Determination of dehydroepiandrosterone and dehydroepiandrosterone sulphate in blood and tissue. Studies of normal women and women with breast or endometrial cancer

J. steroid Biochem. Vol. 26, No. 1, pp. 151-159, 1987

0022-4731/87 $3.00 + 0.00

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Copyright 0 1987 Pergamon Journals Ltd

DETERMINATION OF DEHYDROEPIANDROSTERONE AND DEHYDROEPIANDROSTERONE SULPHATE IN BLOOD AND TISSUE. STUDIES OF NORMAL WOMEN AND WOMEN WITH BREAST OR ENDOMETRIAL CANCER D. L. JONES and V. H. T. JAMES* Department of Chemical Pathology, St Mary’s Hospital Medical School, London W2 IPG, England (Received 26 November 1985)

Summary-Radioimmunoassay methods for measuring dehydroepiandrosterone (DHA) and its sulphate (DHAS) in human plasma and tissues were developed and validated. Plasma levels were measured in men, pre- and postmenopausal women, and patients with endometrial or breast cancer. In the patients, tissue concentrations were also measured. Plasma DHA levels fluctuated synchronously with cortisol, but DHAS levels were less labile, and reached maximum levels during the day and were lowest at night. No obvious pattern was seen in relation to the menstrual cycle. Both DHA and DHAS levels fell with age, but DHA levels reached a plateau at about 60 years. No significant effect of weight on plasma levels was found. Plasma levels in the cancer patients were not significantly different from age-matched controls either when

single samples were compared, or when mean 24 h levels were used. Endometrial tissue levels of DHA fell with age, unlike DHAS. In breast tumour tissue, DHA, but not DHAS concentrations were higher than in normal breast tissue. A good correlation between plasma and tissue DHAS levels was found, and DHA levels were also correlated in plasma and tissue. The correlations between plasma and tissue were not observed in tumour tissue, which may be due to altered tissue metabolism.

INTRODUaION

A role for the adrenal androgens in breast cancer was suggested by the work of Bulbrook[l], with the demonstration of a lower urinary ratio of androsterone:etiocholanone in British women over 40 years old compared to Japanese women, the latter having a much lower incidence of the disease. As dehydroepiandrosterone (DHA) and DHA sulphate (DHAS) are the main plasma precursors to the urinary oxosteroids, many workers have investigated plasma levels of DHA and DHAS in patients with breast cancer. Reported levels have been variable, but the general concensus is that plasma DHA and DHAS levels are low to normal in these subjects. The relevance of measuring the plasma concentration of these steroids may depend on whether or not the tissue concentration is reflected by the plasma concentration. Adams et a1.[2] and Maynard et a1.[3] have measured DHA in breast tissue, but these workers did not comment on the plasma levels. There are no reports on the levels of the conjugated androgens in these tissues. The tissue concentration of DHAS may be important, since, as proposed by Adams[4], DHAS might indirectly influence oestrogen action via an effect on oestradiol metabolism. DHAS also acts as a prehormone to DHA and thence is converted via androstenedione to the oestrogens, or directly to *To whom correspondence

should

be addressed.

androstenediol. Androstenediol has been shown to compete with oestradiol for oestradiol cytoplasmic receptor and to induce biological responses characteristic of oestradiol [5,6,7]. The aim of this study was to develop specific, sensitive and reliable radioimmunoassays (RIA) for plasma and tissue DHA and DHAS, then to investigate the relationships of these hormones in patients with breast and endometrial cancer. EXPERIMENTAL Clinical material

Endometrial tissue and peripheral blood samples were obtained at the time of operation from women aged 20 to 53 years admitted for curretage or hysterectomy for non-malignant conditions and from postmenopausal women aged 49-79 years with endometrial carcinoma. Primary breast adenocarcinomas and normal tissue from the same breast were obtained from eleven postmenopausal patients aged SO-79 years. None of the patients was receiving any form of hormonal treatment up to the time of surgery. Tumours were dissected free from non-tumour tissue and portions of both retained for histological examination. Tissues were transported immediately to the laboratory and stored at - 20°C until required. Blood samples from the postmenopausal and breast cancer subjects (aged 48-83 years) were obtained from patients attending the out-patient clinic

151

152

D. L. JONESand V. H. T.

and were taken at various times of the day. Blood samples used to estimate the mean 24 h plasma steroid levels were taken at 0830, 1100, 1600 and 2200 h. Materials

[7-‘HIDHA (14 Ci/mmol) was obtained from Amersham International PLC. (Amersham, England). [7-3H]DHAS ammonium salt (24Ci/mmol) was obtained from New England Nuclear (Dreieich, W. Germany). Both compounds were regularly tested for purity by thin-layer or paper chromatography and purified if necessary. Stock solutions of label were stored in ethanol at 4°C. Unlabelled steroids were obtained from Steraloids, (Croydon, England) and were tested for purity. Helix pomatia sulphatase (S9626) and human gamma globulin (HGG) were obtained from Sigma (Poole, England), bovine plasma albumin from Armour Pharmaceuticals (Eastbourne, England). All other chemicals were obtained from Fisons (Loughborough, England). Ethanol, methanol and dichloromethane (DCM) were distilled prior to use. Silica gel plates (No. 5333) for thin-layer chromatography (TLC) were obtained from Merck (Darmstadt, Germany). Anti-DHA-7carboxymethyloxime-bovine serum albumin is available from Bioanalysis Ltd. (Cardiff, England). Scintillator was prepared from 12 g p-terphenyl, 0.16 g dimethyl POPOP and 80ml methanol made to 41 with toluene. The borate buffer used for the assay was pH 8.0, 0.5 M and the acetate buffer for hydrolysis was pH 4.8, 0.2 M. DHA assay method

DHA was extracted from aliquots of plasma (50 pl), by the addition of 1.5 ml n-hexanediethyl ether (4: 1, v/v) and mixing on a Buchler vortex evaporator for 10 min. The aqueous phase was frozen in an ethanol-dry ice bath and the organic phase decanted into glass tubes. Doubling concentrations of DHA from 10 to 128 pg in 100 pl of ethanol were pipetted into tubes from individual working solutions. The contents of these tubes and the assay tubes were evaporated to dryness at 3545°C under vacuum. The working solution of antiserum was prepared in borate buffer containing 0.125% (w/v) human gamma globulin 0.08% (w/v) bovine plasma albumin, and [3H]DHA (20,000 dpm/ml). After mixing gently, 300 pl was added to each of two tubes to determine the total counts and non-specific binding. Antibody was added to the remaining volume to give a final dilution of 1:6000 and 300~1 aliquots added to all remaining tubes. Incubation was overnight at 4°C or 30’ at 37°C followed by 1 h at 20°C then 30min at 4°C. Following incubation, separation of bound and free steroid was performed at 4°C by the addition of 1 mg of dextran coated charcoal (DCC) in 250 pl of borate butTer. 250 ~1 of DCC and distilled water were

JAMFS

added to the non-specific blank and total count tubes respectively. After centrifugation at 4°C a 400 pl aliquot of the supernatant was counted for 5 min in a refrigerated Packard liquid scintillation counter using 3 ml of scintillator. DHAS assay method

Aliquots of plasma (20 ~1) were mixed with 150 pl of acetate buffer (pH4.7, 0.2 M). To each tube a recovery tracer of [3HIDHAS (40,000 dpm) in 50 ~1 of redistilled ethanol was added and allowed to equilibrate for at least 1h at room temperature. Three total recovery aliquots were also taken. To each sample 20 units of sulphatase enzyme in 800 ~1 of the acetate buffer, was added and incubated at 37°C for 16 h. After incubation an aliquot (100 ~1) was extracted with 6 ml of redistilled n-hexane. After extraction the aqueous layer was frozen in an ethanoldry ice bath and the organic phase decanted into glass tubes. Three 500 ~1 aliquots of each sample were transferred to glass tubes and the contents evaporated to dryness in a vacuum oven at 35-45”C. The tubes containing the total recovery count and two RIA tubes for background count estimations were similarly treated. To one tube of each sample, the total recovery count and the background tubes of scintillator was added and the activity in each tube counted for recovery estimations which were made after subtraction of the background count. The standard curve was prepared using the same standards as for the DHA assay, including 100 pl of ethanol containing the mean assay recovery count of labelled DHA was also added. The contents of the tubes were evaporated to dryness at 3745°C under vacuum. Antibody mixture (300 ~1) was prepared as for the DHA assay with the exception that the number of DHA counts added was corrected for the amount of labelled tracer steroid present. The remainder of the methodology was identical to the DHA assay. The sample results for DHAS was corrected for methodological losses using the internal recovery and for the molecular weight difference between DHA and DHAS. Tissue preparation

Prior to assay the tissue was washed with glassdistilled water to remove any blood. The wet weight of the tissue was recorded and glass-distilled water added to give approx. 100 mg/ml prior to the tissue being homogenised in a Polytron blender. The breast tissue homogenate was centrifuged for 10min prior to assay to separate tissue and fat. The fat was aspirated and discarded, and the pellet of tissue particles resuspended. Tissue DHA measurement

To

aliquots,

usually

0.8-1.2 ml

containing

Adrenal androgens and cancer

SO-120mg of tissue homogenate (wet weight), 50~1 of ethanol containing 6000 dpm of [‘HIDHA was added as an internal recovery standard. The label was allowed to equilibrate for 30 min prior to extraction of the free steroids with 5 ml of diethyl ether. The aqueous phase was frozen in an ethanol-dry ice bath and the organic phase decanted. The extraction was repeated and the extracts pooled. The aqueous phase was retained for the DHAS estimation The residue remaining after evaporation of the ether at 40°C under nitrogen was applied to silica gel TLC plates with 2 x 100 ,~l of diethyl ether. The TLC plates were developed in dichloromethane-dioxan (96:6, v/v). The areas of [3H]DHA were located, excised and eluted with 5 ml of diethyl ether. The diethyl ether was decanted, evaporated to dryness under nitrogen and the residue reconstituted in 1 ml ethanol. Aliquots (3 x 200 ~1, one for recovery, two for assay) were taken and assayed as described under the plasma DHAS assay, with each tube of the standard curve boosted with an equivalent mass of [3H]DHA as that found for the mean recovery. Tissue DHAS measurement

Tritiated DHAS (SO~1 containing 10,000 dpm) was added to each of the aqueous phases left after the extraction of free steroid, vortex mixed, and allowed to equilibrate overnight at 4°C. The conjugated steroids were extracted from the tissue homogenate using 5 ml of ethyl actate-n-propanol (3 : 1, v/v). The precipitated proteins were removed by centrifugation at 1OOOgfor 10min. The decanted supernatant was evaporated to dryness under nitrogen at 45°C and 1 ml of acetate buffer (pH 4.7, 0.2 M), containing SO units of sulphatase added. After vortex mixing and overnight incubation at 37°C the hydrolysed steroid was extracted using 10 ml of diethyl ether. After collection of the organic phase and evaporation to dryness, the residue was applied to silica gel TLC plates with two 100~1 diethyl ether washes, and developed twice in dicbloromethaneeethyl acetate (93 : 7, v/v). The [ 3HIDHA areas were detected and eluted as described previously, and the residue reconstituted in 2.5 ml of ethanol. Aliquots of 500 ~1 were taken, one for recovery estimation and two for assay. The assay method was then as described for the plasma DHAS assay.

153

Accuracy and sensitivity

Recovery (+ SD) of [3H]DHA from plasma and tissue (both breast and endometrial) was 99 + 3 and 80 f 5% respectively while for [ 3H]DHAS recoveries of 94 f 4 and 42 f 6% were obtained. Good recoveries of unlabelled DHA and DHAS were obtained from spiked plasma, breast and endometrial tissue over the complete range of physiological values studied (Table 2). Non-specific binding in the assay was always less than 3% of total counts. The assay sensitivity, defined as the smallest concentration significantly different from zero at the 5% level was determined from 10 water blanks assayed in duplicate in five separate assays and was 9pg per assay tube. This corresponds to a minimum detectable amount of 0.5 nmol/l DHA and 0.15 pmol/l for DHAS using the plasma assay and 0.5 rig/g and 1.12 rig/g for tissue DHA and DHAS using 100 mg/ml in the tissue assay procedure described above. Charcoal-stripped plasma and water blanks taken routinely through the assay procedure gave results which in all cases were less than the assay sensitivity. Precision

Intra and inter assay precision was measured over a period of 18 months for both hormones with two technicians performing the assays. Results are shown in Table 3 with the intra assay figures representing a random selection of duplicates taken over the 18 month period. Due to the shortage of sample non inter assay CV data is available for the tissue Table

I. Cross-reactions

of antibody

Steroid DHA 16crOH DHA Androstenediol Androstenedione Androsterone DHAS Androstanediol Cortisol

100.0 3.4 I.3 0.7 0.04 0.03 0.003 <0.0015

Percentage cross reaction quoted at 50% displacement of 30pg labelled DHA bound at H final antibody &lotion of I :6000 which gives 60% binding.

Table

2. Recovery after addition

of steroid from tissue and plasma of known amounts of steroid Percentage

RESULTS

Mass/assay

tube

recovery

DHA

(n)

DHAS

(n)

104 IO1 98 103

(4, (6) (7)

96 109 103 109 “’

(8) (13) (7) (8) (9) _

90 94 94

(4) (4) (4)

106 II2 100

(4) (4) (4)

Plasma assay

Antiserum

in this paper were performed using a pooled rabbit antiserum obtained from a number of bleeds with similar titres and specificity at a final titre of I :6000. No change in the binding characteristics of the antiserum were seen over a two year period of regular use. The characteristics of the pooled antiserum are shown in Table 1. A11 results

to DHA

% Cross reaction

shown

40 Pg 50 pg 16Opg 320 pg 500 pg l2gpg

0

Tissue assay l6pg 60 pg 256 pg

154

D. L. JONESand V. H. T. JAMES Table 3. Assay precision

for tissue and plasma methods Plasma

n Intra assay Inter assay

DHA (nmol/l) %C.V. mean

83 38 21 46 57

16.7 8.6 14.4 17.4 18.5

n

7.4 II.5 6.1 8.3 9.9

DHAS (Hmol/l) mean %C.V.

66 38 52 46 18

5.8 0.9 4.4 5.4 1.3

9.7 13.3 9.9 9.3 7.6

27

DHAS Wd 118.4

7.1

Tissue lntra

assay

32

DHA 69.1

Wg) 10.7

methods, however plasma quality control samples were run in each assay and gave similar results to those found with the plasma assay. Assay linearity

Assay linearity was tested by assaying a range of sample volumes (six in quadruplicate for the plasma and five in duplicate for the tissue assays) and calculating the regression lines for the data obtained. Results suggest the assays perform linearly over the volume ranges tested.

subjects studied over a complete cycle (over 2 successive cycles) plasma DHA and DHAS levels can vary significantly 3-fold and 2-fold (Fig. 3). There are not sufficient data to conclude whether the plasma levels of DHA and DHAS are related to the stage of the cycle as assessed by plasma progesterone levels. Efect

of age

The relationship between age and plasma levels of DHA and DHAS in women with either surgical or natural menopause is shown in Fig. 4. For DHA the levels between the age groups <50-60 years old are significantly different (P < 0.005 Student’s t-test), but not for the age groups 5&60 and > 60 years old. For DHAS there was a significant difference between both ~50 vs 5MO year olds and the 50-60 vs > 60 year olds (P < 0.005). 40

r

(4

r Z096

Specificity

The specificity of the plasma assay methods was demonstrated by the measurement of DHA and DHAS on samples with and without an initial purification stage. For both steroids this involved paper chromatography with either Bush A or Bush B3 solvent systems or by TLC with either dichloromethaneeethyl acetate (93 : 7, v/v) or dichloromethane-acetone (80 : 20, v/v). Results are shown in Figs la and lb. Specificity for the tissue assays depends upon the routine use of a TLC stage and a highly specific antiserum. No cross-reacting material was found for either tissue when sections along the TLC strips were eluted and put though the assays.

t. . Ij/ l

y=10461-0.7

.

.

00

‘3

.

.

I 30

I 20

I

10 DHA

nmol/l

wth

I 40

purlfbcotlon

Normal ranges

Normal plasma ranges for the two steroids are shown in Table 5. Normal ranges for the tissue are shown in Table 6. Diurnal variation

Ten adult subjects (7 female, 3 male) were studied over a 24 h period with blood sampling every 30 min. All subjects demonstrated secretory episodes of DHA which occurred synchronously with cortisol. DHAS did not demonstrate secretory episodes. DHAS showed a different pattern; there was a diurnal rhythm of two plateaus, one high between 1100-2200 and a low from 0100-0800. Menstrual cycle levels

With single blood samples taken at varying stages of the menstrual cycle no significant difference can be shown between the plasma levels of DHA or DHAs in the luteal or follicular phases (Table 5). In two

1

2

3 DHAS

with

4

5

6

7

pmo/l

purifkatbn

Fig. 1. Plasma levels of DHA (a) and DHAS (b) measured with and without a TLC (0) or paper (A) purification stage.

Adrenal androgens and cancer

155

Table 4. Linearity data for assay methods Steroid

Volume range x

Plasma DHA Plasma DHAS

25-400jl I 2&200fil

Tissue DHA

250-1500~1

Tissue DHAS

250-1500 ul

9

11

13

r-value

Regression

y = 0.08x + 0.296 Y = 0.005x + 0.018 y=OMx-1.6 v=O.407x+1.46

15

17 clock

19

21

23

Y

I .9-33.3nmol/l

0.998 1.0

0.15-0.99pmol/l

0.98

20&560 pg/assay tube

1.0

1263 neiassav tube

1

3

5

7

9

time hours

Fig. 2. Levels of cortisol, DHA and DHAS measured in plasma samples taken at 30 min intervals from a normal female through 24 h.

The endometrial tissue concentrations of DHA, but not DHAS, are significantly lower in the 5&60 year old group compared to the ~50 group (0.001 < P < 0.01). The number of subjects is too low in the other groups to allow statistical analysis but the same general pattern is seen as was found with the plasma levels (Table 6). Effect of weight Linear regression analysis of the degree of obesity (as % ideal body weight) vs plasma DHA or DHAS levels in 26 subjects showed no relationship between the plasma levels of either steroid with weight or the degree of obesity in single samples or mean 24 h levels taken from normal postmenopausal women. In ten postmenopausal breast cancer subjects a correlation approaching significance (0.05 < P < 0.1) was found between both the mean 24 h plasma DHA and DHAS levels with weight expressed as a percentage of ideal body weight. Breast cancer In 32 postmenopausal women with breast cancer no significant difference was found between the

1

a

12

16 20 day of cycle

24

28

32

Fig 3. Plasma levels of DHA and DHAS measured in samples taken through the menstrual cycles of 4 subjects, one on two successive cycles (0-O and 0-O).

D. L. JONESand V. H. T. Table 5. Plasma DHA

steroid

JAMES

levels

(nmol/l)

mean

range

n

DHAS (rmol/l) _.~__~.. range IWa”

n

Male FclIIalc Postmenopausal follicular luteal

IS.0

7.4-53.0

19

8.7

3.7-15.0

19

19.2 21.2

3.0-35.0 3.443.9

22 33

3.4 3.5

1.5-6.1 1.3-6.7

22 33

Postmenopausal I. 60 yrs old

16.9 8.8 8.3

3.0-43.9 3.3-25.7 1.3-25.0

55 31 37

3.4 2.7 1.48

1.3-6.7 0.67.9 0.1-3.7

55 31 37

Breast cancer (48-83 yrs old)

10.5

1.9-19.1

32

2.6

O&5.0

32

12.6

4.1-32.9

7

2.3

0.7-4.5

8

Endometrial cancer (49-74 yrs old) 1 “S 2 P <0.005. 2 YS 3 P 40.1.

1 “8 2

0.025

plasma levels of either DHA or DHAS in single blood samples compared to age matched normal postmenopausal women. The same was true for the mean 24 h plasma levels of these steroids as determined from 4 samples taken at times of the day that we have found to a good estimate of the 24 h value (Table 7). The mean level of DHA in tumourous tissue was significantly higher than normal tissue taken from the same breast (P < 0.01 n = 11, paired f-test). In 10

similarly matched pairs the mean DHAS levels were not signi~cantly different (Table 6). E~dometr~u~ cancer

The mean plasma levels of DHA and DHAS in 10 postmenopausal women with endometrial cancer were not significantly different (Student’s t-test) normal subjects of the same age. No significant difference was found for the levels of either steroid in normal and cancerous tissue taken from postmenopausal women (Table 6). Correlation of plasma and tissue steroid levels

Linear regression of the concentrations of DHAS and DHA in plasma vs endometrial tissue of 55 normal premenopausal women were signi~cantly correlated (r =O.?, P -=z0.001, and r =0.323, P ~0.01 respectively [Figs Sa and 5b]. In six postmenopausal patients plasma DHAS (Y = 0.889) but not DHA (r = 0.5) levels were correlated to tissue concentrations. With endometrial cancer patients there was no correlation between plasma and tissue levels of either DHAS or DHA (I = 0.3 and 0.35 respectively), but the numbers are low (n = 6) in both these two groups. No data are available for the breast cancer subjects.

Age Fig.

(years)

of age on the plasma DHAS In postmenopausal

4. Effect

levels of women.

DHA and

Results obtained using the methods described here agree well with reported values for both plasma and tissue DHA [3,8,9, IO]. Plasma DHAS values agree with Buster and Abraham[ 1l] and Cattaneo et a[.[ 12). We are not aware of any reports on the measurement of DHAS in breast or endometrial tissue. The synchronous secretion of DHA with cortisol, first shown by Rosenfeld et a/.[91 and confirmed by our data, suggested that ACTH is the principal stimulus to DHA secretion. The episodic nature of DHA secretion demonstrates the necessity of taking more than one sample to obtain a reliabie estimate of the mean 24 h plasma DHA level. The same appfies

Adrenal

androgens

and cancer

157

Table 6. Tissue steroid levels DHA @g/g)

DHAS @g/g)

mean

range

”

mean

range

n

Normal endometrial tissue I. < 50 yrs old 2. 5wo yrs 3. > 60 yrs old 4. Endometrial cancer (49-74 yrs old)

77.6 33.6 32.5 23.4

4.1-343 5.1-94.4 11.5-53.1 2.4-53.1

66 17 3 8

173 154 80 184

3.7-620 9.6-501 29.&131 29.0-530

57 14 2 7

Breast tissue 5. < 50 yrs old 6. 5@60yrs 7. > 60 yrs old 8. Breast cancer (5&79 yrs old)

25. I 8.2 6.5 15.8

4.349.5 6.1-10.2 1.&20.0 3.341.4

3 2 9 II

171 204 92.4 155

73.9-296 41.7-366 6.&554 9.5-857

3 2 8 10

1 “S 2 0.001 c P < 0.01. 2 and 3 vs 4 P ~0.1. 6 and 7 vs 8 P < 0.01.

I “S2 ns. 2 and 3 vs 4 ns. 6 and 7 vs 8 ns.

Table 7. Mean 24 h plasma values in normal women, and women with breast cancer DHA nmol/l

Normal Cancer

Cortisol nmol/l

DHAS pmol/l

mean

range

n

mean

range

n

mean

5.14 6.6

1.37-9.4 1.86-12.8

7 10

1.48 1.78

0.2-3.05 0.29-4.34

7 10

170 262

to DHAS, which although not as variable as plasma DHA, also demonstrates a diurnal rhythm. The significant negative correlation between the plasma levels of DHA and DHAS with age has been demonstrated before [ 131. However, our results agree with the observation by Crilly[l4], that after an initial fall, DHA levels reach a basal level by the age of 60. As Crilly’s subjects were oophorectomised and therefore had no ovarian steroid production, and demonstrated no concomitant decrease in plasma cortisol levels, this is strong evidence for a specific alteration in either the metabolism or the adrenal secretion of DHA and DHAS. The effects of age and diurnal variation are well recognized problems in the comparison of data, but for some homones, e.g. the oestrogens, obesity can also have an effect [15]. Our data show that neither plasma DHA nor DHAS levels are significantly correlated to percentage ideal body weight in normal female subjects, but for breast cancer subjects the correlations were approaching significance. More data are required to test this observation further. When compared to age matched control patients there was no alteration in plasma DHA or DHAS levels in breast cancer patients, which contrasts with the results of Zumoff et a[.[161 and Brownsey et a/.[ 171 who demonstrated in breast cancer patients increased and decreased plasma levels of DHAS respectively. Zumoff also reported increased DHA levels in these patients. With the subjects studied over 24 h, patients with breast cancer had a greater percentage of ideal body weight compared to the control group (111 vs 103%). In view of the possible effect of weight on plasma DHA levels in breast cancer subjects, the slightly elevated DHA levels found for breast cancer subjects (Table 7) may reflect the increased weight. Guerrero[ lo] demonstrated a good correlation be-

range

n

96-264 I 18@-400 10

tween tissue and plasma pregnenolone levels but none for DHA. Our data reveal a relatively good correlation between DHAS levels in plasma and tissue, whilst DHA levels were less well correlated. Plasma DHA levels fluctuate considerably more than DHAS, and if time is required to establish a plasma/tissue equilibrium, this may explain the poorer correlation seen for DHA. It was possible that the steroids would undergo in oitro metabolism in the tissue after excision and prior to assay. To minimise this possibility the tissue samples were placed on ice after excision and frozen as soon as possible. Incubation of tissue with the 3H-labelled DHA and DHAS tracers for 16 h at 4°C did not result in the formation of 3H-labelled metabolic products which would have been located on the TLC scans. This suggests in vitro metabolism may not be a problem. A relationship between tissue and mean 24 h plasma levels of unconjugated steroid may be expected from the observations of Giorgi[l8], that unconjugated steroids cross the cell membrane by passive diffusion. Giorgi[l8] suggests that the intracellular concentration of steroid is related to the amount of unbound steroid in plasma and the solubility of the steroid in the lipid membrane. For DHA and DHAS approx. 5 and 1% of steroid is free in plasma (unpublished data). Using plasma levels found in normal postmenopausal women approx. 144 pg of DHA and 10.6ng of DHAS is unbound in 1 ml of plasma. Expressing the mean tissue concentration of these steroids as a ratio of the plasma concentration, ratios of 230 and 10 are obtained for DHA and DHAS respectively. The partition coefficients of DHA and DHAS between n -octanol and water were obtained using the method of Giorgi[l8] and were 126 and 6.3 re-

D. L. JONESand V. H. T.

158 400-

(a)

300. ~~1.24

x+50.3

r ~0.323

.

.

.

. *

100’

H *.. . . ‘.... .

0-

.

. :

*

.,

:

. *

.

. 4

0

20 nmol

II

40

60

plasma DHA

(b) soc )’

. y=66.6x-50.5

100

0 0

1

2

3

4

5

6

7

p m0l/l plasma

DHAS

Fig. 5. Correlation between plasma and endometrial tissue levels of DHA (a) and DHAS (b) in premenopausal women.

spectively. The partition coefficeint between noctanol and water has been reported to give a good indication of the solutility of the steroid in the cell membrane [19]. The 20-fold higher tissue to plasma

JAMES

ratio of DHA and DHAS corresponds to the relative partition coefficients found for these steroids. These data suggest that for normal endometrial tissue the unbound plasma level and tissue level of DHA and DHAS are related, and that the tissue levels agree with the hypothesis of free diffusion of steroid across the membrane, the degree of diffusion being dependent on the polarity of the steroid. The conclusions presented here assume that endometrial tissue does not contain receptors for either DHA or DHAS or possess the capability to metabolise intracellulary precursors to form these steroids. Our data for breast tissue concentrations of DHAS and DHA demonstrate a significantly increased DHA level in breast tumor tissue. Since the fat tissue is removed prior to the assay procedure the DHA levels expressed here must be regarded as minimal concentrations. As breast tumour tissue contains very little fat tissue, measurements of steroid levels in normal breast tissue containing a large proportion of fat may not be an appropriate control. Elevated breast tumour tissue DHA levels may be due to increased passage of plasma DHA to the tissue, increased passage and metabolism of DHAS or other precursors to DHA, e.g. pregnenolone [20], or a diminished rate of metabolism of DHA to products such as androstenediol and androstenedione. Bonney et a/.[211 have reported that the tissue androstenediol level is directly correlated to the DHA level, suggesting the third possibility may not be valid. There is also no evidence of an increased plasma or tissue level of the possible unconjugated precursors to DHA such as pregnenolone in breast cancer patients [22], which suggests the second possibility may be unlikely. As the tissue concentration of DHA and DHAS are correlated in normal breast but not in the cancerous tissue [23], this suggests an alteration in the metabolism of intracellular DHA and DHAS. The lack of correlation between plasma and tissue DHAS in postmenopausal endometrial carcinoma patients and increased breast tissue DHA and DHAS may result from an increased transport of DHAS across the membrane and metabolism of DHA. In conclusion we have developed radioimmunoassays for plasma and tissue DHA and DHAS and have demonstrated a correlation between plasma and tissue levels of DHA and DHAS in normal endometrial tissue. Plasma levels of DHA and DHAS are not altered in breast or endometrial cancer patients compared to age-matched controls. The correlations between tissue and plasma DHAS and DHA and the relationship of intra-tissue DHA to DHAS has been lost in the cancer patients which may indicate an alteration in the metabolism or transport of these steroids into the tissue. REFERENCES 1.

Bulbrook R. D., Thomas B. S., Utsunomiya J. and Hamaguchi E.: The urinary excretion of 11-deoxy-17-

oxosteroids and 17-hydroxy corticosteroids by normal

Adrenal androgens and cancer Japanese

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