Low Serum Testosterone Increases Mortality Risk among Male

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1 JASN Express. Published on January 14, 2009 as doi: 10.1681/ASN.2008060664 CLINICAL EPIDEMIOLOGY www.jasn.org Low Serum Testosterone Increases Mortality Risk among Male Dialysis Patients Juan Jesus Carrero,* Abdul Rashid Qureshi,* Paolo Parini, Stefan Arver, Bengt Lindholm,* Peter Barany,* Olof Heimburger,* and Peter Stenvinkel* *Divisions of Renal Medicine and Baxter Novum, Department of Clinical Science, Intervention and Technology, Division of Clinical Chemistry, Department of Laboratory Medicine and Center for Andrology and Sexual Medicine, Karolinska Institutet, Stockholm, Sweden ABSTRACT Men treated with hemodialysis (HD) have a very poor prognosis and an elevated risk of premature cardiovascular disease (CVD). In the general population, associations between low testosterone concen- trations and cardiovascular risk have been suggested. We performed a prospective observational study involving a well characterized cohort of 126 men treated with HD to examine the relationship between testosterone concentration and subsequent mortality during a mean follow-up period of 41 mo. Inde- pendent of age, serum creatinine, and sexual hormone binding globulin (SHBG), testosterone levels inversely and strongly associated with the inflammatory markers IL-6 and CRP. Patients with a clinical history of CVD had significantly lower testosterone levels. During follow up, 65 deaths occurred, 58% of which were a result of CVD. Men with testosterone values in the lowest tertile had increased all-cause CLINICAL EPIDEMIOLOGY and CVD mortality (crude hazard ratios [HRs] 2.03 [95% CI 1.24 to 3.31] and 3.19 [1.49 to 6.83], respectively), which persisted after adjustment for age, SHBG, previous CVD, diabetes, ACEi/ARB treatment, albumin, and inflammatory markers, but was lost after adjustment for creatinine. In summary, among men treated with HD, testosterone concentrations inversely correlate with all-cause and CVD- related mortality, as well as with markers of inflammation. Hypogonadism may be an additional treatable risk factor for patients with chronic kidney disease. J Am Soc Nephrol : , 2009. doi: 10.1681/ASN.2008060664 The role of hypogonadism and low testosterone in may be reciprocal (or bidirectional),8 because CVD mens health is still controversial. Although high has also been independently associated with kidney doses of exogenous testosterone or other anabolic function decline.9 Hence, CVD accounts for pre- steroids have been associated with adverse effects mature death in more than 50% of patients on di- on health,1 low concentration of endogenous tes- alysis, contributing to a 10- to 20-fold higher mor- tosterone levels have been linked to worse survival in apparently healthy populations of aged men.2 4 Interestingly, a recent study based on 11,606 men Received June 30, 2008. Accepted September 18, 2008. aged 40 to 79 yr reported that endogenous testos- Published online ahead of print. Publication date available at terone concentrations were inversely related to www.jasn.org. mortality due to cardiovascular disease (CVD), Correspondence: Dr. Peter Stenvinkel, Department of Renal suggesting that low testosterone may be a predictive Medicine K56, Karolinska University Hospital at Huddinge, 141 86 marker for high risk patients.5 Stockholm, Sweden. Phone: 46-8-58582532; Fax: 46-8-711472; E-mail: [email protected]; and Dr. Juan Jesus Carrero, Depart- Chronic kidney disease (CKD) patients have ac- ment of Renal Medicine K56,Karolinska University Hospital at Hud- celerated atherosclerosis,6 and CKD patients are dinge, 141 86 Stockholm, Sweden. Phone: 46-8-58583982; Fax: considered at a greatly increased cardiovascular 46-8-58583925; E-mail: [email protected] risk.7 This relationship between CKD and CVD Copyright 2009 by the American Society of Nephrology J Am Soc Nephrol : , 2009 ISSN : 1046-6673/00- 1

2 CLINICAL EPIDEMIOLOGY www.jasn.org tality rate than in the general population, despite stratification ng/dl]. Sixty-six men (52%) had testosterone deficiency (10 for sex, race, and diabetes.10 nmol/L, or 288 ng/dl), 39 (32%) presented testosterone lev- The altered metabolic milieu in CKD affects the secretion els in the lower normal range (10 to 14 nmol/L, or 288 to 403 of hormones and the response of target tissues, causing en- ng/dl), and only 21 (16%) presented normal testosterone val- docrine dysfunctions.11 As many as 50 to 70% of CKD ues. No differences in testosterone levels were found between stage-5 men have been reported to be hypogonadal on the consumers and nonconsumers of nonsteroidal anti-inflamma- basis of low concentrations of total and free testoster- tory drugs, -blockers, calcium channel blockers or statins one.12,13 Alterations on sex steroid production and metab- (data not shown). Only the use of ACEIs/ARBs had an effect on olism (leading to primary hypogonadism and disturbances the levels of testosterone, and slightly higher levels were ob- of the hypothalamic-pituitary axis) are already seen when served [11.5 (8.8 to 13.2) versus 9.7 (6.5 to 12.0) nmol/L, n moderate reductions in the GFR arise.14 These disorders are 46/80, P 0.01] in treated patients, probably a reflection of a not normalized with initiation of maintenance dialysis borderline significance in age [62 (47 to 70) versus 66 (51 to 76) treatment; instead, they often progress.15 Humoral factors, yr, P 0.06]. which accumulate in uremia, as well as other comorbid con- The general characteristics of the patients according to ditions that frequently accompany CKD and medications low testosterone values are presented in Table 1. Patients may contribute to suppressed sex hormone levels.13 with low testosterone were defined as those below the 33rd Despite the increased prevalence of hypogonadism and its percentile (lower tertile) of distribution in our sample pop- potential consequences, limited data are available on the influ- ulation, which was set at the level of 8.1 nmol/L (or 233 ence of endogenous testosterone and replacement therapy in ng/dl). The 66th percentile was set at 12.0 nmol/L (or 345 CKD patients.16 However, testosterone abnormalities have re- ng/dl). Despite not presenting differences in age, the pa- cently been linked to disorders in bone composition,17 to tients with low testosterone showed an increased number of sRANKL levels,17 and to endothelial dysfunction18 in CKD pa- comorbidities, mainly accounted for by the presence at tients. In light of the association between low testosterone con- baseline of a clinical history of CVD events. Patients with centrations and the risk of developing future cardiovascular low testosterone more often presented signs of protein-en- events recently reported in nonrenal patient groups,5,19 we ergy wasting (PEW) and lower handgrip strength values. evaluated the possible contribution of testosterone levels to the The markers of inflammation [high-sensitivity C-reactive poor prognosis and the elevated CVD risk of male hemodialy- protein (CRP), IL-6, fibrinogen, and plasma long pen- sis (HD) patients. traxin-3] were elevated in this patient group, and the pro- portion of hypochromic red blood cells (RBCs) and the erythropoietin (EPO) requirements were higher. Finally, the levels of sexual hormone binding globulin (SHBG), free RESULTS testosterone, and bioavailable testosterone were also de- creased in the patients with low total testosterone. General Characteristics The male population presented a median age of 63 (inter- Univariate and Multivariate Correlations quartile range 49 to 73, range 29 to 86) years. Thirty-five As expected, testosterone levels decrease with increasing age (27%) of the men presented diabetes mellitus, whereas 85 (Table 2), and move in the same direction as SHBG, free, and (66%) had a clinical history of CVD. Patients were treated bioavailable testosterone. Confirming the observations of Ta- with HD three times a week (4 to 5 h per session) using ble 1, testosterone levels were positively correlated to s-albu- bicarbonate dialysate and high-flux (23%) or low-flux min, s-creatinine, and LDL-cholesterol levels, and a strong (83%) dialysis membranes. Most patients were on antihy- negative correlation was observed for the proportion of hypo- pertensive medications [-blockers, n 69, calcium-chan- chromic RBCs. Of interest, the levels of testosterone were nel blockers, n 33; and angiotensin converting enzyme strongly and negatively related to a variety of surrogate mark- inhibitors (ACEIs)/angiotensin receptor blockers (ARBs), ers of inflammation, especially with IL-6 and CRP. Similar ob- n 46], as well as other commonly used drugs in terminal servations were made for the free and bioavailable estimates CKD (such as phosphate and potassium binders) and vita- (data not shown). min B, C, and D supplementation. Forty-three patients were To test whether the relationships between IL-6, CRP, and on lipid-lowering medication (statins). One hundred and testosterone were a reflection of natural aging, we performed a twenty four patients were receiving erythropoiesis stimulat- multinomial logistic regression analysis for these associations ing agents (ESAs) at time of evaluation. The median ESA correcting for s-creatinine, age, and levels of SHBG (Table 3). equivalent dose was 10,000 (6000 to 14,750) U/wk, which Analyses showed that the associations between testosterone was normalized for body weight and presented as U/kg/wk and inflammatory markers were independent of age, s-creati- in the following analyses. nine, and SHBG concentration. Regression models were re- The levels of total testosterone presented a median value of peated for free and bioavailable testosterone, with results being 9.95 (7.2 to 12.0) nmol/L [equivalent to 286 (206 to 346) the same (data not shown). 2 Journal of the American Society of Nephrology J Am Soc Nephrol : , 2009

3 www.jasn.org CLINICAL EPIDEMIOLOGY Table 1. Demographics and clinical characteristics of the 126 prevalent HD men included in the studya Low Testosterone Non-Low Testosterone Characteristic P Value n 42 n 84 Age, yr 66 (59 to 72) 60 (47 to 74) NS Vintage, mo 33 (16 to 57) 24 (12 to 52) NS Diabetes mellitus, % 31 25 NS CVD, % 79 60 0.05 Wasting (SGA 1), % 54 30 0.01 s-albumin, g/L 33 5 36 4 0.01 s-creatinine, mol/L 776 228 859 206 0.05 Body mass index, kg/m2 23.9 4.7 24.1 3.9 NS Handgrip, kg 22.7 10.8 26.9 11.1 0.05 CRP, mg/L 16.0 (4.3 to 38.5) 6.0 (2.1 to 12.7) 0.0001 IL-6, pg/ml 15.0 (8.1 to 25.8) 7.0 (4.6 to 11.7) 0.0001 Fibrinogen, g/L 4.8 (3.6 to 6.3) 4.1 (3.4 to 5.0) 0.05 Pentraxin 3, ng/ml 16.3 (8.9 to 20.1) 9.2 (6.2 to 14.9) 0.003 Hemoglobin, g/dl 11.9 1.4 12.2 1.2 NS ESA dose, U/Kg/wk 183 (84 to 296) 114 (70 to 171) 0.01 Hypochromic RBCs, % 4.2 (1.1 to 8.5) 0.9 (0.4 to 1.8) 0.0001 SHBG, nmol/L 22.5 (15.0 to 34.0) 29.5 (21.0 to 43.5) 0.01 Total testosterone, nmol/L 6.3 (4.5 to 7.2) 12.0 (9.9 to 13.7) 0.0001 Free testosterone, nmol/L 0.15 (0.09 to 0.19) 0.27 (0.21 to 0.33) 0.0001 Bioavailable testosterone, nmol/L 2.9 (1.6 to 3.5) 5.2 (4.1 to 6.9) 0.0001 Progesterone, nmol/L 3.6 (2.8 to 4.6) 3.2 (2.2 to 4.9) NS a A low testosterone level was defined as below the 33rd percentile (lower tertile) of distribution in our sample population. Data are presented as median (interquartile range) or average SD. For conversion of SHBG to g/dl, divide by 0.025; for conversion of free or bioavailable testosterone to ng/dl, divide by 0.0347; and for conversion of progesterone to ng/dl, divide by 31.8. Table 2. Univariate correlations by Spearmans rank test Table 3. Two different multivariate regression models between total testosterone and selected parameters in 126 predicting for testosterone concentration (nmol/L) prevalent HD men including CRP (model 1) or IL-6 (model 2) levelsa Parameter Spearman P Value Parameter Standard Model/Parameter P Value Age 0.23 0.05 Estimate Error SHBG 0.31 0.001 Model 1: Intercept 8.23 2.62 0.002 Progesterone 0.13 NS age, yr 0.064 0.024 0.008 Free testosterone 0.71 0.001 SHBG, nmol/L 0.087 0.018 0.0001 Bioavailable testosterone 0.70 0.001 CRP, mg/L 0.032 0.011 0.003 Body mass index 0.01 NS s-creatinine, mol/L 0.004 0.002 0.009 s-Albumin 0.23 0.05 Model 2: Intercept 8.33 2.78 0.003 s-Creatinine 0.17 0.05 age, yr 0.066 0.024 0.007 Hypochromic RBCs 0.41 0.0001 SHBG, nmol/L 0.090 0.019 0.0001 ESA dose 0.24 0.01 IL-6, pg/ml 0.043 0.022 0.04 Ferritin 0.09 NS s-creatinine, mol/L 0.004 0.002 0.01 Hemoglobin 0.01 NS a The adjusted r2 of the models were 0.28 and 0.26, respectively. CRP 0.38 0.0001 IL-6 0.42 0.0001 mial logistic regression to test whether this association was Fibrinogen 0.29 0.001 solely a reflection of the process of aging (Table 4). Again, Pentraxin 3 0.24 0.05 analyses showed that the associations between testosterone and clinical CVD were independent of age and SHBG levels. Testosterone Levels and CVD Across decreasing testosterone tertiles, the odd ratios for CVD The levels of testosterone were lower among patients with a were incrementally higher. clinical history of CVD as compared with those with no history of CVD [9.4 (6.5 to 12.0) versus 12.0 (9.5 to 14.2) nmol/L; P Testosterone and Survival in Men Undergoing HD 0.003]. The same was true for free [0.21 (0.15 to 0.29) versus During the follow-up period, 65 (51%) patients died, 38 (58%) 0.25 (0.19 to 0.32) nmol/L; P 0.03] and bioavailable [4.0 (2.8 from CVD-related causes. The baseline testosterone levels in to 5.5) versus 4.9 (3.5 to 7.0) nmol/L; P 0.01] testosterone. the patients who died from CVD [8.0 (6.1 to 11.0) nmol/L] was Because patients with CVD were older [66 (55 to 75) versus 57 lower (P 0.05) than the testosterone levels of patients who (43 to 71) yr, n 84/42; P 0.005], we performed a multino- died from non-CVD-related causes [11.0 (8.3 to 12.0) nmol/ J Am Soc Nephrol : , 2009 Low Testosterone Predicts Outcome in HD Men 3

4 CLINICAL EPIDEMIOLOGY www.jasn.org Table 4. Odds ratios and 95% CI for the presence of CVD at time of inclusion DISCUSSION in 126 men undergoing HDa Parameter Odds Ratio (95% CI) P Value The study presented here reveals a link Intercept 0.0001 between low endogenous testosterone Age 45 to 64 yr 3.58 (1.07 to 11.90) 0.037 concentrations and increased mortality Age 65 yr 5.94 (1.79 to 19.76) 0.003 due to all causes and to CVD in men SHBG 21 nmol/L, lower tertile 0.73 (0.29 to 1.83) 0.511 undergoing HD. Although this relation- Testosterone 8.1 to 12.0 nmol/L, middle tertile 2.60 (0.99 to 6.79) 0.050 ship was independent of age, SHBG, Testosterone 8.1 nmol/L, lower tertile 3.32 (1.16 to 9.48) 0.024 baseline comorbidities (CVD and diabe- a The adjusted r2 of the model was 0.18. For conversion of SHBG to g/dl, divide by 0.025; and for tes), and inflammation, it may be related conversion of testosterone to ng/dl, divide by 0.0347. to muscle depletion (represented by s- creatinine levels). Also, the presence of clinical signs of CVD at the time of inclusion was associated with lower testosterone levels irrespective of age and SHBG. Finally, a strong and inverse association between testosterone and various inflammatory markers was observed. In agreement with previous reports in CKD patients,12,13,16 more than 50% of our patients presented testosterone levels below the normal range, and a further 30% presented testos- terone levels in the lower range of normality. This distribution reflects the high prevalence of hypogonadism in CKD. The mechanisms explaining the high prevalence of hypogonadism in CKD are not completely understood. Dysfunction of the hypothalamic-pituitary-testicular axis exists, and decreased synthesis and secretion of testosterone follows with progressive renal failure. Although some testosterone may be removed by HD, increased clearance cannot explain the low testosterone levels.20,21 In addition, stress, medication, and weight loss may also contribute. Testosterone circulates in plasma freely, bound to specific plasma proteins (e.g., SHBG), or weakly bound to nonspecific proteins such as s-albumin. The SHBG- bound fraction is considered to be biologically inactive because of the high-affinity binding to SHBG. Therefore, one strength of the Figure 1. Kaplan-Meier survival analysis for (A) all-cause and (B) study presented here is represented by the adjustment for SHBG CVD-related mortality in 126 prevalent men undergoing HD ac- in all of our multivariate analyses, indirectly indicating a reduced cording to testosterone levels. Low testosterone levels were de- biologic activity of testosterone in our patients. Moreover, we can fined in our sample population as those below the 33rd percentile also demonstrate that because both the SHBG and the testoster- of testosterone distribution (see results). one levels are decreased, the free and bioavailable fractions re- main decreased in a similar manner. L]. Consequently, Kaplan-Meier analysis (Figure 1) showed Previous studies in male nonrenal patients have shown that that patients with low testosterone (below the 33rd percentile) low endogenous testosterone concentrations are associated have an increased mortality by all causes [log-rank 2 8.37, P with a more harmful CVD risk factor profile and a higher prev- 0.003; hazard ratio (HR) 2.03, 95% confidence interval (CI) alence of metabolic syndrome components.22,23 In addition to 1.24 to 3.31, P 0.004] and CVD-related causes (Log-rank 2 sexual dysfunction, testosterone deficiency is also associated 9.98, P 0.001; HR 3.19, CI 1.49 to 6.83, P 0.008). By using with the loss of muscle mass.16,24 In our study, patients with the Cox proportional hazards model, we corrected these dif- low testosterone levels had lower handgrip strength and lower ferences for potential confounding factors (Table 5). The prog- s-creatinine levels. In stable HD patients, s-creatinine levels are nostic value of low testosterone levels on mortality by all causes a crude reflection of muscle mass and fairly correlate with and CVD-related causes persisted even after adjustment for handgrip strength and lean body mass.25 Because testosterone age, SHBG, baseline comorbidities (CVD and diabetes melli- actively induces muscle protein synthesis,26 and androgens tus), ACEI/ARB medication, s-albumin, and inflammation such as nandrolone decanoate (alone or in combination with (depicted as IL-6 levels). In search of potential mechanisms resistance exercise training) has proved to have anabolic effects behind this observation, further adjustment for s-creatinine in CKD patients,27 these results were not unexpected. Further- resulted in the loss of significance and a considerable loss in the more, our observation that men with low testosterone have magnitude of these effects. increased ESA requirements may be due to loss of the stimula- 4 Journal of the American Society of Nephrology J Am Soc Nephrol : , 2009

5 www.jasn.org CLINICAL EPIDEMIOLOGY Table 5. Crude and adjusted all-cause and CVD-related mortality according to testosterone levels in 126 prevalent HD mena All-Cause Mortality CVD Mortality Model Covariates HR (95% CI) P Value HR (95% CI) P Value 1 Crude 2.03 (1.24 to 3.31) 0.004 3.19 (1.49 to 6.83) 0.002 2 1 age and SHBG 1.94 (1.16 to 3.26) 0.01 2.96 (1.32 to 6.61) 0.008 3 2 baseline CVD, diabetes mellitus, and ACEI/ARB medication 2.21 (1.28 to 3.82) 0.004 2.79 (1.21 to 6.48) 0.01 4 3 IL-6 1.77 (1.06 to 3.70) 0.03 2.53 (1.07 to 5.99) 0.03 5 4 s-albumin 1.74 (1.01 to 3.02) 0.04 2.47 (1.04 to 5.87) 0.04 6 5 s-creatinine 1.51 (0.86 to 2.72) 0.14 2.00 (0.80 to 4.95) 0.13 a Indicated are HRs for all-cause and CVD mortality for patients with testosterone concentration below the 33rd percentile of testosterone distribution crude (model 1) or with various degrees of adjustment (models 2 to 6). tory effect of testosterone on erythropoiesis.28 In fact, testos- which testosterone, per se, influences mortality and specifically terone treatment has a positive effect on anemia in HD patients cardiovascular end points. First, testosterone levels have been and may enhance erythropoiesis even in other types of anemia inversely related to the progression of atherosclerosis in the in which ESA treatment is relatively ineffective.29 This obser- aorta34 and carotid artery,35 perhaps due to an explicit vasodi- vation is of interest and warrants further study, because testos- latory effect.36,37 Second, testosterone actively induces muscle terone assessment and supplementation in deficiency states protein synthesis.26,38,39 Both muscle mass loss in wasted pa- may help to initiate preventive anemia strategies in the CKD tients40 or even rapid weight loss during weight reduction reg- population. imens41 have been correlated with testosterone decline. Thus, An additional novel finding in our study is the strong neg- our observation that adjustment for s-creatinine levels (used ative association between testosterone levels and various in- here as a surrogate marker of muscle mass) resulted in the loss flammatory biomarkers irrespective of age, s-creatinine, and of effect on mortality may indirectly support this hypothesis. SHBG levels. There are a very few studies in the literature in- Although randomized controlled trials using low doses of dicating a relation between testosterone levels and inflamma- exogenous testosterone in aged healthy men38,39 have reported tory markers. Although the normalization of testosterone in favorable increases in muscle mass and other beneficial meta- type 2 diabetic patients leads to concomitant reductions of bolic effects, no studies have yet assessed the effect of testoster- inflammatory cytokines30 results of experimental studies have one supplementation on death or CVD death as the primary been contradictory: Although after renal injury in rats31 both end point. Although the study presented here reports a clear the endogenous and the exogenous testosterone seem to be association between low testosterone levels and increased mor- pro-inflammatory by stimulating TNF- production, they tality, the question whether testosterone therapy would reverse have been reported to favorably contribute to regulation of the increased risk for cardiovascular complications is still atherosclerotic plaque growth and stability by lowering in- open. Also, because our results suggest that increased inflam- flammatory molecules in castrated rabbits.32 Because our matory status is associated with hypogonadism, further studies cross-sectional study cannot provide the direction of this asso- are needed to test whether or not testosterone replacement ciation, intervention studies addressing this issue are needed to may benefit the inflammatory status in these patients. provide evidence for a causal direction. This is the first study addressing testosterone and mortality The main finding of the study presented here is the prog- in CKD men, and the results should be interpreted with cau- nostic value of low testosterone levels for mortality by all tion because several limitations of the study should be high- causes, but especially by CVD. We report that testosterone was lighted. First, the cross-sectional nature of this study precludes a marker of pre-existing CVD and also an independent risk from causality. As the cut-off levels of mortality are based in factor for CVD death. Thus, our study is in agreement with the the 33rd percentile of distribution, the results only apply to our majority,2,3,5,19 but not all33 of the previous studies in nonrenal study population and do not point to the optimal cutpoint for patients supporting the hypothesis that testosterone deficiency risk reduction, which should be derived from larger materials. is implicated in the progression of CVD. In agreement with a Although the careful phenotyping strengthens our analysis, we previous study,2 adjustment for IL-6 levels resulted in an atten- are limited by a relatively small sample size that prevents us uation of the prognostic value of low testosterone on mortality. from taking into consideration all possible confounders. Thus, Whether persistent low-grade inflammation (as commonly our analysis should be understood as a hypothesis-generating observed in CKD) contributes to decreasing testosterone levels study that warrants further confirmation. Moreover, s-creati- or vice versa is currently unknown, and it cannot be ruled out nine levels may not be a perfect reflection of muscle mass in that it represents a vicious circle in which gonadal dysfunction HD patients because this parameter could be influenced by the causes or facilitates increased inflammatory activity, which in diet (meat intake), residual renal function, and/or dialysis turn further suppresses testosterone production. Nevertheless, dose. The classification of CVD, which only considered pa- there is supportive evidence suggesting mechanisms through tients with clinically significant vascular disease, may limit and J Am Soc Nephrol : , 2009 Low Testosterone Predicts Outcome in HD Men 5

6 CLINICAL EPIDEMIOLOGY www.jasn.org underestimate the true prevalence of CVD in this high-risk sudden death, stroke, or complicated peripheral vascular disease. A population. At the same time, cardiovascular death is based on nephrologist blind to other clinical or biochemical patient data regis- medical records, not always confirmed by autopsies. We tered the causes of death. should also acknowledge that the testosterone values were only based on a single measurement. However, sampling at a single Laboratory Analyses time point fairly reliably reflects the mean annual testosterone Blood samples were collected before the HD session after the longest level and is considered a valid marker for population studies.42 interdialytic period. Plasma and serum were separated and kept fro- Finally, estradiol levels were not measured. It would have been zen at 70C, if not analyzed immediately. Serum concentrations of interesting to evaluate whether these beneficial effects of tes- IL-6 were quantified by immunometric assays on an Immulite Ana- tosterone are mediated by the testosterone itself or via aroma- lyzer (Siemens Medical Solutions Diagnostics, Los Angeles, Califor- tization to estradiol. However, such consideration did not ma- nia) according to the instructions of the manufacturers. Plasma long terially change the results in a previous study, in which pentraxin-3 concentration was measured by using a commercially estradiol was less strongly associated with CVD risk factors available ELISA kit (Perseus Preteomics Inc., Japan). High-sensitivi- than testosterone.2 tyCRP, fibrinogen, s-albumin, ferritin, s-creatinine, hemoglobin con- In conclusion, we report for the first time that testosterone centrations, and the percentage of hypochromic RBCs were analyzed concentrations are inversely related to mortality due to both using certified methods in the Department of Laboratory Medicine at CVD and all causes in male HD patients. Because this associa- Karolinska University Hospital or Uppsala Academic Hospital. tion is lost after adjustment for s-creatinine, our data suggest that hypogonadism and muscle wasting are closely related. We Hormonal Assessment also showed that patients with clinical signs of CVD presented Gonadal status was assessed a posteriori from frozen samples. Testos- lower testosterone levels, even after correcting for age and terone, progesterone, and SHBG were measured in all men participat- SHBG. Finally, strong inverse independent correlations were ing in the study, using certified routine methods in the Department of observed between testosterone and various inflammatory Laboratory Medicine at Karolinska University Hospital. Free and bio- markers. Because low testosterone may constitute an addi- available (not SHBG-bound) testosterone were calculated from total tional risk factor in CKD men, data from the observational testosterone, SHBG, and s-albumin concentrations using mass action study presented here encourage further research into the role equations.44,45 Although no uniform definition of hypogonadal tes- of testosterone as a modifiable risk factor in CKD and create a tosterone level is available, values below 10 nmol/L (300 mg/dl) are rationale for randomized controlled trials with testosterone generally considered to represent deficiency.46,47 Accordingly, the fol- supplementation in this patient group. lowing classification was used: (1) normal: testosterone 14 nmol/L (403 ng/dl); (2) low-normal: testosterone 10 to 14 nmol/L (288 to 403 ng/dl); (3) low: testosterone 10 nmol/L (288 ng/dl). CONCISE METHODS Patients and Experimental Design Nutritional Status The study was performed at five dialysis units in Stockholm, and Body mass index and dynamometric measurements were determined one at the Uppsala Academic Hospital in Uppsala, Sweden. This is on a dialysis day. Handgrip strength was measured in the dominant a post hoc analysis of data arising from a cross-sectional study hand (or nonfistula arm) with a Harpenden Handgrip Dynamometer. originally aiming at investigating the variability of inflammatory The highest of three measurements was noted. Subjective global as- markers in HD patients. The protocol has been previously de- sessment was used to evaluate the overall PEW.48 For the purpose of scribed in more detail and patient recruitment took place between this study PEW was defined as subjective global assessment 1. October 2003 through March 2004.43 Out of the 228 prevalent patients included in the study and followed for assessment of over- Statistical Analyses all and cardiovascular mortality, 101 were women. Therefore, tes- All variables were expressed as mean SD or as median (interquartile tosterone and SHBG levels were measured in the remaining 127 range), unless otherwise indicated. Statistical significance was set at men. One male patient was excluded because not enough stored the level of P 0.05. Normality was assessed by Kolmogorov-Smir- plasma was available. The Ethics Committee of Karolinska Institutet nov test. Comparisons between two groups were assessed with the Hospital and Uppsala University Hospital approved the study proto- students unpaired t test and Mann-Whitney test or 2 test, as appro- cols. Signed informed consent was obtained from all patients before priate. Differences between more than two groups were analyzed by inclusion in the study. ANOVA (ANOVA) using one-way ANOVA or Kruskal-Wallis test, as A single clinician, who extracted data pertaining to the underlying appropriate. Spearmans rank correlation () was used to determine CKD and CVD history, other comorbid conditions, and survival, re- correlations of testosterone with other variables. Multinomial logistic viewed each patients medical chart. Survival was determined from regression analyses were used to study the independence from age or the day of examination, with a mean follow-up period of 41 (20 to 47) SHBG levels of some of the observations made. Survival analyses were mo. There was no loss of follow up of any patient. Cardiovascular made with the Kaplan-Meier survival curve and the Cox proportional mortality was defined as death resulting from coronary heart disease, hazard model. The univariate and multivariate Cox regression analy- 6 Journal of the American Society of Nephrology J Am Soc Nephrol : , 2009

7 www.jasn.org CLINICAL EPIDEMIOLOGY sis are presented as (HR; CI). All statistical analyses were performed MJ, Weiner DE: Cardiovascular disease and subsequent kidney dis- using statistical software SAS version 9.1.3 (SAS Campus Drive, Cary, ease. Arch Intern Med 167: 1130 1136, 2007 10. Vanholder R, Massy Z, Argiles A, Spasovski G, Verbeke F, Lameire N: North Carolina). The following conversion factors may be applied: Chronic kidney disease as cause of cardiovascular morbidity and mor- for conversion of SHBG to g/dl, divide by 0.025; for conversion of tality. Nephrol Dial Transplant 20: 1048 1056, 2005 free or bioavailable testosterone to ng/dl, divide by 0.0347; and for 11. Handelsman DJ: Hypothalamic-pituitary gonadal dysfunction in renal conversion of progesterone to ng/dl, divide by 31.8. failure, dialysis and renal transplantation. Endocr Rev 6: 151182, 1985 12. Albaaj F, Sivalingham M, Haynes P, McKinnon G, Foley RN, Waldek S, ODonoghue DJ, Kalra PA: Prevalence of hypogonadism in male ACKNOWLEDGMENTS patients with renal failure. Postgrad Med J 82: 693 696, 2006 13. Gomez F, de la Cueva R, Wauters JP, Lemarchand-Beraud T: Endo- crine abnormalities in patients undergoing long-term hemodialysis. We would like to thank the patients and personnel involved in the The role of prolactin. Am J Med 68: 522530, 1980 creation of this cohort at each of the dialysis units. Also, we are in- 14. Handelsman DJ, Dong Q: Hypothalamo-pituitary gonadal axis in debted to our research staff at KBC (Ann Dreiman-Lif, Annika Nils- chronic renal failure. Endocrinol Metab Clin North Am 22: 145161, son, and Anki Emmoth) and KFC (Bjorn Anderstam, Monika Erics- 1993 son, and Anki Bragfors-Helin). 15. de Vries CP, Gooren LJ, Oe PL: Haemodialysis and testicular function. Int J Androl 7: 97103, 1984 The MIMICK study was supported by an unrestricted project 16. Johansen KL: Testosterone metabolism and replacement therapy in grant from Amgen, Inc. We also benefited from Karolinska Institutet patients with end-stage renal disease. Semin Dial 17: 202208, 2004 Center for Gender-Based Research, Karolinska Institutet research 17. Doumouchtsis KK, Kostakis AI, Doumouchtsis SK, Grapsa EI, Passali- funds, MEC (EX2006-1670), the Swedish Heart and Lung Founda- dou IA, Tziamalis MP, Poulakou MV, Vlachos IS, Perrea DN: The effect tion, the Swedish Medical Research Council, the Swedish Exports of sexual hormone abnormalities on proximal femur bone mineral density in hemodialysis patients and the possible role of RANKL. Association, an unconditional grant from Scandinavian Clinical Nu- Hemodial Int 12: 100 107, 2008 trition AB, the GENECURE project (grant LSHM-CT-2006-037697), 18. Karakitsos D, Patrianakos AP, De Groot E, Boletis J, Karabinis A, and research grants from the ERA-EDTA. 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