TSH, FT3 and FT4 were not associated with changes in body composition in HIV-infected patients on combined antiretroviral therapy.

Mestrado Integrado em Medicina Área: Endocrinologia Trabalho efetuado sob a Orientação de: Dra. Paula Freitas Trabalho organizado de acordo com as normas da revista BMC Infectious Diseases Rita Sofia Bettencourt Silva TSH, FT3 and FT4 were not associated with changes in body composition in HIV-infected patients on combined antiretroviral therapy março, 2012 DEDICATÓRIA Aos meus pais, pelo amor incondicional, suporte emocional e liberdade para fazer as minhas escolhas; À Beatriz, minha irmã, confidente e companheira de casa, com quem partilho diariamente todos os bons e maus momentos; À Maria João Camões e Raquel Marçôa, pela amizade que nos une e pelo apoio prestado durante o desenvolvimento deste trabalho; Ao meu tio Luís, pelos conhecimentos que me transmitiu e pelo seu incentivo para me superar todos os dias. 1 RESUMO INTRODUÇÃO: Os doentes infetados pelo vírus da imunodeficiência adquirida (VIH) em terapêutica antirretrovírica combinada (TARC) podem ter distúrbios metabólicos, lipodistrofia e disfunção tiroideia. Tem sido investigado o impacto potencial de alterações minor da função tiroideia na composição corporal em doente eutiroideus não infetados pelo VIH, mas não na população infetada pelo VIH. Os objetivos deste estudo foram comparar os níveis de TSH, T3L e T4L nos doentes eutiroideus com e sem lipodistrofia [definida pela clínica e pela razão massa gorda (RMG)] e em quatro grupos de composição corporal; e avaliar a possível relação entre a TSH, T3L e T4L e parâmetros demográficos, antropométricos, infeciosos e metabólicos. MÉTODOS: Estudo transversal incluindo 352 adultos caucasianos não institucionalizados infetados pelo VIH-1 sob TARC. Foram avaliados dados demográficos, caracterização da infeção VIH, função tiroideia, características antropométricas, composição corporal por DXA e parâmetros metabólicos. RESULTADOS: Não houve diferenças significativas entre a mediana de TSH, T3L e T4L de acordo com a presença de lipodistrofia (definida pela clínica e pela RMG) e os quatro grupos de composição corporal. A TSH estava positivamente correlacionada com a idade e negativamente com a contagem de células CD4. Na análise multivariada, depois de ajustar para a idade, género e índice de massa corporal (IMC), a TSH permaneceu positivamente associada com a idade e negativamente com a contagem de células CD4. A T3L estava positivamente correlacionada com a duração da infeção por VIH e negativamente com a idade, pressão arterial sistólica (PAS) e pressão arterial diastólica (PAD). Na análise multivariada, não houve associações significativas com a T3L. A T4L estava positivamente correlacionada com a duração da infeção e negativamente com a PAD, colesterol total e C-LDL. Na análise univariada, a FT4 estava negativamente associada com o IMC, colesterol total e C-LDL, mas no modelo de regressão linear múltiplo apenas a associação com o LDL-C permaneceu significativa. CONCLUSÕES: Não houve diferenças nos valores de TSH, T3L e T4L de acordo com a composição corporal em doentes eutiroideus infetados pelo VIH. Os níveis de TSH estavam associados positivamente com a idade e negativamente com a contagem de células CD4, mesmo na análise multivariada. Os doentes eutiroideus podem ter anomalias lipídicas e pressão sanguínea elevada. PALAVRAS-CHAVE: VIH, terapêutica antirretrovírica, tiroide, lipodistrofia, composição corporal, razão massa gorda, pressão sanguínea, dislipidemia. 2 TITLE TSH, FT3 and FT4 were not associated with changes in body composition in HIVinfected patients on combined antiretroviral therapy Author Rita Bettencourt Silva Affiliation Sixth year student of the Integrated Master Degree in Medicine, Faculty of Medicine of Porto University, Porto, Portugal Correspondence Rita Bettencourt Silva Department of Endocrinology, Diabetes and Metabolism, Centro Hospitalar São João Alameda Professor Hernâni Monteiro 4200-319 Porto Portugal Phone: +351 91 939 40 81 E-mail: [email protected] 3 ABSTRACT BACKGROUND: Human immunodeficiency virus (HIV)-infected patients on combined antiretroviral therapy (cART) may have metabolic disorders, lipodystrophy and thyroid dysfunction. It has been investigated the potential impact of minor changes in thyroid function on body composition of euthyroid HIV non-infected patients, but not in HIV-infected population. The aims of this study were to compare TSH, FT3 and FT4 levels in euthyroid patients with and without lipodystrophy [defined by clinical and by fat mass ratio (FMR)] and in four groups of body composition; and to assess the possible relationship between TSH, FT3 and FT4 and demographics, anthropometrics, infectious and metabolic parameters. METHODS: Cross-sectional study including 352 HIV-1-infected non-institutionalized Caucasian adults on cART. Demographic data, HIV infection characterization, thyroid function, anthropometric features, body composition by DXA and metabolic parameters were evaluated. RESULTS: There were no significant differences between the median of TSH, FT3 and FT4 according to the presence of lipodystrophy (defined by clinical and by FMR) and the four groups of body fat composition. TSH was positively correlated with age and negatively with CD4 cell count. In multivariate analysis, after adjustment for age, gender and body mass index (BMI), TSH remained positively associated with age and negatively associated with CD4 cell count. FT3 was positively correlated with duration of HIV infection and negatively with age, systolic blood pressure (SBP) and diastolic blood pressure (DBP). In multivariate analysis, there were no significant associations with FT3. FT4 was positively correlated with duration of infection and negatively with DBP, total cholesterol and LDL-C. In univariate analysis, FT4 was negatively associated with BMI, total cholesterol and LDL-C, however in the multiple linear regression model (with age, gender and BMI as covariates) only the association with LDL-C remained significant. CONCLUSIONS: There were no differences in TSH, FT4 and FT3 values according to body composition in euthyroid HIV-infected patients. TSH levels were positively associated with age and negatively with CD4 cell count, even in multivariate analysis. Euthyroid patients can have lipid abnormalities and raised blood pressure. KEYWORDS: HIV, antiretroviral therapy, thyroid, lipodystrophy, body composition, fat mass ratio, blood pressure, dyslipidemia. 4 BACKGROUND The acquired immune deficiency syndrome (AIDS), primarily reported in 1981, became a leading cause of mortality worldwide [1]. With the successful introduction of combined antiretroviral therapy (cART) in 1996, human immunodeficiency virus (HIV)-related morbidity and mortality have declined substantially [1, 2]. However, and due to an increased survival, there are more and more people living with this infection. At the end of 2010 it was estimated that nearly 34 million adults and children were living with HIV infection worldwide [2]. The use of cART has been associated with many side effects, including lipodystrophy, dyslipidemia, insulin resistance, increased blood pressure, decreased bone mineral density and dysfunction of the adrenal, gonadal and thyroidal axes [3-5]. Önen suggested that these comorbidities are not part of the “normal” aging process [4]. However, the mechanisms responsible for cART-related metabolic disorders are not fully understood and there is a growing recognition that inflammatory cytokines and HIV infection by itself can be the etiologic agents of these metabolic abnormalities [5, 6]. Lipodystrophy is characterized by selective loss of adipose tissue from particular anatomical regions and can be localized or generalized [7]. Acquired lipodystrophies occur more frequently than the inherited types and the most prevalent subtype in the clinic is acquired partial lipodystrophy related to cART in HIV-infected patients [7, 8]. With the beginning of molecular genetic era, congenital and familial types were associated with mutant gene products and not only characterized by clinical features. However, the mechanisms responsible for the relationship between HIV and lipodystrophy remain partially unknown [7]. HIV-associated lipodystrophy syndrome is characterized by loss of subcutaneous fat (lipoatrophy) from the face and limbs with or without deposition of excess fat (lipohypertrophy) in the neck and upper back (causing a double chin and a buffalo hump), abdomen and trunk, resulting in peripheral fat wasting and central adiposity [7-9]. Some patients have peripheral lipoatrophy, others have isolated abdominal prominence and a substantial portion has mixed forms including both phenotypes [9]. HIV-associated lipodystrophy has been linked to components of metabolic syndrome (MS), such as high blood pressure, dyslipidemia and insulin resistance, leading to an increased cardiovascular disease risk [3, 7]. The relationship between cART and fat redistribution is associated with prolonged duration of treatment and is highly linked to the use of protease inhibitors (PI) drugs, 5 although it has been also related to other antiretroviral drugs [3, 8, 9]. In his clinical review, Chen reported that prevalence of lipodystrophy among HIV-infected patients was highly variable according to different authors, ranging between 8% and 84% (due to different definition and diagnosis criteria). He noticed that near 40% of patients treated with PI developed lipodystrophy [8]. Thyroid hormones regulate the metabolism and function of many tissues, such as liver, heart, skin, muscle and adipose tissue. They are involved via thyroid hormone receptors (TRs) in adipogenesis and adipose tissue lipogenesis and lipolysis [10, 11]. All the isoforms of TRs (both TRA1 and TRB1) are expressed in white adipose tissue [12]. Moreover, the monocarboxylate transporter 8 (MCT8) is a thyroid-hormone-specific transporter that is ubiquitously expressed, including in human subcutaneous adipose tissue (SAT) [12]. Overt hypothyroidism is clearly associated with body weight excess and obesity, but the potential impact of minor changes in thyroid function on anthropometric measures of euthyroid HIV non-infected patients remains under investigation [13-16]. So far there are still no studies exploring this relationship among HIV-infected patients. Due to the important role of TH on adipocyte metabolism and as HIV-associated lipodystrophy results from changes in adipose tissue, we hypothesized that minor changes in thyrotropin (TSH), free 3,5,3’-triiodothyronine (FT3) and free thyroxine (FT4) may have a significant role in the peculiar body fat redistribution of these patients. The aims of the current study were (1) to compare the levels of TSH, FT3 and FT4 in euthyroid patients with and without lipodystrophy [(defined by clinical and by fat mass ratio (FMR)] and in four different groups of body composition (no lipodystrophy; isolated central fat accumulation; isolated lipoatrophy; and mixed forms of lipodystrophy); (2) and to assess the possible relationship between TSH, FT3 and FT4 and demographic, anthropometric, infectious and metabolic parameters. 12 partial lipodystrophy (Dunnigan variety) reported that thigh SAT from lipodystrophic patients had lower MCT8 and higher type 2 iodothyronine deiodinase (DIO2) mRNA expression than thigh and abdominal SAT from controls. Then, it was suggested that changes in local thyroid hormones availability occur in lipoatrophic areas [28]. Interestingly in our sample there was no statistically significant correlation between TSH and thyroid hormone levels (both FT3 and FT4). The individual changes on feedback mechanism of pituitary-thyroid axis and the pulsatile TSH secretion pattern can explain this finding [33]. Furthermore, it was suggested that genetic changes can affect the thyrotrophs sensitivity to the feedback regulation by thyroid hormones [34]. Therefore, FT4 and especially FT3 (the active cellular form of thyroid hormone) values may better reflect the physiological actions of thyroid function in different tissues. TSH levels were positively correlated with age, while FT3 levels were negatively correlated with age. In multivariate analysis, there was a statistically significant positive association between TSH and age, regardless the gender and BMI. In previous studies involving both HIV-infected and uninfected populations, the results are controversial. Different authors had reported an inverse association between age and TSH [35], a positive association in both genders [14, 36] and also a positive correlation between TSH and age only in women [33]. In our euthyroid sample, it was found a statistically significant negative correlation between TSH (but not thyroid hormones) and CD4 cell count. Also in multivariate analysis, after adjustment for age, gender and BMI, TSH levels remained negatively associated with CD4 cell count. This relationship with immune system was also described by other authors in HIV-infected population. Madeddu observed a negative correlation between TSH and CD4 cells nadir in HIV-infected patients on cART [26]. On the other hand, Collazos found a positive correlation between FT4 and CD4 cell count [37] and Beltran showed that low CD4 cell count was a risk factor for hypothyroidism [6]. This relationship is likely to be mediated by proinflammatory cytokines as tumor necrosis factor alpha (TNF-α), which has been found increased in HIV patients both before and during therapy and has receptors in thyroid follicular cells [26, 38]. In our study, FT3 and FT4 levels had a statistically significant positive correlation with the duration of HIV infection. However, in univariate and multivariate analysis, there was a lack of association between there parameters. There were no associations of thyroid function 13 regarding to the cART duration, which is not concordant with the positive correlation between TSH value and therapy duration described by Madeddu [26]. The influence of thyroid function on blood pressure homeostasis has been evaluated during the last years. Both overt and subclinical hypothyroidism are often associated with arterial hypertension (from 10 to 25% has diastolic hypertension), due to an increased systemic vascular resistance, increased arterial stiffness and changes in sodium homeostasis [39-41]. On the other hand, hyperthyroidism is a secondary cause of isolated systolic hypertension because FT3 reduces peripheral vascular resistance, increases heart rate and consequently raises cardiac output, leading to a widened pulse pressure [39, 40]. Even in euthyroid patients, thyroid function has been associated with blood pressure homeostasis [4145], but unlike this sample previous studies only included HIV non-infected patients. In our sample, TSH, FT3 and FT4 levels didn’t differ significantly according to the presence of hypertension (only 38.2% had hypertension), but FT3 levels tended to be lower among hypertensive patients [p = 0.051 (data not shown)]. Some authors showed that euthyroid hypertensive patients had higher TSH levels [41-43]. Gumieniak reported that lower FT4 index (an estimative for FT4 values, resulting from the product of T4 total by the thyroid hormone binding ratio) and higher TSH levels within the normal range were linked to hypertension, independently of age, gender, BMI, race, smoking status and insulin sensibility. In his study, the lower FT4 index was an independent predictor of blood pressure salt-sensitivity, one of the key elements in the pathogenesis of hypertension [41]. In our study, it was only able to establish statistically significant correlations between thyroid hormones (but not TSH) and blood pressure. Serum FT3 was negatively correlated with both SBP and DBP and serum FT4 was negatively correlated only with DBP. It has been showed that FT3 and FT4 are direct vasodilator agents that promote relaxation of vascular smooth-muscle cells and of skeletal muscle resistance arterioles, with consequent decrease of systemic vascular resistance [40, 46], which justifies these results. However, in multiple linear regression model (with age, gender and BMI as covariates) there were no associations between FT3, FT4 and blood pressure. It was an expected result because age and BMI are positively associated with blood pressure [43]. Some studies have showed a positive association between TSH within the reference range and SBP and DBP, considering both hypertensive and normotensive subjects [42-44]. Iqbal observed a linear rise in DBP and also in SBP (but only in females) with the increase of normal range TSH quartile, 14 independently of age, BMI and smoking status [43]. In a cross-sectional study, Saltiki reported a positive correlation between TSH and DPB (not SBP), but not in multivariate analysis [44]. On the other hand, controversial studies didn’t show relationship between blood pressure and thyroid function at the normal range [47, 48]. Gumieniak observed an excessive aggregation of high-normal TSH values in hypertensive families and a higher serum TSH levels in healthy subjects with family history of hypertension compared with those with negative family history. He suggested the existence of genetic variants affecting both blood pressure regulation and TSH levels [45]. Looking for BMI, patients with isolated central fat accumulation and with mixed forms of lipodystrophy had a significant higher mean of BMI. As expected, leptin mean levels were higher in patients with central fat accumulation and mixed forms, since leptin concentration is proportional to adipocyte mass [49]. In our sample, thyroid function into normal range didn’t differ according to BMI and there was no significant correlation between these two parameters. It was only observed a negative association between BMI and FT4 in univariate (but not multivariate) analysis. In euthyroid HIV noninfected patients, previous studies found an association between BMI and changes in thyroid function, but this relationship could differ between lower grades of overweight and morbid obesity. Asvold e Nyrnes reported a positive association between BMI and TSH among men and women [13, 14], while Makepeace showed a significant inverse relationship between FT4 concentration and BMI in euthyroid subjects [15]. Michalaki showed that euthyroid morbidity obese subjects have raised T3, FT3, T4 and TSH levels compared with those not overweight [16]. This relationship seems to be changed by smoking status, but it is still controversial [13-15]. Indeed, some studies didn’t found any association between thyroid function and BMI [34, 50]. The relationship between obesity and lipodystrophy in HIV-infected patients was already studied previously [19]. In our population, FT4 levels were negatively correlated with total cholesterol and LDL-C. In univariate analysis there was also found a positive association between FT4 levels and total cholesterol and LDL-C. However in the multiple linear regression model (with age, gender and BMI as covariates) only the association between FT4 and LDL-C remained statistically significant. There was no association between thyroid function and HDL-C and TG. Overt and subclinical hypothyroidism is associated with dyslipidemia [39]. According to previous studies including HIV non-infected patients, TSH within the reference range were positively associated with total cholesterol and LDL-C and 15 negatively with HDL-C [51]. Indeed, a large population-based study including 30000 subjects found that TSH levels were positively correlated with total cholesterol, LDL-C and TG and negatively correlated with HDL-C [52]. This association could be modified by the age, insulin sensitivity, smoking status and time since the last meal [51, 52]. As mentioned previously, this was the first study performed on HIV-infected patients to investigate the association between lipodystrophy and thyroid function at the normal range. One of the strengths of our study was the absence of inter-observer variability because all measurements were taken by the same practitioner. Also there was a careful adjustment for the main confounding factors as age, gender and BMI. However the present study has some limitations. There is a possible selection bias since all patients included were referred by the Infectious Disease specialist specifically for lipodystrophy or metabolic disorders related to cART. It cannot be discarded the influence of pre-HIV body composition, the cumulative exposure of each drug and the nadir value of CD4 cells, factors that could contribute to the risk of lipoatrophy or abdominal prominence that weren’t evaluated. Due to a cross-sectional study, it was only able to establish associations and correlations, but not determine causality or risk factors between thyroid function and other variables. Finally, the studied sample was relatively small, there was homogeneity of age strata compared to other studies (only 15.5% of our sample were 60 years or older) and a lack of comparison with a seronegative control group. Therefore, further prospective larger studies are needed, starting before cART and with a controlled follow-up, to find additional associations and to determine causality between thyroid function and metabolic disorders related to cART. 16 CONCLUSION TSH levels were positively associated with age and negatively with CD4 cell count, even in multivariate analysis. Despite of exclusion of patients with thyroid abnormalities from analysis, these results raised the interesting possibility that even in the normal range of thyroid function patients can have lipid abnormalities and raised blood pressure. So, thyroid function must be considered as a continuum spectrum from the euthyroid status to subclinical and overt thyroid dysfunction. Our data suggest that thyroid function doesn’t seem to be involved in body fat redistribution among HIV-infected patients at least at global serum level, but we do not discard that changes in local thyroid hormones availability occur in lipoatrophic or lipohypertrophic areas. However further studies are required at this issue. 17 LIST OF ABBREVIATIONS AIDS - acquired immune deficiency syndrome; BMI - body mass index; cART - combined antiretroviral therapy; CDC - Centers for Disease Control and Prevention; CL - clinical lipodystrophy; DIO2 - type 2 iodothyronine deiodinase; DXA - dual-energy X-ray absorptiometry; FMR - fat mass ratio; FT3 - free 3,5,3’-triiodothyronine; FT4 - free thyroxine; HIV - human immunodeficiency virus; IDF - International Diabetes Federation; IQR - interquartile range; LDL-C - low density lipoprotein cholesterol; HDL-C - high-density lipoprotein cholesterol; MCT8 - monocarboxylate transporter 8; MS - metabolic syndrome; NRTI - nucleoside reverse transcriptase inhibitors; NNRTI - non-nucleoside reverse transcriptase inhibitors; PI - protease inhibitors; REE - resting energy expenditure; SAT - subcutaneous adipose tissue; SD - standard deviation; TG - triglycerides; TNF-α - tumor necrosis factor alpha; TRs - thyroid hormone receptors; TSH - thyrotropin. 18 COMPETING INTERESTS The author declares that she has no competing interests. ACKNOWLEDGMENTS The author would like to sincerely thank M.D. Paula Freitas, for her excellent guidance throughout this project, with all her invaluable ideas and suggestions. Her dedication and encouragement were a strong motivation. The author also expresses her gratitude to Professor Ana Cristina Santos for her continuous support in the statistical analysis, which made this project possible, and for her critical review of this manuscript. The most sincere thank to Professor Davide Carvalho for the opportunity to develop this study in Department of Endocrinology, Diabetes and Metabolism of Centro Hospitalar São João. Finally, the author is grateful to Professor António Sarmento for the possibility to study patients who were referred from Infectious Diseases Outpatient Clinic, and to M.D. Jorge Pereira for proceeding DXA in Department of Nuclear Medicine. 19 REFERENCES 1. HIV surveillance--United States, 1981-2008. 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Bonnet E, Delpierre C, Sommet A, Marion-Latard F, Herve R, Aquilina C, Labau E, Obadia M, Marchou B, Massip P et al: Total body composition by DXA of 241 HIV-negative men and 162 HIV-infected men: proposal of reference values for defining lipodystrophy. Journal of Clinical Densitometry : the official journal of the International Society for Clinical Densitometry 2005, 8(3):287-292. 25. Hoffmann CJ, Brown TT: Thyroid function abnormalities in HIV-infected patients. Clinical Infectious Diseases : an official publication of the Infectious Diseases Society of America 2007, 45(4):488-494. 26. Madeddu G, Spanu A, Chessa F, Calia GM, Lovigu C, Solinas P, Mannazzu M, Falchi A, Mura MS, Madeddu G: Thyroid function in human immunodeficiency virus patients treated with highly active antiretroviral therapy (HAART): a longitudinal study. Clinical Endocrinology 2006, 64(4):375-383. 28 Table 3: Thyroid function according to the four groups of body fat distribution. Lipodystrophy classified by the four groups of body fat distribution No lipodystrophy Isolated central fat accumulation Isolated lipoatrophy Mixed forms of lipodystrophy p TSH [mUI/mL, median (IQR)] 1.40 (1.09) 1.52 (1.50) 1.51 (0.94) 1.71 (1.21) 0.294 FT3 [pg/mL, median (IQR)] 2.97 (0.76) 2.98 (0.75) 3.07 (0.73) 2.84 (0.67) 0.126 FT4 [ng/dL, median (IQR)] 0.96 (0.16) 0.94 (0.84) 0.97 (0.24) 0.91 (0.18) 0.223 29 Table 4: Median levels of each parameter of thyroid function according to the presence of metabolic syndrome and its individual features. TSH [mUI/mL, median (IQR)] FT3 [pg/mL, median (IQR)] FT4 [ng/dL, median (IQR)] High blood pressure (mmHg) Absent Present P 1.53 (1.21) 1.53 (1.10) 0.885 3.05 (0.68) 2.85 (0.69) 0.030 0.95 (0.20) 0.92 (0.19) 0.389 High fasting glucose (mg/dL) Absent Present P 1.65 (1.36) 1.51 (1.08) 0.108 2.97 (0.61) 3.01 (0.81) 0.429 0.96 (0.24) 0.94 (0.19) 0.154 Low HDL-C (mg/dL) Absent Present P 1.52 (1.20) 1.60 (1.15) 0.696 3.00 (0.69) 2.96 (0.75) 0.177 0.95 (0.18) 0.94 (0.21) 0.669 High TG (mg/dL) Absent Present P 1.49 (1.22) 1.57 (1.15) 0.613 2.97 (0.69) 3.00 (0.74) 0.788 0.96 (0.19) 0.94 (0.20) 0.680 Waist circumference (cm) Absent Present P 1.54 (1.12) 1.50 (1.20) 0.942 3.04 (0.76) 2.93 (0.71) 0.131 0.96 (0.19) 0.92 (0.20) 0.429 Metabolic syndrome Absent Present P 1.52 (1.25) 1.56 (1.09) 0.845 3.00 (0.68) 2.96 (0.80) 0.270 0.96 (0.21) 0.92 (0.20) 0.092 30 Table 5: Association between TSH levels and demographic and clinical characteristics. β p Adjusted β* p TSH (mUI/mL) Age (years) 0.006 0.008 0.007 0.005 Gender -0.020 0.726 0.0004 0.994 BMI (kg/m2) -0.005 0.391 -0.005 0.408 CD4 (cells/mm3) -0.108 0.005 -0.107 0.006 *-The coefficients were adjusted for age, gender and BMI. 31 Table 6: Association between FT3 levels and demographic and clinical characteristics. β p Adjusted β* p FT3 (pg/mL) Age (years) -0.022 0.424 -0.002 0.162 Gender -0.002 0.001 -0.016 0.572 BMI (kg/m2) -0.003 0.269 -0.002 0.395 SBP (mmHg) -0.001 0.074 -0.001 0.325 DBP (mmHg) -0.001 0.103 -0.001 0.200 Duration of infection (years) 0.024 0.229 0.020 0.330 *-The coefficients were adjusted for age, gender and BMI. 32 Table 7: Association between FT4 levels and demographic and clinical characteristics. β P Adjusted β* p FT4 (ng/dL) Age (years) -0.001 0.575 -0.001 0.745 Gender -0.062 0.096 -0.059 0.122 BMI (kg/m2) -0.008 0.035 -0.007 0.057 DBP (mmHg) -0.002 0.158 -0.002 0.258 Total cholesterol (mg/dL) -0.001 0.018 -0.001 0.064 LDL-C (mg/dL) -0.001 0.002 -0.001 0.006 Duration of infection (years) 0.052 0.059 0.047 0.093 *-The coefficients were adjusted for age, gender and BMI. Anexo I – Normas editoriais da Revista “BMC Infectious Diseases” BMC Infectious Diseases Instructions for authors – Research article Assistance with the process of manuscript preparation and submission is available from BioMed Central customer support team. See 'About this journal' for information about policies and the refereeing process. We also provide a collection of links to useful tools and resources for scientific authors on our page. Criteria Research articles should report on original primary research, but may report on systematic reviews of published research provided they adhere to the appropriate reporting guidelines which are detailed in 'About this journal'. Submission process Manuscripts must be submitted by one of the authors of the manuscript, and should not be submitted by anyone on their behalf. The submitting author takes responsibility for the article during submission and peer review. 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Overview of manuscript sections for Research article Manuscripts for Research article articles submitted to BMC Infectious Diseases should be divided into the following sections (in this order): • Title page • Abstract • Keywords • Background • Methods • Results and discussion • Conclusions • List of abbreviations used (if any) • Competing interests • Authors' contributions • Authors' information • Acknowledgements • Endnotes • References • Illustrations and figures (if any) • Tables and captions • Preparing additional files The Accession Numbers of any nucleic acid sequences, protein sequences or atomic coordinates cited in the manuscript should be provided, in square brackets and include the corresponding database name; for example, [EMBL:AB026295, EMBL:AC137000, DDBJ:AE000812, GenBank:U49845, PDB:1BFM, Swiss-Prot:Q96KQ7, PIR:S66116]. The databases for which we can provide direct links are: EMBL Nucleotide Sequence Database (EMBL), DNA Data Bank of Japan (DDBJ), GenBank at the NCBI (GenBank), Protein Data Bank (PDB), Protein Information Resource (PIR) and the Swiss-Prot Protein Database (Swiss-Prot). You can download a template (Mac and Windows compatible; Microsoft Word 98/2000) for your article. For reporting standards please see the information in the About section. Title page The title page should: • provide the title of the article • list the full names, institutional addresses and email addresses for all authors • indicate the corresponding author Please note: • the title should include the study design, for example "A versus B in the treatment of C: a randomized controlled trial X is a risk factor for Y: a case control study" • abbreviations within the title should be avoided Abstract The Abstract of the manuscript should not exceed 350 words and must be structured into separate sections: Background, the context and purpose of the study; Methods, how the study was performed and statistical tests used; Results, the main findings; Conclusions, brief summary and potential implications. Please minimize the use of abbreviations and do not cite references in the abstract. 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Authors must disclose any financial competing interests; they should also reveal any non-financial competing interests that may cause them embarrassment were they to become public after the publication of the manuscript. Authors are required to complete a declaration of competing interests. All competing interests that are declared will be listed at the end of published articles. Where an author gives no competing interests, the listing will read 'The author(s) declare that they have no competing interests'. When completing your declaration, please consider the following questions: Financial competing interests • In the past five years have you received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Is such an organization financing this manuscript (including the article-processing charge)? 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Authors' contributions In order to give appropriate credit to each author of a paper, the individual contributions of authors to the manuscript should be specified in this section. An 'author' is generally considered to be someone who has made substantive intellectual contributions to a published study. To qualify as an author one should 1) have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) have been involved in drafting the manuscript or revising it critically for important intellectual content; and 3) have given final approval of the version to be published. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Acquisition of funding, collection of data, or general supervision of the research group, alone, does not justify authorship. We suggest the following kind of format (please use initials to refer to each author's contribution): AB carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. JY carried out the immunoassays. MT participated in the sequence alignment. ES participated in the design of the study and performed the statistical analysis. FG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. All contributors who do not meet the criteria for authorship should be listed in an acknowledgements section. Examples of those who might be acknowledged include a person who provided purely technical help, writing assistance, or a department chair who provided only general support. Authors' information You may choose to use this section to include any relevant information about the author(s) that may aid the reader's interpretation of the article, and understand the standpoint of the author(s). This may include details about the authors' qualifications, current positions they hold at institutions or societies, or any other relevant background information. Please refer to authors using their initials. Note this section should not be used to describe any competing interests. Acknowledgements Please acknowledge anyone who contributed towards the article by making substantial contributions to conception, design, acquisition of data, or analysis and interpretation of data, or who was involved in drafting the manuscript or revising it critically for important intellectual content, but who does not meet the criteria for authorship. Please also include the source(s) of funding for each author, and for the manuscript preparation. Authors must describe the role of the funding body, if any, in design, in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. Please also acknowledge anyone who contributed materials essential for the study. If a language editor has made significant revision of the manuscript, we recommend that you acknowledge the editor by name, where possible.