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Free testosterone levels in plasma and saliva as determined by a direct solid-phase radioimmunoassay: A critical evaluation
Clinica Cltimico Acta. 191 (1990) 21-30 Elsevier
21
CCA 04806
Free testosterone levels in plasma and saliva as determined by a direct solid-phase radioimmunoassay: a critical evaluation Franqois
Rey, Giovanni Chiodoni, Karine Braillard, and ThCr&e Lemarchand-BCraud
Christine
Berthod
Division d’Endocrinologie et Biochimie Unique, Centre Hospitalier Universitaire Vaudois, Laurame (Switzerland) (Received
6 April 1989; revision
Key words: Free testosterone;
received 29 May 1990; accepted
Saliva testosterone;
Free hormone
5 June 1990)
radioimmunoassay
Summary We compared unbound (free) testosterone radioimmunoassay concentrations in plasma and saliva from men, using a direct radioimmunoassay kit involving a ligand analog of testosterone as tracer. The assay failed to reveal detectable testosterone concentrations in saliva. In plasma the free testosterone levels were about 4 times lower than those obtained by calculation or ultr~iltration methods. Moreover, unexpected similar free testosterone levels were obtained in samples comparable in their total testosterone content but distinct in their steroid binding protein content (buffered testosterone dilutions). We suspect that free testosterone levels determined with this direct radioimmunoassay probably do not reflect the true free testosterone values and conclude that their significance remains to be established.
Introduction The direct relationship between the free fraction of testosterone (T) in plasma and the biological activity of the hormone has been recently reevaluated in view of the suggestion that part of the protein bound T might be transported across microcirculatory barriers [l]. This latter process may involve a mechanism of enhanced dissociation of hormone from plasma-binding proteins induced by transient conformational changes.
Correspondence Clinique, Centre
and requests for reprints to: F. Rey, Ph.D. Division Hospitalier Universitaire Vaudois, CH-1011 Lausanne,
0009-8981/90/%03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
d’Endocrinologie Switzerland.
Division)
et Biochimie
22
In men and women, salivary T concentrations closely correlate with plasma free T measured either by dialysis [2], ultrafiltration [3] or when estimated by calculation [4]. However in women, the total salivary T concentrations exceed by 2- to 3-fold those of plasma free T estimated by these methods. This observation may support the view that some albumin-bound T is available to peripheral tissues [5] or that saliva contains steroid-binding proteins (like SHBG) or albumin which could bind T [6]. Nevertheless we have recently suggested that the discrepancy between saliva and plasma free T might be due to local conversion of androstenedione to T in salivary glands [7]. Whatever, salivary T determination provides an excellent approach for the evaluation of androgenic activity. To further evaluate the relationship between circulating free T levels and saliva content without methodological bias, we chose to use a commercial RIA kit designed for direct free T determination in serum and plasma. Materials and methods Samples collection Blood and/or saliva were collected from 2 groups of healthy male volunteers (aged 20 to 32). In the first group (n = 9) samples were obtained at 08.00 after an overnight fasting. In the second group (n = 10) samples were collected at various times of the day and in some cases after an injection of testosterone in order to provide a broad pattern of total T levels. Blood was obtained by venepuncture using lithium heparinate monovettes (Sarstedt, Sevelen, Switzerland). Individuals were asked to salivate 2 to 5 ml into disposable tubes in about 5 minutes. Blood and saliva samples were immediately centrifuged (3000 x g, 4O C, 5 min) and decanted. Supematants were stored at - 20 o C until assayed. Assays Free T, direct solid-phase RIA Free T determinations in plasma, saliva and buffered T dilutions were performed by the direct solid phase commercial RIA from Diagnostics Products Corporation (DPC, Los Angeles, CA) designed to leave essentially unchanged the original equilibrium between free and protein bound testosterone in serum and heparinized plasma samples (Coat-A-Count Free Testosterone lz51 RIA). Before assays, samples were centrifuged 5 min at 3 000 X g, 4°C. We chose also to treat saliva samples using the protocol recommended for serum. In regards of the ratio between sample and reagent volumes (50 ~1 vs. 1000 ~1) we considered that the matrix effect would be negligible. Total T ZtZA Total T levels were measured in plasma, saliva and calibrators from the RIA kit by a specific and sensitive RIA method developed in our laboratory and previously described [8].
23
We measured free T fractions in plasma, Free T, determination by ultrafiltration saliva and buffered T dilutions by ultrafiltration according to Vlahos et al. [9]. We used the micropartition system MPS-1 from Amicon (Danvers, MA, USA) with YMT membranes (molecular cut off 30000 Da). Free T. estimation by calculation Knowing total T, SHBG and albumin levels, free T concentrations were calculated in plasma, saliva and RIA kit calibrators according to S&lergard et al. [lo]. SHBG was measured by IRMA commercial kit (Farmos Diagnostica, Oulunsalo, Finland). For dete~nation in saliva, the lower limit of the standard curve was extended from 6.25 to 0.390 nmol ’ 1-l and the dilution step of samples was omitted. Plasma albumin was measured with bromocresol green [ll]. Salivary albumin was determined using a micro-albumin RIA validated for urine (Pharmacia AB, Uppsala, Sweden). Evaluation
of the DPC free testosterone
RIA
The following 4 calibrators Calculated free T vs. indicated levels in kit calibrators given as 0, 2.08, 58.9 and 191.0 pmol *1-l were choosen for free T levels estimation by calculation (calibrators lot numbers TFC3UO4, TFC4004, TFC6004 and TFCSOO4, respectively; kits lot number 090bl). Free T determination in various media at constant total T levels In order to evaluate the influence of sample matrix in the RIA we determined free T levels in 4 different matrix conditions, each condition being tested at 10 distinct concentrations of total T. 1. Human plasma: 10 samples containing respectively 50.2, 45.1, 40.2, 35.4, 30.6, 26.0, 20.5, 16.0, 10.4 and 6.7 nmol *1-l of total T and physiological concentrations of SHBG and albumin were selected to cover progressively the range of clinically observable values. 2. 0.1 mol/l Tris buffer pH 7.8, protein free: 10 samples constituted by T dilutions at levels similar to those observed in the 10 selected plasma samples. 3. 0.1 mol,/l Tris buffer, pH 7.8, gelatin 1 g - 1-r (Merck 4078): 10 samples constituted by T dilutions as above mentioned. 4. Krebs original Ringer phosphate buffer, pH 7.4, enriched with 20 g. 1-i human serum albumin (albumin dry powder, Red Cross, Bern, Switzerland): 10 samples constituted by T dilutions as above described. Results
Table I reports the basal plasma and saliva free T levels determined in 9 healthy men by ultrafiltration, calculation and direct RIA, as well as total T and binding proteins in plasma and saliva. Plasma-free T levels obtained by ultrafiltration and calculation were similar. Values obtained by direct RIA were significantly lower than those determined by
24 TABLE
I
Plasma-
and saliva-free
T levels obtained
by ultrafiltration, Plasma-free
Ultrafiltration Calculation RIA direct
T a % of total plasma
538.1+ 142.7 523.8 + 131.6 128.2 f 29.2
2.74+ 0.29 2.53kO.18 0.65 f 0.09
a The corresponding
7
T ’
pmol.l-’
% of total saliva T
182.1 f 53.9 390.8 f 99.6 undetectable
44.69 + 4.48 99.65 k 6.94
Total T and binding
Testosterone (nmol.l-r) SHBG (nmol.l-‘) Albumin (g.l-‘)
and RIA in 9 men (mean + SD)
pmol-1-l
Saliva-free
Ultrafiltration Calculation RIA direct
calculation
proteins
Plasma
Saliva
20.5 + 4.5 23.4k6.1 47.2 + 3.7
403.3 * 101.0 x lo- 3 5.6+ 5.7x10-’ 51.1+ 29.7x10-’
levels of total T, SHBG
and albumin
are indicated
in the lower panel.
the former methods. Direct RIA free T levels were positively correlated to those determined by ultrafiltration or calculation and also to total T levels with the same degree of significance (P < 0.01). In saliva, calculated free T levels were similar to those determined by our own RIA designed for total T measurements, indicative of a negligable binding of T to SHBG and albumin. In contrast, free T concentrations determined in saliva by ultrafiltration showed an important binding of about 55%. With the direct RIA no significant saliva free T levels were detected ( < 0.5 pmol . l- ’ ). Figure 1 shows free T levels measured by the free T RIA in the 4 series of 10 samples, each series being characterized by the nature of sample matrix. In all cases, free T values appeared directly dependent of total T levels but almost independent of the protein content of diluting media. Table II allows comparison of free T values (expressed in percent of total T) estimated by ultrafiltration and RIA in the same 4 series of 10 samples. In Tris buffer, without or with addition of gelatin, free T fraction determined by ultrafiltration was over 80% (probably high non specific binding to membranes). In Krebs original Ringer buffer, albumin 20 g. 1-l) the free T fraction was constant and equal to 11% whatever the total T concentration. The respective values of 80% and 11% must be considered as minimal. Indeed it is not possible to estimate the true values in media deprived or totally free of proteins since non specific absorption to membranes is higher in such media than in total plasma. Table III shows total T, SHBG, albumin and calculated free T levels determined in the DPC RIA calibrators. Determination of binding proteins levels revealed
II
’ The 4 different total T levels.
RIA
RIA 4. Krebs, albumin Ultrafiitrat~on
I _’
media compositions
20 g. 0.68
0.97
have been choosen
0.75
0.99
1.03
1.13
RIA 3. Tris, gelatin 1 g.lUltrafiltration
’
3.12 0.65
2 45.1
0.73
the influence
0.64
0.67
11%
80% 0.88
80% 0.88
2.22 0.38
6 26.0
0.62
0.84
0.78
3.39 0.82
8 16.0
and the concentration
0.64
0.82
0.55
2.16 0.45
7 20.5
levels of total T in 4 different
of the quality
~nimum
minimum 0.87
minimum 0.86
1.03
0.99
2.54 0.70
5 30.6
2.20 0.46
4 35.4
and by direct RIA at 10 distinct
in order to evaluate
0.12
0.94
0.88
3.23 0.82
3 40.2
by ultrafiltration
3.51 0.17
1 50.2
of total T determined
Ultra~ltration RIA 2. Tris protein free Ultrafiltration
1. Plasma
% free T in:
Total T a (nmol,l-‘)
Free T values in percent nature of their matrix
TABLE
in protein
0.62
0.19
0.77
2.37 0.72
9 10.4
contents
by the
at constant
0.69
0.78
0.79
2.23 0.57
10 6.7
media distinguishable
e-.-
Plasma
-
Trls, proteins free
c-c-
Tris,gebtin Krebs, albumin
67 L
0
to.4 I
16.0
10
x).5 I
26.0
30.6 I
30
20
Total
35.4
40.2 L
40 testosterone
45.1
50 2 I
50 nm-oi.l-*
Fig. 1. DPC free T levels determined at 10 levels of total T in 4 different media characterized by the nature of their matrix. In all T dilutions performed in Tris buffer and in most T dilutions made in Krebs-albumin buffer (total T z 20.5 nmol.l-‘), direct free T RIA should have been totally saturated. On the contrary free T levels appeared assessable and well correlated (P < 0.01) whatever the milieu.
SHBG concentrations ranging from 5.2 to 5.8 nmol. 1-l and constant albumin levels of 38.0 g. I-‘. Total T concentrations ranged from 0.03 to 39.41 nmol *1-l. Calculated free T levels were totally in discordance with those indicated: 1.1 vs. 0.0,
TABLE III DPC direct free T RIG kit: indicated vs calculated free T levels in 4 calibrators as well as contents in total T, SHBG, and albumin
Indicated free T (pmol .l- ’ ) Calculated free T (pmol.l-‘) Testosterone (nmol.l-t) SHBG (nmol.l-‘) Albumin (gel-‘)
Level 1
Level 2
Level 3
Level 4
0.0 1.1 0.03 5.2 38.0
2.08 22.2 0.59 5.4 38.0
58.9 391.0 10.23 5.8 38.0
191.0 1569.1 39.41 5.7 38.0
27
22.2 vs. 2.08, 391.0 vs. 58.9 and 1569.1 vs. 191.0 pmol- I-‘. This comparison is relevant only if the affinity constant of SHBG is similar in calibrators and in biological samples. If it is not the case, our estimation must be considered as erroneous but at the same time it has to be acknowledged that kit calibrators do not react as serum or plasma samples. In calibrators, the ratio between calculated and indicated free T levels showed values higher than those observed in plasma between calculated and RIA free T levels. Discussion
The present results confirm that free T levels estimated by the direct RIA in heparinized plasma and serum are totally equivalent but distinct from those determined by ultrafiltration (comparable to dialysis) or theoretically calculated according to the law of mass knowing SHBG, albumin and total T levels. As estimated in 9 normal male volunteers in basal conditions, the mean value of plasma-free T levels determined by RIA was 4 times lower than those obtained by the conventional methods. Like Cheng et al. 1121,W&e and Utley recently reported a similar bias in women between direct RIA and dialysable or calculated plasma-free T levels [13]. Nevertheless, these authors noted that the relative amp~tude of this bias is not constant, showing that in pregnant women particularly, RIA-free T levels are only one half of calculated levels. In view of the general positive correlation between free T levels determined by direct RIA and calculation, and despite the later observation in pregnant women, the authors concluded to the reliability of the RIA. In the present study, we found also good correlations between free T levels obtained by RIA and either ultrafiltration or calculation. However we found the same positive correlation between RIA free T and total T levels. We are therefore not able to confirm that RIA free T reflect circulating free T levels depending from total T and protein binding concentrations. In women, total saliva T concentrations exceed free plasma T levels particularly when estimated by ultrafiltration or calculation [3,4,7]. We have previously suggested that this discrepancy might be due to a local transformation of androstenedione into T [7]. The high ratio between plasma androstenedione and T might be responsible for this transformation. In normal men, such a discrepancy is not observed, free plasma and total saliva T levels being similar. However, we cannot exclude that the same process exists, but hidden by the probable and concomitant transformation of T into 5cw-dihydrotestosterone [14]. In saliva, T exists both in bound and free forms. Shaw indicated that about 10% of T binds to macromolecules, i.e. to undialysable compounds [15]. In the present study, the comparison between total and free saliva T levels determined by ultrafiltration is suggestive of a more important binding of T to protein of about 50%. This high binding cannot be related to the minor cont~nation of saliva by SHBG and albumin, total and calculated saliva-free T levels being strictly comparable, This observation is indicative of the presence of unidentified steroid binding proteins in
28
saliva. In any case, free saliva T levels must represent at least one half of total saliva T, i.e. of free plasma T levels. A priori, such saliva levels should be accurately estimated using a method designed for the measurement of free serum T. However, the direct free T RIA developed for measuring T in serum failed to reveal any detectable free saliva T levels despite the fact that saliva samples represented only 5% of volume during RIA incubation (lower detectable concentration: 0.5 pmol 1 1-Q. To further estimate free T RIA reliability, we determined the real influence of T proteins binding at 10 levels of the hormone in 4 media with different protein contents. Unexpectedly, RIA free T concentrations appeared almost independent of media composition. Particularly with T dilutions ranging from 6.7 and 50.2 nmol .ll’ performed in Tris buffer protein free, in which T remains in free form, RIA gives free T levels ranging from 53 to 568 pmol .1-i. These values are similar to those obtained in complete plasma samples with the same T concentrations. This observation is suggestive that the RIA provides results which are not related to the true free T values but are merely reflective of total T concentration. The high free T values determined in pregnant women by Wilke et al. [13] and the lack of a negative and significant correlation between SHBG levels and free T values found by the same authors, support this view. The method of standardization used by the DPC firm is unknown. RIA calibrators are dramatically depleted in SHBG when compared to plasma. The reference method used to calibrate the standards is doubtful and in any case different from that used by others. Consequently the adequacy of DPC RIA calibrators is highly questionable. In view of our results and without the purpose to further contribute to hot debate on the validity of analog free hormone immunoassays [16,17] we are of the opinion that the so-called free T values determined using the DPC direct RIA are misleading since they are not representative of true free T levels. The precise significance of these values remains to be established. Acknowledgements The authors wish to thank Dr. M. Markert for plasma albumin determinations, Dr. D. Jallut for technical assistance and Mrs. M.-C. Evraere for editorial assistance. References Pardridge WM. Plasma proetin-mediated transport of steroid and thyroid hormones. Am J Physiol 1987;252:E157-E164. Baxendale PM, Jacobs HS, James VHT. Salivary testosterone: relationship to unbound plasma testosterone in normal and hyperandrogenic women. Clin Endocrinol 1982;16:595-603. Sanmkka E, Terho, P, Suominen J, Santti R. Testosterone concentration in human seminal plasma and saliva and its correlation with non-protein-bound and total testosterone levels in serum. Int J Androl 1983;6:319-330.
29 4 Rey F, Bumand B. Gomez, F. Free testosterone and clinical hyperandrogenism in women: correlation of sex hormone binding globulin, calculated free testosterone and salivary testosterone with hirsutism score. In: Forest MG, Pugeat M, eds. Binding proteins of steroid hormones. Inserm-John Libbey, 1986; 636 (abstract). 5 Ruutiainen K, Sannikka E, Santti R, Erkkola R, Adlercreutz H. Salivary testosterone in hirsutism: correlations with serum testosterone and the degree of hair growth. J Clin Endocrinol Metab 1987;64:1015-1020. 6 Selby C, Lobb PA, Jeffcoate WJ. Se.x hormone binding globulin in saliva. J Clin Endocrinol 1988;28:19-24. 7 Rey F, Chiodoni G, Gomez F, Felber JP. Interpretation of the discrepancy observed between plasma free and salivary testosterone levels in man. Steroids 1988;52:371-372. 8 Magrini G, Chiodoni G, Rey F, Felber JP. Further evidence for the usefulness of the salivary testosterone radioimmunoassay in the assessment of androgenecity in man in basal and stimulated conditions. Horm Res 1986;23:65-73. 9 Vlahos I, MacMahon W, Sgoutas D, Bowers W, Thompson J, Trawick W. An improved ultrafiltration method for determining free testosterone in serum. Clin Chem 1982;28:2286-2291. 10 Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and oestradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem 1982;16:801-810. 11 Doumas BT, Watson WA, Briggs HG. Albumin standards and the measurement of serum albumin with bromocresol green. CIin Chim Acta 1971;31:87-96. 12 Cheng RW, Reed MJ, James VHT. Plasma free testosterone: equilibrium dialysis vs direct radioimmunoassay. Clin Chem 1986;32:1411. 13 WiIke TJ, Utley DJ. Total testosterone, free-androgen index, calculated free testosterone, and free testosterone by analog RIA compared in hirsute women and in otherwise-normal women with altered binding of sex-hormone-binding globulin. CIin Chem 1987;33:1372-1375. 14 Coffey JC, Crutchfield WC. In vitro metabolism of 4-androsten-3,17-dione by human submaxillary gland homogenates. J Dent Res 1977;56:332-334. 15 Shaw MA. Steroid binding in human saliva. Clin Chem 1985;31:345-346. 16 Ekins R. Validity of analog free thyroxin immunoassays. Chn Chem 1987;33:2137-2144. 17 Midgley JEM, Moon CR, Wilkins TA. Validity of analog free thyroxin immunoassays. Part II. Clin Chem 1987;33:2145-2152.
21
CCA 04806
Free testosterone levels in plasma and saliva as determined by a direct solid-phase radioimmunoassay: a critical evaluation Franqois
Rey, Giovanni Chiodoni, Karine Braillard, and ThCr&e Lemarchand-BCraud
Christine
Berthod
Division d’Endocrinologie et Biochimie Unique, Centre Hospitalier Universitaire Vaudois, Laurame (Switzerland) (Received
6 April 1989; revision
Key words: Free testosterone;
received 29 May 1990; accepted
Saliva testosterone;
Free hormone
5 June 1990)
radioimmunoassay
Summary We compared unbound (free) testosterone radioimmunoassay concentrations in plasma and saliva from men, using a direct radioimmunoassay kit involving a ligand analog of testosterone as tracer. The assay failed to reveal detectable testosterone concentrations in saliva. In plasma the free testosterone levels were about 4 times lower than those obtained by calculation or ultr~iltration methods. Moreover, unexpected similar free testosterone levels were obtained in samples comparable in their total testosterone content but distinct in their steroid binding protein content (buffered testosterone dilutions). We suspect that free testosterone levels determined with this direct radioimmunoassay probably do not reflect the true free testosterone values and conclude that their significance remains to be established.
Introduction The direct relationship between the free fraction of testosterone (T) in plasma and the biological activity of the hormone has been recently reevaluated in view of the suggestion that part of the protein bound T might be transported across microcirculatory barriers [l]. This latter process may involve a mechanism of enhanced dissociation of hormone from plasma-binding proteins induced by transient conformational changes.
Correspondence Clinique, Centre
and requests for reprints to: F. Rey, Ph.D. Division Hospitalier Universitaire Vaudois, CH-1011 Lausanne,
0009-8981/90/%03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
d’Endocrinologie Switzerland.
Division)
et Biochimie
22
In men and women, salivary T concentrations closely correlate with plasma free T measured either by dialysis [2], ultrafiltration [3] or when estimated by calculation [4]. However in women, the total salivary T concentrations exceed by 2- to 3-fold those of plasma free T estimated by these methods. This observation may support the view that some albumin-bound T is available to peripheral tissues [5] or that saliva contains steroid-binding proteins (like SHBG) or albumin which could bind T [6]. Nevertheless we have recently suggested that the discrepancy between saliva and plasma free T might be due to local conversion of androstenedione to T in salivary glands [7]. Whatever, salivary T determination provides an excellent approach for the evaluation of androgenic activity. To further evaluate the relationship between circulating free T levels and saliva content without methodological bias, we chose to use a commercial RIA kit designed for direct free T determination in serum and plasma. Materials and methods Samples collection Blood and/or saliva were collected from 2 groups of healthy male volunteers (aged 20 to 32). In the first group (n = 9) samples were obtained at 08.00 after an overnight fasting. In the second group (n = 10) samples were collected at various times of the day and in some cases after an injection of testosterone in order to provide a broad pattern of total T levels. Blood was obtained by venepuncture using lithium heparinate monovettes (Sarstedt, Sevelen, Switzerland). Individuals were asked to salivate 2 to 5 ml into disposable tubes in about 5 minutes. Blood and saliva samples were immediately centrifuged (3000 x g, 4O C, 5 min) and decanted. Supematants were stored at - 20 o C until assayed. Assays Free T, direct solid-phase RIA Free T determinations in plasma, saliva and buffered T dilutions were performed by the direct solid phase commercial RIA from Diagnostics Products Corporation (DPC, Los Angeles, CA) designed to leave essentially unchanged the original equilibrium between free and protein bound testosterone in serum and heparinized plasma samples (Coat-A-Count Free Testosterone lz51 RIA). Before assays, samples were centrifuged 5 min at 3 000 X g, 4°C. We chose also to treat saliva samples using the protocol recommended for serum. In regards of the ratio between sample and reagent volumes (50 ~1 vs. 1000 ~1) we considered that the matrix effect would be negligible. Total T ZtZA Total T levels were measured in plasma, saliva and calibrators from the RIA kit by a specific and sensitive RIA method developed in our laboratory and previously described [8].
23
We measured free T fractions in plasma, Free T, determination by ultrafiltration saliva and buffered T dilutions by ultrafiltration according to Vlahos et al. [9]. We used the micropartition system MPS-1 from Amicon (Danvers, MA, USA) with YMT membranes (molecular cut off 30000 Da). Free T. estimation by calculation Knowing total T, SHBG and albumin levels, free T concentrations were calculated in plasma, saliva and RIA kit calibrators according to S&lergard et al. [lo]. SHBG was measured by IRMA commercial kit (Farmos Diagnostica, Oulunsalo, Finland). For dete~nation in saliva, the lower limit of the standard curve was extended from 6.25 to 0.390 nmol ’ 1-l and the dilution step of samples was omitted. Plasma albumin was measured with bromocresol green [ll]. Salivary albumin was determined using a micro-albumin RIA validated for urine (Pharmacia AB, Uppsala, Sweden). Evaluation
of the DPC free testosterone
RIA
The following 4 calibrators Calculated free T vs. indicated levels in kit calibrators given as 0, 2.08, 58.9 and 191.0 pmol *1-l were choosen for free T levels estimation by calculation (calibrators lot numbers TFC3UO4, TFC4004, TFC6004 and TFCSOO4, respectively; kits lot number 090bl). Free T determination in various media at constant total T levels In order to evaluate the influence of sample matrix in the RIA we determined free T levels in 4 different matrix conditions, each condition being tested at 10 distinct concentrations of total T. 1. Human plasma: 10 samples containing respectively 50.2, 45.1, 40.2, 35.4, 30.6, 26.0, 20.5, 16.0, 10.4 and 6.7 nmol *1-l of total T and physiological concentrations of SHBG and albumin were selected to cover progressively the range of clinically observable values. 2. 0.1 mol/l Tris buffer pH 7.8, protein free: 10 samples constituted by T dilutions at levels similar to those observed in the 10 selected plasma samples. 3. 0.1 mol,/l Tris buffer, pH 7.8, gelatin 1 g - 1-r (Merck 4078): 10 samples constituted by T dilutions as above mentioned. 4. Krebs original Ringer phosphate buffer, pH 7.4, enriched with 20 g. 1-i human serum albumin (albumin dry powder, Red Cross, Bern, Switzerland): 10 samples constituted by T dilutions as above described. Results
Table I reports the basal plasma and saliva free T levels determined in 9 healthy men by ultrafiltration, calculation and direct RIA, as well as total T and binding proteins in plasma and saliva. Plasma-free T levels obtained by ultrafiltration and calculation were similar. Values obtained by direct RIA were significantly lower than those determined by
24 TABLE
I
Plasma-
and saliva-free
T levels obtained
by ultrafiltration, Plasma-free
Ultrafiltration Calculation RIA direct
T a % of total plasma
538.1+ 142.7 523.8 + 131.6 128.2 f 29.2
2.74+ 0.29 2.53kO.18 0.65 f 0.09
a The corresponding
7
T ’
pmol.l-’
% of total saliva T
182.1 f 53.9 390.8 f 99.6 undetectable
44.69 + 4.48 99.65 k 6.94
Total T and binding
Testosterone (nmol.l-r) SHBG (nmol.l-‘) Albumin (g.l-‘)
and RIA in 9 men (mean + SD)
pmol-1-l
Saliva-free
Ultrafiltration Calculation RIA direct
calculation
proteins
Plasma
Saliva
20.5 + 4.5 23.4k6.1 47.2 + 3.7
403.3 * 101.0 x lo- 3 5.6+ 5.7x10-’ 51.1+ 29.7x10-’
levels of total T, SHBG
and albumin
are indicated
in the lower panel.
the former methods. Direct RIA free T levels were positively correlated to those determined by ultrafiltration or calculation and also to total T levels with the same degree of significance (P < 0.01). In saliva, calculated free T levels were similar to those determined by our own RIA designed for total T measurements, indicative of a negligable binding of T to SHBG and albumin. In contrast, free T concentrations determined in saliva by ultrafiltration showed an important binding of about 55%. With the direct RIA no significant saliva free T levels were detected ( < 0.5 pmol . l- ’ ). Figure 1 shows free T levels measured by the free T RIA in the 4 series of 10 samples, each series being characterized by the nature of sample matrix. In all cases, free T values appeared directly dependent of total T levels but almost independent of the protein content of diluting media. Table II allows comparison of free T values (expressed in percent of total T) estimated by ultrafiltration and RIA in the same 4 series of 10 samples. In Tris buffer, without or with addition of gelatin, free T fraction determined by ultrafiltration was over 80% (probably high non specific binding to membranes). In Krebs original Ringer buffer, albumin 20 g. 1-l) the free T fraction was constant and equal to 11% whatever the total T concentration. The respective values of 80% and 11% must be considered as minimal. Indeed it is not possible to estimate the true values in media deprived or totally free of proteins since non specific absorption to membranes is higher in such media than in total plasma. Table III shows total T, SHBG, albumin and calculated free T levels determined in the DPC RIA calibrators. Determination of binding proteins levels revealed
II
’ The 4 different total T levels.
RIA
RIA 4. Krebs, albumin Ultrafiitrat~on
I _’
media compositions
20 g. 0.68
0.97
have been choosen
0.75
0.99
1.03
1.13
RIA 3. Tris, gelatin 1 g.lUltrafiltration
’
3.12 0.65
2 45.1
0.73
the influence
0.64
0.67
11%
80% 0.88
80% 0.88
2.22 0.38
6 26.0
0.62
0.84
0.78
3.39 0.82
8 16.0
and the concentration
0.64
0.82
0.55
2.16 0.45
7 20.5
levels of total T in 4 different
of the quality
~nimum
minimum 0.87
minimum 0.86
1.03
0.99
2.54 0.70
5 30.6
2.20 0.46
4 35.4
and by direct RIA at 10 distinct
in order to evaluate
0.12
0.94
0.88
3.23 0.82
3 40.2
by ultrafiltration
3.51 0.17
1 50.2
of total T determined
Ultra~ltration RIA 2. Tris protein free Ultrafiltration
1. Plasma
% free T in:
Total T a (nmol,l-‘)
Free T values in percent nature of their matrix
TABLE
in protein
0.62
0.19
0.77
2.37 0.72
9 10.4
contents
by the
at constant
0.69
0.78
0.79
2.23 0.57
10 6.7
media distinguishable
e-.-
Plasma
-
Trls, proteins free
c-c-
Tris,gebtin Krebs, albumin
67 L
0
to.4 I
16.0
10
x).5 I
26.0
30.6 I
30
20
Total
35.4
40.2 L
40 testosterone
45.1
50 2 I
50 nm-oi.l-*
Fig. 1. DPC free T levels determined at 10 levels of total T in 4 different media characterized by the nature of their matrix. In all T dilutions performed in Tris buffer and in most T dilutions made in Krebs-albumin buffer (total T z 20.5 nmol.l-‘), direct free T RIA should have been totally saturated. On the contrary free T levels appeared assessable and well correlated (P < 0.01) whatever the milieu.
SHBG concentrations ranging from 5.2 to 5.8 nmol. 1-l and constant albumin levels of 38.0 g. I-‘. Total T concentrations ranged from 0.03 to 39.41 nmol *1-l. Calculated free T levels were totally in discordance with those indicated: 1.1 vs. 0.0,
TABLE III DPC direct free T RIG kit: indicated vs calculated free T levels in 4 calibrators as well as contents in total T, SHBG, and albumin
Indicated free T (pmol .l- ’ ) Calculated free T (pmol.l-‘) Testosterone (nmol.l-t) SHBG (nmol.l-‘) Albumin (gel-‘)
Level 1
Level 2
Level 3
Level 4
0.0 1.1 0.03 5.2 38.0
2.08 22.2 0.59 5.4 38.0
58.9 391.0 10.23 5.8 38.0
191.0 1569.1 39.41 5.7 38.0
27
22.2 vs. 2.08, 391.0 vs. 58.9 and 1569.1 vs. 191.0 pmol- I-‘. This comparison is relevant only if the affinity constant of SHBG is similar in calibrators and in biological samples. If it is not the case, our estimation must be considered as erroneous but at the same time it has to be acknowledged that kit calibrators do not react as serum or plasma samples. In calibrators, the ratio between calculated and indicated free T levels showed values higher than those observed in plasma between calculated and RIA free T levels. Discussion
The present results confirm that free T levels estimated by the direct RIA in heparinized plasma and serum are totally equivalent but distinct from those determined by ultrafiltration (comparable to dialysis) or theoretically calculated according to the law of mass knowing SHBG, albumin and total T levels. As estimated in 9 normal male volunteers in basal conditions, the mean value of plasma-free T levels determined by RIA was 4 times lower than those obtained by the conventional methods. Like Cheng et al. 1121,W&e and Utley recently reported a similar bias in women between direct RIA and dialysable or calculated plasma-free T levels [13]. Nevertheless, these authors noted that the relative amp~tude of this bias is not constant, showing that in pregnant women particularly, RIA-free T levels are only one half of calculated levels. In view of the general positive correlation between free T levels determined by direct RIA and calculation, and despite the later observation in pregnant women, the authors concluded to the reliability of the RIA. In the present study, we found also good correlations between free T levels obtained by RIA and either ultrafiltration or calculation. However we found the same positive correlation between RIA free T and total T levels. We are therefore not able to confirm that RIA free T reflect circulating free T levels depending from total T and protein binding concentrations. In women, total saliva T concentrations exceed free plasma T levels particularly when estimated by ultrafiltration or calculation [3,4,7]. We have previously suggested that this discrepancy might be due to a local transformation of androstenedione into T [7]. The high ratio between plasma androstenedione and T might be responsible for this transformation. In normal men, such a discrepancy is not observed, free plasma and total saliva T levels being similar. However, we cannot exclude that the same process exists, but hidden by the probable and concomitant transformation of T into 5cw-dihydrotestosterone [14]. In saliva, T exists both in bound and free forms. Shaw indicated that about 10% of T binds to macromolecules, i.e. to undialysable compounds [15]. In the present study, the comparison between total and free saliva T levels determined by ultrafiltration is suggestive of a more important binding of T to protein of about 50%. This high binding cannot be related to the minor cont~nation of saliva by SHBG and albumin, total and calculated saliva-free T levels being strictly comparable, This observation is indicative of the presence of unidentified steroid binding proteins in
28
saliva. In any case, free saliva T levels must represent at least one half of total saliva T, i.e. of free plasma T levels. A priori, such saliva levels should be accurately estimated using a method designed for the measurement of free serum T. However, the direct free T RIA developed for measuring T in serum failed to reveal any detectable free saliva T levels despite the fact that saliva samples represented only 5% of volume during RIA incubation (lower detectable concentration: 0.5 pmol 1 1-Q. To further estimate free T RIA reliability, we determined the real influence of T proteins binding at 10 levels of the hormone in 4 media with different protein contents. Unexpectedly, RIA free T concentrations appeared almost independent of media composition. Particularly with T dilutions ranging from 6.7 and 50.2 nmol .ll’ performed in Tris buffer protein free, in which T remains in free form, RIA gives free T levels ranging from 53 to 568 pmol .1-i. These values are similar to those obtained in complete plasma samples with the same T concentrations. This observation is suggestive that the RIA provides results which are not related to the true free T values but are merely reflective of total T concentration. The high free T values determined in pregnant women by Wilke et al. [13] and the lack of a negative and significant correlation between SHBG levels and free T values found by the same authors, support this view. The method of standardization used by the DPC firm is unknown. RIA calibrators are dramatically depleted in SHBG when compared to plasma. The reference method used to calibrate the standards is doubtful and in any case different from that used by others. Consequently the adequacy of DPC RIA calibrators is highly questionable. In view of our results and without the purpose to further contribute to hot debate on the validity of analog free hormone immunoassays [16,17] we are of the opinion that the so-called free T values determined using the DPC direct RIA are misleading since they are not representative of true free T levels. The precise significance of these values remains to be established. Acknowledgements The authors wish to thank Dr. M. Markert for plasma albumin determinations, Dr. D. Jallut for technical assistance and Mrs. M.-C. Evraere for editorial assistance. References Pardridge WM. Plasma proetin-mediated transport of steroid and thyroid hormones. Am J Physiol 1987;252:E157-E164. Baxendale PM, Jacobs HS, James VHT. Salivary testosterone: relationship to unbound plasma testosterone in normal and hyperandrogenic women. Clin Endocrinol 1982;16:595-603. Sanmkka E, Terho, P, Suominen J, Santti R. Testosterone concentration in human seminal plasma and saliva and its correlation with non-protein-bound and total testosterone levels in serum. Int J Androl 1983;6:319-330.
29 4 Rey F, Bumand B. Gomez, F. Free testosterone and clinical hyperandrogenism in women: correlation of sex hormone binding globulin, calculated free testosterone and salivary testosterone with hirsutism score. In: Forest MG, Pugeat M, eds. Binding proteins of steroid hormones. Inserm-John Libbey, 1986; 636 (abstract). 5 Ruutiainen K, Sannikka E, Santti R, Erkkola R, Adlercreutz H. Salivary testosterone in hirsutism: correlations with serum testosterone and the degree of hair growth. J Clin Endocrinol Metab 1987;64:1015-1020. 6 Selby C, Lobb PA, Jeffcoate WJ. Se.x hormone binding globulin in saliva. J Clin Endocrinol 1988;28:19-24. 7 Rey F, Chiodoni G, Gomez F, Felber JP. Interpretation of the discrepancy observed between plasma free and salivary testosterone levels in man. Steroids 1988;52:371-372. 8 Magrini G, Chiodoni G, Rey F, Felber JP. Further evidence for the usefulness of the salivary testosterone radioimmunoassay in the assessment of androgenecity in man in basal and stimulated conditions. Horm Res 1986;23:65-73. 9 Vlahos I, MacMahon W, Sgoutas D, Bowers W, Thompson J, Trawick W. An improved ultrafiltration method for determining free testosterone in serum. Clin Chem 1982;28:2286-2291. 10 Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and oestradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem 1982;16:801-810. 11 Doumas BT, Watson WA, Briggs HG. Albumin standards and the measurement of serum albumin with bromocresol green. CIin Chim Acta 1971;31:87-96. 12 Cheng RW, Reed MJ, James VHT. Plasma free testosterone: equilibrium dialysis vs direct radioimmunoassay. Clin Chem 1986;32:1411. 13 WiIke TJ, Utley DJ. Total testosterone, free-androgen index, calculated free testosterone, and free testosterone by analog RIA compared in hirsute women and in otherwise-normal women with altered binding of sex-hormone-binding globulin. CIin Chem 1987;33:1372-1375. 14 Coffey JC, Crutchfield WC. In vitro metabolism of 4-androsten-3,17-dione by human submaxillary gland homogenates. J Dent Res 1977;56:332-334. 15 Shaw MA. Steroid binding in human saliva. Clin Chem 1985;31:345-346. 16 Ekins R. Validity of analog free thyroxin immunoassays. Chn Chem 1987;33:2137-2144. 17 Midgley JEM, Moon CR, Wilkins TA. Validity of analog free thyroxin immunoassays. Part II. Clin Chem 1987;33:2145-2152.