Photonometric correction in the determination of urinary 17-ketosteroids

566 CCA

CLINICA

CHIMICA

ACTA

4815

Photonometric** 17-ketosteroids*

correction in the determination

of urinary

Most of the calorimetric methods used in the determination of r7-ketosteroids are modifications of the basic reaction discovered by Von Bittol and adapted to steroid ketones by Zimmermann”. The reaction depends upon the development of a red-purple color with an absorption maximum at 520 nm when the steroid co~ltainillg a CH,CO configuration is reacted with nz-dinitrobenzene in an alkali. Examples of such steroids are the neutral r7-ketosteroids. Many modifications of this reaction have been made with the purpose of either simplifying it or rendering it more specific. These modifications concern primarily the type and concentration of the alcohol and alkali to be employed. In the Callow et aL3 method the alcohol concentration is IOO~/~ and the concentration of potassium hydroxide is 0.8 N, whereas in the HoltorffKoch4 method the alcohol concentration is 50% and that of the potassium hydroxide is 1.7 12’. A comparison of the two methods resulted in the finding that while the former was reasonably accurate for the assay of urine extracts, the Holtorff-Koch method did not obey Beer’s law for urine extracts. Since alcoholic potassium hydroxide solutions are relatively unstable, yielding high reagent blanks, the use of organic bases instead of potassium hydroxide has been suggested5*g. The 3-, II-, and zo-ketosteroids give some color with alkaline nz-dinitrobenzene, but much less than the r7-ketosteroids. Substitution at C-II by a hydroxy-group results in a partial suppression of the r7-keto-derived chromophore. In other words, the color equivalences of the various neutral r7-ketosteroids extracted from hydrolyzed urines differ, depending on the components that make up the existing mixtures. Brownish pigments which absorb strongly at 400 to 440 nm are formed during the Zimmermann reaction with either pure x7-ketosteroids or the residues derived from urine extracts. The latter, of course, are present in large amounts, sometimes, dependent on the type of hydrolysis, the type of solvent used for extraction and the degree of purification of the extract. Ketone groups at other positions of the steroid nucleus may also yield brown-colored pigments. The combinations of these other ketonic steroids, nonsteroidal ketones and non-ketonic, non-specific chromogens complicates estimations of urinary r7-ketosteroids. Several procedures have been utilized to correct for the nonspecific brown pigments, varying from the use of “color correction equations” to solvent extraction of the red-purple chromophore prior to spectrophotometry7~8~s. The non-ketonic material also can be separated from the ketonic steroids by the aid of Girard reagent ‘10; during the Girard purification, however, some of the chromogenic material is destroyed. In order to correct for both the non specific chromogeneity and the variable extinction co-efficients of neutral 17 ketosteroids, a simple but effective procedure is described. It was discovered that the “fading” of Zimmermann reaction mixtures was proportional to the amount of r7-ketosteroid involved, as long as the exposure to * This work was supported in part by the John A. Hartford Foundation, Inc. * * Photonometry is the name applied to photochemical reactions that can be used for the detection or determination of certain chemical substances. Clin. Chinz. Ada,

36 (1972)

566-569

BRIEF NOTES

567

Fig. I. Photonometer. A: Light source, zoo watt light bulbs; B: 2 rev./min micro-motor; C: Extcnsion of mat or shaft; D: Plastic perforated tube holders; E: Plexiglass pnrtititions; F: Cover with airvents; G: Base-board.

light was controlled in relation to time and intensity. A simple wooden box was lined with aluminum foil to create reflectance, and a 200 W light bulb was placed in front of one end and a small fan in front of the other end. This method of exposure was slightly improved by providing a turntable, increased illuminating power with a second 200 IV light bulb and air circulation as shown in Fig. I. The Plexiglass partitions (E) protect the contents of the cuvettes from evaporation due to the heat from the light bulbs. Conveniently, a timer may be inserted in the line controlling the On/Off switch of the light source. The correction procedure here proposed consists of: (I) reading the extinction at gzo nm (ES,,- I), (2) exposing the cuvettes, including reagent blank and standard(s) to the light for a predetermined period of time (i.e. IO min), and (3) repeating the reading of the extinction at 520 nm (E,,0-2). Calculate the change Es20-~-E520-z = d E,,,. Concentration of the U1;KNOWiS =

d

E,,, unknown X concentration ---

A E,,

STANDARD

STANDARD

i.e. the

E,,, is directly proportional to the amount of IT-ketosteroid present. Table I illustrates the photonometric equivalences of various compounds in comparison to the variable absorptivities of the same compounds under the conditions of assay employed. The advantage of the proposed method is apparent. In Table II the effectiveness of the procedure to correct for non-specific chromogens, as well as the elimination of most of the differences in color equivalence of representative compounds when included in urine extracts, is demonstated. In order to further substantiate our claim as to the validity of the proposed correction procedure, a number of chloroform :ethyl acetate-extracts of acid-hydrolized urines were submitted to Zimmermamr quantitation, (a) as described in the Clin. Chim. AC&Z, 36 (1972) 566-569

568 TABLE

BRIEF

XOTES

I

ABSORPTIVITIES

(E)

AND

PHOTONOMETRIC

EQUIVALEKCES

Compound

(dE&

E

Dehydroepiandrosterone ~~~~~~e~i;-Ipndrost-5-en-

IO0

6300

4

6800 5900 4000 I2 200 IO 200 IO 500 I4 800 5600 7000 9700

(3a-hydroxy-5/%androstan-I7-one) Androstan-17.one I r-OH-etiocholanolone I I-K&~-etiocholanolone Androstane-3, I 7-dione d4-Androstene-3, I 7.dione d4-i\ndrostene-3,rr,r7-trione d4-Androstcne-II-01,3,17-dione Androstan-3-01, I 7.one Etiocholane-3,r7-dione

PURE

17.KETOSTEROIDS*

AEm

(DHA) 17-one)

~;;~h~;~;;~zzandrostan-~7-one)

OF

0,

:0

O.IOj

IO0

91

0.100

95

98

0.100

95 95 90 II0 100 IO0 II5 90 95 IO0

56 64 ‘77 148 I53 215 81 I02 ‘41

0.100

0.095 0.115 0.105 0.105 O.IZO 0.095 0.100 0.105

* 25 pugof each compound, in triplicate, reacted with 0.5 ml of a reagent consisting of 2 parts of I o/0 ethanolic m-dinitrobenzene and I part of benzyltrimethyl-ammonium-methoxide (4036 in methanol) for 60 min at 25’. At the end of this period the reaction mixture uas diluted with 3.0 ml of 50% (v/v) ethanol. Measurements were made in 12 mm round cuvcttes in a B & L Mod. IOO Spectrophotomcter. TABLE

II

RECOVERY

OF

IF-KETOSTEROIDS

ADDED

qKetostwoid

TO

URINE

EXTRACTS*

Amount added (pg)

“/o mean ,.q IT-KS cow. (ref. 9)

L&g-IT-IiS

Urine extract

without

additives

-

32

30

Urine extract

without additives

-

30

Dehydroepiandrosterone

15 25

56 54

96

31 55 56

II-OH-etiocholanolone

25 25

45 46

57

I I-0-etiocholanolone I I-OH-etiocholanolonc

25 25 12.5 of each

75 7S 60.5

I r-0-etiocholanolone

12.5 of each

60.5

IS2

00 nwan

this method

52 54 57 5S

IO0 94 108

55 118

55

9S

* Acid hydrolyzed normal urine (200 ml) was brought to pH S and extracted with 2 volumes of a 2 : I mixture of chloroform and ethyl acetate. The extract was divided into 20 equal portions, recovery standard compounds were added, the solvent evaporated and the dry residues reacted as described in the footnote to Table I.

footnote to Table I; (b) as suggested by the photonometric correction procedure; and (c) following a simple column chromatographic separation and purification as proposed by Goldzieher and Axelrod12. Table III shows clearly that the photonometric correction approaches the values obtained following chromatography and uncorrected Zimmermann quantitation, while the so-called standard method’3 may lead to entirely different results. An attempt has also been made to exclude the possible interference of non-steroidal urinary constituents by treating a number of urine specimens with sodium borohydride to reduce the carbonyl groups at carbons II, 20 and 17. These IT-ketosteroid-free urines resulted in Zimmermann equivalences which were negligible, whether assayed by photonometry or by the standard procedure. Clin. Chim. Acta, 36 (1972) 566-569

BRIEF NOTES

569

TABLE III EXAMPLES OF URINARY 17.KETOSTEROID METHODS OFQUANTITATION Patient

Sex

Diagnosrs

M.&f.

I.

2. R.F.

DETERMINATIONS

BY

Total 17-KS

A@

Gushing’s syndrome preoperative spec. #I preoperative spec. #z postoperative spec. postoperative spec. following 48 h ACTH stimulation

F

Hermaphrodite

M

I4

APPLYING

THREE

DIFFERENT

mg1z.f h

!a)

(bf

180

129

26 20

155 8

99

20

I4

‘5

13

21

20

IO2

6

5

3. A.W.

Stein Leventhal syndrome

F

40

14

26

25

4. M.T.

Stein Leventhal syndrome

F

30

26

33

34

5. GM.

4 days postthyroidectomy

F

37

I

5

6

6. A.G.

Adrenogenital syndrome specimen #I specimen # 2

F

33 IO

15

7

II

16 II

7-23

“Normal

subjects“

12 i 4

24-38

“Normal

subjects”

23 f

6

9 f

2.5

19 + 3.5

10 f

2

20 rt 4

In summary, therefore, it is suggested that by the simple application of photonometry it is possible to correct effectively for the presence of non-specific chromogens, as well as for differences in absorptivity of various x7-ketosteroids when using the Zimmermann reaction on extracts from normal and pathological urines.

~ep~~~~ten~of Swgevy, Washington University School of Medicine,St. Louis, MO. 63110

HARRYW.MARGRAF LESLIE WISE WALTER F. BALLINGER

I B. VON BITTO, Ann. Chem.. 269 (1892) 377. 2 Mr. ZIMMERMANN, Z. Physiol.Chem., 233 (1935)257. 32 (1938) 1.312. 3 N. H. CALLOW, R. K. CALLOW AND C.W. EMMENS, Biochem.J., A. F. HOLTORFF AND F. C. KOCH, J. Biol. Chem., 135 (1940) 377. 5” A. &I. BONGIOVANNI, W. R. EBBRLEIN AND P. Z. THOMAS, J.CZin. Endocrinol., 17 (1957) 331, 6 S. BJERREAND R.Kr~n,Clin. Chem., 13(x967)717. W. EVI. ALLEN, 1. C&n. E~doc~~nol~, IO (rggo) 71. AND SUNDERMAX (Eds.), L+%ds aad the : R. E. PETERSON AND C.E.PIERCE, in: SUNDERMAN Stsroid Hormonrs in Clinical Medicine, Lippincott, Philadelphia, 1960. 147. 9 A. G. WARE,J. A. DEMETRION, SNOTRICA, R.SEACY, C. WALBERG AND F. Cox,CZa’n.Chem.,

5 (1959)

10 H.P. II

479.

J.Lab.Clin.M&.,45

SCHEDL.W.B.BEAN,B.M.STEVENSONANDE.R.SCHUMACHER,

(‘9.55) 191. C. E. BRICKER

AND S. S. SCHONBERG, Anal.

Chem..

30 (1958)

J.W. GOLDZIEHER AND I..R. AXELROD,J. Clin.Endocrinol. 13 R. E. PETERSON, in D. SELIGSON (Ed.),Standard Methods 12

Press, NewYord,

922. M&ab., z2(1g6z) 1234. of Cli&al Chemistry,

Academic

4 (1963) 151.

Received June 16,1971 C&t.Chim.Acta,

36 (1972)

566-569