Further Studies on the Origin of Pregnanetriol in Adrenal Carcinoma
DAVID K. FUKUSHIMA, PH.D., H. LEON BRADLOW, PH.D., LEON HELLMAN, M.D. AND T. F. GALLAGHER, PH.D. Sloan-Kettering Institute for Cancer Research and Montefiore Hospital, New York City
I N AN EARLIER study, the endog- enous production of pregnanetriol (PT) from 17-hydroxyprogesterone1 in a patient with metastatic adrenal carci- noma was found to be small compared with the large amount of that metabolite present in the urine (1). It was suggested, therefore, that 38,17-dihydroxy-45-preg- nene-20-one (17-hydroxypregnenolone) was a major precursor of PT in that pa- tient and, based on results obtained from the metabolism of tritium-labeled 17- hydroxypregnenolone in other subjects, an estimate of the amount secreted was derived. However, the wide range (54 mg-2 g/day) of the approximation prompted the present study.
17-Hydroxyprogesterone-4-C14 and 38,17-dihydroxy-45-pregnene-20-one-7a- H3 were administered simultaneously to a patient with adrenal carcinoma. The specific activities of the C21 metabolites
Received September 24, 1962.
This investigation was supported in part by a grant from the American Cancer Society and a research grant (CY-3207) from the National Cancer Institute of the National Institutes of Health, USPHS.
1 The following trivial names and abbrevia- tions are used in this paper: pregnanetriol, PT =pregnane-3a,17,20a-triol; 17-hydroxypreg- nanolone, 17-OHP =3a,17-dihydroxypregnane- 20-one; pregnenetriol, 45 -PT = 45-pregnene- 36,17-20a-triol; 17-hydroxypregnenolone, 45- 17-OHP=36,17-dihydroxy-45-pregnene-20-one; 17-hydroxyprogesterone-17-hydroxy-44-preg- nene-3,20-dione; androsterone = 3a-hydroxy- androstane-17-one; etiocholanolone =3a-hy- droxyetiocholane-17-one; dehydroisoandroster- one =38-hydroxy-45-androstene-17-one.
₼ ABSTRACT. The biotransformation of 17- hydroxyprogesterone-4-C14 and 38,17-dihy- droxy-45-pregnene-20-one-7a-H3 administered simultaneously was studied in a patient with adrenal carcinoma. The specific activities of the metabolites studied varied widely from each other. 38,17-Dihydroxy-45-pregnene-20- one appeared to be the major precursor of urinary pregnane-3a,17,20a-triol in this pa- tient; the evidence indicated that compound (s) other than 36,17-dihydroxy-45-pregnene-20- one contributed to 45-pregnene-38,17,20a- triol. 38,17-Dihydroxy-45-pregnene-20-one- 7a-H3 was also studied in a “normal” subject.
showed clearly that the precursors stud- ied were not the only sources of the metabolites examined. Cleavage of the side chain to yield 17-ketosteroids oc- curred to only a limited extent, as previ- ously described for each precursor given separately (1-3). Judging from the very low specific activities of these 17-keto- steroids, 17-hydroxyprogesterone and 17-hydroxypregnenolone are minor pre- cursors of these metabolites.
A study of the metabolism of 33,17- dihydroxy-45-pregnene-20-one in a nor- mal subject is included in this report, since previous isotopic studies have been with patients with adrenal tumor (2, 3). The results show that the normal subject metabolized the tracer in almost the same manner as the carcinoma patient.
Materials and Methods
A solution of 36, 17-dihydroxy-45-preg- nene-20-one-17a-H3 (4.74×10º cpm, 5.2 µg) and 17-hydroxyprogesterone-4-C14 (1.64 X106 cpm, 35 µg) in 1 ml of propylene glycol
| Day | Isotope | Enzymic hydrolysate A | Cold acid hydrolysate B | Hot acid hydrolysate C | ||||
|---|---|---|---|---|---|---|---|---|
| 104 cpm | % dose | 104 cpm | % dose | 104 cpm | % dose | |||
| 1 | C14 | 51.0 | 31.1 | 5.5 | 3.3 | 5.5 | 3.3 | |
| H3 | 73.4 | 15.5 | 77.0 | 16.3 | 15.2 | 3.2 | ||
| 2 | C14 | 7.0 | 4.3 | 0.79 | 0.5 | 1.4 | 0.9 | |
| H3 | 23.3 | 4.9 | 5.56 | 1.2 | 2.6 | 0.5 | ||
| 3 | C14 | 1 3.0 | 1.8 | 0.82 | 0.5 | |||
| H3 | 15.3 | 3.2 | 4.3 | 0.9 | ||||
| 4 | C14 | 1.0 | 0.6 | 0.02 | 0.01 | |||
| H3 | 5.5 | 1.2 | 1.5 | 0.3 | ||||
| 5 | C14 | 0.3 | 0.2 | |||||
| H3 | 1.5 | 0.3 | 1.5 | |||||
| Total | C14 | 62.3 | 38.0 | 7.1 | 4.3 | 6.9 | 4.2 | |
| H3 | 119.0 | 25.0 | 88.4 | 18.6 | 17.8 | 3.8 | ||
was administered intravenously over a period of 10 min to a 12-yr-old girl (CN) with metastatic adrenal carcinoma. Com- plete urine collections were made for the 5 following days. Her daily creatinine excre- tion was 0.7 g during the period of study. Each day’s urine was treated with @-glu- curonidase2 at pH 5.0 for 5 days at 37 C, continuously extracted with ether for 48 hr, and neutral steroid extracts (A) were pre- pared. The residual urine and the aqueous washes following the enzyme treatment were made to IN with sulfuric acid, continously extracted with ether for 48 hr, and neutral extracts (B) were prepared. Residual urines and aqueous washes following B from days 1 and 2 were re-acidified to 1N, boiled for 30 min, and continuously extracted with ether for 48 hr. Neutral steroid fractions (C) were prepared from these extracts. The radio- activity in the various fractions is reported in Table 1. Extract A represented material principally conjugated as glucosiduronate; extract B represented material chiefly con- jugated as sulfate. It is recognized that the method of hydrolysis and extraction would lead to the inclusion of some material con- jugated as sulfate in extract A, but it is believed there would be only a minor amount of glucosiduronate in extract B.
Half of each of the enzyme hydrolysates
A were combined, recounted (3.27×10 cpm-C14, and 6.03 ×105 cpm-H3) and treated with Girard’s reagent T. The ketonic and nonketonic fractions were separated into a and ß subfractions with digitonin. Half of extracts B from each day were combined (3.7×103 cpm-C14, and 4.23×105 cpm-H3) and processed in the same manner. The radioactivity in the various fractions in terms of the total extract is recorded in Table 2. A portion of each subfraction was used for analysis of the endogenous steroids; the results are reported in Table 3. The ex- tracts C were not examined because of known artifact formation under the condi- tions of hydrolysis. The neutral steroid frac- tions were analyzed separately for andros- terone, etiocholanolone, dehydroisoandros- terone, 45-PT, 17-OHP and PT. The ex- tracts were separated by chromatography on paper, using the systems described in this as well as in another report (4). The 17- ketosteroids were measured by the Zimmer- mann reaction (5) and the triols by vapor phase chromatography (6) on the material eluted from the paper. The triols were also measured by the method of Cox (7), and satisfactory agreement was obtained by the 2 procedures. The value for 17-OHP was obtained by vapor phase chromatography after separation on paper (6).
In order to obtain the specific activity of androsterone, etiocholanolone and 3a,17- dihydroxypregnane-20-one (17-OHP), re- ported in Table 4, the a ketonic fraction of extract A was chromatographed on silica gel
2 B-Glucuronidase, known as Ketodase, was obtained from Warner-Chilcott Laboratories, a division of Warner-Lambert Pharmaceutical Company, New York, N. Y.
| Enzymic hydrolysate A | Cold acid hydrolysate B | |||
|---|---|---|---|---|
| C14 105 cpm | H3 105 cpm | C14 105 cpm | H3 105 cpm | |
| Crude neutral extract | 6.54 | 12.06 | 0.74 | 8.46 |
| Ketonic fraction | 4.10 | 3.82 | 0.33 | 1.92 |
| a | 3.94 | 2.94 | 0.27 | 0.92 |
| B | 0.17 | 0.87 | - | 0.82 |
| Nonketonic fraction | 2.60 | 8.16 | 0.35 | 5.01 |
| a | 2.66 | 5.76 | 0.33 | 0.92 |
| ₿ | 0.06 | 2.77 | 0.06 | 3.47* |
* Portion lost.
containing ethanol, as previously described (8). The fractions containing androsterone were combined, rechromatographed on acid washed alumina and then on paper in Sys- tem A for 16 hr to give 4 mg of product. Sublimation and crystallization from acetone afforded androsterone which was measured by the micro-Zimmermann method (5), and the specific activity was calculated from this value. The fractions containing etio- cholanolone were combined, purified by sub- limation in vacuo and recrystallized from hexane to yield 11 mg of etiocholanolone, mp 153.5-154 C; 122 cpm-C14/mg and 384 cpm-H3/mg. A second recrystallization from ether yielded 6 mg of etiocholanolone with the same mp and a specific activity of 118 cpm-C14/mg and 386 cpm-H3/mg. The aver- age specific activity is reported in Table 4. The fractions containing 17-OHP were com- bined and chromatographed on paper in System B for 4 hr. The product con- tained 3a,116-dihydroxyandrostane-17-one, as judged by both the Zimmermann reaction and vapor phase chromatogram. The amount of 17-OHP present in the sample measured
by vapor phase chromatography (6) was 2.6 mg. The specific activity based on this weight of steroid, assuming there was no contribution of radioactivity from other metabolites, was 11,300 cpm-C14/mg and 2,700 cpm-H3/mg. A portion of this sample of 17-OHP was reduced with sodium boro- hydride. The product was chromatographed on paper in System C for 16 hr and preg- nene-3a,17,200-triol so obtained was ana- lyzed both by vapor phase chromatography (11,400 cpm-C14/mg, and 2,200 cpm-H3/mg) and by the acetaldehydrogenic method of Cox (7) (12,800 cpm-C14/mg, and 2,400 cpm-H3/mg). The average specific activity derived for 17-OHP was 11,800 cpm-C14/ mg, and 2,400 cpm-H3/mg (Table 4).
The a nonketonic fraction from extract A was chromatographed on silica gel (8). The fractions containing PT were combined and recrystallized from methanol and from ethyl acetate, and 17 mg of product, mp 250-251 C, was obtained. The triol was acetylated and the diacetate was recrystallized from ether-petroleum ether and from ethanol to give mp 160.5-163 C and specific activities of
| Steroid | mg/day | Total radioactivity (5 days, A +B) | |||||
|---|---|---|---|---|---|---|---|
| Neutral extract | Total A +B | 103 cpm | % of dose | ||||
| A | B | C14 | H3 | C14 | H3 | ||
| Androsterone | 2.4 | 2.9 | 5.3 | 6.1 | 21.2 | 0.4 | 0.5 |
| Etiocholanolone | 23.7 | 3.3 | 27.0 | 16.2 | 52.0 | 1.0 | 1.1 |
| Dehydroisoandrosterone | 9.0 | 56.3 | 65.3 | - | 81.5 | 1.7 | |
| PT | 16.0 | 16.0 | 144. | 312. | 8.8 | 6.6 | |
| 45-PT | 10.4 | 32.8 | 43.2 | 531. | 11.2 | ||
| 17-OHP | 2.2 | - | 2.2 | 130. | 26.4 | 7.9 | 0.6 |
1,440 and 1,430 cpm-C14/mg, and 3,120 and 3,160 cpm-H3/mg, from successive crystal- lizations of the diacetate. The average spe- cific activity for PT was calculated to be 1,800 cpm-C14/mg and 3,900 cpm-H3/mg (Table 4). In order to check these values, PT diacetate was hydrolyzed and then oxidized with periodic acid. Etiocholanolone thus obtained was chromatographed on paper in System A for 17 hr and sublimed in vacuo. After recrystallization, the product had mp 151-152 C; 2,100 cpm-C14/mg, and 4,500 cpm-H3/mg, corresponding to 1,800 and 3,950 cpm/mg, respectively, for PT.
Chromatography on silica gel of the nonketonic fraction obtained from extract A afforded 19 mg of 45-PT. Recrystallizations from methanol-ethyl acetate and ethyl ace- tate gave the unsaturated triol, mp 223.5- 225 C, which was shown to be homogeneous by thin layer chromatography on silica gel G in methanol-ethyl acetate (1:9). Acetylation of this product afforded the 3,20-diacetate which was recrystallized from methanol, mp 198-200 C; 2,680 cpm-H3/mg. Further re- crystallizations from methanol and ethanol gave 45-PT diacetate, mp 203-204.5 C, with specific activity of 2,650 and 2,640 cpm-H3/ mg, respectively. The average specific activ- ity of 45-PT was calculated to be 3,300 cpm- H3/mg.
The ß nonketonic fraction from extract B by the same treatment described above for extract A yielded 45-PT diacetate, mp 203- 204 C, and specific activities of 1,740 and 1,760 cpm-H3/mg, corresponding to an aver- age specific activity of 2,200 cpm/mg for 45-PT. The average specific activity calcu- lated for the total 45-PT found in both ex- tracts A and B was 2,460 cpm-H3/mg. The compound from both extract A and B was devoid of C14.
| Specific activity, cpm/mg | ||
|---|---|---|
| Enzymic hydrolysate A | C14 | H3 |
| PT | 1,800 | 3,900 |
| 45-PT | 3,300 | |
| 17-OHP | 11,800 | 2,400 |
| Etiocholanolone | 120 | 385 |
| Androsterone | 230 | 800 |
| Cold acid hydrolystate B 45-PT | - | 2,200 |
| Dehydroisoandrosterone | - | 250 |
The @ ketonic fraction obtained from ex- tract B was chromatographed on silica gel containing 40% of ethanol, and 50 mg of dehydroisoandrosterone was obtained by elution with 1% ethanol in petroleum ether- methylene chloride (1:1). In order to remove traces of saturated steroids from this com- pound, the material was treated with 1 ml of formic acid and 0.2 ml of 30% hydrogen peroxide (9) at room temperature for 16 hr. The product was isolated and saponified with 0.1 ml of 25% aqueous potassium hy- droxide in 3 ml of methanol under reflux for 10 min. The neutral product was acetylated and chromatographed on alumina to yield 20 mg of 36,66-diacetoxy-5x-hydroxyandros- tane-17-one. The diacetate was recrystallized from acetone-isooctane, mp 215-215.5 C; [a]D22+1.2 (acetone); 177 cpm-H3/mg. A second recrystallization gave a product with specific activity of 178 cpm-H3/mg, corre- sponding to 250 cpm-H3/mg for dehydroiso- androsterone (Table 4). The product was devoid of C14.
The total radioactivity present in each metabolite was calculated from the respec-
| Day | Urine | Neutral Extracts | ||||||
|---|---|---|---|---|---|---|---|---|
| 105 cpm | % dose | A | B | C | ||||
| 105 cpm | % urinary H3 | 105 cpm | % urinary H3 | 105 cpm | % urinary H3 | |||
| 1 | 23.20 | 37.1 | 9.31 | 40.1 | 5.38 | 23.2 | 1.66 | 7.1 |
| 2 | 15.60 | 25.0 | 2.93 | 18.8 | 1.25 | 7.1 | 0.43 | 2.8 |
| 3 | 5.75 | 9.2 | 2.05 | 35.7 | 0.83 | 14.4 | ||
| 4 | 0.90 | 1.4 | ||||||
| Totals | 45.45 | 72.7 | 14.29 | 31.5 | 7.46 | 16.4 | 2.09 | 4.6 |
| Wt of carrier mg | Specific activity cpm/mg | Total activity cpm | % Extract | |
|---|---|---|---|---|
| Extract A (299,000 cpm) | ||||
| 17-OHP | 46.8 | 338 | 15,800 | 5.3 |
| PT | 46.5 | 1,320 | 61,300 | 20.4 |
| 45-17-OHP* | 44.0 | 59 | 2,600 | 0.9 |
| 45-PT | 47.9 | 1,150 | 55,100 | 18.4 |
| Extract B (143,000 cpm) | ||||
| PT | 44.0 | 41 | 1,800 | 1.3 |
| 45-17-OHP* | 49.1 | 19 | 900 | 0.6 |
| 45-PT | 49.5 | 1,110 | 55,000 | 39.0 |
* 38, 17-dihydroxy-45-pregnene-20-one.
tive H3- and C14-specific activity and the total endogenous production of each com- pound during 5 days after administration of the tracer steroids. The results are shown in Table 3.
Normal Subject. A solution of 36,17-dihy- droxy-45-pregnene-20-one-7a-H3 (6.25×106 cpm, 4.5 µg) in 90 g of 5% glucose and a small amount of ethanol was administered intravenously over a period of 10 min to a 58-yr-old man (FD) whose daily creatinine excretion was 1.3 g. All the urine was col- lected for 4 days and counted. The first 3 days’ urine was processed as in the preceding study and the results are recorded in Table 5.
For reverse isotope dilution studies, 20% portions of each daily neutral steroid extract A were combined. The carrier steroids in the amount shown in Table 6 were added. Three carrier steroids in the amount shown in Table 6 were also added to the combined 20% por- tions of the daily extracts B. Extract C was not examined.
Each extract plus carrier was dissolved in 8 ml of ethanol and separated into a and B fractions with 400 mg of digitonin in 12 ml of 80% ethanol. Each subfraction was chromat- ographed on 80 g of silica gel containing 32 ml of ethanol. Dihydroxy ketones were eluted with 3% and triols with 5% ethanol in meth- ylene chloride. The column eluates were examined by paper chromatography and appropriate fractions were combined. Since the compounds were not sufficiently soluble in the phosphor solution, they were acety- lated and recrystallized repeatedly from ethanol and methanol to give acetates with specific activities which were constant through at least 2 successive crystallizations.
The results, calculated to the “free” steroid, are recorded in Table 7. Only small amounts of radioactivity were obtained with 1% ethanol in methylene chloride, in which androsterone, etiocholanolone and dehydro- isoandrosterone were eluted.
Counting Procedures. Tritium-labeled sam- ples were counted in a Packard Tricarb man- ual liquid scintillation counter equipped with model 314E electronics, with the high voltage set at tap 7.50 and the pulse height discrimi- nators 10-100 volts with the gain at 100. Toluene containing 6 g/liter of 2-phenyl-5- (4-biphenylyl)-1,3,4-oxadiazole (PBD), and 40 mg/liter of 1,4-di-[2-(5-phenyloxazolyl)]- benzene (POPOP) was used for counting most samples. Quenching was corrected by addition of internal standards. Aqueous samples were counted in diotol (10), a solvent composed of 1 liter of dioxan, 1 liter of tolu- ene, 600 ml of methanol, 208 g of naphtha- lene, 13 g of diphenyloxazole and 260 mg of 1,4-di-[2-(5-phenyloxazolyl)]-benzene. The results were normalized to correct for the difference in counting efficiencies of the 2 scintillant solutions.
Samples with 2 isotopes were counted in the same scintillant mixture used above. The high voltage was set at tap 9.25. The pulse height discriminators in channel A were set at 30-90 volts, with the gain set at 100. The pulse height discriminators in channel B were set at 40-100 volts, with the gain set at 14. Under these conditions, 99.4% of the tritium count was in channel A and 0.6% in channel B. Eighteen and four-tenths per cent of the C14 counts were in channel A and 81.6% in channel B. The H3- and C14-activities were calculated from the usual simultaneous
equations (11) and the distribution of counts in the 2 channels. The results were normal- ized against a sample of the dose mixture to correct for any minor fluctuations or drift in the instrument.
Paper Chromatography Systems :
System A: 2,2,4-Trimethylpentane : meth- anol:water (10:10:1).
System B: 2,2,4-Trimethylpentane : tolu- ene (1:3), methanol:water (4:1).
System C: 2,2,4-Trimethylpentane : tolu- ene (3:5), methanol:water (4:1).
Discussion
This study of steroid transformation in metastatic adrenal carcinoma shows clearly a) the H3- or C14- specific activity of each metabolite was different; b) the H&-specific activity of “sulfate” 45-PT was distinctly lower than that of “glucu- ronic” 45-PT; c) the H3-specific activity of 45-PT in its two conjugates was lower than that of PT in spite of the fact that the saturated triol was derived from at least one other precursor. These unex-
| Metabolite | neutral extracts % of total (A +B) | % of dose |
|---|---|---|
| 17-OHP | 3.7 | 1.3 |
| 45-17-OHP | 0.8 | 0.3 |
| PT | 14.3 | 5.1 |
| 45-PT | 25.4 | 8.8 |
pected results are shown in Fig. 1, with suggested metabolic paths. If the 17- hydroxyprogesterone (I) mixed com- pletely with the endogenously secreted compound, it should have been metabo- lized by route A to 17-OHP (III) and PT (IV), each with the same specific activity before dilution from other precursors. Similarly, 17-hydroxypregnenolone-H3 (II), after mixing, would be transformed by route B to III and IV, each with the same H3-specific activity before dilution from other precursors. If it be presumed that the conversion of 45-PT(V) to PT is negligible, it is possible to calculate the amount and respective H3- and C14- specific activity of these labeled metabo-
CH3 1
CH3
C= O
!
C = 0
OH
OH
0
HO
CH3
I
II
c
H-C-OH
OH
A
B
HO
CH3 1
CH3
V
C=0
1
H-C-OH
G, 3,300 H3
OH
OH
S, 2,200 H3
HO
* 2,460 H3
H
HO
H
III
IV
G,
2,400 H3
3,900 H 3
11,800 C14
G,
1,800 c14
lites before dilution with each other from the general equation:
Total radioactivity =total weight of
metabolite X specific activity. (1)
Application of this equation to the present problem affords:
RI= WXSe (2)
R2= YXSc (3)
R3= (11-W) XSh (4)
R4 = (80-Y) XSh (5)
Where
R1 and R2 = Total C14-radioactivity in 17-OHP and PT, respec- tively.
R3 and R4 =Total H3-radioactivity in 17-OHP and PT, respec- tively.
W = Weight of 17-OHP with specific activity Sc.
Y = Weight of PT with specific ac- tivity Sc.
So = C14-specific activity of metabolite before dilution with H3-metabo- lites.
Sh = H3-specific activity of metabolite before dilution with C14-metabo- lites.
The values R1 and R4 are recorded in Table 3.
Total production of 17-OHP and PT in five days was 11 and 80 mg, respectively (Table 3).
Upon solving the simultaneous equa- tions 2-5, it was found that Sc =27,300 cpm/mg, Sh =4,200 cpm/mg, W=4.7 mg and Y =5.3 mg. Thus, 4.7 mg of 17-OHP with a C14-specific activity of 27,300 cpm/mg was diluted with 6.3 mg of the metabolite having an H3-specific activity of 4,200 cpm/mg, while 5.3 mg of PT with 27,300 cpm-C14/mg was diluted with 74.7 mg of PT with 4,200 cpm-H3/mg.
These derived amounts and specific activities of metabolites may be used to estimate the turnover of 17-hydroxypro- gesterone and 17-hydroxypregnenolone during the five days of the study from the equation:
Amount of steroid
==
Total radioactivity injected S (6)
where S =Se or Sh.
For 17-hydroxypregnenolone the esti- mate is 1.13 g or 226 mg per day; for 17-hydroxyprogesterone the value is 60 mg or 12 mg per day. From these calcula- tions it can be concluded that a very significant portion of the steroid produc- tion of this tumor was not measured by the means employed in this study.
There is a further meaningful conclu- sion to be drawn from the different spe- cific activity of the “sulfate” and “glu- curonic” 45-PT, as well as the specific activity of this metabolite, which was lower than that of PT with respect to tritium. The results indicate that there was another source of 45-PT than 17-hy- droxypregnenolone in this patient. The nature of this precursor can be suggested by analogy with the results of Baulieu (12), who found that a functional adrenal carcinoma secreted dehydroisoandros- terone sulfate. It is tempting to suggest that this tumor or its metastases pro- duced the analogous 17-hydroxypreg- nenolone sulfate which could not mix with the radioactive hormone injected, but would, nevertheless, lower the spe- cific activity of the urinary metabolites. It is equally possible that the neoplastic tissue produced either conjugated or unconjugated 45-PT, or perhaps even both, which would also fail to mix but would lower the specific activity of the radioactive metabolite. Other possibili-
ties could be suggested; it is evident that the steroid production of metastatic adrenal carcinoma can be so complex that it is difficult to devise studies which will lead to a clear and meaningful con- clusion during the short time these pa- tients are available for study.
Acknowledgments
The authors gratefully acknowledge the in- valuable technical assistance of Mrs. Shirley Dobriner, Mrs. Thelma Freeman, Miss Ruth Jandorek and Mrs. Tamara Salumaa. We also wish to thank Dr. Robert S. Rosenfeld for the vapor phase chromatography results.
References
1. Fukushima, D. K., H. L. Bradlow, L. Hell- man and T. F. Gallagher, J. Clin. Endocrinol. & Metab. 22: 765, 1962.
2. Roberts, K. D., R. L. Vande Wiele and S. Lieberman, J. Clin. Endocrinol. & Metab. 21: 1522, 1961.
3. Solomon, S., A. C. Carter and S. Lieberman, J. Biol. Chem. 235: 351, 1960.
4. Fukushima, D. K., T. F. Gallagher, W. Greenberg and O. H. Pearson, J. Clin. Endocrinol. & Metab. 20: 1234, 1960.
5. Wilson, H., Arch. Biochem. 52: 217, 1954.
6. Rosenfeld, R. S., M. C. Lebeau, R. D. Jandorek and T. Salumaa, J. Chromat. 8: 355, 1962.
7. Cox, R. I., J. Biol. Chem. 234: 1693, 1959.
8. Fukushima, D. K., H. L. Bradlow, L. Hell- man, B. Zumoff and T. F. Gallagher, J. Clin. Endocrinol. & Metab. 21: 765, 1961.
9. Fieser, L. F. and S. Rajagopalan, J. Amer. Chem. Soc. 71: 3938, 1949.
10. Herberg, R. J., Anal. Chem. 32: 42, 1960.
11. Okida, G. T., J. J. Kabara, F. Richardson and V. LeRoy, Nucleonics 15: 111, 1957.
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