TY - JOUR
AU - Arregger, Alejandro L.
AB - Abstract Background. Hypogonadism is frequent in patients with end-stage renal disease (ESRD). Salivary testosterone (Sal-T) is a non-invasive tool to screen androgen deficiency in adult male with normal renal function. However, available data on its utility in ESRD are not conclusive. Objectives. The objectives of the study were: (i) to compare free testosterone fractions in saliva (SAL-T) and serum (Free-T); (ii) to establish the correlation of Sal-T with circulating total (TT) and bioavailable testosterone (Bio-T); (iii) to detect androgen deficiency through Sal-T; (iv) to determine the correlation of Sal-T with clinical parameters. Methods. The study included: 60 adult ESRD men on haemodialysis (20–60 years old) with decreased libido referred from two dialysis centres; 112 eugonadic and 40 hypogonadic adult men with normal renal function as controls. Simultaneous morning saliva and serum samples were obtained for testosterone measurements by liquid RIA (SAL-T; TT). Free-T and Bio-T were calculated by the Vermeulen equation. Results. Sal-T (0.338 ± 0.177 nM) and Free-T (0.338 ± 0.165 nM) did not differ (P > 0.900) in ESRD as well as in control (0.337 ± 0.182 and 0.337 ± 0.172 nM, respectively; P > 0.900). Sal-T levels correlated positively (P < 0.0001) with Free-T (r = 0.95), TT (r = 0.80) and Bio-T (r = 0.76) in ESRD. Sal-T negatively correlated with age and years on dialytic therapy. Sal-T showed 100% sensitivity and specificity to differentiate patients with androgen deficiency (22%) from those with normal androgen levels (78%). Hypogonadism was hypergonadotrophic in 69% cases and hypogonadotrophic in 31%. Conclusions. These data demonstrate the value of morning Sal-T testing as a non-invasive approach to screen androgen status in ESRD patients. end-stage renal disease, haemodialysis, male androgen deficiency, salivary testosterone, serum testosterone Introduction A number of metabolic and endocrine disorders linked to chronic uraemic syndrome remain a challenge in spite of the progresses in renal disease therapy [1,2]. As reported, two-thirds of men on haemodialysis (HD) have serum testosterone in the hypogonadal range [3,4]. This deficiency was ascribed to a combination of primary and secondary testicular failure [3]. Clinical features associated with hypogonadism include low libido, erectile dysfunction, mood changes, fatigue, decreased bone-mineral density, anaemia and loss of muscle mass and strength [5]. None of these signs and symptoms are specific of low androgen concentration but may raise suspicion [6]. Changes in prolactin, gonadotrophins and gonadal hormone levels are found in HD patients [7,8]; these alterations along with vascular, neurologic, psychogenic and other factors, such as medications, contribute to the development of sexual dysfunction [9]. In the general population, low levels of endogenous testosterone in men are related to higher risk of cardiovascular disease, depression and decreased survival [10]. Circulating testosterone is routinely measured in clinical laboratories for investigation of androgen-related disorders. In healthy adult men, testosterone circulates in plasma either free (2.23%) or bound to serum proteins [albumin: 49.9%, sex hormone binding globulin (SHBG): 44.3%, CBG: 3.56%] [11]. Bioavailable testosterone, including both the measurable free and albumin bound fractions, might reflect more accurately the clinical situation when total serum testosterone is borderline or steroid binding protein levels are impaired [12]. Saliva is a widely accepted sample source for steroid analysis and offers a non-invasive and stress-free alternative to plasma and serum [13]. It is particularly useful in anaemic and high-risk patients as those with end-stage renal disease (ESRD) [14]. Salivary testosterone (Sal-T) reflects the free fraction of plasma testosterone that is able to diffuse passively across the salivary glands, independently of salivary flow-rate [13,15]. A highly significant correlation between salivary and free testosterone (Free-T) was described in adult men [16–20]; also, SAL-T positively correlated with circulating total (TT) and bioavailable (Bio-T) testosterone in eugonadal and hypogonadal subjects with normal renal function [20]. Recently, Sal-T was suggested as a biomarker for the diagnosis of male androgen deficiency, representing an attractive alternative to plasma [13,18–20]. Early studies [21,22] assessed Sal-T in uraemic men, but data on its clinical utility were not conclusive. Since SHBG levels have been reported to be low, normal or increased in uraemic patients [23–25], measurements of the free steroid fraction in serum or saliva may become the most accurate approach. Unfortunately, the usefulness of SAL-T was not further investigated. As in other countries, the population on haemodialysis in Argentina has been steadily increasing. Male ESRD with decreased libido are frequently referred to our department for endocrine evaluation. The aim of this study was to investigate if Sal-T could become a reliable non-invasive tool to screen the androgen status in these patients. For this purpose, the following steps were performed: (i) to compare free testosterone fractions in saliva (SAL-T) and serum (Free-T); (ii) to establish the correlation of Sal-T with circulating TT and Bio-T; (iii) to detect androgen deficiency through Sal-T; (iv) to determine the correlation of Sal-T with clinical parameters. Materials and methods Study population Sixty male ESRD (aged 20–60 years old) who complained of decreased libido were evaluated. They were all on maintenance HD (three times a week) in two dialysis centres of the Buenos Aires metropolitan area. They had no evidence of active psychiatric disease, infection, uncontrolled congestive heart failure or acute complications from uraemia at the time of the study. Exclusion criteria were cognitive impairment and history of alcohol or substance abuse. Patients taking drugs that might affect testosterone concentrations (anabolics, glucocorticoids, estrogens, ketoconazole, spiranolactone) or induce hyperprolactinaemia (haloperidol, metoclopramide, phenothiazine, chlorpromazine) within 3 months prior to the study were also excluded. Most patients were on drugs commonly used in ESRD (vitamins B, C, D supplementation, folic acid, phosphate and potassium binders). Other specific medications were: β-blockers, angiotensin converting enzyme inhibitors and angiotensin receptor blockers (n = 21); human insulin (n = 16); subcutaneous recombinant human erythropoietin (rHuEpo) (n = 31; mean dose ± SD = 4355 ± 2882 U/week). Table 1 displays the clinical and biochemical findings in ESRD. Saliva samples were obtained after confirming the integrity of salivary gland function as previously described [20]. Table 1 Clinical and biochemical data of 60 male patients on haemodialysis Patient characteristic    Age (median and range; years)  39.5 (18–60)   Body mass index (mean ± SD; kg/m2)  23.8 ± 3.2   Married (n, %)  49 (80%)   Paternity (n, %)  20 (33%)   HDRT (mean ± SD; years)  5.7 ± 4.1    CRF aetiology   Hypertension  35%   Diabetes mellitus  27%   Policystic renal syndrome  10%   Haemolytic uraemic syndrome  4%   Kidney stones  3%   Other  8%   Unknown  13%    Laboratory parameters (mean ± SD)   Serum albumin (g/dL)  4.0 ± 0.2   Serum haemoglobin (g/dL)  11.0 ± 1.5   Serum SHBG (nM)  24.6 ± 9.9   Total serum calcium (mg/dL)  8.96 ± 1.06   Intact serum PTH (pg/mL)  512.0 ± 356.0   Kt/V  1.17 ± 0.27  Patient characteristic    Age (median and range; years)  39.5 (18–60)   Body mass index (mean ± SD; kg/m2)  23.8 ± 3.2   Married (n, %)  49 (80%)   Paternity (n, %)  20 (33%)   HDRT (mean ± SD; years)  5.7 ± 4.1    CRF aetiology   Hypertension  35%   Diabetes mellitus  27%   Policystic renal syndrome  10%   Haemolytic uraemic syndrome  4%   Kidney stones  3%   Other  8%   Unknown  13%    Laboratory parameters (mean ± SD)   Serum albumin (g/dL)  4.0 ± 0.2   Serum haemoglobin (g/dL)  11.0 ± 1.5   Serum SHBG (nM)  24.6 ± 9.9   Total serum calcium (mg/dL)  8.96 ± 1.06   Intact serum PTH (pg/mL)  512.0 ± 356.0   Kt/V  1.17 ± 0.27  HDRT, haemodialysis replacement therapy; CRF, chronic renal failure; the index Kt/V is a measure of dialysis adequacy, value of 1.2 was considered appropriately. Reference range values: BMI: 20.0–25.0 kg/m2; albumin: 3.6–5.0 g/dL; haemoglobin: 12.0–14.0 g/dL; SHBG: 10.0–56.0 nM; calcium: 8.90–10.50 mg/dL; PTH: 10.0–70.0 pg/mL. View Large Androgen deficiency was established following the approach suggested by the Endocrine Society using a cut-off value of total testosterone < 10.4 nM with Free-T ≤ 0.23 nM and Bio-T ≤ 5.2 nM in subjects with decreased libido [26,27]. Total testicular volume was determined using Prader’s orchidometer (normal total volume ≥ 30.0 mL). The reference population (C) consisted of 112 eugonadic subjects (aged 20–60 years old, BMI = 23 ± 2.0 kg/m2) and 40 known hypogonadic subjects (n = 22, 25–70 years old, BMI = 24 ± 3.0 kg/m2) with normal renal function. Patients treated with testosterone enantathe (250 mg every 2–3 week i.m.) were evaluated 1 month after the last injection. The protocol was approved by the Human Research Ethics Committee of the School of Medicine, University of Buenos Aires, Argentina, and all subjects gave informed consent to participate in the study. Study design Sample collection All subjects obtained saliva in fasting conditions. They were instructed not to brush their teeth 2 h before the saliva collection. Subjects collected 3.5 mL of whole saliva by directly spitting into polypropylene tubes between 07.00 and 09.00 hours, and simultaneous blood samples were drawn in each case. In ESRD patients, samples were obtained before starting the second dialytic procedure of the week. All patients were normotensive, without peripheral edema, had normal serum albumin levels, and weight gained interdialysis did not exceed 2.5 L. To minimize preanalytical errors, saliva was discarded if contaminated with blood. In case of any possible pink coloration, a dipstick test was used to detect haemoglobin contamination. When necessary, a second saliva and blood sample were obtained 1 to 3 months after. The supernatants of saliva and serum obtained after centrifugation (1000 g, 10 min) were kept at − 20°C for hormone analysis. Salivary testosterone Testosterone in saliva was determined by an adapted 125I double antibody test for the quantification of total testosterone in serum (DSL 4100, Diagnostic System Laboratories, Inc. Webster, TX) with the modifications previously described [20]. Aliquots of 200 μL of saliva samples were processed by duplicate following the described steps [20]. The sensitivity of the SAL-T assay was 3.47 pM. The intra-assay and inter-assay coefficients of variation (CVs) were < 6.8% and < 9.5%, respectively. Blood assessments Sex hormone binding globulin SHBG, expressed as the maximal dihydrotestosterone (DHT) binding capacity, was performed using 50 μL of serum as described [20]. Serum Hormones Serum total testosterone and estradiol levels were assessed by radioimmunoassay (DSL 4100 and DSL 4400, Diagnostic System Laboratories Inc, Webster, TX, respectively) following the manufacturer’s guidelines. The analytical sensitivities were 0.28 and 0.017 nM, respectively. The intra- and inter-CVs were < 6.0% and 9.0% for TT and < 5.4% and 9.4% for estradiol. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) levels were measured by coat-a count immunoradiometric assays (Diagnostic Products Corporation, Los Angeles, CA) with sensitivities of 0.15 mUI/mL, 0.10 mUI/mL and 0.1 ng/mL (2.12 mUI/mL), respectively. The intra- and inter-CVs were: < 1.7% and < 7.2% for LH, < 3.8% and < 5.9% for FSH and < 3.8% and < 5.9% for PRL. Whenever hyperprolactinaemia was found, native and bioactive monomeric PRL were estimated. To assess monomeric PRL, equal volumes (200 μL) of a 25% (wt/vol) solution of polyethyleneglycol (PEG 6000 kD) and patient serum were mixed for 1 h at 4°C and centrifuged at 4500 rpm for 15 min. Immunoreactive PRL was measured in the supernatant and unprecipitated serum by RIA. A PRL recovery ≤ 40% was indicative of macroprolactinaemia, while the condition was unlikely to be present at values > 60% [28]. Albumin Albumin concentration was measured using a Technicon RA500 analyzer (Technicon Instruments Corporation, New York, USA) following the manufacturer’s recommended protocol. Other analytes included in Table 1 were performed by the clinical chemistry laboratories for patients’ regular monitoring. Circulating free testosterone and bioavailable testosterone concentrations They were both calculated as described by Vermeulen [29] using a second-order equation based on SHBG, total testosterone and albumin concentrations [30]. Statistical analysis Results are expressed as mean ± standard deviation unless otherwise specified. Correlations between serum and salivary steroid levels were evaluated by Spearman analysis. The receiver operating characteristic (ROC) curve was employed to graphically demonstrate the sensitivities and specificities of the different diagnostic tests and cut-off values optimized for sensitivities. Statistical analysis was performed with program Medcalc for Windows version 7.4.3.1. P-values < 0.05 were considered statistically significant. Results Free testosterone levels in saliva and serum in ESRD and controls In morning samples, Sal-T (0.338 ± 0.177 nM) and Free-T (0.338 ± 0.165 nM) did not differ (P > 0.900) in ESRD as shown in C (0.337 ± 0.182 and 0.337 ± 0.172 nM, respectively; P > 0.900). In addition, a positive and significant correlation (r = 0.95, 95% confidence interval r = 0.95–0.98, P = 0.0001) was found between Sal-T and Free-T in ESRD and in C (r = 0.97, 95% CI for r = 0.96–0.98, P = 0.0001) (Figure 1). Fig. 1 View largeDownload slide Correlation between salivary (Sal-T) and serum free testosterone (Free-T) levels from simultaneously obtained morning samples in 60 male patients with end-stage renal disease and in 152 subjects without renal failure (reference group). Significant correlation (P = 0.0001) between Sal-T and Free-T was observed in ESRD patients (closed circles; r = 0.95) and in the reference group (closed diamonds; r = 0.97) constituted by 112 eugonadic and 40 hypogonadic subjects with normal renal function. Fig. 1 View largeDownload slide Correlation between salivary (Sal-T) and serum free testosterone (Free-T) levels from simultaneously obtained morning samples in 60 male patients with end-stage renal disease and in 152 subjects without renal failure (reference group). Significant correlation (P = 0.0001) between Sal-T and Free-T was observed in ESRD patients (closed circles; r = 0.95) and in the reference group (closed diamonds; r = 0.97) constituted by 112 eugonadic and 40 hypogonadic subjects with normal renal function. Correlation between SAL-T and serum testosterone concentrations in ESRD TT and Bio-T were 12.94 ± 4.53 and 7.47 ± 2.96 nM, respectively. SAL-T correlated positively and significantly with both in ESRD (r = 0.80 and r = 0.76) as observed in C (r = 0.91 and r = 0.93); P < 0.0001 for all. Salivary and serum testosterone levels in the reference group (C) Androgen levels in eugonadic subjects were (mean ± SD): TT = 17.9 ± 5.3 nM, Bio-T = 9.32 ± 3.13 nM; Free-T = 0.410 ± 0.138 nM; Sal-T = 0.413 ± 0.148 nM. Hypogonadic subjects showed significantly lower androgen levels than eugonadic (P < 0.0001): TT = 6.14 ± 2.40 nM; Bio-T = 3.1 ± 1.43 nM, Free-T = 0.136 ± 0.06 nM, Sal-T = 0.134 ± 0.05 nM. ROC analysis defined TT ≤ 10.1 nM, Bio-T ≤ 5.3 nM, Free-T ≤ 0.229 nM and Sal-T ≤ 0.195 nM as the cut-off values with 100% sensitivity and specificity able to discriminate hypogonadic from eugonadic subjects [AUCROC: (95% CI) = 1.0 (0.976–1.00) in all cases]. Salivary and serum testosterone levels in ESRD: diagnosis of androgen deficiency Figure 2shows individual data of Sal-T, Free-T, Bio-T and TT in ESRD. In 47 ESRD, circulating testosterone fractions and SAL-T lay within the eugonadic range. Thirteen patients (22%) had Sal-T levels (0.129 ± 0.003 nM) in the hypogonadic range, confirmed by an additional second salivary sample (0.132 ± 0.003 nM, P = 0.792). Clinical and biochemical features of hypogonadal ESRD are shown in Table 2. High concentrations of LH and FSH were found in nine patients not associated to elevated monomeric PRL. In two ESRD with inappropriate gonadotrophin levels, high monomeric PRL concentrations were found. Fig. 2 View largeDownload slide Morning salivary testosterone (A), serum free testosterone (B), serum bioavailable testosterone (C) and serum total testosterone (D) levels in simultaneous samples obtained from eugonadic and hypogonadic male patients with end-stage renal disease. Dotted lines showed the cut-off values of SAL-T, Free-T, Bio-T and TT with 100% sensitivity and specificity able to discriminate hypogonadic (n = 40) from eugonadic subjects (n = 112) in the reference group. Fig. 2 View largeDownload slide Morning salivary testosterone (A), serum free testosterone (B), serum bioavailable testosterone (C) and serum total testosterone (D) levels in simultaneous samples obtained from eugonadic and hypogonadic male patients with end-stage renal disease. Dotted lines showed the cut-off values of SAL-T, Free-T, Bio-T and TT with 100% sensitivity and specificity able to discriminate hypogonadic (n = 40) from eugonadic subjects (n = 112) in the reference group. Table 2 Clinical and biochemical findings in 13 hypogonadal patients on haemodialysis Patient  Age (years)  Diagnosis  Paternity  Time (years)  TTV (mL)  TT (nM)  FreeT (nM)  Sal-T (nM)  LH (mUI/mL)  FSH (mUI//mL)  PRL (ng/mL)  PRL PEG recovery (%)  48  29  HBP  No  7.0  24.0  7.6  0.152  0.149 ± 0.003  7.4  11.2  16.0  n.d.  49  29  UHS  No  15.0  16.0  7.3  0.120  0.099 ± 0.001  1.0  5.8  90.0  70.0a  50  36  PKD  No  6.0  14.0  5.2  0.141  0.131 ± 0.003  8.0  16.0  16.0  n.d  51  37  PKD  No  9.0  18.0  7.3  0.095  0.119 ± 0.001  7.5  9.5  39.7  47.0  52  39  HTA  No  8.0  20.0  6.6  0.174  0.166 ± 0.010  0.6  3.0  4.3  n.d.  53  46  Unknown  Yes  8.0  22.0  10.1  0.120  0.118 ± 0.002  11.0  13.0  16.0  n.d.  54  46  DM  Yes  2.0  25.0  6.9  0.087  0.092 ± 0.007  7.0  10.0  92.8  41.0  55  48  HTA  No  10.0  27.0  8.3  0.166  0.168 ± 0.003  10.0  5.0  20.0  n.d.  56  55  HTA  Yes  6.0  27.0  7.0  0.134  0.113 ± 0.012  16.0  10.0  5.0  n.d.  57  55  Unknown  No  2.0  16.0  5.9  0.070  0.064 ± 0.005  4.4  3.9  40.9  53.0  58  56  HTA  Yes  11.0  25.0  9.4  0.140  0.133 ± 0.008  9.0  4.0  12.0  n.d  59  59  HTA  Yes  13.0  12.0  5.0  0.170  0.179 ± 0.009  6.0  7.8  45.8  68.0a  60  60  DM  No  3.0  20.0  5.6  0.153  0.164 ± 0.015  30.0  17.0  6.0  n.d.  Refrence values (range)          30.0–50.0  10.4–33.0  0.23–0.73  0.200–0.729  0.5–6.0  1.0–9.3  1.8–16.0    Patient  Age (years)  Diagnosis  Paternity  Time (years)  TTV (mL)  TT (nM)  FreeT (nM)  Sal-T (nM)  LH (mUI/mL)  FSH (mUI//mL)  PRL (ng/mL)  PRL PEG recovery (%)  48  29  HBP  No  7.0  24.0  7.6  0.152  0.149 ± 0.003  7.4  11.2  16.0  n.d.  49  29  UHS  No  15.0  16.0  7.3  0.120  0.099 ± 0.001  1.0  5.8  90.0  70.0a  50  36  PKD  No  6.0  14.0  5.2  0.141  0.131 ± 0.003  8.0  16.0  16.0  n.d  51  37  PKD  No  9.0  18.0  7.3  0.095  0.119 ± 0.001  7.5  9.5  39.7  47.0  52  39  HTA  No  8.0  20.0  6.6  0.174  0.166 ± 0.010  0.6  3.0  4.3  n.d.  53  46  Unknown  Yes  8.0  22.0  10.1  0.120  0.118 ± 0.002  11.0  13.0  16.0  n.d.  54  46  DM  Yes  2.0  25.0  6.9  0.087  0.092 ± 0.007  7.0  10.0  92.8  41.0  55  48  HTA  No  10.0  27.0  8.3  0.166  0.168 ± 0.003  10.0  5.0  20.0  n.d.  56  55  HTA  Yes  6.0  27.0  7.0  0.134  0.113 ± 0.012  16.0  10.0  5.0  n.d.  57  55  Unknown  No  2.0  16.0  5.9  0.070  0.064 ± 0.005  4.4  3.9  40.9  53.0  58  56  HTA  Yes  11.0  25.0  9.4  0.140  0.133 ± 0.008  9.0  4.0  12.0  n.d  59  59  HTA  Yes  13.0  12.0  5.0  0.170  0.179 ± 0.009  6.0  7.8  45.8  68.0a  60  60  DM  No  3.0  20.0  5.6  0.153  0.164 ± 0.015  30.0  17.0  6.0  n.d.  Refrence values (range)          30.0–50.0  10.4–33.0  0.23–0.73  0.200–0.729  0.5–6.0  1.0–9.3  1.8–16.0    HBP, high blood pressure; UHS, uraemic haemolytic syndrome; PKD, policystic kidney disease; DM, diabetes mellitus; paternity, prior to haemodialysis replacement therapy; time, time elapsed since the first haemodialytic procedure; TTV, total testicular volume; TT, serum total testosterone; Free-T, serum free testosterone; Sal-T, salivary testosterone (mean ± SD) from two samples obtained in non-consecutive days; n.d., not determined. a PRLPEG recovery,< 40% was indicative to macroprolactinaemia and > 60% was indicative that hyperprolactinaemia is due to monomeric prolactin. View Large Interestingly, a significant increase in mean serum LH (4.9 ± 1.1 mUI/mL) was observed in eugonadal ESRD compared to normogonadic C (2.4 ± 1.52 mUI/mL, P = 0.001) although lying within the normal range. Serum estradiol in ESRD (eugonadic: 0.14 ± 0.04 nM and hypogonadic = 0.128 ± 0.05 nM) did not differ from C (0.15 ± 0.037 nM) P > 0.06 for all. Salivary and circulating testosterone in relation to clinical and biochemical parameters Salivary testosterone correlated negatively with age in ESRD (r = − 0.312, P = 0.012) (Figure 3) as in normogonadic C (r = − 0. 465, P = 0.001). Circulating TT, Bio-T and Free-T levels also correlated negatively with age in ESRD (r = − 0.261; r = − 0.315; r = − 0.383, respectively; P ≤ 0.04 in all cases) and in normogonadic C (r = − 0.480; r = − 0.454; r = − 0.384, respectively, P < 0.001 in all cases). Fig. 3 View largeDownload slide Correlation between morning salivary testosterone levels (Sal-T) and age (years) in 60 male with end-stage renal disease and 112 eugonadic healthy subjects. Sal-T declines significantly with age (r = − 0.312, P = 0.012) in HD (closed circles) as in 112 eugonadic healthy subjects (closed diamonds; r = − 0.465, P < 0.001). Fig. 3 View largeDownload slide Correlation between morning salivary testosterone levels (Sal-T) and age (years) in 60 male with end-stage renal disease and 112 eugonadic healthy subjects. Sal-T declines significantly with age (r = − 0.312, P = 0.012) in HD (closed circles) as in 112 eugonadic healthy subjects (closed diamonds; r = − 0.465, P < 0.001). In ESRD patients, SAL-T had a weak negative correlation with number of years on dialytic therapy (r = − 0.270, P = 0.038). Circulating TT, Bio-T and Free-T also correlated negatively with this variable (r = − 0.316, r = − 0.311 and r = − 0.358, respectively, P < 0.02 for all). Salivary and circulating testosterone in ESRD did not correlate significantly with either BMI or haemoglobin, albumin and parathormone levels. In addition, adequacy to dialysis (Kt/V) and rHuEpo dose administered did not show correlation with androgen levels. In rHuEpo-treated eugonadic patients (n = 22), TT (14.5 ± 3.0 nM), Bio-T (8.6 ± 2.0 nM), Free-T (0.381 ± 0.09 nM) and Sal-T (0.380 ± 0.157 nM) did not differ from those without rHuEpo (n = 25) (14.5 ± 4.2 nM, 8.43 ± 2.8 nM, 0.374 ± 0.137 nM, 0.428 ± 0.132 nM, respectively), P ≥ 0.260 in all cases. In nine hypogonadic patients treated with rHuEpo TT (7. 1 ± 1.5 nM), Bio-T (3.7 ± 1.0 nM), Free-T (0.157 ± 0.04 nM) and Sal-T (0.125 ± 0.04 nM) were not different from those in four patients who were not treated (7.05 ± 1.75, 3.9 ± 0.1, 0.173 ± 0.01 and 0.136 ± 0.03 nM, respectively), P ≥ 0.227 in all cases. Discussion This is the first study that demonstrates the value of Sal-T in the diagnosis of androgen deficiency in male ESRD. Morning Sal-T agreed with serum Free-T levels and correlates positively with all circulating testosterone fractions. Sal-T testing was able to differentiate patients with androgen deficiency (22%) from normogonadic patients (78%) with 100% sensitivity and specificity. Hypogonadism was hypergonadotrophic in 69% and hypogonadotrophic in 31%. Sal-T and all serum testosterone fractions correlated negatively with age and years on dialysis. In the early 1980s, two studies described low TT levels in ESRD, but data on SAL-T where controversial [21,22]. Differences could be ascribed to the small number of patients in both series, sampling (timing, collection device) or in the in-house radioimmunoassay methodology [21,22]. In this study, we proved in ESRD that Sal-T agrees with Free-T and correlates positively with all circulating testosterone fractions as described in healthy men [20,31]. The correlation between salivary and serum free steroid concentrations in our ESRD could be due to the normal protein levels of this population (SHBG and albumin). Testosterone levels in biological fluids may change with different methodologies, and troubles in the dosage may appear when levels are very low [27]. The Sal-T assay used in this study had a detection limit of 1 pg/mL in agreement with others [32,33], proving its utility when known hypogonadic adult men (reference group) where evaluated. Whenever SAL-T was low, a second saliva sample was obtained. The reproducibility of SAL-T levels in hypogonadic ESRD did not differ from other hypogonadic population [20]. Literature describes a high (26–56%) prevalence of hypogonadism in unselected males with chronic renal failure with TT levels ≤ 10.4 nM [2,4,21,22,34–36]. However, in younger ESRD patients with decreased libido, we found a lower percentage (22%) of androgen deficiency using similar criteria. In the remaining 78%, symptoms could be related to uraemia, chronic illness, diabetes, vascular disease, depression, medications, inadequate psychological and physical response to dialysis, etc. [37]. Interestingly, all patients who reported paternity had fathered before starting dialysis therapy. As previously described [38], high PRL levels were found in 10% of our patients; increased monomeric isoforms were confirmed in two hypogonadal ESRD patients. These patients started cabergoline, improving symptoms while decreasing PRL and normalizing androgens levels. As reported, this medical approach may benefit uraemic patients, although resistance and side effects to dopaminergic agents have been described [39]. Our next goal is to investigate benefits of testosterone therapy in hypogonadic ESRD [35]. As reported in healthy men, a negative correlation between circulating testosterone levels, SAL-T and age was confirmed in ESRD [4,36,40]. Anaemia and secondary hyperparathyroidism are frequent in ESRD. However, as reported by others [4], neither haemoglobin nor parathormone levels correlated with circulating and salivary testosterone concentrations. The treatment of anaemia with rHuEpo in male renal failure patients may affect sexual performance, although the mechanism of action remains controversial [41]. EPO stimulates Leidig cell steroidogenesis in vitro (rats [42]) and in vivo (healthy young males [43]). However, in agreement with Lawrence et al. [44], we could not confirm this effect in haemodialysis male patients. When focusing on the number of years on dialysis, a negative correlation was found with testosterone levels. Disorders of the pituitary–gonadal axis rarely normalize with dialysis; in fact, they often progress [7,39]. Since there is evidence that testosterone is not substantially cleared from circulation during haemodialysis [21,34], hypogonadism in these patients seem to depend on reduced production of this hormone by the testes. The assessment of SAL-T in ESRD met the criteria for diagnostic salivary steroid testing proposed recently [31]: there was a constant and predictable correlation between salivary and serum free steroid concentrations, the diagnostic accuracy of SAL-T was similar to that of serum testosterone, and a single saliva sample was just as informative as a single serum sample. This study supports the usefulness of morning Sal-T testing as a non-invasive approach to screen androgen status in men with end-stage renal disease. Acknowledgement We thank Mrs. Adriana M. Pena (CEREHA) and Mrs. Sonia Montini (Department of Nephrology, IDIM A. Lanari) for their technical assistance. This study was supported in part by a grant from the University of Buenos Aires M825, and Fundación Glándulas Salivales 2007. Conflict of interest statement. None declared. References 1 Meyer TW,  Hostetter TH.  Uremia,  N Engl J Med ,  2000, vol.  357 (pg.  1316- 1325) Google Scholar CrossRef Search ADS   2 Sy Lim V,  Fang VS.  Gonadal dysfunction in uremic men. A study of the hypothalamo-pituitary-testicular axis before and after renal transplantation,  Am J Med ,  1975, vol.  58 (pg.  655- 662) Google Scholar CrossRef Search ADS PubMed  3 Handelsman DJ.  Hypothalamic–pituitary gonadal dysfunction in renal failure, dialysis and renal transplantation,  Endocr Rev ,  1985, vol.  6 (pg.  151- 182) Google Scholar CrossRef Search ADS PubMed  4 Albaaj F,  Sivalingham M,  Haynes P, et al.  Prevalence of hypogonadism in male patients with renal failure,  Postgrad Med J ,  2006, vol.  82 (pg.  693- 696) Google Scholar CrossRef Search ADS PubMed  5 Palmer BF.  Outcomes associated with hypogonadism in men with chronic kidney disease,  Adv Chronic Kidney Dis ,  2004, vol.  11 (pg.  342- 347) Google Scholar CrossRef Search ADS PubMed  6 Wang C,  Nieschlag E,  Swerdloff R, et al.  Investigation, treatment and monitoring of late-onset hypogonadism in males,  Int J Androl ,  2008, vol.  32 (pg.  1- 10) Google Scholar CrossRef Search ADS PubMed  7 Sy Lim V,  Kathpalia SC,  Henriquez C.  Endocrine abnormalities associated with chronic renal failure,  Med Clin North Am ,  1978, vol.  62 (pg.  1341- 1361) Google Scholar CrossRef Search ADS PubMed  8 Schmidt A,  Luger A,  Hörl WH.  Sexual hormone abnormalities in male patients with renal failure,  Nephrol Dial Transplant ,  2002, vol.  17 (pg.  368- 371) Google Scholar CrossRef Search ADS PubMed  9 Anantharaman P,  Schmidt RJ.  2007 Sexual function in chronic kidney disease,  Adv Chronic Kidney Dis ,  2007, vol.  14 (pg.  119- 125) Google Scholar CrossRef Search ADS PubMed  10 Snyder PJ.  Might testosterone actually reduce mortality?,  J Clin Endocrinol Metab ,  2008, vol.  93 (pg.  32- 33) Google Scholar CrossRef Search ADS PubMed  11 Dunn JF,  Nisula BC,  Rodbard D.  Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid globulin in human plasma,  J Clin Endocrinol Metab ,  1981, vol.  53 (pg.  58- 68) Google Scholar CrossRef Search ADS PubMed  12 Gheorghiu I,  Moshyk A,  Lepage R, et al.  When is bioavailable testosterone a redundant test in the diagnosis of hypogonadism in men?,  Clin Biochem ,  2005, vol.  38 (pg.  813- 818) Google Scholar CrossRef Search ADS PubMed  13 Gröschl M.  Current status of salivary hormone analysis,  Clin Chem ,  2008, vol.  54 (pg.  1759- 1769) Google Scholar CrossRef Search ADS PubMed  14 Arregger AL,  Cardoso EM,  Tumilasci O, et al.  Diagnostic value of salivary cortisol in end stage renal disease,  Steroids ,  2008, vol.  73 (pg.  77- 82) Google Scholar CrossRef Search ADS PubMed  15 Rilling JK,  Worthman CM,  Campbell BC, et al.  Ratios of plasma and salivary testosterone throughout puberty: production versus bioavailability,  Steroids ,  1996, vol.  61 (pg.  374- 378) Google Scholar CrossRef Search ADS PubMed  16 Wang C,  Plymate S,  Nieschlag E, et al.  Salivary testosterone in men: further evidence of a direct correlation with free serum testosterone,  J Clin Endocrinol Metab ,  1981, vol.  53 (pg.  1021- 1024) Google Scholar CrossRef Search ADS PubMed  17 Sannikka E,  Terho P,  Suominen J, et al.  Testosterone concentrations in human seminal plasma and saliva and its correlation with non-protein-bound and total testosterone levels in serum,  Int J Androl ,  1983, vol.  6 (pg.  319- 330) Google Scholar CrossRef Search ADS PubMed  18 Goncharov N,  Katsya G,  Dobracheva A, et al.  Diagnostic significance of free salivary testosterone measurement using a direct luminescence immunoassay in healthy men and in patients with disorders of androgenic status,  Aging Male ,  2006, vol.  9 (pg.  111- 122) Google Scholar CrossRef Search ADS PubMed  19 Morley JE,  Perry HMIII,  Patrick P, et al.  Validation of salivary testosterone as a screening test for male hypogonadism,  Aging Male ,  2006, vol.  9 (pg.  165- 169) Google Scholar CrossRef Search ADS PubMed  20 Arregger AL,  Contreras LN,  Tumilasci OR, et al.  Salivary testosterone: a reliable approach to the diagnosis of male hypogonadism,  Clin Endocrinol ,  2007, vol.  67 (pg.  656- 662) Google Scholar CrossRef Search ADS   21 de Vries CP,  Gooren LJG,  Oe PL.  Haemodialysis and testicular function,  Int J Androl ,  1984, vol.  7 (pg.  97- 103) Google Scholar CrossRef Search ADS PubMed  22 Joven J,  Villabona C,  Rubiés-Prat J, et al.  Hormonal profile and serum zinc levels in uraemic men with gonadal dysfunction undergoing haemodialysis,  Clin Chim Acta ,  1985, vol.  148 (pg.  239- 245) Google Scholar CrossRef Search ADS PubMed  23 Van Kammen E,  Thijssen JHH,  Schwarz F.  Sex hormones in male patients with chronic renal failure. The production of testosterone and of androstenedione,  Clin Endocrinol ,  1978, vol.  8 (pg.  7- 14) Google Scholar CrossRef Search ADS   24 Chen JC,  Vidt DG,  Zorn EM, et al.  Pituitary-Leydig cell function in uremic males,  J Clin Endocrinol Metab ,  1970, vol.  31 (pg.  14- 17) Google Scholar CrossRef Search ADS PubMed  25 Gupta D,  Bundschu HD.  Testosterone and its binding in the plasma of male subjects with chronic renal failure,  Clin Chim Acta ,  1972, vol.  36 (pg.  479- 484) Google Scholar CrossRef Search ADS PubMed  26 Bhasin S,  Cunningham GR,  Hayes FJ, et al.  Testosterone therapy in adult men with androgen deficiency syndromes: an Endocrine Society Clinical Practice Guideline,  J Clin Endocrinol Metab ,  2006, vol.  91 (pg.  1995- 2010) Google Scholar CrossRef Search ADS PubMed  27 Rosner W,  Auchus RJ,  Azziz R, et al.  Position statement: utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement,  J Clin Endocrinol Metab ,  2007, vol.  92 (pg.  405- 413) Google Scholar CrossRef Search ADS PubMed  28 Olukoga AO,  Kane JW.  Macroprolactinaemia: validation and application of the polyethylene glycol precipitation test and clinical characterization of the condition,  Clin Endocrinol ,  1999, vol.  51 (pg.  119- 126) Google Scholar CrossRef Search ADS   29 Vermeulen A,  Verdonck L,  Kaufman JM.  A critical evaluation of simple methods for the estimation of free testosterone in serum,  J Clin Endocrinol Metab ,  1999, vol.  84 (pg.  3666- 3672) Google Scholar CrossRef Search ADS PubMed  30 de Ronde W,  Van der Schouw YT,  Pols HAP, et al.  Calculation of bioavailable and free testosterone in men: a comparison of 5 published algorithms,  Clin Chem ,  2006, vol.  52 (pg.  1777- 1784) Google Scholar CrossRef Search ADS PubMed  31 Wood P.  Salivary steroid assays—research or routine?,  Ann Clin Biochem ,  2008, vol.  46 (pg.  183- 196) Google Scholar CrossRef Search ADS   32 Granger DA,  Schwartz EB,  Booth A, et al.  Salivary testosterone determination in studies of child health and development,  Horm Behav ,  1998, vol.  35 (pg.  18- 27) Google Scholar CrossRef Search ADS   33 van Anders SM,  Watson NV.  Relationship status and testosterone in North American heterosexual and non-heterosexual men and women: cross-sectional and longitudinal data,  Psychoneuroendocrinology ,  2006, vol.  31 (pg.  715- 723) Google Scholar CrossRef Search ADS PubMed  34 Singh AT,  Norris K,  Modi N, et al.  Pharmacokinetics of a transdermal testosterone system in men with end stage renal disease receiving maintenance hemodialysis and healthy hypogonadal men,  J Clin Endocrinol Metab ,  2001, vol.  86 (pg.  2437- 2445) Google Scholar PubMed  35 Johansen KL.  Treatment of hypogonadism in men with chronic kidney disease,  Adv Chronic Kidney Dis ,  2004, vol.  11 (pg.  348- 356) Google Scholar CrossRef Search ADS PubMed  36 Carrero JJ,  Qureshi AR,  Parini P, et al.  Low serum testosterone increases mortality risk among male dialysis patients,  J Am Soc Nephrol ,  2009, vol.  20 (pg.  613- 620) Google Scholar CrossRef Search ADS PubMed  37 Martín Díaz F,  Reig-Ferrer A,  Ferrer-Cascales R.  Sexual functioning and quality of life of male patients on hemodialysis,  Nefrología ,  2006, vol.  26 (pg.  452- 460) Google Scholar PubMed  38 Yavuz D,  Topcut G,  Özener C, et al.  Macroprolactin does not contribute to elevated levels of prolactin in patients on renal replacement therapy,  Clin Endocrinol ,  2005, vol.  63 (pg.  520- 524) Google Scholar CrossRef Search ADS   39 Karagiannis A,  Harsoulis F.  Gonadal dysfunction in systemic diseases,  Eur J Endocrinol ,  2005, vol.  152 (pg.  501- 513) Google Scholar CrossRef Search ADS PubMed  40 Wu FCW,  Tajar A,  Pye SR, et al.  ,  The European Male Aging Study Group.  Hypothalamic pituitary testicular axis disruptions in older men are differentially linked to age and modifiable risk factors: the European Male Aging Study Group,  J Clin Endocrinol Metab ,  2008, vol.  93 (pg.  2737- 2745) Google Scholar CrossRef Search ADS PubMed  41 Ayub W,  Fletcher S.  End-stage renal disease and erectile dysfunction. Is there any hope?,  Nephrol Dial Transplant ,  2000, vol.  15 (pg.  1525- 1528) Google Scholar CrossRef Search ADS PubMed  42 Foresta C,  Mioni R,  Bordon P, et al.  Erythropoietin and testicular steroidogenesis: the role of second messengers,  Eur J Endocrinol ,  1995, vol.  132 (pg.  103- 108) Google Scholar CrossRef Search ADS PubMed  43 Foresta C,  Mioni R,  Bordon P, et al.  Erythropoietin stimulates testosterone production in man,  J Clin Endocrinol Metab ,  1994, vol.  78 (pg.  753- 756) Google Scholar PubMed  44 Lawrence IG,  Price DE,  Howlett TA, et al.  Erythropoietin and sexual dysfunction,  Nephrol Dial Transplant ,  1997, vol.  12 (pg.  741- 747) Google Scholar CrossRef Search ADS PubMed  © The Author 2010. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org Oxford University Press
TI - Salivary testosterone for the diagnosis of androgen deficiency in end-stage renal disease
JO - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/gfq439
DA - 2010-07-20
UR - https://www.deepdyve.com/lp/oxford-university-press/salivary-testosterone-for-the-diagnosis-of-androgen-deficiency-in-end-RF1wEOeJnA
SP - 677
EP - 683
VL - 26
IS - 2
DP - DeepDyve
ER -