Low Testosterone and High C-Reactive Protein Concentrations Predict Low Hematocrit in Type 2 Diabetes

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1 Pathophysiology/Complications O R I G I N A L A R T I C L E Low Testosterone and High C-Reactive Protein Concentrations Predict Low Hematocrit in Type 2 Diabetes VISHAL BHATIA, MBBS, MD AJAY CHAUDHURI, MBBS, MRCP RASHMI TOMAR, MBBS SANDEEP DHINDSA, MBBS HUSAM GHANIM, PHD PARESH DANDONA, MD, PHD OBJECTIVE After the demonstration that one-third of male patients with type 2 diabetes have hypogonadotrophic hypogonadism, we have shown that patients with hypogonadotrophic hypogonadism also have markedly elevated C-reactive protein (CRP) concentrations. We have now hypothesized that type 2 diabetic subjects with hypogonadotrophic hypogonadism may have a lower hematocrit because testosterone stimulates, whereas chronic inflammation suppresses, erythropoiesis. RESEARCH DESIGN AND METHODS Seventy patients with type 2 diabetes at a tertiary referral center were included in this study. RESULTS The mean hematocrit in patients with hypogonadotrophic hypogonadism (n 37), defined as calculated free testosterone (cft) of 6.5 ng/dl, was %, whereas that in eugonadal patients (n 33) was % (P 0.011). The hematocrit was related to cft concentration (r 0.46; P ); it was inversely related to plasma CRP concentration (r 0.41; P ). Patients with CRP 3 mg/l had a higher hematocrit ( %) than those with CRP 3 mg/l ( %; P 0.05). The prevalence of normocytic normochromic anemia (hemoglobin 13 g/dl) was 23% in the entire group, whereas it was 37.8% in the men with hypogonadotrophic hypogonadism and 3% in the eugonadal men (P 0.01). Erythropoietin concentration was elevated or high normal in all 11 patients with anemia in whom it was tested. CONCLUSIONS We conclude that hypogonadotrophic hypogonadism in male type 2 diabetic subjects is associated with a lower hematocrit and a frequent occurrence of mild normocytic normochromic anemia with normal or high erythropoietin concentrations. In these patients, hematocrit is also inversely related to CRP concentration. Thus, low testosterone and chronic inflammatory mechanisms may contribute to mild anemia. Such patients may also have a high risk of atherosclerotic cardiovascular events in view of their markedly elevated CRP concentrations. After our previous observations that one-third of patients with type 2 diabetes have hypogonadotrophic hypogonadism (1), that type 1 diabetic subjects do not suffer from this condition (2), and that the patients with hypogonadotrophic hypogonadism have markedly elevated plasma C-reactive protein (CRP) Diabetes Care 29: , 2006 concentrations (V.B., R.T., S.D., A. Chandel, A.C., H.G., P.D., unpublished observations), an index of systemic inflammation, we have now studied whether patients with hypogonadotrophic hypogonadism have lower hemoglobin concentrations. Testosterone is known to exert a stimulatory effect on From the Division of Endocrinology, Diabetes and Metabolism, State University of New York at Buffalo and Kaleida Health, Buffalo, New York. Address correspondence and reprint requests to Paresh Dandona, MD, PhD, Diabetes-Endocrinology Center of Western New York, 3 Gates Circle, Buffalo, NY pdandona@kaleidahealth.org. Received for publication 23 March 2006 and accepted in revised form 22 June Abbreviations: cft, calculated free testosterone; CRP, C-reactive protein; GFR, glomerular filtration rate; FSH, follicle-stimulating hormone; LH, luteinizing hormone; SHBG, sex hormone binding globulin. A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances. DOI: /dc by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. erythropoiesis in the bone marrow (3). Inflammation, on the other hand, is known to suppress erythropoiesis, partly through its direct action on erythropoiesis and partly through its suppression of erythropoietin secretion (4 7). Thus, we hypothesized that hematocrit in patients with type 2 diabetes is lower in patients with hypogonadotrophic hypogonadism who also have an elevated CRP concentration, an index of systemic inflammation. RESEARCH DESIGN AND METHODS The study was conducted in the Diabetes-Endocrinology Center of Western New York, a tertiary referral center affiliated with the State University of New York and Kaleida Health in Buffalo, New York, and in an endocrinology specialty clinic in Midland, Texas. The study was carried out in 70 male patients (50 from Buffalo and 20 from Midland) referred to the centers for management of type 2 diabetes. Patients with a known history of primary or secondary hypogonadism, hypopituitarism, renal failure, cirrhosis, glucocorticoid therapy, or known HIV infection were excluded from the study. Demographic parameters were collected, and height, weight, glucose, and HbA 1c (A1C) were measured. The age of patients (means SE) was years (range 24 78), weight was kg ( ), BMI was kg/m 2 ( ), and A1C was % (4.7 14). The clinical and biochemical features of the patients are summarized in Table 1. Fasting blood samples were obtained to measure plasma hemoglobin, hematocrit, serum total testosterone, sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, glucose, A1C, and plasma CRP. Serum total iron, ironbinding capacity, ferritin, vitamin B 12, and folate concentrations were measured in all 16 patients who had anemia. Total testosterone was measured by a solid-phase radioimmunoassay (Coat-A- Count; Diagnostic Products, Los Angeles, CA). The lower limit of normal for total testosterone in our clinical laboratory is 10.4 nmol/l (300 ng/dl). SHBG was tested DIABETES CARE, VOLUME 29, NUMBER 10, OCTOBER

2 Low testosterone anemia and CRP in diabetes Table 1 Characteristics of patients with low cft and normal cft values at Specialty Laboratories (Santa Monica, CA) by an immunochemiluminometric assay. Anemia was defined as hemoglobin 13 g/dl or hematocrit 39.0% as per the World Health Organization definition. Calculated free testosterone (cft) was determined from SHBG and total testosterone using the method of Vermeulen et al. (8), using a computer program and Web site address ( freetesto.htm) provided by Dr. T. Fiers, University Hospital Ghent, Ghent, Belgium. This cft value has been shown to correlate very well (r 0.9) with free testosterone measured by equilibrium dialysis (9). For cft, nmol/l (6.5 ng/dl) was taken as the lower limit of normal (10). It is known that cft values are generally 10 15% higher than those for free testosterone measured by equilibrium dialysis. Bioavailable testosterone (non SHBG-bound testosterone) was also calculated similarly using SHBG and testosterone. The lower limit of normal was considered to be 5.2 nmol/l (150 ng/dl) (10). LH and FSH were measured by chemiluminescent immunometric assays. The lower limit of total testosterone was 300 ng/dl, that for cft was 6.5 ng/dl, and that for bioavailable testosterone was 150 ng/dl in normal subjects. Hypogonadism cft 6.5 ng/dl (HH) cft 6.5 ng/dl (eugonadal) n Age (years) A1C (%) BMI (kg/m 2 ) * Hematocrit (%) CRP (mg/l) Total testosterone (ng/dl) cft (ng/dl) HDL (mg/dl) * LDL (mg/dl) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) Serum creatinine (mg/dl) GFR (ml/min) FSH (miu/ml) LH (miu/ml) ACE inhibitor therapy 23 (62.1) 21 (63.6) Angiotensin receptor blocker therapy 6 (16.2) 5 (15.1) Statins 26 (70.2) 23 (69.7) Aspirin 30 (81.1) 25 (75.8) Insulin 27 (73) 22 (66.7) Data are means SE or n (%). Note that patients with hypogonadotrophic hypogonadism (HH) were significantly heavier. *P 0.05; P 0.01; P 0.01; P 0.014; P was defined as cft 6.5 ng/dl. Hypogonadotrophic state was defined as inappropriately low FSH and LH concentrations for the concomitant subnormal cft concentrations. Thus, LH and FSH concentrations in the normal reference range would be considered to be low when associated with low cft concentrations. CRP levels 3 mg/l were termed high because those concentrations are known to be associated with increased cardiovascular events. Plasma CRP concentrations were measured using a high-sensitivity enzyme-linked immunosorbent assay kit from Alpha Diagnostics International (San Antonio, TX). Erythropoietin was measured in duplicate using the Advantage erythropoietin chemiluminescence immunoassay (Nichols Institute Diagnostic, San Clemente, CA), which has a sensitivity of 1.2 mu/ml and a coefficient of variation 6%. The mean of the two assays was used in the analysis. Glomerular filtration rate (GFR) was estimated by calculating creatinine clearance from the serum creatinine concentration using the Cockcroft-Gault formula. Statistical methods Data are presented as means SE. Student s t test was used to compare parametric data, and the Mann-Whitney ranksum test was used to compare nonparametric data. Fisher s exact test or the 2 test was also used to compare the groups whenever appropriate. Spearman correlation (for nonparametric data) and Pearson correlation (for parametric data) were used to establish correlations. Multiple regression analysis between variables was performed if there was more than one independent variable. P 0.05 was considered significant. Sigma Stat software was used for analysis. RESULTS The plasma total testosterone and cft concentrations (means Table 2 Characteristics of patients with CRP concentrations < and >3 mg/l CRP 3 mg/l CRP 3 mg/l n Age (years) A1C (%) BMI (kg/m 2 ) Hematocrit (%) * Total testosterone (ng/dl) cft (ng/dl) Bound testosterone (ng/dl) HDL (mg/dl) LDL (mg/dl) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) FSH (miu/ml) LH (miu/ml) Serum creatinine (mg/dl) GFR (ml/min) Data are means SE. *P 0.05; P 0.01; P 0.05; P 0.032; P DIABETES CARE, VOLUME 29, NUMBER 10, OCTOBER 2006

3 Figure 1 Mean hematocrit in the hypogonadal (cft 6.5 ng/dl; n 37) versus the eugonadal (cft 6.5 ng/dl; n 33) group. *P using the Mann-Whitney rank-sum test. SE) in hypogonadal men (n 37) were ng/dl (range ) and ng/dl ( ), respectively, and were significantly lower (P 0.01) than those in eugonadal men (n 33; ng/dl [305 1,428] and ng/dl [ ], respectively). The mean CRP concentrations were 6.5 mg/l in hypogonadal patients and 3.2 ng/dl in eugonadal patients (P 0.001); 40 patients (57.1%) had CRP 3 mg/l and 30 (42.9%) had CRP 3 mg/l. Table 2 summarizes the characteristics of patients with low and high CRP concentrations. Mean hematocrit was % in hypogonadal men, whereas it was % in eugonadal men (P 0.011) (Fig. 1). The hematocrit was % in patients with CRP 3 mg/l compared with % in patients with CRP 3 mg/l (P 0.05) (Fig. 2). Anemia was observed in 16 of 70 (23%) patients. In 15 of these 16 anemic patients, the anemia was normocytic and normochromic and was not associated with iron, folate, or vitamin B 12 deficiency. In one patient, it was microcytic and hypochromic and was associated with iron deficiency. Fourteen of 15 (93%) patients with normocytic normochromic anemia had low cft concentrations. Erythropoietin concentrations were normal or elevated in all of the 11 patients with normocytic normochromic anemia in whom they were measured. Bone marrow biopsy was available in one patient with hypogonadism and normocytic normochromic anemia. It demonstrated normal iron stores and had normal cellularity (40%) with normal erythroid, myeloid, and megakaryocytic cell lines. The prevalence of normocytic normochromic anemia was 37.83% in hypogonadal men, whereas it was 3% in eugonadal men (P 0.05). The prevalence of anemia was 13.2% in patients with CRP 3 mg/l, whereas it was 33.3% in those with CRP 3 mg/l (P 0.05). The frequency of anemia in those with both low cft and high CRP was 50%. SHBG concentrations tended to be lower in patients with anemia and in those with elevated CRP. Hematocrit was related to total testosterone (r 0.36; P 0.001) and cft (r Bhatia and Associates 0.46; P ) (Fig. 3). There was an inverse relationship between plasma CRP concentrations (r 0.41; P ) and hematocrit (Fig. 4). There was also an inverse relationship between plasma cft and CRP concentrations (r 0.27; P 0.02) (Fig. 5). Serum creatinine concentrations were not significantly different between hypogonadotrophic hypogonadism and eugonadal patients, nor was there a relationship between serum creatinine clearance and hematocrit. GFR was estimated from serum creatinine concentration of the patients using the Cockcroft- Gault formula. The mean calculated creatinine clearance was ml/ min in the hypogonadal group, whereas it was ml/min in the eugonadal groups, and the difference was not significant (P 0.33). No significant relationship was found between GFR and hematocrit (r 0.2; P 0.8). On multivariate analysis, both cft (P 0.001) and CRP (P 0.01) but not BMI were related independently to hematocrit. In an another multivariate analysis using CRP as the dependent variable and cft, BMI, and age as independent variables, only cft and age were independently related to CRP (P and 0.006, respectively). SHBG concentrations were inversely related to CRP (r 0.23; P 0.02) but were not related to either the hematocrit or cft. CONCLUSIONS This demonstration of a significantly lower hematocrit in hypogonadal men and a direct relation- Figure 2 Mean hematocrit in patients with CRP 3 mg/l versus CRP 3 mg/l. *P 0.05 using the Mann-Whitney rank-sum test. DIABETES CARE, VOLUME 29, NUMBER 10, OCTOBER

4 Low testosterone anemia and CRP in diabetes Figure 3 The relationship between cft and hematocrit (r 0.46; P ). There was also a significant relationship between total testosterone and hematocrit (r 0.36; P 0.002). ship between the hematocrit and testosterone in type 2 diabetic subjects, described for the first time, suggests that a low testosterone concentration may contribute to the pathogenesis of the mild anemia in these patients. Testosterone is known to stimulate erythropoiesis in the bone marrow and to increase the hematocrit (3). Consistent with a diminished erythropoietic drive was the fact that in 15 of 16 anemic patients, the anemia was normocytic normochromic and 14 of 15 patients had a subnormal cft. Only 1 patient of 16 had iron deficiency with a hypochromic microcytic anemia. None of the 15 patients with normocytic normochromic anemia had iron, folate, or vitamin B 12 deficiency. Eleven of 15 patients with normocytic normochromic anemia had erythropoietin concentrations measured; all 11 had high normal or elevated erythropoietin concentrations. Thus, the anemia was not due to erythropoietin deficiency. The highly significant inverse relationship between CRP concentrations and the hematocrit, independent of testosterone concentrations, suggests that inflammatory processes in type 2 diabetes probably also suppress the hematocrit. CRP has for a long time been considered a marker of systemic inflammation; therefore, it is also a prognosticator of cardiovascular events (11 13) because atherosclerosis is a chronic inflammation of the arterial wall. However, there is now evidence that CRP may be a mediator of inflammation (14 16). It induces intercellular adhesion molecule-1 and macrophage inflammatory protein-1 in endothelial cells in vitro and probably exerts this effect through the FcII receptor (17). More recently, it has been shown to exert a proinflammatory effect after an injection into normal human subjects in vivo (18). Thus, the relationship of CRP with hematocrit may either be through the direct action of CRP or through other inflammatory mediators associated with CRP or both. Inflammatory mechanisms may affect the hematocrit in two ways. First, they may suppress erythropoietin secretion (4), and second, they may cause increased apoptotic death of red cell precursors, resulting in no increase in erythropoiesis despite elevated erythropoietin levels (4,19 21). These mechanisms are relevant to the pathogenesis of anemia of chronic inflammatory disease. Thus, the mild anemia of type 2 diabetes may in part be attributable to processes similar to those involved in chronic inflammatory disease in addition to the contribution by low testosterone concentrations. The overwhelming predominance of the normocytic normochromic picture in the anemic patients is also consistent with marrow suppression secondary to inflammatory mechanisms. Elevated erythropoietin levels associated with anemia in these patients suggest an inadequate response to erythropoietin rather than a deficiency of erythropoietin. The relationship of inflammatory mechanisms with hematocrit becomes even more intricate because these mechanisms may be involved in the pathogenesis of hypogonadotrophic hypogonadism itself in patients with type 2 diabetes. We have previously reported and confirmed again in this study that there is a highly significant inverse relationship between plasma testosterone and CRP concentrations (V.B., R.T., S.D., A. Chandel, A.C., H.G., P.D., unpublished observations). It has previously been shown that insulin may facilitate gonadotropin-releasing hormone release from hypothalamic neurons in vitro and that interference with insulin signal transduction may reduce gonadotropin-releasing hormone secretion (22). Because inflammatory mediators interfere with insulin signal Figure 4 The inverse relationship between CRP and hematocrit (r 0.41; P ) DIABETES CARE, VOLUME 29, NUMBER 10, OCTOBER 2006

5 Bhatia and Associates Figure 5 The inverse relationship between cft and CRP (r 0.27; P 0.02). transduction, they may contribute to the pathogenesis of hypogonadotrophic hypogonadism. Consistent with our previous report, LH and FSH concentrations in patients with hypogonadotrophic hypogonadism were significantly lower than those in eugonadal patients. One recent European study (23) showed a mild elevation of hematocrit in patients with type 2 diabetes. The lowest quartile of hematocrit in this study had values that were similar to those in patients with hypogonadotrophic hypogonadism in our study. Although we cannot confirm elevated hematocrit in type 2 diabetes in our study, it would be worth investigating whether low hematocrit in other studies is associated with hypogonadotrophic hypogonadism. We conclude that the hematocrit is significantly lower in hypogonadal men with type 2 diabetes compared with eugonadal men and that there is a significant direct relationship between cft and hematocrit. There is also an inverse relationship between hematocrit and CRP. Thus, both a low testosterone concentration and inflammatory mechanisms may play an important role in the pathogenesis of the low-grade anemia observed in patients with type 2 diabetes. Because high CRP concentrations are known to be associated with atherosclerosis and there are early data showing that low testosterone concentrations may be associated with increased cardiovascular events (24), the anemic diabetic patient may have an increased risk of atherogenesis. References 1. Dhindsa S, Prabhakar S, Sethi M, Bandyopadhyay A, Chaudhuri A, Dandona P: Frequent occurrence of hypogonadotropic hypogonadism in type 2 diabetes. 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6 Low testosterone anemia and CRP in diabetes immortalized conditionally are activated by insulin: implication of the MAP kinase pathway. Endocrinology 147: , Natali A, Toschi E, Baldeweg S, Casolaro A, Baldi S, Sironi AM, Yudkin JS, Ferrannini E: Haematocrit, type 2 diabetes, and endothelium-dependent vasodilatation of resistance vessels. Eur Heart J 26: , Smith GD, Ben-Shlomo Y, Beswick A, Yarnell J, Lightman S, Elwood P: Cortisol, testosterone, and coronary heart disease: prospective evidence from the Caerphilly study. Circulation 112: , DIABETES CARE, VOLUME 29, NUMBER 10, OCTOBER 2006