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Departments of Emergency Medicine (E.C., O.O., C.B.), Biochemistry (H.Y., E.O.A., M.K.E.), and Gastroenterology (Z.Y.), Gulhane Military Medical School, Ankara, Turkey
Address all correspondence and requests for reprints to: Dr. Erdinc Cakir, Department of Emergency Medicine, Gulhane Military Medical School, 06010, Etlik, Ankara, Turkey. E-mail: erdcakir{at}yahoo.com.
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Abstract |
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Introduction |
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Arterial functions, which are modulated by the endothelial cell layer, may also be subject to androgenic influence (10). Endothelial dysfunction is an early event in atherogenesis, appears to have detrimental functional consequences as well as adverse long-term effects, including vascular remodeling, and seems to predict adverse coronary outcomes; thus, gender differences in endothelial function and the effects of hormonal therapy on vascular function have been the focus of considerable research interest (11, 12, 13, 14). T also causes coronary, mesenteric, renal, and iliac vasodilatation in anesthetized animals, the mechanism of which was reported to involve nitric oxide (NO) release and ATP-sensitive K+ channels (15, 16).
NO, synthesized from L-arginine, accounts for the powerful vasodilator effects of endothelium-derived relaxing factor (17, 18) and consequently plays a decisive role in determining vasomotor tone (19, 20). Asymmetric NG,NG-dimethyl-L-arginine (ADMA), a guanidino-substituted analog of L-arginine, is synthesized endogenously and can act as inhibitor of NO synthase (21), the enzyme responsible for the formation of NO from L-arginine. The closely related compound symmetric NG,NG’-dimethyl-L-arginine, a stereoisomer of ADMA, has no inhibitory effect on NO synthase. The plasma levels of these L-arginine analogs are significantly increased in various pathological conditions, including end-stage chronic renal failure (22), congestive heart failure, preeclampsia, peripheral arterial occlusive disease, and hypertension (23, 24).
The purpose of the present study was to evaluate the effect of single dose depot testosterone on plasma L-arginine and dimethylarginine concentrations and their relationship to NO production, measured as plasma nitrite-plus-nitrate (NOx) concentration, in patients with idiopathic hypogonadotropic hypogonadism (IHH), compared with control subjects.
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Subjects and Methods |
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Eighteen newly diagnosed men with IHH and 20 healthy males matched for age and body mass index were enrolled in the study. The diagnosis of IHH was based on failure to undergo spontaneous puberty before 18 yr of age and was confirmed by decreased serum T concentrations below the normal range for adults, FSH and LH levels within or below the normal range, absence of a pituitary or hypothalamic mass lesion on computed tomography or magnetic resonance imaging, presence of a gonadotropin response to repetitive doses of GnRH, normal smell test, and normal karyotypes (46,XY).
None of the patients had hyposomia, anosomia, or a family history of IHH. All patients had scrotal testes. All controls had normal gonadal development, and their physical and biochemical findings were normal. Patients and controls with a history of hormone replacement therapy; diabetes mellitus; hypertension; hyperlipidemia; renal, hepatic, and vascular diseases; malignancies; anemia; hypothyroidism; intake of methotrexate, phenytoin, carbamazepine, NO, theophyline, metformin, colestipol, niacin, penicillamine, thiazide diuretics, vitamin B6, cobalamin, and/or folate supplements; excessive coffee consumption; chronic alcohol intake; and current smoking were excluded. Strenuous physical activity was not allowed before collection of blood samples. All participants were informed about the study and gave their written consent. The study was approved by the ethics committee of Gulhane Military Medical Academy.
Therapy and analysis
Patients with IHH were treated with a single dose im injection of Sustanon 250 (Organon, Oss, The Netherlands) that contained 30 mg T propionate, 60 mg T phenylpropionate, 60 mg T isocaproate, and 100 mg T decanoate.
Fasting blood samples were collected from patients and controls between 0800–0830 h after overnight fasting. Posttreatment blood samples were drawn 10 d after the injection of Sustanon. Blood samples were put on ice. Venous blood samples were centrifuged within less than 60 min. The plasma and serum was stored at –80 C until tHcy, ADMA, L-arginine, and NOx levels could be measured.
Measurement of L-arginine and ADMA was accomplished by HPLC, using the method described by Chen et al. (25). In brief, to 1 ml serum, 20 mg 5-sulfosalisilic acid was added, and the mixture was left in an ice bath for 10 min. The precipitated protein was removed by centrifugation at 2000 x g for 10 min. Ten microliters of the supernatant which was filtered through a 0.2-µm pore size filter was mixed with 100 µl derivatization reagent (prepared by dissolving 10 mg o-phtaldialdehyde in 0.5 ml methanol, 2 ml 0.4 M borate buffer (pH 10.0), and 30 µl 2-mercaptoethanol were added) and then injected into the chromatographic system. Separation of L-arginine and ADMA was achieved with a 150 x 4-mm interior diameter Nova-Pak C18 column with a particle size of 5 µm (Waters, Millipore Corp., Milford, MA) using 50 mM sodium acetate (pH 6.8), methanol, and tetrahydrofurane as mobile phase (A, 82:17:1; B, 22:77:1) at a flow rate of 1.0 ml/min. The areas of peaks detected by fluorescent detector (excitation, 338 nm; emission, 425 nm) were used for quantification. The variability of the method was less than 7%, and the detection limit of the assay was 0.1 µM.
Plasma NOx levels were measured in triplicate after conversion of nitrate to nitrite by nitrate reductase, and nitrite was measured using the Griess reaction as described previously (26) The intra- and interassay coefficients of variation were 3% and 7%, respectively. Recoveries of both nitrites and nitrates in our samples were greater than 95%.
Total plasma cholesterol and triglyceride levels were measured by enzymatic colorimetric method with an Olympus AU 2700 autoanalyzer using reagents from Olympus Diagnostics, GmbH (Hamburg, Germany). Serum creatinine levels were determined using a modified kinetic Jaffe method.
Plasma tHcy concentrations were measured by HPLC (HP Agilent 1100, Agilent Technologies, Palo Alto, CA) (27). Cobalamin and folate levels were measured by RIA with reagents from Diagnostic Product Corp. (Los Angeles, CA). Serum FSH, LH, estradiol (E2), and prolactin concentrations were measured by immunoradiometric assay with reagents from Radim Techland (Angleur, Belgium). Serum total T concentrations were determined by a solid phase 125I RIA with reagents from Diagnostic Product Corp. The free T values were obtained by calculation from T and SHBG values according to the method proposed by Vermeulen et al. (28). Serum total T was determined by RIA with reagents from Diagnostic Systems Laboratories, Inc. (Webster, TX; active testosterone kit). The intra- and interassay coefficients of variation for total T were 8.7% and 10.5%. Serum SHBG levels were measured by RIA with a reagent from Radim Techland. The normal ranges in our laboratory are less than 15 IU/liter for FSH, less than 20 IU/liter for LH, less than 60 pg/ml for E2, 15–45 pg/ml for free T, and 9–38 nmol/liter for SHBG.
Statistical analyses
All of the statistical analyses were performed using the SPSS 11.0 (SPSSFW, SPSS, Inc., Chicago, IL) statistical package. Descriptive statistics were given as the arithmetic mean ± SD. For the pairwise comparisons, we used a paired sample t test when the normality assumptions were held, and Wilcoxon signed rank test otherwise. For comparisons of the values of two different groups, we used an independent sample t test. P 
0.05 was considered statistically significant.
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Results |
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. Pretreatment levels of FSH, LH, E2, and free T were lower in patients than in controls; posttreatment levels of free T were statistically significantly increased, and FSH, LH, and E2 levels were not changed. SHBG was higher than control levels before therapy, whereas it did not change after treatment. Prolactin levels did not differ significantly between groups.
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The pretreatment folate and cobalamin levels were statistically significantly higher in patients compared with controls, and they did not change significantly after treatment. The IHH patients had lower tHcy levels than controls. Plasma fasting tHcy concentrations was not statistically significant after 10 d of treatment.
The pretreatment ADMA and L-arginine levels were significantly higher, and NOx levels were lower than those in the control group. After 10 d of treatment, ADMA and L-arginine levels were significantly reduced, and NOx levels were significantly increased. There was a significant positive correlation (P < 0.01) between ADMA and L-arginine, and a negative correlation between ADMA and NOx levels in patients and controls. There is not a significant, direct relationship between free T levels and plasma ADMA or between free T levels and NOx.
IHH patients exhibited a significant increase in L-arginine compared with both control and posttreatment groups (P < 0.01), whereas L-arginine remained unaltered in the posttreatment group compared with the controls. The pretreatment group also revealed a significant increase in ADMA plasma levels (P < 0.01).
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Discussion |
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The finding of elevated ADMA levels in patients with IHH that are returned to the control group values after acute T treatment shows a possible relationship between T and ADMA metabolism. However, the mechanisms involved in the rise in ADMA concentrations in the present study have not been defined. Plasma dimethylarginines arise mainly from degradation of intracellular methylated proteins and are eliminated via urinary excretion (29) ADMA is also metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (30). At least three possibilities exist for an elevation of plasma ADMA: a decrease in renal filtration, an increased synthesis of ADMA, and a decreased enzymatic hydrolysis. Elevated ADMA, attributable to reduced renal excretion, is unlikely, because creatinine plasma levels in the groups were within normal values.
Several lines of evidence indicate that ADMA is synthesized from the degradation of methylated proteins rather than from the methylation of free arginine. The specific enzyme protein arginine N-methyltransferase (protein methylase I) has been shown to methylate internal arginine residues in a variety of polypeptides. Catabolism of these polypeptides generates ADMA and NG,NG’-dimethyl-L-arginine (29, 31). It could be hypothesized that T would down-regulate protein methylase I activity or decrease dimethylarginine dimethylaminohydrolase activity, offering a putative mechanism for elevated ADMA levels associated with hypogonadism. Moreover, in a recent study elevation of T levels in hypogonadal men, whether endogenously by Leydig cell stimulation or exogenously by injection of T, caused endothelium-dependent vasoreactivity to decrease into the range found in eugonadal men (32).
The clinical relevance of these findings has to be judged from the general perspective of other known effects of T on cardiovascular risk factors. In men, the influence of androgens on body composition and insulin resistance and the modulation of factors involved in clotting and fibrinolysis have to be considered beneficial. The decreasing effect of androgen substitution on high density lipoprotein cholesterol has, at least theoretically (8), to be regarded as adverse, although clinical evidence is lacking. As demonstrated in this trial, elevated ADMA levels lowered by parenteral T administration in hypogonadal men may contribute to the diminished endothelial function that was previously reported (32).
Gyurko et al. (33) have shown that NO generated by neuronal NOS is necessary for normal reproductive function in the mouse. Furthermore, in the male, NO is required for normal mating behavior, FSH secretion, and testicular development. Because NOS is also expressed in the gonads, we considered the possibility that ADMA may cause local NOS inhibition that is responsible for the infertility.
We have also shown that the plasma fasting tHcy concentrations in patients with IHH were lower in pre- and posttreatment groups than in the control group. Short-term T therapy had no effect on plasma tHcy levels. However, it was previously reported that long-term T treatment increases tHcy values (34).
In conclusion, these data show that an elevation of plasma ADMA levels in patients with IHH associated with a reduction in NO production may contribute to some cardiovascular alterations. Single dose parenteral T administration lowered ADMA concentrations and increased NO production to the control group values. Additional studies will be required to understand the mechanisms responsible for the observed effects and their consequences.
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Footnotes |
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Abbreviations: ADMA, Asymmetric NG,NG-dimethyl-L-arginine; E2, estradiol; IHH, idiopathic hypogonadotropic hypogonadism; NO, nitric oxide; NOx, plasma nitrite plus nitrate; T, testosterone; tHcy, total homocysteine.
Received October 15, 2004.
Accepted December 13, 2004.
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