i\NALYTICAL BIOCHEMISTRY 13, 85-90 (1965)

Absorption Spectra of As-3P-Hydroxy Steroids

in Two Reagents’

ALFRED M. BONGIOVANNI

From the Children’s Hospital of Philadelphin, Department of Pediatrics, School of Medicine, University of Pennsylvania, Philndclphio. Pcnnsylunrria

Received April 1, 1965

In the course of studies of A5-3P-hydroxy steroids in human urine, the absorption spectra of these compounds in two sulfuric acid reagents proved of value in arriving at their identification (1). Wilson (2) has previously reported spectra of 11 steroids in sulfuric acid reagents. Smith et al. (3) have compiled spectra for a large number of steroids in sulfuric acid. This report extends these observations to 10 additional compounds and includes 5 previously described. These methods arc helpful for the partial characterization of steroids in small quantity and as noted below one pair of isomers may be distinguished by these techniques.

MATERIALS AND METHODS

Reagents and Equipment

Concentrated sulfuric acid: Mallinckrodt, analytical grade. Ethanol, absolute: Commercial Solvents Corporation. Oertel reagent (4) : 2 vol concentrated sulfuric acid was added to 1

vol of 95% absolute ethanol with cooling. Cuvets: Pyrocell Corporation, 1.5-ml capacity, lo-mm light path. Spectrophotometer: Beckman model DB with Photovolt model 43

. varicord electronic recorder, set with a scanning speed of 40 mp/min covering the region from 220 to 700 mp.

Steroids: Some pure steroids employed were obtained from urine and fully characterized as described (I). Others were purchased from Mann Research Laboratories, 136 Liberty St., New York, N. Y., and Steraloids, Pawling, N. Y. Generous gifts of certain compounds were received from Dr. .J. .J. ‘Schneider and Dr. D. K. Fukushima.

‘These studies have been supported by grants from the U. S. P. H. S. National Institutes of Health #HD 00371-11 and Tl-AM-5197.

85

TABL

E 1

M

Ster

oid

Tiie

(m

ins.

)

oerte

l re

agen

t

Max

ima

mw

(K

)* M

inim

a Ti

me

WI

(IO

(min

s.)

5

Min

ima

WI

(K)

3B-h

ydro

~-A~

andr

oste

ne-1

7-on

e 21

0 4%

ndro

sten*

3&17

&diol

30

90

38,1

7~ihy

drox

y-As

-and

roste

ne-1

6-

one

30

240

38,1

6odih

ydro

xy-A

~~nd

roste

nel7-

on

e 30

90

38,7

0-dih

ydro

xy-A

s--a

ndro

stene

-17-

on

e 30

180 30

240

38,1

7adih

ydro

xy-A

s-pr

egne

ne-2

0-on

e 15

90

Ai

pRgn

en43

8,17

a.20

8-tri

ol 30

305(

412)

, 40

5(27

65)

325(

460)

, 40

5(21

57),

485(

60)1

29

5(76

5),

405(

1670

). 48

5(17

38)1

, 52

5(26

0)1,

600(

313)

33

0(21

9)1.

40

5(22

25)

330(

246)

1,

405(

2105

). 60

5(16

5)

375(

1672

), 40

0(17

75).

610(

617)

, 65

5(58

7)1

255(

587)

, 37

5(13

80).

400(

572X

61

0(17

15).

655(

1334

)1

260(

220)

, 29

3(16

5).

570(

1000

) 25

5(22

9),

295(

201)

, 57

0(94

0)

335(

651)

. 40

7(70

0).

530(

615)

. 61

0(31

8).

690(

149)

1 33

5(48

6).

410(

658)

. 49

5(58

0)1,

526(

608)

, 60

8(43

8)

265(

644)

, 40

5(23

80),

485(

765)

1 26

5(55

3),

405(

2345

), 49

0(24

7),

525(

213)

1

310(

366)

1,

345(

459)

. 42

5(10

90).

525(

899)

, 61

5(11

18)

340(

318)

31

5(37

4),

450(

58)

335(

365)

, 44

5(14

8),

545(

226)

325(

223)

330(

270)

, 44

5(54

7)

385(

1600

), 44

0(22

0)

315(

470)

, 39

5(57

2),

44om

o)

280(

155)

, 32

5(W

)

255(

248)

, 37

0(44

5),

470(

382)

, 57

5(29

0),

650(

141)

37

0(35

4),

470(

495)

. 50

5(57

4),

565(

365)

325(

281)

325(

306)

, 45

5(20

4).

505(

212)

320(

365)

, 37

5(44

6),

465(

473)

. 55

0(58

5)

210

305(

1338

), 40

0(24

25)

340(

453)

. 44

5(11

0)

240

310(

875)

. 40

8(13

00),

440(

845)

1,

545(

226)

1 34

5(42

3),

4300

06)

30

240 240 30

130

240 240 30

385(

1070

)1,

405(

1250

). 48

0(21

30),

595(

200)

1,

655(

267)

37

5(10

40).

405(

775)

1.

430(

510)

1,

595(

640)

, 66

5(58

7H

450(

173)

510(

133)

330(

561)

,420

(614

), 46

0 (3

74)

270(

102)

, 37

0(15

2).

405(

144)

. 55

5(13

4L

600(

121)

1.

660(

87)1

23

1(45

7)1,

29

6(29

3),

381(

604)

23

0(65

0)1,

29

5(73

0).

393(

192)

320(

84),3

85(1

39),

450(

84)

250(

220)

,324

(145

)

249(

302)

,330

(110

)

310(

334)

, 41

0(18

6).

450(

139)

1 37

0(15

1),4

00(1

77)

305(

791)

, 34

0(71

2)1.

41

0(10

00),

445(

802)

1,

490(

836)

, 54

0@52

)1

335(

820)

, 40

5(11

90),

445(

597)

1,

495(

657)

. 60

0(13

1)1

360(

587)

, 46

0(76

9)

360(

676)

, 46

5(56

0)

38,1

6a-d

ihydr

oxy-

As-p

regn

ene-

20-o

ne

30

38,1

7a,2

1-tri

hydr

oxy-

46-~

regn

ene-

20

-one

18

0

Al-p

regn

ene-

38,1

7~,2

0,21

-tetro

l 30

240

3,9,

11@

,17a

,21-

tetra

hydr

oxy~

AL

preg

nene

-20-

one

30

210

30

180

38,2

1-dih

ydro

uy-u

s-pr

egne

ne-2

0-on

e

90

30

90

180 30

210

310(

372)

1,

345(

459)

. 42

1(64

5),

522(

1562

), 61

5(11

10)

310(

382)

,415

(123

5),

520@

30),5

75C3

87)1

. 61

5(42

0)

310(

386)

,405

(933

). 42

1(80

2)1,

51

5(13

04),

5W42

4)1,

615(

466)

29

0(98

8),4

05(2

380)

, 48

5(44

3),6

05(1

01)

375(

366)

, 45

5(61

9),

555(

772)

255(

270)

,325

(326

), 46

5(49

0),

560(

350)

. 59

5(38

7)

335(

240)

,455

(690

), 57

5(41

9),5

95(4

29)

325(

517)

,440

(277

), 54

5(92

)

290(

1190

), 40

5(21

18),

325(

535)

,440

(312

), 48

5(53

5).6

05(2

77)

545(

203)

315(

1120

),405

(241

5),

475(

745)

, 57

5(86

0)1,

81

5(98

5)

310(

1308

),400

(997

), 47

5(10

60),

575(

2010

)1,

615(

2428

) 32

0(53

4),4

05(1

475)

, 48

5(29

6),5

30(2

50)1

305(

106)

,405

(253

5).

480(

674)

30

5(12

40),4

05(2

220)

, 48

0(87

1)

310(

753)

,350

(767

), 41

0(12

20).5

10(1

182)

1,

550(

1250

) 30

5(90

0),

470(

1170

)1,

510(

1250

), 55

5(11

33)1

28

0(41

0)1,

40

1(23

10),

480(

157)

, 52

0(12

6)1

345(

904)

.435

(530

), 51

0(43

6),

590(

857)

355(

701)

,430

(639

), 51

0(87

4)

350(

409)

,440

(247

), 50

5(24

9)

335(

885)

,440

(594

)

345(

924)

,440

(739

)

335(

715)

,360

(747

), 44

5(84

5),

531(

1200

)

360(

590)

440(

126)

260(

505)

, 37

0(12

40)1

, 40

0076

5),

520(

651)

, 60

0(15

7)1

320(

440)

,435

(242

)

210 30

30

130

180

180 18

0 30

If30

310(

774)

.335

(675

). 40

5(@

33),

445(

760)

1,

495(

657)

, 5%

X336

) 31

0(50

5),3

35G

O5)

, 40

5(96

5),

445@

93)1

. 49

5(43

9)

295(

684)

, 34

0(63

3)1,

38

5(&3

1),4

05(7

35),

485(

470)

, 53

5(29

4).

640(

40)1

29

5(72

4),

370(

882)

, 40

5(62

9)1,

475(

544)

, 53

5(29

8)1,

65

0(55

)1

308(

1110

), 38

0(90

9),

470(

985)

, 50

0(73

6)1

320(

775)

1,

335(

779)

, 40

5(82

0),4

85(4

21),

660(

106)

1

265(

675)

1,31

5(66

5),

400(

1465

),495

(772

) 28

5(96

6)1,

33

4(15

30),

354(

1470

)1,

370(

1385

)1,

405(

6YJ)

,480

(346

)1

285(

1200

),371

(125

0).

405(

975)

,484

(630

)1

325(

662)

,360

(630

), 43

0(76

5),4

75(6

30).

560(

308)

32

0(49

5),3

60(3

93),

435(

419)

,465

(360

)

320(

595)

, 40

0(72

7),

435(

346)

,520

(286

)

320(

610)

,430

(426

), 58

0(81

)

355(

840)

,425

(675

)

365(

520)

,465

(414

), 57

0(15

2)

290(

647)

,345

(531

), 45

0(70

5)

360(

1410

),390

(630

)

320(

1060

),386

(890

)

The

Tabl

e ind

icate

s wa

vele

ngth

an

d in

pare

nthe

ws

abso

rban

ce

of

0.1

M

conc

entra

tion

(K)*.

I

= inf

lectio

n.

ASSAY FOR CYCLODEXTRIN TRAKSGLCCOSIDASE 95

subtilis enzyme has attributes b, c, and d listed above, it produces a significantly smaller amount of reducing sugar than the other enzymes and hence limits the sensitivity of the method. Because the sensitivity of reducing sugar assays is frequently the limiting factor in assessing initial steady-state velocities, we suggest the use of pig pancreatic cY-amylase for enzyme kinetics at a level of 0.125 mg/ml as the hydrolytic enzyme (cf. Fig. 3) even though the extent of hydrolysis is somewhat dependent upon the enzyme concentration. Thus for precise analysis it will be imperative that the amylase concentration be carefully controlled for each run, but this is a small price to pay for high sensitivity. Obviously a glucosidase would give the highest reducing value, but unfortunately none of sufficient purity and activity is commercially available. The increase in t,he color yield at high (Y- and /3-amylase concentration may result from the slow cleavage of the small oligosaccharides resulting from the degra- dation of the maltoheptaose (11, 12). Under the conditions of the assay it was found that none of these enzymes produces a significant amount of hydrolysis of either the cyclic dextrin or the acceptor, methyl-Y-n- glueoside.

Endogenous Activators or Inhibitors. To test for the presence of endogenous activators or inhibitors in the crude enzyme, the activity was studied as a function of CT concentration at, 25” and 35°C. The linearity of the two curves in Fig. 4 indicates the absence of observable endogenous activators or inhibitors in the CT preparation.

Time Course of Reaction. The time course of the production of reduc- ing sugar in the CT digest is depicted in Fig. 5. The data were obtained

0 0.2 0.4 06 CT IN ARBITRARY “NITS

FIG. 4. Activity vs. CT concentration at 25” (A) and 35°C (0). Except for change

in temperature, standard assay conditions were employed.

ABSORPTION SPECTRA

d-PREGNENE-38,174,20-TRIOL

I5 - 90 MIN fi -2QALPHA ! I

FIG. 2. Differing spectra of isomers of pregnenetriol in the Oertel reagent, 0.1 M concentration. at 90 mins.

simple means for the partial identification of these compounds after their isolation from mixtures. Special features of their absorption spectra in reagents of several types containing sulfuric acid have been informative. Characteristics of staining on paper chromatograms have also been helpful (6, 7).

As noted earlier, most but not all steroids with this grouping demon- strate high peaks of absorption in the region of 400415 rnp in the Oertel reagent. It would appear that an oxygen at C-7 whether ketonic, as re- ported by Wilson (Z), or a hydroxyl as noted herein deIetes this typical peak. In addition, among the two isomers of pregnenetriol the 20~~ demonstrates a notable peak near 410 rnp although not the major one, whereas the 2Ob has a very weak and barely detectable maximum in this region (Fig. 2). This characteristic is somewhat dependent on time and in some instances is better observed earlier, since it fades thereafter while other peaks are augmented (Fig. 1). The spectra in concentrated sulfuric acid are considerably different from those in the Oertel reagent and the combination of both tests reinforces the ability to identify these compounds. In sulfuric acid there is often a maximum in the region of 406415 rnp but it is frequently surpassed by other peaks.

SUMMARY

Absorption spectra of several A5-3&hydroxy steroids in concentrated sulfuric acid and the Oertel reagent have been reported. These spectra, when determined in two different reagents and at two time intervals, may aid in identification and yet require little material. The charac-

90 ALFRED M. BONGIOVANNI

teristic alterations in the spectra with time are often sufficient to render characterization more exact. It has been demonstrated that one pair of isomers may be distinguished by these methods.

REFERENCES

1. BONGIOVANNI, A. M., J. Clin. Invest. 41, 2086 (1962). 2. WILSON, H., Anal. Biochem. 1, 402 (1960). 3. SMITH, L. L., AND BERNSTEIN, S., in “Physical Properties of the Steroid Hor-

mones” (L. L. Engel, ed.). Macmillan, New York, 1963, p. 321. 4. OERTEL, G. W., AND EIK-NES, K., Anal. Chem. 31, 98 (1959). 5. BERNSTEIN, S., AND LENHARD, R. H., J. Org. Chem. 18, 1146 (1953). 6. FUKUSHIMA, D. K., AND GALLAQHER, T. F., J. Biol. Chem. 229, 85 (1957). 7. EBERLEIN, W., J. Clin. Endocrinol. Metab. 25, 288 (1965).