Sterol metabolism. XXVI. Pyrolysis of some sterol allylic alcohols and hydroperoxides

627

STEROL METABOLISM. XXVI.

PYROLYSIS OF SOME STEROL ALLYLIC

ALCOHOLS AND HYDROPEROXIDESl Leland L. Smith, Martin J. Kulig, 2 and Jon I. Teng Division of Biochemistry,

Department of Human Biological Chemistry

and Genetics, University of Texas Medical Branch, Galveston, Texas 77550. Received:

8117173

SUMMARY

The thermal decomposition of the allylic alcohols Sa-cholest -6-ene -3p, 5 diol, cholest-5-ene-3J3,7a-diol, and cholest-S-ene-3l3,7P-diol and of the allylic hydroperoxides 3J3-hydroq -5a -cholest -6 -ene -5 -hydroperoxide , 3J3-hydroxycholest -5 -ene -7a -hydroperoxide, and 3@hydroxycholest -5 ene-7J3-hydroperoxide to six common major pyrolysis products cholest5-ene-3J3,7a-diol, cholest-5 -ene-SJ3,7l3-diol, 3J3-hydroxycholest-5-en-7one, cholesta-3,5-dien-7-one, cholesta-4,6-dien-3-one, and cholesta2,4,6 -triene was established. In distinction to side-chain substituted hydroxycholesterol

derivatives which are

stable to pyrolysis conditions (3), the cholesterol B-ring allylic alcohols Ib, IIb, and IIIb, the side -chain substituted (20a-, 24 -, 25 -, and 26 -) cholesterol hydroperoxides, the B-ring hydroperoxides

Ia, IIa, and IIIa are thermally decomposed.

and

We have de-

scribed the pyrolysis behavior of the side-chain substituted cholesterol hydroperoxides in detail (4,5),

and we present details herein of the thermal decomposition of the allylic

sterols I, II, and III (5-7).

Six major components IIb, IIIb, IV, V, VI, and VII formed

from each parent sterol were identified by comparison of physical properties with those of authentic reference sterols. The A6-36, Sa-diol Ib did not survive pyrolysis but was dehydrated to the 2,4,6triene VII as the major reaction, with isomerization to IIb as a second prominent mode of alteration.

The ketones IV, V, and VI were formed in small amounts.

the epimeric A5 -3J3,7-diols IIb and IIlb survived pyrolysis to the 2,4,6-triene

in large part.

By contrast Dehydration

VII was the major reaction pathway, but small amounts of the

ketones IV, V, and VI also formed.

Thermal interconversion of IIh and IIIb was not

628

22:5

STEROIDS

observed@.

Differentiation

between the epimeric

on the basis of their different tion between the 3p, Sa-diol higher proportion

retention

times

3p, 7-diols

on gas chromatography

Ib and the 3p, 7a-diol

of the dehydration

product

IIb and IIIb is readily (6).

made

Differentia-

IIb is best made on the basis of the

VII to IIb formed

on pyrolysis

of Ib.

6R la.

R *OH

Ila.

R = OH

Illa.

R=OH

=H

Ilb.

R

=H

Illb.

R=H

lb. R

IV.

V.

Rationalization thus requires only),

of the pyrolysis

three reaction

and dehydrogenation

probably derived

derived

from IV (3,9),

(formation

of the allylic

of an initially

pyrolysis

involving

for cholesta-4,6

formed

-dien-3P-ol

is dehydrated

Ia was degraded

dehydrogenation

A496 -3 -ketone VI predominated. double loss of the elements in this regard.

mixture

Formation

of water,

(Ib

V and VI are product,

V being

-en-3 -one

An alternative

path-

of Ib to cholesta-4,6-dien-3P-01 almost

exclusively

to the 2,4,6

is un-triene

VI can be detected.

Ia, Ha, or IIIa survived

to a complex

Ib, IIb and IIIb

rearrangement

The dienones

and which was not detected.

initial dehydration

None of the hydroperoxides

ing analogous

alcohols

(to VII), allylic

of IV, V, and VI).

VII, and only very small amounts of the dienone

peroxide

VII.

VI from the putative analog 5 -hydroxy-Sa-cholest-6

which should not survive

likely,

behavior dehydration

modes:

by dehydration

way of VI formation

VI.

The Sa-hydro-

pyrolysis.

in which the 3p, 7a-diol of VI from Ia represents

the A4 ’ 6-3-ketone

VI and the A

Since VI was of such prominence

IIb and the formally

395 -7-ketone

the V be-

among the pyrolysis

629

STEROIDS

Nov. 1773

products of Ia, neither Ib nor the 7-oxygenated sterols II and III are likely intermediates. Rather the pyrolysis may proceed via the putative intermediate S-hydroxy-Sa-cholest6-en-3-one

or more probably via a special process such as the sequence shown below.

Ho@= Ho@c8H I i

I

I H

c8H17 0

Other identified pyrolysis products of Ia were the 3l3,7P-diol IIIb (most probably formed by rearrangement of Ia to IIa, epimerization of IIa to IIIa, with subsequent reduction of IIIa to IIIb), the 7-ketone IV (derived by formal dehydration of either IIa or IIIa formed from Ia), the A 395 -7-ketone V (formed by dehydration from IV), and the A2 ‘4 ’ 6-triene VII (formed by dehydration of Ib, IIb or IIIb).

An unidentified component

(No. 2 described in the Experimental) recognized as most probably being a cholestatriene isomeric with VII was regularly formed from Ia but not from IIa or IIIa.

Formation

of this rapidly

ketone VI confers

examined

eluted cholestatriene

uniqueness

The pyrolysis

behavior

together.

of the epimeric

Dehydration

of IIa and of IIb among

7-hydroperoxides,

since

was not regularly

interconversion

rearrangement

IIa and IIIa may be

IV and reduction

reactions

observed

small amounts

to the corre-

Small amounts of V,

of IIib among pyrolysis

thermal

of the corresponding

interconversion 7-alcohols

In distinction

Ia, IIa, and IIIa thus involves

(of Ia to IIa), epimerization

of the

IIb and IIIb

double dehydration

to pyrolysis

behavior

(4,5) carbon-carbon

six reactions:

(of IIa and IIIa), reduction

to a ketone (IIa and IIIa to IV), alcohol

IIb and IIIb to VII), and formal

hydroperoxides

from Ia.

7-hydroperoxides

those of IIIa suggested

of the hydroperoxides

IIIa to IIIb), dehydration

V).

products

observed@).

Pyrolysis allylic

Moreover,

A 4&3_

with the prominent

of both to the 7-ketone

and VII were also formed.

products

together

to the pattern of pyrolysis

sponding 3p, 7 -dial IIb or IIIb were the major VI,

22:5

STEROIDS

630

to a dienone

of the cholesterol

bond scission

dehydration

(IIa to IIb, (IV to V,

(Ia to VI, IIa and IIIa to

20a-, 24-, 25 -, and 26-

reaction

were not observed

for

Ia,

IIa, and IIIa. Our results VI frequently cholesterol

suggest

reported

a basis for the presence

in sterol

in such samples,

preparations

whether

tion would afford these sterols. some samples esterol

by attack of excited-state

ing an endogenous erythrocyte tion-induced

by thermal

oxidation

7p-hydroperoxide

of cholesterol

IIIa as chief product

Exposure

or solid state,

molecular

(11).

presence

decomposition

Very recently

ghosts has been reported

sources.

or dispersed

the characteristic

singlet

photosensitizer.

IIb, IIIb, IV, V, and

Ia, IIa, and IIIa whose subsequent

Thus,

(10)may be accounted

from biological

in solution

tion in air may yield the hydroperoxides

of the sterols

such an example

to radia-

decomposi-

of IIb and VI in

of Ia formed

oxygen formed

from

in samples

processes

(12) with subsequent

of Ia formation

IIIb, IV, and V could account for their presence

in other tissue

of the

decomposition

samples

(13).

in

radia-

leading the formation thermal

chol-

contain-

On the other hand, the unsensitized

by radical

of

to

In the

Nov. 1973

STEROIDS

631

absence of demonstrated enzymic origins for IIb, IIIb, IV, V, and VI their natural product status should be viewed with reservation. ACKNOWLEDGEMENT The authors are grateful to the Robert A. Welch Foundation, Houston, Texas, and to the U.S. Public Health Service (via research grants AM-13520 and HE-10160) for financial support of these studies. EXPERIMENTAL Analytical thin -layer ~b~matography was conducted using 0.25 mm thick 20 x 20 cm chromatoplates of Silica Gel HF254 (E. Merck GmbH. , Darmstadt) and techniques previously reported in detail (14). Mobility data are given in terms of RF or RC (where cholesterol was used as unit mobility). Analytical gas chromatography was conducted on 4 mm I.D. silanized glass U-tube columns packed with 2-3% SP-2401 or with 2-3% OV-210 fluoroalkyl silicone liquid phases on loo-120 mesh Supelcoport (Supelco Inc. , Bellefonte, Pa.) (6) as well as with 3% SE-30 and 3% QF-1 phases previously described (3). Flash injector zone temperature was 250” , column 230”) and detector 250”. Nitrogen was used as carrier gas at a flow rate of 20 mllmin. Retention times (tR) are given relative to cholesterol as unit retention time. Preparative gas chromatography was conducted on 6 mm I.D. silanized glass U-tube columns packed with 2% OV-210 on loo-120 mesh Supelcoport. Effluxing pyrolysis products were collected in glass capillary tubes as individual components where possible, as previously described (9). Sterols thus collected were rinsed from the collecting capillary with acetone and crystallized for melting point, spectral, and chromatographic analyses. Melting points were determined on a Kofler block under microscopic magnification. Ultraviolet light absorption spectra were determined on a Gary Model 14 spe~trophotometer. Infrared absorption spectra were obtained on 1.5 mm diameter KBr disks incorporating the sample, using a Perkin-Elmer Model 337 spectrophotometer equipped with a beam condensing lens. Reference Sterols. Reference sterols were demonstrated to be of high purity by thin-layer and gas chromatographic, melting point, and infrared spectral criteria. The sterol hydroperoxides were prepared by published methods: Ia, mp 144-146” [lit. mp 142” (15b), mp 145-148” dec (16a), mp 148-149” dec (lSe), mp 149.5150.5” (15d)] by photosensitized oxidation of cholesterol in pyridine (15); IIa, mp 152-153” [lit. mp 154-156.5” dec (16a), 154-155” (16b)] by isomerization of Ia in chloroform solution (16); IIIa, mp 147-149” [lit. mp 148-150” (7)] by 6oCo gamma-radiation induced autoxidation of crystalline cholesterol (12). The correspond= ing allylic alcohols prepared by sodium borohydride reduction of the hydroperoxides were: Ib, mp 147-148” [lit. 181” (17), mp 170-175”, f66-171”, and 134-135” (15b, 15c), mp 147-150” (1541; IIb, mp 182-183” flit. mp 186-187” (18a), 188-189” (1891; IIIb, mp 174-175” [lit. mp 174-176” (18a), 172-176”/ 180-181” (18b)]. Pyrolysis Conditions. Solutions of each parent allylic sterol I, If, and III in chloroform-methZiiZiwere injected into the flash heater zone of the gas chromatograph. Routine analysis of the major pyrolysis products was conducted with 5 pg of sterol sample; detection of the minor components was achieved using lo-20 pg of sterol; preparative gas chromatography was with 3-5 mg of sterol. Pyrolysis of Ia produced the most complex gas chromatographic elution curve composed of at least ten regularly resolved components catalogued by their relative retention times on 3%

22:5

STEROIDS

632

OV-210 columns as follows: Component No. 1, 0.43 (identified as VII); No. 2, 0.51; No. 3, 1.56; No. 4, 1.90; No. 5, 2.17 (V); No. 6, 2.25 (IIb); No. 7, 2.45 (IIIb); No. 8, 2.87; No. 9, 3.57 (VI); No. 10, 5.04 (IV). Pyrolysis of IIa and IIIa gave slightly less complex elution curves with the following components: Nos. 1,3,5,6,7,9, and 10. Pyrolysis of Ib and IIb gave components Nos. 1,5,6,9, and 10; IIIb gave Nos. 1,5,7,9, and 10. Initial gas chromatographic resolution of each component was not always possible, and groups of components were collected for rechromatography. Typically, such collections were: Zone 1, with retention time O-5 min., containing chiefly components No. 1,2; Zone 2, 5-12 min, components No. 3,4; Zone 3, 12-18 min, components No. 5 -8; Zone 4, 18-25 min, components No. 8,9; Zone 5, 25 -80 mm, component No. 10. Each pyrolysis product from the six parent sterols I, II, and III was further purified and characterized, as typified in the following experimental details. Cholesta-2,4,6 -triene (VII). Component No. 1 obtained from Ia, Ib, IIa, IIb, IIIa, IIIb, and from cholesta-4,6-dien-36-01 was chromatographed on a 0.25 mm thick chromatoplate irrigated with hexane -benzene (1: 1)) and the RPO. 75 zone was excised from the plate, the sterols eluted with chloroform, and the solvent evaporated under vacuum to give VII, typically characterized by mp 72-76” i lit. mp 71-72” (19a), 7274” (19c),

71.5-72.5”

nm (8,020)

] lit. x max 295-300

ca. 295 inflection, 305 (13,640), (15,400),

(19d)];

Acmc$hexane inflection,

296.5

306 (& 15,70(l),

ca. 306, 320 nm inflection

320 nm(8,720,

321 nm (10,080)

inflection) (19d); c

(E 11,900),

(19~);

yarx 1680,

(19b);

307.5

(12,600),

322.5

ca. 320 nm (19a); x Kt,“”

Amiszctane

296 (( 14,380),

304-

307 h ’ y clohexane 297 (6 13,400), max 1600, 1420, 1345, 865, 750, 685, 615

-l* R 0 75 in hexane-benzene (1-l) R 1 32 in benzene-ethyl acetate (3:l). F,“,ediatFe biue Color with 50% sulfuric acids (2b); tR 0.42 (3%, CV-210), 0.41 (3i 2401).

SP-

Unidentified Cholestatriene. Component No. 2 derived from Ia was collected with VII-from which it could not be adeauatelv purified desuite additional thin-laver chromatography. Component No. 2 was ‘characterized: RF 0.73 in hexane-benzene (1: 1); RC 1.32 in benzene-ethyl acetate (3: 1); immediate blue color with 50% sulfuric acid; tR 0.50 (3% OV-210), 0.50 (3% SP-2401); A,,, 296.5, 308.5, 322.5 nm (obtained on a mixture

of component

No. 2 and VII);

2 m:;,

1620 cm-‘.

Unidentified Component No. 3. Component No. 3 derived from Ia, IIa, and IIIa was collected along with component No. 4 in Zone 2 as described. The sterol mixture was chromatographed on a 0.25 mm thick chromatoplate using hexanebenzene (l:l), the RF 0.34 zone excised from the chromatoplate and the sterol therein eluted with chloroform. Evaporation of the chloroform under vacuum gave unidentified com3 Xcyclohexane 1640, 1490, 1400, 1350, 1265, 1235, 288 nmi G kz ponent No. ; RF 0.34 in hexane -benzene (1: l), RC 1.24 in 1145, 1015, ?95,?%, 635, 545 cm benzene-ethyl a.cetate (3: 1); yellow color with 50% sulfuric acid; tR 1.59 (3% CV-210). Component No. 4 was characterized solely by its relative retention time 1.91 on 3% ov-210. Cholesta-3,5-dien-7-one (V). Component No. j; obtained from Ia, Ib, IIa, IIb, IIIa, and IIIb was generally collected together with components No. 6 and 7 (IIb and chromatography using hexane-benzene (1:l) IIIb respectively) in Zone 3. Thin-layer resolved the 3,5 -dien-7-one well ahead of other sterols in the sample. Elution with chloroform and evaporation under vacuum gave pure V, mp 110-112” [lit. mp 109-

STE

Nov. 1773

111” (21), 114-114.5”

(lo),

633

ROIDS

114” (13)];

x zyohexane

280 nm [lit.

x max 280 nm

(1: 1); 1590, 875 cm-l; RF 0.24 in hexane-benzene _. RF 0.73 in benzene-ethyl acetate (1:3), RC 1.40 in benzene -ethyl acetate (3: 1); yellow color with 50% sulfuric acid; tR 2.15 (3% OV-210), 2.01 (3% SP-2401); identical in these properties with those of an authentic sample of V. (10,13,21)];

;

?a;

1640,

1610,

In the case of pyrolysis of IIa there was another sterol component isolated with component No. 5, but which was resolved from V on thin-layer chromatography with This unidentified component was characterized: benzene -ethyl acetate (1: 3). -1 c KBr ; RF 0.54 in benzeneY max 3350, 1600, 1410, 1340, 1085, 1050, 860, 795 cm ethyl acetate (1:3); yellow color with 50% sulfuric acid. Cholest lysis of Ia, Ib, tive thin-layer atography with

-5 -ene -3p, 7a-diol (IIb). The polar sterols of Zone 3 derived by pyro IIa, IIb, and IIIa were recovered after removal of V from the preparachromatogram irrigated with benzene-ethyl acetate (1:l). Rechrombenzene-ethyl acetate (3:l) and elution of the more pals sterol gave

pure component No. 6 identified as the 3P,7a-diol IIb, mp 181-183”; Y “mB,‘,3330, 1550, 1060, 945 cm-l; R 0.52 in benzene-ethyl acetate (3:l); immediate blue color with 50% sulfuric acid (205 ; tR 2.26 (3% OV-210), 2.22 (3% SP-2401); identical in these properties with those of an authentic sample of IIb. Cholest -5 -ene -3p, 7/3-diol (III@. The more mobile polar sterol giving an im mediate blue color with 50% sulfuric acid and found in close association with IIb described above was eluted as a pure sterol and identified as IIIb, mp 175-178”; 77;;

3310,

1550,

1050,

810, 795, 615-l;

RC 0.60 in benzene-ethyl

acetate

immediate blue color with 50% sulfuric acid (20); tR 2.44 (3% OV-210), 2401); identical in these properties with those of an authentic reference

(3: 1);

2.38 (3% SPsample of IIIb.

Unidentified Component No. 8. Component No. 8 from pyrolysis of Ia eluted from preparative thin-layer chromatograms run with benzene-ethyl acetate (3:l) was characterized:

5 kz

3350,

1640, 1430,

1350,

1055, 810, 795, 615 cm-‘;

RC 0.79

in benzene-ethyl acetate (3:2); yellow color with 50% sulfuric acid; tR 2.88 (3% OV210), 2.64 (3% SP-2401). Rechromatography of component No. 8 on preparative 3% OV-210 columns gave, among other components, component No. 3, as a major product . Cholesta -4,6-dien-3 -one (VI). Component No. 9 obtained by pyrolysis of Ia, Ib, IIa, IIb, IIIa, IIIb, and cholesta-4,6-dien-3@-ol was purified by thin-layer chromatography using benzene -ethyl acetate (3: l), the strong ultraviolet light absorbing zone eluted with chloroform, to provide pure VI, mp 76-79” [lit. mp 79.5-81” (lo), 79-80”

(13)];

1 zzFhexane

285 nm [lit.

x max 285 nm (10,13)];

c

Fat

1620,

1590, 1560, 868, 665 cm -l; RC 1.09 in benzene -ethyl acetate (3: 1); tR 3.51 (3% OV210), 3.30 (3% SP-2401); identical in these properties with those of an authentic reference sample of VI. 3p -Hydroxycholest -5 -en-7 -one (IV). Component No. 10 recovered in Zone 5 from pyrolysis of Ia, Ib, IIa, IIb, IIIa, and IIIb was chromatographed on a 0.25 mm thick chromatoplate using double ascending irrigation with benzene-ethyl acetate (3:l). The ultraviolet light absorbing zone was eluted with chloroform, the solvent evaporated, and the sterol recrystallized from hexane to afford pure IV, mp 169-171”

2215

STEROIDS

634

[lit. mp 173” (16a), 170-172” (21)];

xEay238

N “,“,‘, 3500, 1640, 1620, 1055 cm-‘; V

nm (lit.

Amax 238 nm (16a,21)];

RC 0.75 in benzene-ethyl acetate (3:l); tR 5.03

(3% OV-210), 4.78 (3% SP-2401); identical in these properties with those of an authentic sample of IV. REFERENCES 1.

Paper XXV of the series: M. J. Kulig and L. L. Smith, J. Org. Chem., in press.

2.

Robert A. Welch Foundation Post-Doctoral

3.

J. E . van Lier and L. L. Smith, Anal. Biochem. , -24, 419 (1968).

4.

(a) J. E. van Lier and L. L. van Lier and L. L. Smith, J. and G. Kan, J. Org. Chem. ,

5.

J. E. van Lier and L. L. Smith, Steroids, -15, 485 (1970).

6.

J. I. Teng, M. J. Kulig, and L. L. Smith, J. Chromatography, -75, 108 (1973).

7.

J. I. Teng, M. J. Kulig, L. L. Smith, G. Kan, and J. E. van Lier, J. Org. C&em., -38, 119 (1973).

8.

Gas chromatography of 4-14C-Ib, IIb, and IIIb (7000-9000 cpm) on 2% OV-210, followed by repeated thin-layer chromatography of pyrolysis products suggested that some interconversion of IIb and IIIb occur. Purification to constant specific radioactivity was not attempted, and the values for interconversion are thus maximum ones. From 4-14C-Ib: VII, 61.2%; VI, 15.0%; V, 3.8%; IV, 1.4%; IIb, 1.4%; IIIb, 0.5%; other components, 16.7%. From 4-14C-Iti VII, 67.0%; VI, 8.1%; V, 2.0%; IV, 2.3%; IIb, 3.0%; IIIb, 1.0%; other components, 16.6%. From4-14C-IIIb: VII, 43.3%; VI, 11.4%; V, 8.6%; IV, 4.3%; IIb, 0.1%; IIIb, 4.1%; other components, 28.2%.

9.

(a) J. E. van Lier and L. L. Smith, Biochemistry, 6, 3269 (1967); (b) J. E. van Lier and L. L. Smith, J. Chromatography, -36,7 (1968).

Fellow, 1971-1973.

Smith, J. Org. C&em., 35, 2627 (1970); (b) J. E. Org. C&em., 36, 1007 (i-971); (c) J. E. van Lier 37, 145 (1972)F -

10.

E. Hardegger, (1943).

L. Ruzicka, and E. Tagmann, Helv. Chim. Acta, -26, 2205

11.

A. A. Lamola, T. Yamane, and A. M. Trozzolo,

12.

L. L. Smith, J. I. Teng, M. J. Kulig, and F. L. Hill, J. Org. Chem., -38, 1763 (1973).

13.

V. Prelog, L. Ruzicka, and P. Stein, Helv. Chim. Acta, -26, 2222 (1943).

14.

L. L. Smith, W. S. Matthews, J. C. Price, R. C. Bachmann, and B. Reynolds, J. Chromatog., -27, 187 (1967).

Science, 179, 1131 (1973).

Nov. 1973

STEROIDS

635

15.

(a) G. 0. Schenck, Angew. Chem., 69, 579 (1957); (b) G. 0. Schenck, K. Gollnick, and 0. -A. Neumilller, h., 603, 46 (1957); (c) G. 0. Schenck and 0. -A. Neumtiller, Ann., 618, 194 (1958);(d) A. Nickon and J. F. Bagli, J. Amer. Chem. Sot., -83, 14pB(1961).

16.

(a) G. 0. Schenck, 0. -A. Neumtiller, and W. Eisfeld, Angew. Chem., 70, 595 (1958); Ann., 618, 202 (1958); (b) B. Lythgoe and S. Trippett, J. Chem. sot., 471 (1959).

17.

H. B. Henbest and E. R. H. Jones, J. Chem. Sot. , 1792 (1948).

18.

(a) L. F. Fieser, J. E. Hen, M. W. Klohs, M. A. Romero, and T. Utne, J. Amer. Chem. Sot., 74, 3309 (1952); (b) C. W. Shoppee and B. C. Newman, J. Chem. Sot., (C), 981 (1968).

19.

(a) J. Schmutz, H. Schaltegger, and M. Sanz, Helv. Chim. Acta, 34, 1111 (1951); (b) D. H. Gould, K. H. Schaaf, and W. L. Ruigh, J. AmerZhem. sot., 73, 1263 (1951); (c) W. R. Nes, R. B. Kostic, and E. Mosettig, J. Amer. 78, 436 (1956); (d) G. A. Selter and K. D. McMichael, J. Org. Chem.Soc., Chem., -32, 25X6 (1967).

20.

The 2,4,6 -triene VII herein demonstrated to be formed from IIIa, and from cholesta -4,6 -dien-3J3-01 is most probably that species giving the intense blue colors with a variety of acidic ing the classic Lifschtitz color test, cf. S. Bergstrtim and 0. J. Biol. Chem., 145, 309 (1942).

21.

S. Bergstrbm and 0. Wintersteiner,

IIb, IIIb, Ia, IIa, chromogenic reagents, includ Wintersteiner,

J. Biol. C&em., 141, 597 (1941).