Steroid metabolism in human and boar testis tissue. Steroid concentrations and the position of the sulfate group in steroid sulfates

STEROID METABOLISM IN HUMAN AND BOAR TESTIS TISSUE.

STEROID CON-

CENTRATIONS AND THE POSITION OF THE SULFATE GROUP IN STEROID SULFATES

A. Ruokonen

Steroid University Clinical

Received:

Research

Laboratory,

of Helsinki, Chemistry,

Department

SF-00170

University

and R. Vihko

Helsinki of Oulu,

of Medical

Chemistry,

17, and Department SF-90220

of

Oulu 22, Finland

10/12/73

ABSTRACT The steroid composition of and steroid conjugation in human cadaver and boar testes were investigated by analyzing the endogenous steroids . Gas-liquid chromatography and gas chromatography-mass spectrometry were used to identify the steroids and to determine the position of the sulfate group in sulfate conjugates. For the latter purpose, the steroids were first acetylated and subsequently solvolyzed and converted to trimethylsilyl ethers. In addition to the compounds previously identified as endogenous components, human testis was also found to contain progesterone, 17a-hydroxyprogesterone and 20a-hydroxy-4-pregnen-3-one in the free steroid fraction and 3B-hydroxy-17a-5-pregnen-20-one in the monosulfate fraction.

231

STEROIDS

steroid In both of the species investigated, monosulfated C diols with a 17f3-hydroxyl group were conjugated at C-3, 38ereas the 17a epimers were conjugated at C-17. Thus it is probable that there are sulfokinases with different specificities in testis tissue. The formation of testosterone or its sulfate from 38-hydroxy-5-androstene precursors may be regulated by the activity of the steroid sulfatase. INTRODUCTION Analyses shown that

many of the

-conjugates of both

of endogenous

(1,2).

secrete

gland,

the capacity

Most of the tissue

they might -5-ene

(6),

steroids

present

serve

as precursors

through

activity present

the

of testosterone

(9,lO).

regulation

On the other

in human testis

in

seems to lack

Therefore,

in the

3@-hydroxy-

testosterone

biosynthesis

of testicular hand,

may be formed

steroid

the testosterone via

hydrolysis

of the conjugate.

of steroid

sulfate

metabolism

have been demonstrated

in 11).

steroid,

(1,2).

without

(summarized

steroid

human or boar

--i.e.

tissues

is

(7,8).

structure

that

Another

the ovary

as sulfates

is possible

the testes secretion

a sulfated

whereas

have

of sulfate-

steroids,

(3-5).

secretes

have a 3B-hydroxy-5-ene

may be regulated sulfatase

also

form

and this

gonadotropin

sulfated

steroids

Thus it

way",

conjugates,

sulfate

pathway.

sulfate

sulfate

to secrete

are in the

to unconjugated

the adrenal,

dehydroepiandrosterone

in human and boar testes

present

by human chorionic

endocrine

testis

steroids

In addition

species

influenced

steroids

a "direct

Direct

path-

pathways in several

Jan. 1974

STEROIDS

Steroid the

diol

hydroxyl

elucidation

groups. of the

monosulfates additional

monosulfates This

might

be conjugated

investigation

position

was directed

of conjugation

in human cadaver information

3

and boar

on the endogenous

of the

testis

at either

of

at the steroid

diol

and at providing

steroids

in these

tissues.

MATERIAL

AND METHODS

Tissue sam les. Human testis samples were obtained from threea le 1). The boar testes were obtained from three Finnish landrace boars (ages 10, 10 and 24 months) immediatelx after slaughter.All tissue samples were frozen and stored at -20 C until analyzed. Table

Subject

1. Data on subjects

Age (years)

studied.

Time interval after death

(h)

Weight of combined testes (g)

Cause of death

BT

24

30

34.5

Cerebral hemorrhage

LR

29

5

48.0

Rupture of spinal cord

ML

41

14

22.2

Cerebral hemorrhage

Trivial and systematic names of steroids. Androsterone, 3a-hydroxy-5a-androstan-l/-one; epiandrosterone, 38-hydroxy-5adehydroepiandrosterone, 3B-hydroxy-5-androsten-androstan-17-one; -17-one; testosterone, 17B-hydroxy-4-androsten-3-one; pregnenolone, 3B-hydroxy-5-pregnen-20-one; progesterone, 4-pregnene-3,20-dione. Reference compounds. The free steroids used as starting material in the synthesis of reference compounds were obtained Israel) or Schering AG (Berlin, Germany). from Ikapharm (Ramat-Gan,

4

STEROIDS

Dehydroepiandrosterone and pregnenolone Sulfates were Purchased from Steraloids, Inc.( Pawling, N.Y., U.S.A.),androsterone sulfate from Ikapharm, and epiandrosterone sulfate was synthesized and has been characterized previously (12). The purity of the compounds (in the case of sulfates after solvolysis) was checked by gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS) of their trimethylsilyl (TMS) ethers. 5-Androstene-36,17@-diol 3-OAc (acetate) 17-OTMS, 5~ -androstane-3a,l7B-diol 3-OAc 17-OTMS, 5u-androstane-38,17B-dial 3-OAc 17 0-TMS and 5-pregnene-3@,20a(and B)-diol 3-OAc PO-OTMS were prepared from dehydroepiandrosterone, androsterone, epiandrosterone and pregnenolone, respectively, by acetylating the starting material (13) and then reducing the keto group with sodium borohydride (14). The products were finally silylated. 5-Androstene-38,17B-diol 3-OTMS 17-OAc, 5a-androstane-3a,l76-dial 3-OTMS 17-OAc , 5a-androstane-3@,176-diol 3-OTMS 17-OAc and 5-pregnene-36,20a(and 8) 3-OTMS 20-OAc were prepared from the sulfates of dehydroepiandrosterone, androsterone, epiandrosterone and pregnenolone , respectively, by sodium borohydride reduction followed by acetylation, solvolysis (see 12) and silylation. 5-Androstene-38,17a-diol 3-OAc 17-OTMS and 3-OTMS 17-OAc, 5a-androstane-3a,l7a-diol 3-OAc 17-OTMS and 3-OTMS 17-OAc as well as 5a-androstane-3B,17a-diol 3-OAc 17-OTMS and 3-OTMS 17-OAc were prepared from thg respective parent diols employing 5-min. acetylation at 27 C terminated by addition of a few drops of water. After evaporation to dryness, TMS ether derivatives were prepared. In the case of these steroid diols, all the four possible derivatives (3-OAc 17-OTMS, 3-OTMS 17-OAc, 3,17di-OTMS and 3,17-di-OAc) were formed. Identification of the products was accomplished by GLC and GC-MS. 5-Androstene-3a,l7B-diol 3-OAc 17-OTMS and 3-OTMS 17-OAc were prepared from 3a-hydroxy-5-androsten-17-one by sodium borohydride reduction followed by acetylation for 5 min. and silylation. All of the four possible derivatives were identified. Gas-liquid chromatography (GLC) of the steroid derivatives was carried out on QF-1 and SE-30 columns in instruments equipped with flame ionization detectors, as described previously (12). Gas chromatography-mass spectrcmetry was carried out using a computerized (Varian, Spectra System 100 MS) combined gas chromatograph-mass spectrometer (Varian , Model CH 7). The energy of the bombarding electrons was 70 eV and the ionizing current 300 VA. Procedure. In certain analyses, samples of both testes from a cadaver were combined. In the case of the boar testes, both testes were dissected into small pieces and combined into a single pool of which 20-30 g were used in each analysis.

231

STEROIDS

Jan.1974

The tissue samples were processed as described previously (1). Briefly, the method involves extraction of the tissue and fractionation of the steroids into unconjugated, monosulfated, glucuronide-conjugated and disulfated compounds on Sephadex LH-20. For the quantification of endogenous steroids, the conjugates were cleaved by solvolysis and the free steroids purified and fractionated by chromatography on silicic acid and hydroxythe TMS or methyloxime-trimethylalkoxypropyl Sephadex. Finally, silyl (MO-TMS) ethers of the compounds isolated were analysed by GLC and, when needed, by GC-MS. The position of the sulfate moiety in steroid diol monosulfates was determined as described.by Cronholm (13). The monosulfate fraction was acetylated, solvolyzed and finally fractionated according to polarity on a 200 mg silicic acid column. The fractions were silylated and analyzed by GLC and GC-MS. RESULTS Position Using

an acetyl

in steroid site

of the

diol

group

moiety

as a marker

monosulfates,

of conjugation

spectra

sulfate

published

stane-3a,l7a-diol

in steroid for

it

free

is possible

monosulfates.

hydroxyl

(13).

In addition

by Cronholm

(13),

the mass spectra

the

to the mass of 5a-andro-

3-OAc 17-OTMS, 5a-androstane-3B,l7a-diol 3-OTMS 17-OAc,

3-OAc

5-androstene-

3-OTMS 17-OAc and 5-androstene-3B,17a-diol

17-OTMS were investigated mentation

group

to determine

of a diol

17-OTMS, 5a-androstane-3B,17B-diol -3u,l78-diol

the

diol

was observed

our mass spectra

in this with

are very

all

study. of these

similar

3-OAc

The same basic compounds

to those

published

frag-

(Fig.

1) and

by Cron-

holm (13). In man, 17B-hydroxy-C,g jugated

at C-3 (Tables

5-pregnene-3@,20a-diol. monosulfates

steroid

2 and 3, Fig. In contrast,

were conjugated

diols 2). the

were exclusively The same was true

confor

17a-hydroxysteroid

at C-17 (Tables

2 and 3, Fig.

diol 2).

23:l

STEROIDS

100

200

m/e

300

200

m/e

300

400

400

spectra of 5-androstene-3a,l78-diol 3-OTMS 17-OAc d 5-androstene-3B,l7a-diol 3-OAc 17-OTMS (lower panel).

In addit ion to the boar testis

a lso

stene-3a,l78-diol in the

boar was exactly group

17a-hydroxyl

group

addition, c-3.

contains (2).

17khydroxyl

steroid

monosulfates

found

5a-androstane-3a,l7a-diol

The conjugation as found

are sulfated are

diol

sulfated

5-pregnene-38,20a-diol

of the

and 5-androdiol

monosulfates

in man. Cl9 steroid at C-3,

whereas

at C-17 (Tables is conjugated

in man,

those

diols with

2 and 4). exclusively

with a In at

a

7

STEROIDS

Jan. 1974

retention times Table 2. Relative identlfled in human and boar testis of reference compounds. Conditions = 1.00.

of steroid diol monoacetate monosilyl ethers tissue and,those of correspondieg derivatives 3% QF-1 215 C and 2.2% SE-30 210 C. Cholestane

Compound Compound from testis 5a-Androstane-3u,l7a-diol-3-OTMS,17-OAc 5a-Androstane-3a,l7a-diol 17-OTMS 5a-Androstane-3a,l7B-diol 17-OAc 5a-Androstane-3a,l7B-diol 17-OTMS 5,-Androstane-3B,17a-diol 17-OAc 5a-Androstane-3B,17U-diol 17-OTMS 5,-Androstane-3B,17B-diol 17-OAc 5a-Androstane-3B,17B-diol 17-OTMS 5-Androstene-3a,17B-diol 17-OAc 5-Androstene-3a,17B-diol 17-OTMS 5-Androstene-3B,17a-diol 17-OAc 5-Androstene-3B,17a-diol 17-OTMS 5-Androstene-3B,17B-diol 17-OAc 5-Androstene-3B,17B-diol 17-OTMS 5-Pregnene-3B,20,-diol ZO-OAc 5-Pregnene-3B,20a-diol 20-OTMS

QF-1 Reference compound

SE-30 Compound Reference from testis compound

0.99 1.04

1.04

0.49

0.50

B

3-OTMS,

1.14

1.14

0.54

0.55

H,B

3-OAc,

-

1.32

0.64

3-OTMS,

-

1.39

0.68

3-OAc,

1.25

1.26

0.61

0.61

H,B

3-OTMS,

1.51

1.51

0.71

0.71

H,B

3-OAc,

-

1.48

3-OTMS,

1.06

1.07

3-OAc,

-

1.13

0.58

3-OTMS,

-

1.29

0.67

3-OAc,

1.23

1.22

0.61

0.61

H,B

3-OTMS,

1.37

1.38

0.70

0.70

H,B

-

1.41

2.26

2.25

-

2.45

3-OTMS, 3-OAc,

in human testis

B = present

in boar testis

0.49

3-OAc,

3-OAc,

H = present

Source

0.72 0.53

0.53

B

0.71 1.22

1.23 1.29

H,B

23:l

STEROIDS

8

Table 3. Position of the sulfate group and concentrations of steroid dio'lnosulfates in hunan testis. The values are expressed as micrograms of the free steroid in 100 g of tissue and are uncorrected for methodological losses.

Compound BT

Subjects LR

ML

5a-Androstane-3a,l76-diol

3-SO4

-l-2*

-1"

5a-Androstane-3B,l7a-diol

17-SO4

-l-2*

-2"

-1-3"

5a-Androstane-3B,l76-diol

3-SO4

-2-3"

-3*

-2-3'

5-Androstene-38,17a-diol

17-SO4

37

37

53

5-Androstene-3B,l7B-diol

3-SO4

189

71

173

10

8

110

5-Pregnene-3B,20a-diol

*

Minor

3-SO4

impurities

Table 4. Position dial monosulfates in Table 2.

or other

steroids

of the sulfate in boar testis

disturb

group tissue.

exact

-2*

quantificatio;.

and concentrations The values

Compound

of steroid are expressed as

Animals (10 mkths)

5a-Androstane-3a,l7a-diol

17-SO4

5a-Androstane-3a,l7@-diol

3-SO4

5a-Androstane-36,17a-diol 5a-Androstane-3B,l78-diol

(102months)

(2;

months)

6

*

24

3

17-SO4

39

7

7

3-SO4

93

112

66

2

*

5-Androstene-3a,l7$-diol

3-SO4

5-Androstene-36,17a-diol

17-SO4

27

15

21

5-Androstene-38,17@-diol

3-SO4

41

65

84

25

25

5

5-Pregnene-3@,20a-diol

3-SO4

* Only trace amounts detected. - Steroid not found.

STEROIDS

Jan. 1974

30

20

10

MINUTES

Figure 2. Gas chromatographic analysis of a steroid monosulfate fraction from human testis after silicic acid fractionation. Subject LM. The following compounds were identified in this fraction: 1 = 5a-androstane-3a,l78-diol 3-OTMS 174Ac; 2 = 5-androstene-38, 3-OAc 17-OTMS; 17a-diol 3-OAc 17-OTMS; 3 = 5a-androstane-36,17a-diol 4= 5-androstene-3B,17B-diol 3-OTMS 17-OAc (t a small amount of dehydroepiandrosterone TMS ether); 5 = 5a-androstane-3@,17B-diol 6 = 3B-hydroxy-17a-5-pregnen-20-one TMS ether; 3-OTMS 17-OAc; 7 = pregnenolone TMS ether; 8 = cholesterol TMS ether; 9 = 5-pregnene-3B,20a-dial 3-OTMS 20-OAc; 10 = stigmasterol TMS ether standard). Column and conditions: 3% QF-1 isothermally

Mass spectrum of the TMS ether derivative of 3B-hydroxyFigure 3. -17a-5-pregnen-20-one isolated from human testis tissue.

23:l

STEROIDS

10

Steroids

detected

pounds

previously

samples

studied

identified samples (Table also

found (1).

studied

in human testes

but

it

TMS ether

being

respectively

(peak

smaller

for

than

3) had close

1.63

pregnenolone

the spectrum

very

relative

2).

these its

of pregnenolone were identical

TMS ether

(16).

Therefore,

the

pregnenolone,

which

have

of this sulfates

NO 38-hydroxy-17a-

was formed.

In one of the

subjects

were found free

of

To

and their

medium (1).

(15)..

those

formation

16a-hydroxypregnenolone

(Fig.

with

steroid

artificial

are

TMS ether

of the

testicular

(RRT)

mass spectrum

the

concentrations

fraction

values

exclude

-5-pregnen-20-one

of the

on QF-1 and SE-30 columns,

and RRT values

solvolytic

was

times

as 38-hydroxy-17a-5-pregnen-20-one.

in the

in the

monosulfate

compound was identified

were incubated

com-

low concentrations

Although

to that

possibility

the

in all

retention

TMS ether,

38-hydroxy-17a-5-pregnen-20-one

2)

steroid

and 0.70

6 in Fig.

similarities

in

of the the

were present

2, Table

was present

a compound,

All

5a-androstane-38,17a-diol

In addition,

GL chromatograms

revealed

However,

concentrations.

by GLC and GC-MS (Fig.

3).

of its

and their

steroid

studied (Tables

fraction

not been previously

(ML, Tab le 1) very 3 and 5).

of this found

in

high

In GLC analyses

subject

three

human testicular

stero id of the

compounds, tissue,

Jan. 1974

STEROIDS

11

Table 5. Concentrations of free and monosulfated steroids in human testis tissue. The values are expressed as micrograms of the free steroid in 100 g of tissue and are uncorrected for methodological losses. Concentrations for steroid diol monosulfates are given in Table 2.

Compound

Fraction

Subject LR

BT Androsterone Epiandrosterone Dehydroepiandrosterone

4 5

1;

13; la3 a a

Androstenedione Pregnenolone 38,17a-Dihydroxy-5-pregnen-20-one Progesterone 20a-Hydroxy-4-pregnen-20-one 17a-Hydroxy-4-pregnene-3,20-dione 3B-Hydroxy-17a-5-pregnen-20-one M F a b

7; a a a

= = = =

monosulfate fraction free steroid fraction sample lost steroid identified , but quantification - = steroid not found

appeared.

Their

GC-MS analysis revealed

relative

-4-pregnen-3-one

impurities

retention

times

did

identical (for (Fig.

reference

with

not

allow

1: 9

see 17),

37

an exact

in Table

of the

the corresponding

spectrum,

1; 7

are given

of the MO or MO-TMS derivatives

structures

of progesterone

2

M

99" 9

1: 4 335 72 140 b 270 902 129

155 48 9

Testosterone

ML

6.

compounds derivatives

20a-hydroxy-

4) and 17a-hydroxy-4-pregnene-3,20-dione

STEROIDS

12

23:l

Table 6. Relative retention times (RRT) of MO and MO-TMS derivatives ofC21-3-keto-4-ene steroids isolated from human testis tissue and those of correspondin 8 derivatives of relevant reference compounds. Conditions 2.2% SE-30 210 C and 3% QF-1 215OC. Cholestane = 1.00.

Identification

QF-1

SE-30 Compound from testis ;M

Progesterone

Compound from testis

Reference compound

Reference compound MO-T S

-

1.16

1.16

1.77; 1.81

1.75; 1.79

17a-OH-Progesterone

1.53

1.52

1.83

1.83

20r-OH-Progesterone

1.33

1.32

1.70; 1.77

1.71; 1.78

(Fig.

5).

compounds

Thus,

GLC and GC-MS data

as indicated

was identified

in Table

in the testis

100

Figure 4. Mass spectrum 4-pregnen-3-one isolated

200

confirm

the

structures

6. In addition,

sample of subject

mle

of the

three

17a-hydroxyprogesterone LR (Table

300

of MO-TMS ether derivative from human testis tissue.

5).

400

of 2Q-hydroxy-

Figure 5. progesterone

Mass spectrum of MO-TMS ether derivative isolated from human testis tissue.

Steroid ject

13

STEROIDS

Jan. 1974

disulfates

were analyzed

LM. The disulfates

-3B,176-diol tions

in the

of 17&-hydroxy-

testis

sample

of 5-androstene-3f3,17a-diol,

and 5-pregnene-38,20a-diol

of 9, 8 and 5 pg/lOO

5-androstene-

were detected

g tissue,

of sub-

in concentra-

respectively.

DISCUSSION In a number of species in testis -5-ene with

tissue

are known to exist,

pathways

(18).

are present

preferably

conjunction

with

the

findings

fates

may be precursors

and,

as sulfate

conjugates.

When considered

that

Notation

and Ungar

steroid

38-sulfates

cursors

and that

of a number of certain

may be regulated

(19),

Using

the

the

regulation

to

of their

compounds

in addition,

incubation

It

is also

rat

as the

hydrolysis

in

steroid

(9,lO) sul-

possible

that

of the

activity

experimental

conclusion

unconjugated

they

studies

by regulation

however , came to the

were secondary

testes,

3B-hydroxy-5-ene

of testosterone.

sulfatase.

(2)

and 38-hydroxy-

predominate

formation steroid

3-keto-4-ene

formation

structure

suggest

testicular

testosterone

and boar

results

these

the

for

In human (1)

a 36-hydroxy-5-ene

testosterone

two pathways

steroid

animal,

that pre-

does not con-

of.

23:l

STEROIDS

14

stitute

a primary

man, however, steroid

mode of action

HCG administration

sulfate

secretion

sulfate-conjugated indicates

must be very

steroid

Cl9 steroid

Several

as monosulfates

this

demonstrate

are conjugated its

sulfate,which

is also

present

sults

are in agreement

fatase

arises

reaction

could

formation direct

necessitates

sulfate

via

the sulfated

sulfate

could

When the sulfates

is

is possible

compared

with

in blood

differences

are evident.

precursor.

These rethat

(19,21). the

rate

plasma

testoThe sul-

of testosterone

pregnenolone

from

sulfated

intermediates.

of testicular

In both

steroid

of the corresponding certain

diol

tissue

mono-

conjugates

similarities

plasma and testis

and 5-pregnene-36,20a-diol

and

monosulfate.

in human testis

(13),

A

sulfate

present

that

or 3@-sul-

is also

pattern

are

human and boar testis.

from

of

diolq

(l),from

to 5-androstene-3@,178-diol

conjugation

diols

17a-hydroxy

demonstrating

in

immediate

of testosterone

testosterone

pathway

species

The results

tissue

of the

which

(2).

steroid

formation

regulate

exclusively

circulating

-38,176-diol

free

sulfate,which

be formed

(ZO),

, are present

the

in testis

efficiently

even cholesterol

(1),

the

findings

from

pathway

Epitestosterone

3, whereas

hydrolysis with

sulfated

and boar

178-hydroxy-Cl9

at carbon Thus,

diol

sterone

the

in

in this

human and boar testis.

at carbcnl7.

fated

testes

regulation

in man (1)

In

no endogenous

, among them the probable

in

that

conjugated

Further,

5-androstene-3B,17B-diol

preferably

exclusively

increase

in rat

and its

that

diols

of testosterone,

study

(4).

were detected

from

hormones.

to a large

testes

metabolism

different

gonadotrophic

leads

by the

steroids

that

precursor

for

as well

as

5-androstene-

are exclusively

conjugated

15

STEROIDS

Jan. 1974

at C-3 and 5-androstene-36,17a-diol -androstane-3a,17B-diol in plasma

only

conjugated

25% is present

in steroid

to disulfates. strict

in this

ular

further

metabol

steroid

diols.

diol

-pregnen-3-one

monosulfates

it

samples.

This

indicates

--in vivo.

A steroid

not

the

as the monosulfate unconjugated

was incubated

been postulated precursors 17,-side 16-ene

chain steroids

may thus

ever,

the to

with

that

rat

16-ene

(16,

23).

(24,25) be the

precursovsand be investigated.

caecal

in the

pathway from

precursor eventual

exists and

also

operates

biological

sources,

was identified

. The formation

microorganisms formed

place

from

formation

16a-Hydroxylation takes

there

of the

when 38-hydroxy-5,16-pregnadien-

steroids

are intermediates

alone

remain

tissue

compound was observed

conjugated

human testis

(isopregnenolone)

in human testis

groups

and disulfated

in certain

isolated

3B-hydroxy-17a-5-pregnen-20-one

hydroxyl

and 20U-hydroxy-4-

3-keto-4-ene

previously

in blood

formation

monosulfated

endogenously that

present

in testis

in the

17a-hydroxyprogesterone were found

the

that

compartmentalization

whereas being

can be further

is obvious

5~t-

5-androstene-

Thus,

sm of unconjugated,

Progesterone,

remainder

are also

(1,13,22).

T herefore,

intracell

-20-one

form the

tissue

at C-3,

5-Androstene-3B,17a-diol,

as disulfates

sulfated

In testis

conjugated

and 5-pregnene-36,20u-diol

and in testis not

is exclusively

at C-17 (13).

-36,17@-diol

at C-17.

in

(16).It 16,-hydroxylated

of steroids (1)

has

with

and formation

human testis

tissue

a of

and pregnen-

of 3B-hydroxy-17a-5-pregnen-20-one. physiological

importance

of this

Howsteroid

STEROIDS

16

23:l

ACKNOWLEDGMENTS The skilful technical assistance of Mrs. Salme Ollikainen is gratefully acknowledged. This investigation was supported by a grant from the Ford Foundation. REFERENCES 1. 2. 3. 4. 5. ;: 8. 1;: 11.

K: 14. 15. 16. 17. 18. 19. 20.

23. 24. 25.

Ruokonen, A., Laatikainen, T., Laitinen, E.A., and Vihko, R., BIOCHEMISTRY 11, 1411 (1972). Ruokonen, A.,