Impact of antiretroviral therapy on bone metabolism markers in HIV-seropositive patients

Bone 57 (2013) 62–67

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Original Full Length Article

Impact of antiretroviral therapy on bone metabolism markers in HIV-seropositive patients Erika Grasiela Marques de Menezes Barbosa a,⁎, Francisco Jose Albuquerque de Paula b, Alcyone Artioli Machado b, Francisco de Assis Pereira b, Fernando Barbosa Júnior c, Anderson Marliere Navarro b a

Rodovia Araraquara - Jaú Km 1, Cep: 14801-902, Araraquara, SP, Brazil Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, FMRP-USP, Avenida Bandeirantes, 3900, Bairro: Monte Alegre, CEP: 14049, 900, Ribeirão Preto, SP, Brazil c Department of Clinical, Toxicological and Bromatological Analyses, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University de São Paulo, FCFRP-USP, Avenida Bandeirantes, 3900, Bairro: Monte Alegre, CEP: 14049, 900, Ribeirão Preto, SP, Brazil b

a r t i c l e

i n f o

Article history: Received 14 December 2012 Revised 7 July 2013 Accepted 9 July 2013 Available online 25 July 2013 Edited by: Felicia Cosman Keywords: HIV ART Bone metabolism Osteopenia Osteoporosis

a b s t r a c t Objective: To evaluate the impact of antiretroviral therapy (ART) on bone and mineral metabolism and to determine the occurrence of osteopenia and/or osteoporosis in HIV-infected patients taking ART or not. Methods: A cross-sectional study was conducted on 50 HIV-seropositive adult men treated with or not treated with ART. Dual energy X-ray absorptiometry (DXA) was performed and biochemical analyses of the following markers were carried out: FSH, LH, testosterone, total calcium, phosphorus (Pi), magnesium (Mg), albumin, 24 h calcium, creatinine, urea, parathormone (PTH), insulin-like growth factor 1 (IGF-I), 25 hydroxyvitamin D (25-OH-D), osteocalcin, and urinary deoxypyridinoline (DPD). The participants were divided into two groups according to ART use or not: Group A, 10 treatment-naive subjects; Group B, ART use for N2 years, subdivided into: Group B1, 10 subjects treated with protease inhibitors (PIs) and nucleoside/nucleotide analog reverse transcriptase inhibitors (NRTIs) and Group B2, 10 subjects treated with NRTIs and non-nucleoside analog reverse transcriptase inhibitors (NNRTIs); and Group C, subjects treated with ART b2 years, subdivided into: Group C1, 10 subjects treated with PIs and NRTIs and Group C2, 10 subjects treated with NRTIs and NNRTIs. Results: The values of the bone formation marker, osteocalcin, were normal in all groups, whereas urinary DPD values were increased in all groups. Whole body DXA revealed a higher percentage of osteopenia (80%) in Group B2. Lumbar spine DXA showed osteoporosis in Groups A and B1 (10%) and total femur DXA in Group B2 (10%). Conclusion: The increased bone reabsorption marker indicated a high reabsorptive activity of bone tissue. These data indicate a greater osteoclastic activity in bone loss in HIV-infected patients on ART. © 2013 Elsevier Inc. All rights reserved.

Introduction Advances in antiretroviral therapy (ART) have resulted in a decline of mortality for individuals infected with human immunodeficiency virus (HIV) and an increase in life expectancy. Despite the progress obtained with ART, concern has arisen about the long-term adverse effects of chronic HIV infection and of ART, including complication of bone and mineral metabolism [1]. Complications of bone metabolism and reduced bone mass have been demonstrated in young and adult HIV-infected individuals on ART [2]. HIV-infected patients have been reported to be a population with risk factors for osteopenia and/or osteoporosis, such as weight

⁎ Corresponding author at: Avenida Dr. Carlos Botelho, 1813, loja 5, Centro, 13560-250, São Carlos, São Paulo, Brazil. Fax: +55 16 3372 3556. E-mail address: [email protected] (E.G.M. de Menezes Barbosa). 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.07.019

loss, smoking, increased basal metabolism, vitamin D deficiency, inadequate calcium intake, physical inactivity, and other diseases [3]. A recent study [4] showed that HIV-infected persons may present manifestations of premature aging compared to the general population, with the onset of cardiovascular diseases, cognitive disorders, cancer, and metabolic complications. Clinical studies have suggested that the physiopathology of this premature aging can be multifactorial, including HIV-infection, immune activation (leading to immunodeficiency) and some antiretroviral agents, which may contribute to systemic inflammation leading to tissue senescence and to the onset of degenerative and proliferative diseases [5,6]. In addition to the premature aging leading to disorders of bone metabolism in HIV-infected patients, several studies have demonstrated that bone metabolism is altered by direct effects of HIV on osteoclasts and osteoblasts or indirectly by generalized inflammation promoting osteoblast apoptosis, reducing the functionality of these cells and promoting bone reabsorption [7].

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Several studies comparing HIV-infected and non-infected individuals have shown a higher prevalence of reduced bone mineral density (BMD) and a higher incidence of fracture among HIV-infected persons, aggravated by the beginning of ART, with increased bone remodeling and significant increases in bone formation and reabsorption markers [9,10]. A recent study compared the bone loss occurring with treatment schemes based on different ART classes such as nucleoside/nucleotide analog reverse transcriptase inhibitors (NRTIs), non-nucleoside analog reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). The three different classes led to BMD loss but NRTIs were associated with a significantly greater BMD loss in the hip and spine [11]. Another study showed that the NRTI class resulted in perturbation of gene osteoblast expression and changes implying osteoblast dysfunction leading to reduced bone formation and resulting in reduced BMD [12]. To detect the early onset of changes in the activity of bone formation and reabsorption, evaluation of the biochemical markers of bone remodeling has been proposed, among them the bone formation marker osteocalcin and the bone reabsorption marker urinary deoxypyridinoline [10] and the major hormone involved in the regulation of calcium metabolism, i.e., parathyroid hormone which acts on reabsorption regulating calcium and phosphate release into the extracellular fluid, and 25 hydroxyvitamin D [25(OH)D] which acts together with PTH in the maintenance of these extracellular levels [13]. In addition to the above hormones, reduced concentrations of testosterone and thyroid stimulating hormone (TSH) have been reported to lead to bone loss [14]. The role of insulin-like growth factor type 1 (IGF-I) in the regulation of bone metabolism in men has been studied, and reduced serum concentrations have been found to be associated with an increased risk of fractures [15]. It is important to point out that an imbalanced interaction of osteoblasts and osteoclasts due to pathological conditions such as infection and hormonal, immunological and metabolic changes has a negative effect on bone mass and bone structure, resulting in greater bone fragility and risks of fracture [8]. Thus, studies have demonstrated that HIV-infected individuals show loss of bone mineral density and are more aggravating with the use of ART regimens and time associated with the use of this therapy [9,16]. On this basis, the objective of the present study was to assess the impact of ART on the changes in bone and mineral metabolism and the occurrence of osteopenia and/or osteoporosis in HIV-infected patients treated with or not treated with ART.

Anthropometric evaluation

Methodology

Bone evaluation

A cross-sectional study was conducted on 50 adult HIV-seropositive men with different viral loads, under treatment or not with ART. Exclusion criteria were: female gender, use of calcium and vitamin D supplements, use of medications for the treatment of osteoporosis, a previous history of thyroidectomy, and the presence of renal and/or hepatic insufficiency [18]. The volunteers were recruited at the Special Unit for the Treatment of Infectious Diseases (UETDI) of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCFMRP-USP) and at the Cuiabá Teaching Health Center, Ribeirão Preto, SP, in order to complete the sample. The study was approved by the Ethics Committee of HCFMRP-USP (protocol no. 2605/2010), and all subjects gave written informed consent to participate. Data regarding the duration of HIV infection, the therapeutic scheme and the time of ART use, T CD4+ cell count and the quantitation of viral load were obtained from the medical records of the patients. The volunteers were divided into the following groups according to the use or not of ART: Group A, 10 treatment-naive subjects; Group B, patients on ART N2 years, subdivided into: Group B1, 10 subjects treated with PIs and NRTIs and Group B2, 10 subjects treated with NRTIs and NNRTIs; and Group C: patients on ART b2 years, subdivided into: Group C1, 10 subjects treated with PIs and NRTIs and Group C2, 10 subjects treated with NRTIs and NNRTIs.

DXA was applied to the following bone sites: whole body, total femur and lumbar spine and the procedure was carried out in the service of Radiology of HCFMRP-USP using a Hologic QDR 4500A scanner (Hologic Inc., Waltham, MA, USA). BMD was evaluated according to the classifications proposed by the WHO (1994) [34]. Standard procedures were used to position the volunteers during the execution of the exam, wearing no shoes or metal objects. The exam was carried out by total scanning of the patient's body.

Weight and height were measured and the body mass index (BMI) was calculated by the formula weight/height2. The patients were classified according to the recommendations of the WHO, 1998 [19]. Body composition (lean mass and fat mass) was determined by dual energy X-ray absorptiometry (DXA). Biochemical analyses For the biochemical analyses, peripheral blood samples were collected after a 12 hour fast in the morning. Twenty-four hour urine was collected into 3 liter flasks containing 50% hydrochloric acid (HCl). For the collection of the 2nd morning urine sample, a universal sterile 80 mL collector was used. After collection, the blood samples were centrifuged in a Universal 320R Hettich® centrifuge for 10 min at 23 °C at 3500 rpm. The serum obtained was stored in a freezer at −80 °C and the urine samples were stored in a freezer at −20 °C until the time for analysis. Total calcium, 24 h calcium and magnesium concentrations were determined using an equipped mass spectrometer. Albumin, creatinine, urea and phosphorus concentrations were determined by colorimetry using Labtest® kits. FSH, LH, testosterone, PTH and osteocalcin were determined by immunochemiluminescence (ICMA) using an IMMULITE® 1000 analyzer. Serum 25(OH)D and IGF-I were determined by chemiluminescence (CLIA) using the LIAISON® 25-OH vitamin D test and the DiaSorin® 4000th Series analyzer for IGF-I, and DPD was determined by ELISA using a Quidel® kit. The analysis of urinary creatinine, used to calculate correction for DPD, was performed by an automatic enzymatic method using the Wiener® kit for liquid AA kinetic creatinine using the BT 3000 Plus apparatus of the Laboratory of Nutrition and Metabolism of HCFMRP-USP. Viral load was determined by chemiluminescence using a Versant® HIV-1 RNA 3.0 kit. CD4+ T lymphocytes were quantitated by flow cytometry using the Multitest® kit. The reference values published by Young in 1987 [38] were used for serum calcium and urinary calcium, and the reference values published by the Health Ministry [36] were used for T CD4+ lymphocyte counts. The reference ranges proposed in the analytical kits were used for the remaining determinations being shown in Table 2.

Statistical analysis Mean, standard deviation and 95% confidence interval were used to describe the data regarding the quantitative variables (age, weight, BMI, fat and lean mass, duration of HIV infection, CD4+ cell count, time of ART use, energy, macronutrient and calcium intake, total calcium, 24 h calcium, total femur BMD, lumbar spine BMD and whole body BMD) [22]. The Fisher exact test was applied to determine the association between categorical variables (viral load, total femur BMD, lumbar spine BMD and whole body BMD and the groups) [22]. The Kruskal–Wallis test was used for group comparison regarding continuous variables (age, weight, BMI, lean and fat mass,CD4+ cell

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Table 1 General characterization of HIV-seropositive patients according to study group. Variables

A: without ART

B1: PIs + NRTIs

B2: NRTIs + NNRTIs

C1: PIs + NRTIs

C2: NRTIs + NNRTIs

Age, years Weight (kg) BMI (kg/m2) Fat mass (%) Lean mass (%) Duration of infection (months) Undetectable VL CD4 (cells/mm3) Time of ART use (months)

30.8 ± 9.4a 72.2 ± 11.2 20.4 ± 6.7 24.9 ± 7.5 69.4 ± 7.0 34.8 ± 20.7a 20% 521.9 ± 82.6 0

45.6 ± 9.5b 65.9 ± 7.3 22.3 ± 2.4 21.1 ± 5.2 73.7 ± 4.8 177.6 ± 54.5b 100% 605.4 ± 205.9 136.3 ± 43.9

48.4 ± 11.9c 68.1 ± 10.8 23.6 ± 2.6 25.6 ± 4.7 69.5 ± 4.8 146.4 ± 68.7b 100% 580.7 ± 271.1 118.2 ± 64.1

35.0 ± 12.6a,b,c 66.4 ± 16.5 22.1 ± 4.1 22.3 ± 7.1 71.5 ± 6.4 37.2 ± 28.5c 50% 378.2 ± 279.1 8.6 ± 7.3

33.3 ± 8.6a,b,c 73.7 ± 12.6 24.4 ± 3.9 25.4 ± 6.5 68.8 ± 5.7 31.2 ± 16.2c 90% 470.1 ± 279.2 11.4 ± 6.1

Note: Kruskal–Wallis test. Data are reported as mean ± SD. a,b,cDifferent letters: p b 0.05. BMI = body mass index; VL = undetectable viral load b50 copies/mL; ART = antirretroviral therapy; PIs = protease inhibitors; NRTIs = nucleoside/nucleotide analog reverse transcriptase inhibitors; NNRTIS = non-nucleoside analog reverse transcriptase inhibitors.

count, duration of HIV infection, total calcium, and 24 h calcium). When a difference was detected between some group pairs, the Dunn posttest was applied [23]. For group comparison regarding the variables osteocalcin, urinary DPD, femur BMD, lumbar spine BMD and whole body BMD, we first used a method for the selection of variables from a set of independent variables. This selection method was adjusted for R2, which selects a set of variables presenting the highest R2 adjusted for the regression model. Analysis of covariance (ANCOVA) was then proposed starting from the selected variables. This methodology permits us to incorporate into ANOVA one or more quantitative variables presenting a linear relation with the response variable [23]. Statistical analysis was performed using the SAS® 9.0 and SAS® 9.2 software [22,23] at the Center of Quantitative Methods — CEMEQ, of FMRP-USP. Results The body mass index (kg/m2) in all groups was found to be healthy in terms of the BMI classification of the WHO [19] and there was no statistical difference between the groups. Percent fat mass was higher in Group B2: 25.6 ± 4.7 and, on the basis of the classification of body fat percentages, none of them was at risk for diseases associated with obesity. The groups with longer duration of HIV infection and of time of ART use (Groups B1 and B2) had an undetectable viral load (b50 copies/mL blood) and a higher CD4+ cell count (mm3). There was a significant difference in age between Groups A and B1 and B2 (p b 0.05) and in time of duration of HIV infection between all groups (p b 0.05) (Table 1).

Regarding the laboratory parameters related to bone metabolism, the serum concentrations of phosphorus, urea, FSH, testosterone and albumin, urinary magnesium, IGF-I and the bone formation marker osteocalcin were within normal limits (Table 2). Calcemia was within normal limits except for Groups A (8.5 ± 0.6 mg/dL) and C1 (8.6 ± 0.4 mg/dL) which showed reduced values, with a significant difference between Groups A and B1 (p b 0.05). Serum magnesium concentration was reduced in Groups A and C2. Most groups had serum creatinine levels above normal limits, except for Group A, which showed normal values (1.2 ± 0.3 mg/dL). Serum PTH concentration was elevated in all groups except Group C2 (8.5 ± 9 pg/mL) which presented normal values. Serum PTH levels differed significantly between Groups B1 and C2 (p b 0.05). LH was altered only in B1, with a value of 8.1 ± 6.4 mIU/mL. All groups had normal urinary calcium concentrations b250 mg/24 h. 25 (OH) vitamin D values were below acceptable limits in all groups. The bone reabsorption marker was above normal reference values in all groups. According to the number of participants showed 10% osteoporosis obtained by DXA for the lumbar spine of Groups A and B1 and for the total femur of Group B2. Osteopenia was detected at all bone sites in each group, with a higher percentage for whole body: 40% for Groups A and C1, 50% for Groups B1 and C2, and 80% for Group B2. There was no significant difference between groups and categorical variables (Table 3). The linear regression models were adjusted between groups to determine the possible effects of duration of HIV infection, age, BMI, fat mass and lean mass, calcium and protein intake, and serum and urinary

Table 2 Serum and urinary biochemical concentrations according to study group. Variables

A: without ART

Total calcium (mg/dL) Phosphorus (mg/dL) Magnesium (mg/dL) Albumin (mg/dL) Creatinine (mg/dL) Urea (mg/dL) PTH (pg/mL) FSH (mIU/mL) LH (mIU/mL) Testosterone (ng/dL) IGF-I (ng/mL) Osteocalcin (ng/mL) Urinary calcium (mg/24 h) Urea (g/24 h) 25 OH vitamin D (ng/mL) DPD (nmol/mmol)

8.5 3.5 1.7 4 1.2 22.4 18.8 2.6 3.5 461.4 221.7 0.56 96.7 30.0 29.1 7.5

± 0.6a ± 0.9 ± 0.2 ± 0.3 ± 0.3 ± 5.1 ± 13.6 ± 2.5 ± 2.6 ± 141.5 ± 53.7 ± 0.4 ± 47.1 ± 12.3 ± 11.3 ± 2.3

B1: N2 years PIs + NRTIs

B2: N2 years NRTIs + NNRTIs

C1: b2 years PIs + NRTIs

C2: b2 years NRTIs + NNRTIs

Reference values

9.6 ± 0.4b 3.6 ± 0.8 1.9 ± 0.2 3.9 ± 0.4 1.7 ± 0.3 37.6 ± 15.1 34.6 ± 23.3a 11.2 + 13.6 8.1 ± 6.4 521.0 ± 191.6 153.4 ± 68.8 0.88 ± 1.3 88.7 ± 65.1 36.7 ± 11.3 27.7 ± 8.0 9.2 ± 3.1

9.1 ± 1.4 3.7 ±0.6 1.8 ± 0.3 4 ± 0.3 1.5 ± 0.3 32.9 ± 8.7 15.2 ± 8.2 5.8 ± 3.9 6.1 ± 4.7 427.9 ± 149.1 214.7 ± 35.1 0.31 ± 0.1 91.1 ± 58.3 44.4 ± 23.4 25.9 ± 7.7 7.9 ± 3

8.6 ± 0.4 4.1 ± 0.7 1.8 ± 0.3 4.4 ± 0.7 1.3 ± 0.3 26.1 ± 10.1 23.7 ± 6.7 4.9 ± 4.1 4.0 ± 1.7 588.1 ± 212.2 267.9 ± 113.2 0.43 ± 0.2 67.0 ± 41.8 34.3 ± 23.4 29.6 ± 11.5 9.67 ± 3.7

8.8 ± 0.8 3.9 ± 0.5 1.7 ± 0.3 3.9 ± 0.4 1.3 ± 0.3 26.5 ± 7.5 8.5 ± 9.0b 4.7 ± 6.5 4.6 ± 3.2 495.5 ± 298.1 200.1 ± 38.9 0.41 ± 0.2 95.8 ± 72.0 32.8 ± 13.1 28.7 ± 7.5 10.5 ± 2.6

8.8–10.3 2.5–4.8 1.8–3.0 3.5–5.5 0.70–1.20 15–45 6.7–11 0.7–11.1 0.8–7.6 160–726 160–318 b2.0–21 b250 26–43 30–100⁎ 2.3–5.4

Note: Kruskal–Wallis test. Data are reported as mean ± SD. a,bDifferent letters: p b 0.05. ART = antiretroviral therapy; PIs = protease inhibitors; NRTIs = nucleoside/nucleotide analog reverse transcriptase inhibitors; NNRTIS = non-nucleoside analog reverse transcriptase inhibitors; PTH = parathormone intact molecule; FSH = follicle stimulating hormone; LH = luteinizing hormone; IGF-I = insulin-like growth factor 1; DPD = urinary deoxypyridinoline. ⁎ Values for the US population.

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Table 3 Classification of bone mineral density at the following bone sites: femur, lumbar spine and whole body, according to the criteria of the World Health Organization, for each study group. Bone sites

A: without ART

B1: N2 years PIs + NRTIs

B2: N2 years NRTIs + NNRTIs

C1: b2 years PIs + NRTIs

C2: b2 years NRTIs + NNRTIs

Femur Osteopenia [n (%)] Osteoporosis [n (%)]

2 (20) 0

3 (30) 0

4 (40) 1 (10)

3 (30) 0

3 (30) 0

Lumbar spine Osteopenia [n (%)] Osteoporosis [n (%)]

3 (30) 1 (10)

4 (40) 1 (10)

6 (60) 0

4 (40) 0

6 (60) 0

Whole body Osteopenia [n (%)] Osteoporosis [n (%)]

4 (40) 0

5 (50) 0

8 (80) 0

4 (40) 0

5 (50) 0

Note: ART = antiretroviral therapy; PIs = protease inhibitors; NRTIs = nucleoside/nucleotide analog reverse transcriptase inhibitors; NNRTIS = non-nucleoside analog reverse transcriptase inhibitors.

markers on the five indicators of bone health evaluated, i.e., osteocalcin, urinary DPD, total femur BMD, lumbar spine BMD, and whole body BMD. To adjust the linear regression models for dietary calcium and protein intake, a three-day food record was filled out and the nutritional analysis Diet Pro® software, version 5i [20,35] was used to determine the possible effects on the indicators of bone health evaluated. The data regarding each indicator of bone health evaluated, i.e., osteocalcin (ng/mL), urinary DPD (nmol DPD/mmol creatinine), femur BMD (g/cm2), lumbar spine BMD (g/cm2) and whole body BMD (g/cm2), were adjusted to two models. In the first, adjustment was based on the biochemical variables of serum concentrations and in the second, adjustment was based on the variables of urinary concentrations. A tendency of a reduction of osteocalcin levels was observed in the present study when the effect of FSH was analyzed (each 1 m IU/mL led to a reduction of 0.03 ng/mL in osteocalcin levels, p = 0.001), in

Table 4 Linear regression models for osteocalcin values (models I, II) e for urinary deoxypyridinoline values (models III, IV). Model

Parameters

I

Duration of HIV infection (months) PTN consumption (g) FSH (mIU/mL) PTH (pg/mL) Magnesium (mg/dL) Phosphorus (mg/dL) CD4 (cells/mm3) Femur BMD (g/cm2) Lumbar BMD (g/cm2) Duration of HIV infection (months) PTN consumption (g) Ca consumption (mg) CD4 (cells/mm3) Lumbar BMD (g/cm2) Duration of HIV infection (months) BMI (kg/m2) BMI (kg/m2) Lean mass (%) Fat mass (%) Magnesium (mg/24 h) Whole body BMD (g/cm2)

Estimate

p-Value

LL

II

III

IV

0.004

0.02

R2

95% CI UL

0.001

0.007

0.61

0.003 −0.03 0.02 0.39 0.13 −0.001 −1.05 1.01 0.003

0.02 0.001 0.001 0.21 0.25 0.10 0.13 0.20 0.09

0.001 −0.05 0.009 −0.23 −0.10 −0.001 −2.44 −0.57 −0.001

0.007 −0.01 0.034 1.01 0.37 0.001 0.33 2.6 0.007

0.39

0.001 −0.001 −0.001 0.73 −0.02

0.002 0.18 0.14 0.37 0.06

0.002 −0.001 −0.001 −0.91 0.06

0.01 0.001 0.002 2.37 −0.03

0.27

0.26 0.57 −1.10 −1.17 0.37 −7.82

0.03 0.003 0.08 0.05 0.03 0.17

0.03 0.21 −2.34 −2.37 0.03 −19.2

0.02 0.94 0.14 0.02 0.71 3.56

0.36

Note: The linear regression models I and III were adjusted according to serum concentration variables. The linear regression models II and IV were adjusted according to the variables of urinary concentrations. Statistically significant: p b 0.05. CI = confidence interval; LL = lower limit; UL = upper limit; R2 = coefficient of variation; BMD = bone mineral density; PTN = protein; Ca = calcium; BMI = body mass index.

contrast to PTH, the duration of HIV infection and protein intake, which were related to an increase in osteocalcin levels (p b 0.05). This tendency was observed by the increased percentage of osteocalcin variability in the first linear regression model (Table 4). Urinary DPD was reduced when the effect of fat mass was analyzed in a linear regression model (each one percent led to a reduction of 1.17 nmol/mmol in DPD levels, p = 0.05). BMI and urinary magnesium showed increased levels of urinary DPD (p b 0.05), as observed by the increase in the percentage of DPD variability in the second model (Table 4). However, the linear regression models for the bone densitometry values revealed a reduction of femoral BMD when the effect of age (p b 0.05) and fat mass (p b 0.05) was analyzed. A reduced lumbar BMD was observed when the effect of calcemia was analyzed (p = 0.05), in contrast to BMI, which increased the values of femoral and lumbar BMD (p b 0.05). Analysis of the effect of lean mass and fat mass on whole body BMD showed that each 1% leads to a reduction of 0.05 g/cm2 of whole body (p = 0.001), in contrast to BMI, for which each 1 kg/m2 increases by 0.02 g/cm2 whole body BMD (p b 0.001) (Table 5). Discussion The present study investigated the possible changes in bone and mineral metabolism influenced by the time of use of different ART schemes in HIV-infected patients and the secondary causes of osteopenia and osteoporosis such as body weight, age, CD4+ T cell count, HIV infection and reduced calcium intake. Most the participants in the present study had healthy body weight and were younger than 50 years. It is known that advanced age and low body weight are usually associated with risks for osteoporosis as reported in several studies [13,30]. For example Arnsten et al. [24] evaluated the BMI of HIV-infected men and detected an association between lean mass and BMD compared to excess weight. In the present study, analysis of the coefficients of determination (R2) of the linear regression models revealed that BMI had a greater influence on the bone reabsorption marker and BMD of the femur, lumbar spine and whole body. Studies involving bone evaluation in HIV-infected men have reported a greater prevalence of bone loss compared to the general population [24]. An important factor contributing to the occurrence of reduced BMD is the premature aging caused by HIV and by some antiretroviral agents which contributes to systemic inflammation leading to tissue senescence [4–6]. Cross-sectional studies have demonstrated that the reduction of CD4+ T cells or virologic factors are associated with the loss of BMD [26] and in this study the majority of participants had normal CD4+ T cell count and undetectable viremia, not considered a factor that contributes to bone changes. It is interesting to note that Group B2 had an undetectable viral load and a normal CD4+ T cell count, but a greater

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Table 5 Linear regression models for total femur bone densitometry values (models I, II) for bone densitometry values of the lumbar spine (models III, IV) and for whole body bone densitometry values (models V, VI). R2

Model

Parameters

Estimate

p-Value

95% CI LL

UL

I

Age (years) BMI (kg/m2) Fat mass (%) Ca consumption (mg) FSH (mIU/mL) Calcium (mg/dL) IGF-I (ng/mL) Age (years) BMI (kg/m2) Lean mass (%) Fat mass (%) BMI (kg/m2) Fat mass (%) PTH (pg/mL) Calcium (mg/dL) IGF-I (ng/mL) 25(OH)D (ng/mL) Age (years) Duration of HIV infection (months) BMI (kg/m2) Lean mass (%) Fat mass (%) Magnesium (mg/24 h) Age (years) Duration of HIV infection (months) BMI (kg/m2) Lean mass (%) Fat mass (%) PTH (pg/mL) Testosterone (ng/dL) Calcium (mg/dL) Age (years) Duration of HIV infection (months) BMI (kg/m2) Lean mass (%) Fat mass (%) Magnesium (mg/24 h)

−0.002 0.02 −0.006 8.43 −0.002 −0.03 −0.002 −0.003 0.03 −0.02 −0.03 0.01 −0.002 0.001 −0.03 −0.001 −0.002 0.002 0.001

0.05 b0.001 0.02 0.07 0.16 0.06 0.28 0.007 b0.001 0.11 0.05 0.01 0.32 0.20 0.05 0.23 0.09 0.14 0.08

−0.005 0.01 −0.01 −6.5 −0.006 −0.06 −0.005 −0.005 0.02 −0.06 −0.06 0.002 −0.007 −0.001 −0.06 −0.001 −0.005 −0.001 −7.8

−1.5 0.03 0.03 0.001 0.001 0.001 0.002 −0.001 0.03 0.006 0.004 0.02 0.002 0.003 −0.001 0.0001 0.001 0.004 0.001

0.01 −0.03 −0.03 0.01 −0.001 0.001

0.001 0.11 0.07 0.09 0.42 0.29

0.01 −0.06 −0.06 −0.001 −0.003 −0.001

0.02 0.01 0.003 0.02 0.001 0.001

0.02 −0.05 −0.05 0.001 2.36 −0.02 −0.001 0.001

b0.001 0.001 0.001 0.85 0.96 0.06 0.32 0.15

0.01 −0.08 −0.08 −0.01 −0.001 −0.05 −0.003 −0.001

0.03 −0.02 −0.02 0.001 0.001 0.001 0.001 0.001

0.02 −0.05 −0.05 −0.001

b0.001 0.001 0.001 0.9

0.01 −0.08 −0.08 −0.01

0.02 −0.02 −0.02 0.01

II

III

IV

V

VI

0.69

0.65

0.39

0.31

0.55

0.50

Note: The linear regression models I, III and V were adjusted according to serum concentration variables. The linear regression models II, IV, and VI were adjusted according to the variables of urinary concentrations. Statistically significant: p b 0.05. CI = confidence interval; LL = lower limit; UL = upper limit; R2 = coefficient of variation; BMI = body mass index; IGF-I = insulin-like growth factor 1; Ca = calcium.

loss of whole body BMD. This bone loss may be associated with other factors such as duration of HIV infection and the use of ART for a longer period of time, in agreement with a clinical study by Rivas et al. [18] in which the CD4+ cell count was not associated with BMD. The evaluation of bone and mineral metabolism revealed that the participants in the present study presented factors that might interfere with bone formation and reabsorption, including HIV infection itself and ART use. Several studies have suggested that the loss of bone mass is mainly due to an impairment of bone formation and reabsorption activity [26]. In the present study, osteocalcin levels were normal and urinary DPD levels were increased in all groups and the data regarding the markers of bone formation and reabsorption showed the presence of a more marked activity of bone remodeling. Thus, a correlation was detected here between the indicators of bone health osteocalcin and PTH and the duration of HIV infection. There was a correlation between urinary DPD and BMI values, suggesting that these factors interfered with the markers of bone formation and reabsorption. Haskelberg et al. [27] detected an increase of the bone reabsorption marker carboxyterminal telopeptide (C-Tx) after more than 96 weeks

of ART use, especially with the NRTI scheme. Studies have reported that increased bone reabsorption is associated with HIV infection itself and becomes more severe with the use of ART [28,32]. In the present study, most groups showed increased PTH and decreased serum concentration of 25 (OH) vitamin D and were considered to have vitamin D insufficiency according to the reference values for the North American population [37]. Increased PTH levels favor bone reabsorption and reduced vitamin D levels impair the PTH production and reduce calcium absorption in the intestinal tract, thus favoring dysfunction of bone metabolism [27]. Several studies have shown that reduced testosterone levels may lead to bone loss [14,28], a factor that was not observed in the present study, since all participants had normal testosterone levels. Most groups presented normal total calcium concentrations [17], except for Groups A and C1 which presented reduced values. This may have been associated with inadequate calcium intake, reduction of serum vitamin D, increased PTH, and HIV infection itself, factors that are known to be associated with the loss of bone mass [39]. Studies have shown that urinary calcium concentration can be affected by inadequate calcium intake, by renal dysfunction and by disorders of the action of hormones and that increased urinary calcium is related to bone loss in men [31]. In contrast, in the present study the groups showed normal calciuria values that did not contribute to loss of bone mass. Groups A and C2 showed reduced serum magnesium values. Studies have shown that minerals are implicated in bone metabolism and that magnesium deficiency reduces calcium absorption and metabolism, with consequent impairment of bone mass [21,33]. Regarding the therapeutic scheme, it was observed that the group of patients using NRTIs and NNRTIs for an average time of 118 months showed a greater reduction of BMD compared to the remaining groups. Studies by Horizon et al. [17], Ofotokun et al. [26] and Rivas et al. [18] have recently shown that HIV-infected patients treated with NRTIs suffered frequent fractures. A recent in vitro study showed that PIs increase osteoclast activity. Similarly, NRTIs stimulate osteoclastogenesis and alter the expression of the osteoblast gene, implying a loss of osteoblast function [29]. In the present study, regarding bone densitometry, HIV-seropositive ART-naive patients demonstrated a loss of BMD. Womack et al. [25] investigated the association between HIV infection and risk of fractures and detected a higher risk of fractures among HIV-infected men compared to non-HIV-infected men. Brown et al. [16] showed that chronic HIV infection is associated with systemic inflammation, which may have adverse effects on BMD. However, HIV infection and ART use, especially with the NRTI regimen, are associated with changes in bone reabsorption markers and with 25 (OH) vitamin D deficiency, leading to dysfunction of bone remodeling and a consequent reduction of bone mass. These facts, therefore, may help explain that among the 50 participants we were able to determine the presence of osteopenia and osteoporosis.

Conclusion There is an increase in the bone reabsorption marker, indicating high bone tissue reabsorptive activity in HIV-infected patients on ART. Patients with longer use of ART found greater bone loss compared with the treatment-naive group of antiretroviral. In addition, we identified possible predictors of reduced bone mass such as duration of HIV infection, detectable viremia, time of use and scheme of ART, age, BMI, lean mass, PTH, FSH, protein intake, vitamin D insufficiency, and inadequate calcium intake. However, further studies are needed to correlate the changes in bone metabolism and the risks of fracture in order to prevent or minimize the changes in BMD. Monitoring of this population is necessary in order to detect early signs of damage to bone health, as already reported in various studies.

E.G.M. de Menezes Barbosa et al. / Bone 57 (2013) 62–67

Conflict of interest Divulgation of the authors: Erika Grasiela Marques de Menezes Barbosa, Francisco Jose Albuquerque de Paula, Alcyone Artioli Machado, Francisco de Assis Pereira, Fernando Barbosa Júnior, Anderson Marliere Navarro declare they have no conflict of interest. Acknowledgments The authors wish to thank the Special Unit for the Treatment of Infectious Diseases of the University Hospital of Ribeirão Preto, University of São Paulo, Brazil. We are grateful to “Fundação de Apoio ao Ensino, Pesquisa e Assistência”, FAEPA, HCFMRP-USP and to the Clinical Research Unit, UPC, HCFMRP-USP for their financial support. Thanks are also due to the Quantitative Methods Center, CEMEQ, Faculty of Medicine of Ribeirão Preto, University of São Paulo, and to our collaborators Prof. Dr. Alceu Afonso Jordão Junior and the laboratory technician Paula Payão Ovidio. Finally, we also thank all the men who participated in this study.

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