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Original Article |
Departamentos de Fisiología (C.H., P.M., M.P., G.S., J.M.V., S.L.) and Cirugía (J.O.), Universidad de Valencia and Research Unit (C.H.), Hospital Clínico Universitario de Valencia, E-46010 Valencia, Spain
Address all correspondence and requests for reprints to: Dr. Pascual Medina, Departamento de Fisiología, Facultad de Medicina y Odontología, Blasco Ibáñez 17, 46010 Valencia, Spain. E-mail: medinap{at}post.uv.es.
Abstract
Cardiovascular manifestations are frequent findings in patients with thyroid hormone disorders. Nitric oxide (NO) plays a key role in vascular, endothelial-mediated relaxation. NO is synthesized from L-arginine by NO synthase, an enzyme inhibited by endogenous compounds, mainly asymmetric dimethylarginine [asymmetric NG,NG-dimethyl-L-arginine (ADMA)]. The aim of our work was to investigate whether plasma L-arginine and dimethylarginine concentrations and NO production are altered in hypo- and hyperthyroid patients, compared with control subjects. L-arginine, ADMA and symmetric dimethylarginine were analyzed by HPLC. NO was measured as plasma nitrite plus nitrate (NOx) concentration by a colorimetric method based on Griess reagent. L-arginine, ADMA, and symmetric dimethylarginine plasma levels in the hypothyroid group were similar to those of the control group; whereas in hyperthyroidism, these values were significantly increased. However, the L-arginine/ADMA ratio was decreased in hyperthyroid patients, resulting in diminished NOx production. When all subjects were analyzed together, free T4 levels were directly correlated with ADMA and inversely correlated with NOx.
THYROID HORMONES HAVE multiple effects on the cardiovascular system, exerted through both direct and indirect mechanisms of action (1, 2, 3). Patients with thyroid disease, especially those with hyperthyroidism, often have clinical manifestations suggesting changes in cardiovascular hemodynamics (3).
The mechanisms by which thyroid hormones affect vascular physiology are largely unknown. It has been shown that thyroid hormones cause rapid relaxation of vascular smooth muscle cells isolated from rat (4) and produce a fall in systemic vascular resistance in patients after coronary artery bypass surgery (5). Nevertheless, few data are available regarding the effects of thyroid hormones on endothelial function. In experimental animals, there are many conflicting reports. For instance, it has been reported that aorta obtained from hyperthyroid rats had less relaxation response to acetylcholine (6), while others have reported increased relaxation responses to acetylcholine (7). A recent report demonstrated that forearm resistance vessel relaxation response to acetylcholine is increased in hyperthyroid patients, suggesting that endothelium is a specific target of thyroid hormones (8).
Nitric oxide (NO), synthesized from L-arginine, accounts for the powerful vasodilator effects of endothelium-derived relaxing factor (9, 10) and consequently plays a decisive role in determining vasomotor tone (11, 12). 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 (13), the enzyme responsible for the formation of NO from L-arginine. The closely related compound symmetric NG,NG’-dimethyl-L-arginine (SDMA), 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 (14), congestive heart failure, preeclampsia, peripheral arterial occlusive disease, and hypertension (15, 16).
The purpose of the present study was to evaluate plasma L-arginine and dimethylarginine concentrations and their relationship to NO production, measured as plasma nitrite-plus-nitrate (NOx) concentration, in patients with hyperthyroidism or hypothyroidism, compared with control subjects.
Subjects and Methods
Subjects
Patients with either hyperthyroidism or hypothyroidism were included in the study. The diagnoses were based on basal plasma TSH values less than 0.3 mIU/liter and greater than 15 mIU/liter, respectively, and were ascertained by determination of free T4.
Thirty-one outpatients with no past history or current symptoms of coronary or peripheral arterial disease were recruited from the Outpatient Central Service at Hospital Clínico Universitario of Valencia. Nineteen patients with hyperthyroidism and 12 with hypothyroidism were enrolled. Causes of hyperthyroidism were Graves’ disease (n = 14) and toxic multinodular goiter (n = 5). Causes of hypothyroidism were chronic autoimmune thyroiditis (n = 9), thyroidectomy (n = 2), and 131I therapy (n = 1). The control group consisted of a population of 16 healthy hospital employees who undergo a routine visit at the preventive healthy service on the same days as the patients. Informed consent was obtained from each participant, and the study protocol was approved by the ethics committee of our institution.
Analytical methods
Blood sampling. Fasting blood samples (10 ml) were drawn into vacuum tubes containing EDTA. Plasma was prepared by centrifugation within 60 min and was stored at -80 C until analysis. A routine analysis of blood samples was performed, using enzymatic methods, by an autoanalyzer. Free T4 and TSH were measured by enzyme immunoassay kits free [Biotrol Diagnostic, Chennevières-lès-Louvres, France; free T4 (fT4) and TSH , respectively]. The intraassay and interassay coefficients of variation for these methods were less than 10%.
Isolation and measurement of dimethylarginines. Measurement of L-arginine, ADMA, and SDMA was accomplished by HPLC, using a modification of the method described by Vallance et al. (13). In brief, dimethylarginines from 1 ml plasma were purified with Bond Elut SCX columns (Varian, Inc., Palo Alto, CA) and eluted with 4 ml methanol containing 30% distilled water and 2% triethylamine. The eluent was then evaporated to dryness at 60 C, and the dried extract was redissolved in running buffer. HPLC was carried out on a Shimadzu chromatography system (Shimadzu Corporation, Kyoto, Japan). Separation of dimethylarginines was achieved with a 250 x 4.6-mm (inner diameter), 5-µm Kromasil C18 analytical column (Scharlau, Barcelona, Spain) using 25 mM phosphoric acid containing 10 mM hexane sulfonic acid and 1% (vol/vol) acetonitrile (pH 5.0). The analysis was carried out at a flow rate of 1.3 ml/min, and the absorbance was monitored at 200 nm. Concentrations of L-arginine, ADMA, and SDMA in the samples were determined by comparison with standards. The variability of the method was less than 7%, and the detection limit of the assay was 0.1 µM.
Determination of NOx. NO is rapidly converted to nitrite and nitrate in human plasma. In our study, plasma NOx levels were measured in triplicate after conversion of nitrate to nitrite by nitrate reductase, and nitrite was measured by using the Griess reaction, as described previously (17). 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%.
Materials
Phosphoric acid, hexane sulfonic acid, and acetonitrile were purchased from Scharlau. All other materials were purchased from Sigma (St. Louis, MO).
Statistical analysis
Values shown in the text, tables, and figures are means ± SEM. Repeated-measures ANOVA and Student t test were applied for comparisons of means of study groups. P values 
0.05 for a two-sided test were considered significant. Regression analyses were performed and Pearson correlation coefficients (r) were calculated to quantify the relationships between members of each pair of the following plasma parameters: T4, ADMA, and NOx. The statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL), version 10.0 for Windows.
Results
The composition of the three study groups, presented in Table 1
, was standardized according to the demographic characteristics of the subjects. The median age (54 yr) of hypothyroid patients was 9 yr higher than that of hyperthyroid patients, although the difference was not statistically significant (P = 0.072). Women were overrepresented in both groups of patients (75.0% in the hypothyroid group and 73.7% in the hyperthyroid group); and therefore, the control group was gender-matched by the inclusion of more control women (68.8%) than men. Body mass index was slightly greater in hypothyroidism, as expected, but there were no statistically significant differences between groups. Systolic blood pressure and heart rate were significantly higher in hyperthyroid patients. Diastolic blood pressure was not significantly different in the three groups. Creatinine levels of the three groups remained within normal values. Total cholesterol levels in the hypothyroid group were higher than in control and hyperthyroid groups. That increment was attributable to a significant increase in low-density lipoprotein cholesterol in hypothyroidism, compared with both control and hyperthyroidism (P < 0.05), given that high-density lipoprotein cholesterol levels were indistinguishable in the three groups. All other measured biochemical parameters (blood glucose, urea, uric acid, and triglycerides) did not reveal significant differences.
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. L-arginine remained unaltered in hypothyroidism, compared with controls; whereas hyperthyroid patients exhibited a significant increase of L-arginine when compared with both control and hypothyroid groups (P < 0.01). The hyperthyroid group also revealed a significant increase of ADMA and SDMA plasma levels. ADMA concentration in hyperthyroidism was more than twice that measured in the control group and hypothyroid group (1.30 ± 0.12 µM vs. 0.58 ± 0.06 and 0.57 ± 0.07, respectively; P < 0.01). SDMA levels of hyperthyroid patients were also significantly higher than in control subjects (0.54 ± 0.07 µM vs. 0.27 ± 0.04, respectively; P < 0.01). The L-arginine/ADMA ratio in hyperthyroid patients was 116 ± 10, significantly lower than the values obtained in control subjects and in hypothyroid patients (190 ± 14 and 189 ± 28, respectively; P < 0.01).
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To determine the relationship among thyroid status, ADMA, and NO production, we plotted the individual values of ADMA and NOx against free T4 values. The results in Fig. 2 illustrate the direct relationship that exists between free T4 and plasma ADMA concentration, T4 levels being predictive of ADMA concentration (r = 0.60; P < 0.001). In a similar way, free T4 values demonstrated a good, inverse correlation with NO production (r = 0.31; P < 0.05). As expected, an inverse correlation existed between plasma ADMA and NO levels, NO concentration being related to ADMA levels by the following formula: NO concentration = -14.69 ADMA + 60.09 (r = 0.36; P < 0.005) (data not shown).
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The major findings of the present study are that: 1) L-arginine, ADMA, and SDMA levels are increased, whereas NOx levels are decreased in plasma from hyperthyroid patients; 2) the hypothyroid group does not reveal any difference in L-arginine, dimethylarginines, or NOx concentrations, compared with the control group; 3) there is a significant, direct relationship between free T4 levels and plasma ADMA; and 4) there is also a significant, but inverse, relationship between free T4 levels and NOx.
Plasma concentrations of methylarginines and L-arginine/ADMA ratios obtained in our control subjects remained within previous reported ranges (18, 19). Our results showed that the hypothyroid group had methylarginine levels similar to those of the control group, thus resulting in the same levels of NOx.
The increase in methylarginines observed in hyperthyroid patients was moderate (2-fold elevation in ADMA), even though the values are similar to those in individuals with other known vascular risk factors (19, 20, 21). Hyperthyroidism has been associated with tachycardia, systolic hypertension, atrial fibrillation, heart failure, and evidence of increased probability of cardiovascular and cerebrovascular mortality (3, 22). Clinical studies revealed that endothelial dysfunction seems to be the possible cause of such complications (23). In the present study, NOx levels were significantly lower in hyperthyroidism than in control subjects. Moreover, there was a significant, direct relationship between plasma T4 and ADMA levels and also a significant, but inverse, relationship between T4 and NOx. Taken together, a decreased L-arginine/ADMA ratio, along with diminished plasma NOx concentration, might reflect an impaired L-arginine-NO pathway in hyperthyroidism that could be related to some of its cardiovascular alterations.
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 (24). ADMA is also metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (25). Then, at least three possibilities exist for an elevation of plasma ADMA: a decrease in renal filtration, an increased synthesis of ADMA, or 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 NG-monomethyl-L-arginine, ADMA, and SDMA (24, 26). Thyroid hormone up-regulates protein methylase I activity (27), offering a putative mechanisms for elevated ADMA levels associated with hyperthyroidism. Also, it could be hypothesized that hyperthyroidism would decrease DDHA activity through increased production of oxygen free radicals and increased lipid peroxidation (28).
Caution should be taken to interpret the pathophysiological consequences of our results. The decreased systemic vascular resistance observed in patients with hyperthyroidism is attributable to the well-known action of thyroid hormone to increase peripheral oxygen consumption and to a direct effect on vascular smooth muscle (4, 29). The role played by ADMA and NO plasma levels in the changes in peripheral vascular resistance in hyperthyroidism are still elusive. It has been shown that ADMA and other arginine analogs increase the tone of peripheral vessels by inhibiting the basal release of NO from the endothelium (30). In addition, inhibition of NO synthesis enhances vascular contractile responses induced by adrenergic agonists or sympathetic stimulation (31, 32). Therefore, it is conceivable that an increase in ADMA in hyperthyroidism might represent a compensatory mechanism to decrease NO production and, consequently, to counterbalance excessive peripheral vasodilatation. It is also possible that only a high serum triiodothyronine concentration is the specific stimulus to trigger an increase formation of ADMA. If this were the case, the vascular changes typical of hypothyroidism (mild hypertension and increased systemic vascular resistance) would seem to be unrelated to the L-arginine-NO system.
In summary, the present study demonstrated an elevation of ADMA plasma levels of patients with hyperthyroidism associated with a reduction in NO production, which may contribute to some cardiovascular alterations. Hypothyroid patients did not display any change in these parameters. Further studies will be required to understand the mechanisms responsible for the observed effects and their consequences.
Acknowledgments
Footnotes
This work was supported by Grant 01/0917 from the Fondo de Investigación Sanitaria and Grant GV01-69 from the Generalitat Valenciana and Comisión Interministerial de Ciencia y Tecnología. G.S. was the recipient of a Fellowship of the Instituto de Salud Carlos III (99/9016).
Abbreviations: ADMA, Asymmetric NG,NG-dimethyl-L-arginine; NO, nitric oxide; NOx, NO measured as plasma nitrite plus nitrate concentration; r, correlation coefficient(s); SDMA, symmetric dimethylarginine.
Received June 10, 2002.
Accepted August 29, 2002.
References
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, P < 0.05 vs. control values.