Dairy calcium intake and lifestyle risk factors for bone loss in hiv-infected and uninfected mediterranean subjects

The study participants were Caucasian HIV-infected patients, consecutively enrolled between January 2011 and January 2012, who were being followed-up prospectively at the AIDS Center of the University of Palermo.

BMD was measured in all subjects by dual X-ray absorptiometry (DXA) [9]. Exclusion criteria included kidney disease, gastrointestinal disorders and steroid and sex steroid therapy.

Data on age, gender, drug addiction, antiretroviral therapy (ART), protease inhibitors (PI) and non-nucleoside reverse-transcriptase inhibitor (NNRTI) exposure were all recorded in a database designed for this study. Specific tenofovir exposure > 1 year was also recorded. Exposures to ART and PI were graded 0 (no exposure) to 3 (1: < five years; 2: from five to nine years; 3: ≥ ten years).

Body mass index (BMI) was calculated as weight (in kg) divided by height squared (m²). Subjects were considered underweight when BMI was ≤ 18.5 kg/m², normal weight when BMI was between 18.6-24.9 kg/m², overweight when BMI was between 25–29.9 kg/m² and obese when BMI was ≥ 30 kg/m².

Diabetes mellitus was defined in accordance with the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus criteria [10].

Seventy-six HIV-uninfected subjects referred by family doctors to our Center for laboratory and BMD assessment were enrolled as the control group. Age and sex of these subjects were comparable with the patients and the same exclusion criteria were applied.

At the start of the study, the HIV-infected subjects filled in a health and lifestyle questionnaire considering medical and drug abuse history, lifestyle habits (including smoking status, alcohol consumption, Italian espresso coffee drinking, physical activity) and bone fractures. Smoking status was categorized as non-smokers, former smokers and current smokers. Alcohol intake > 20 g per day either at the time of the study or in the past as well as Italian espresso coffee intake > 4 cups per day were also recorded. Physical activity was defined as either non-occupational or occupational when physical exercise was performed during free time or at work, respectively. Non-occupational physical activity was considered active when > 2 sessions of 20 minutes per week were performed. Occupational physical activity was defined as heavy when manual work was carried out.

In order to assess calcium intake, the consumption of foods representing the major sources of daily calcium intake in the Italian diet, such as typical Italian aged cheese (i.e. Parmesan), ricotta cheese and yogurt was recorded in a weekly food-frequency questionnaire, in accordance with the tables of nutrient values issued by the Italian National Institute of Nutrition [11‐13]. This was administered through an individual face to-face interview [11]. A fixed range of food containers, i.e. a glass for milk and a cup for yogurt, was used to standardize portion sizes, each containing ≈ 300 mg of calcium. A similar amount of calcium was contained in the reference servings of aged and ricotta cheese (≈ 25 and 100 g, respectively). Color photographs were shown to the subjects to demonstrate the standard sizes of the cheese servings. In addition, the number of servings eaten weekly was recorded and calcium intake was categorized according to quartiles of weekly servings (1: ≤8, 2: 9–10, 3: 11–14, and 4: ≥15), based on the distribution of the HIV-population. The level of education was also determined for all subjects. Three options were used: primary school (which in Italy represents a five-year compulsory education period), lower secondary school (three additional compulsory years) and higher levels of education, including upper secondary school, professional schools or university.

In all the HIV-patients CD4+ T-cell count (most recent value and nadir) and plasma HIV-RNA levels were assessed as previously reported [14]. Serum bone alkaline phosphatase, 25-hydroxyvitamin D, phosphorus and calcium levels were obtained in both HIV -infected cases and un-infected controls.

Vitamin D assessment was performed in the sunnier months from April through October, since it is known that vitamin D formation is not possible or inadequate [15] in Italy during the less sunny months, due to its latitude.

For both HIV-infected cases and uninfected controls, BMD was assessed at baseline by DXA, using a QDR Discovery Hologic DXA in the femoral neck and DXA in the lumbar spine by total body DXA [16]. For each scan, BMD and T-scores were recorded. T-scores compare BMD with the mean of a healthy young (age 20–30 years) reference population, matched for sex and race, and were expressed as the number of standard deviations above or below the reference mean. Osteopenia was defined when at least one of the two DXA T-scores was less than −1. Osteoporosis was diagnosed when either femoral neck or lumbar spine DXA T-scores were less than −2.5, as recommended by the World Health Organization (WHO) and the National Osteoporosis Foundation [17‐19].

We calculated the 10-year fracture risks according to the standardized WHO FRAX equation, computed with BMD (T-score) at the femoral neck [20].

Data were expressed as mean ± standard deviation when distribution was Gaussian. Differences were calculated using Student’s t-test. Otherwise, data were expressed as median and range and analyzed with the Mann–Whitney U test. Fisher’s exact and χ² tests, Pearson’s correlation and Spearman's rank correlation were used where appropriate. Multiple logistic regression analysis was performed to estimate the independence of the association between lumbar spine and femoral neck T-scores as well as variables significant at univariate analysis in the HIV-infected subjects. Variables contributing significantly to fit the logistic equation were then selected by a step-wise procedure. P < 0.05 was considered significant.

All analyses were performed using the SPSS software package (version 16.0; Chicago, IL, USA).

The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

The study was approved by the local Ethics Committee and informed consent was obtained from all subjects.

The main characteristics of the 112 HIV-infected and control subjects are shown in Table 1.

The average age of the recruited patients was 47 ± 9.7 years. Sixty-three individuals were male (56%). Among the 112 subjects, 28 (25%) were overweight, 6 (5%) were underweight (BMI < 18.5) and 7 (6%) were obese. As regards smoking status, drug addiction and Italian espresso coffee drinking we found that half of the study subjects regularly smoked cigarettes, one third drank coffee > 4 times a day and one third used cocaine and/or heroin.

About half of this population did physical activity in their free time and had a higher level of education.

Concerning eating habits, the median daily calcium intake was 454 mg/die, range (96–1359). More than half of the participants interviewed reported that they drank a glass of milk in the morning, whereas yogurt intake was less frequent. Parmesan cheese intake, according to our definition, was reported in about 90% of subjects.

Most subjects were on ART (93%), seventy-five (67%) had nadir CD4+ T-cell count < 200 (cells/μL) and 41 (36.6%) were co-infected with hepatitis C virus (HCV) (Table 1).

Osteopenia was present in 38 (34%) HIV-infected vs 19 (25%) HIV-uninfected subjects (p = ns). In detail, at the lumbar site osteopenia was present in 37 (33%) HIV-infected vs 17 (22%) HIV-uninfected subjects (p < 0.02); at the femoral site osteopenia was present in 42 (37.5%) HIV-infected vs 18 (24%) HIV-uninfected subjects (p = ns). In the setting of HIV-infection, osteopenia was present in 18 subjects at both the lumbar and femoral sites.

Osteoporosis was present in 34 (30.3%) HIV-infected vs 8 (10%) HIV-uninfected subjects (p < 0.05). At the lumbar site osteoporosis was present in 34 (30%) HIV-infected vs 8 (10%) HIV-uninfected subjects (p < 0.05); at the femoral site osteoporosis was present in 9 (8%) HIV-infected vs 4 (5%) HIV-uninfected subjects (p = ns).

In the setting of HIV-infection osteoporosis was present in 9 subjects at both the lumbar and femoral sites.

Serum bone alkaline phosphatase was significantly higher in HIV-infected (54.7 range 19–170 IU/L) than uninfected subjects (28.2 range 5–84 IU/L) (p < 0.0001), while phosphorus levels were lower in HIV-infected (3.1 range 1.4-4.4 mg/dL) than in uninfected subjects (3.5 range 2.1-6.8 mg/dL) (p < 0.0001). 25-hydroxyvitamin D levels were also lower in HIV-subjects (16.4 range 0.7-74 ng/mL) than in controls (20 range 9–40.2 ng/mL) (p < 0.02).

Calcium and parathyroid hormone levels were not significantly different in the two groups of patients (data not shown).

The correlations between the studied risk factors and lumbar spine and femoral neck DXA T-scores are presented in Table 2. Age was negatively correlated with BMD measured in both the lumbar spine and femoral neck (p < 0.05). Higher BMI values were positively correlated with BMD values measured in the femoral neck (p < 0.05).

By stratifying patients into two groups (milk or milk + yogurt) we found that osteopenia and osteoporosis were significantly higher in the patients with milk only than in those with milk + yogurt (χ_MH= ² 5.6; p < 0.02).

As regards biochemical markers, increasing values of bone alkaline phosphatase were negatively correlated with lumbar spine and femoral neck DXA T-scores (p < 0.002; p < 0.005, respectively).

Duration of HIV-infection was negatively correlated with BMD in the femoral neck (p < 0.01). BMD in both lumbar spine and femoral neck DXA T-scores was negatively correlated with drug addiction (p < 0.04; p < 0.02, respectively), nadir CD4+ T-cell count < 200 (cells/μL) (p < 0.05; p < 0.04, respectively), ART exposure score (p < 0.002; p < 0.0001, respectively), PI exposure score (p < 0.05; p < 0.001, respectively) and HCV co-infection (p < 0.001; p < 0.005, respectively) (Table 2).

The mean of the 10-year risk of fracture, calculated in 76 HIV-infected subjects, was 1.2% for hip fracture and 4.7% for major osteoporotic fracture.

Figure 1 shows the positive correlation between BMI values and quartiles of dairy intake (ρ = 0.36; p < 0.0001). Lower dairy intake quartiles were more frequent in subjects with osteoporosis than without, although the difference was not significant (ρ = −0.17; p = ns).

No correlations between calcium intake and overall fracture risk at 10 years (r = −0.12; p = ns) and between calcium intake and risk of hip fracture at 10 years were found (r = −0.13; p = ns).

At multiple linear regression analysis, age and HIV/HCV co-infection were negatively correlated with BMD measured in the lumbar spine (p < 0.0001; p < 0.005, respectively), while an independent positive correlation was found between yogurt intake and BMD in the same site (p < 0.05). In the femoral neck T-score, age, nadir CD4+ T-cell count < 200 (cells/μL) and drug addiction (p < 0.01; p < 0.05; p < 0.0001, respectively) were negatively correlated with BMD (Table 3).