after intravenous injection of human REFERENCES subjects with testosterone. J. biol. Chem. 1951, 193, 219. 1. Isselbacher, K. J., and Axelrod, J. Enzymatic for- 16. Dutton, G. J. Foetal and gastro-intestinal glucuro- mation of glucuronides. J. Amer. nide synthesis. Biochem. J. 1958, 69, 39P. chem. Soc. 1955, 77, 1070. 17. Lipschitz, W. L., and Bueding, E. Mechanism of 2. Clayton, G. W., Bongiovanni, A. M., and Papadatos, the biological formation of conjugated glucuronic C. Preliminary investigations in the nature of acids. J. biol. Chem. 1939, 129, 333. neutral 17-ketosteroids in human plasma. J. clin. 18. Bush, I. E. Discussion on in blood. Endocr. 1955, 15, 693. Recent Progr. Hormone Res. 1954, 9, 449. 3. Migeon, C. J., and Plager, J. E. Neutral 17-keto- 19. Bondy, P. K., Abelson, D., Scheuer, J., Tseu, T. K. L., steroids in human plasma. Recent Progr. Hor- and Upton, V. Determination of in hu- mone Res. 1954, 9, 235. man plasma by quantitative paper chromatography. 4. Purdy, R. H., Engel, L. L., and Oncley, J. L. The J. biol. Chem. 1957, 224, 47. chemical nature of some human plasma . 20. Rubin, B. L., Dorfman, R. I., and Pincus, G. A Fed. Proc. 1959, 18, 305. method for the quantitative analysis of 17-keto- 5. Cohn, G. L., and Bondy, P. K. The isolation, char- steroid mixtures. J. biol. Chem. 1953, 203, 629. acterization, and measurement of steroid glucuro- 21. Bush, I. E., and Willoughby, M. The excretion of nides in human plasma. J. biol. Chem. 1959, 234, allotetrahydrocortisol in human urine. Biochem. 31. J. 1957, 67, 689. 6. Oertel, G. W., and Eik-Nes, K. B. Dehydroepiandro- 22. Schneider, J. J., and Lewbart, M. L. Fractionation sterone in blood. I. Cleavage of dehydroepiandro- and isolation of steroid conjugates. Recent Progr. sterone conjugates by phosphatases. Acta endocr. Hormone Res. 1959, 15, 201. (Kbh.) 1958, 28, 293. 23. Lewbart, M. L., and Schneider, J. J. Paper chro- 7. WVeichselbaum, T. E., and Margraf, H. XV. Identi- matography of steroid glucuronosides and sul- fication of "free" tetrahydrocortisone in human phates. Nature (Lond.) 1955, 176, 1175. peripheral plasma. J. clin. Endocr. 1959, 19, 1011. 24. ILakshmanan, T. K., and Lieberman, S. An im- 8. Cohn, G. L., and Bondy, P. K. The isolation and proved method of gradient elution chromatography measurement of corticosteroid glucuronides ini the and its application to the separation of urinary plasma of patients with Laennec's cirrhosis. Clin. ketosteroids. Arch. Biochem. 1954, 53, 258. Res. 1958, 6, 300. 25. Isselbacher, K. J. Enzymatic mechanisms of hor- 9. Dubrulle, P., and Claeys, C. Place et interet du dos- mone metabolism. II. Mechanism of hormonal age des 17-cetosteroides sanguins. Presse med. glucuronide formation. Recent Progr. Hormone 1955, 63, 1877. Res. 1956, 12, 134. 10. Bondy, P. K., Cohn, G. L., Herrmann, W., and 26. Lehtinen, A., Nurmikko, V., and Hartiala, K. Du- Crispell, K. R. The possible relationship of etio- odenal glucuronide synthesis. Acta chem. scand. cholanolone to periodic fever. Yale J. Biol. Med. 1958, 12, 1585. 1958, 30, 395. 27. Klyne, W. The Chemistry of the Steroids. London, 11. Bondy, P. K., Cohn, G. L., and Castiglione, C. Etio- Methuen, 1957, p. 32. cholanolone fever, a new clinical entity. Trans. 28. Barton, D. H. R. The stereoclhemistry of cyclohexane Ass. Amer. Phycns 1960, 73. In press. derivatives. J. chem. Soc. 1953, 1027. 12. Slaunwhite, W. R., Jr., and Sandberg, A. A. Metabo- 29. Bongiovanni, A. M., and Eberlein, W. R. The renal lism of 4-C'4-testosterone in human subjects. III. clearance of neutral 17-ketosteroids in man. J. clin. Fate of androsterone and etiocholanolone. J. clin. Endocr. 1957, 17, 238. Endocr. 1958, 18, 1056. 30. Kellie, A. E., and Smith, E. R. Renal clearance of 13. Fukushima, D. K., Bradlow, H. L., Dobriner, K., 17-oxo steroid conjugates found in human periph- and Gallagher, T. F. The fate of testosterone in- eral plasma. Biochem. J. 1957. 66. 490.