Assessment of Plasma Levels of Bone Metabolism Biomarkers among HIV Infected Adult Patients in Ethiopia

Background: Bone diseases are common complications in HIV infected patients with increased fracture rates. Factors, calcium and Vitamin D deficiency, depression and smoking are contributed to increased prevalence of this disease. Some studies reported that antiretroviral therapy may also relate to bone diseases in HIV patients. Aim of the study: To assess plasma levels of bone metabolism biomarkers in HIV infected patients before and after ART treatment. Materials and methods: Leftover blood samples from advanced clinical monitoring project were used for this study. 156 individuals with HIV infection before and after ART at different stages of treatment (0, 6 and 12 months) were investigated. Plasma level of bone metabolism biomarkers like PTH, phosphate, Vitamin D and Osteocalcin were measured using Cobas 6000 chemistry machine. Data analysis was carried out by means of statistical package for social science version 16. Results: The mean level of PTH and Osteocalcin increased significantly in HIV infected patients taking ART compared to HIV patients not taking ART (p value<0.05). Viral load was negatively correlated with PTH, Osteocalcin, Vitamin D and Phosphate. Conclusions: As the duration of treatment with ART extended, further increase of the higher rate of bone turnover in HIVinfected patients was observed.


Bone metabolic disorder in HIV patients
Bone diseases are common complications in HIV infected patients with fracture rates three times higher than HIV uninfected individuals [7][8][9].
Bone loss in HIV infected persons can be aggravated by pathophysiological disruptions in the body such as hormonal imbalances, inflammatory cytokines action and kidney pathological processes [10].Other studies revealed that bone loss in HIV infected individuals occurs as a consequence of HIV viral protein and antiretroviral therapy [11][12][13][14][15][16][17][18].

Demographic characteristics
Out of 156 study participants, 45.5% were female.All study participants were in the age group between 18-45 years.

Specimen collection, handling, storage and processing
Plasma samples stored at -80°C was used for this study.The samples were de-frozen and centrifuged at 3000 rpm for 10 minutes before analysis.

Laboratory sample analysis
Blood levels of bone turnover biomarkers including Osteocalcin (OC), Phosphate, intact Parathyroid hormone (iPTH) and Vitamin D were determined using standard procedures [33,34] by Cobas 6000 chemistry analyzer.Quality controls and calibrators were run before sample analysis to confirm the quality of the testing reagents and proper functioning of the machine.

Antiretroviral drugs and bone metabolic complications
Antiretroviral Drugs (ART) are medication for treatment of infection by HIV.These drugs block various steps of the HIV replication cycle and inhibit the progression of the virus [19].
ART has significantly improved the morbidity and mortality in HIV-infected patients by preventing opportunistic infections, restoring and retaining immune function, increasing CD4+ cell counts, and delaying progression to AIDS [20].These benefits are compromised by numerous side effects, adverse clinical events and toxicities [21].
Due to complex metabolic complications of HIV infection and its treatment, the reduction in bone mineralization was observed in a large percentage of HIV-infected patients.This might be as the result of interplay of host, viral and specific antiretroviral factors [22].
One longitudinal cohort study revealed a marked incidence of low BMD in a large number of patients with long-term HIV infection and prolonged antiretroviral therapy [22].

Bone metabolism biomarkers
Bone is made of collagen, Calcium and phosphate [23].Parathyroid Hormone (PTH) is a polypeptide hormone which regulates plasma Ca 2+ and PO 4 [24][25][26] by sensing protein in the cell membrane of the parathyroid cells [27,28].
Osteocalcin is the main non-collagen protein of bone matrix which is synthesized during the bone formation [7,29].The deficiencies of calcium and phosphate lower the formation of the hydroxyapatite crystals, which make the free osteocalcin to circulate in the blood.This results in increased concentrations of osteocalcin in the plasma of osteoporotic patients [8].
Due to its tissue specificity; its wide availability and its relatively low variation within person, osteocalcin can be considered as a clinical index of bone turnover.In general, a high bone turnover rate can be characterized by elevated levels of plasma osteocalcin [29].
Bone mineralization and calcium homeostasis can be maintained by the action of Vitamin D. Low Vitamin D concentrations might lead to secondary hyperparathyroidism that results in bone resorption and fracture risk [30].

Statement of Problem
Though, the introduction of potent antiretroviral drugs resulted in significant reduction of mortality and morbidity in HIV patients, the new problems due to HIV infection and adverse effects of antiretroviral drugs have been critical [31].Currently, bone metabolic disorders have been emerged as a worrisome complication in HIV infected patients.It is not clear that if HIV infection itself or antiretroviral treatment or both are causes of bone loss; however, most of the studies have shown a high prevalence of osteoporosis in HIV/AIDS patients [32].In this area, no studies were conducted in Ethiopia and this study supports to understand the relation between bone

Discussion
In this study prolonged exposure to antiretroviral drugs resulted in increased bone resorption of HIV patients.This finding aligned with the study of Anna et al. [21] that indicated around 75% of study participants under long follow-up of ART treatment has shown some grade of low bone mass, followed by one third having developed osteoporosis.
In the current study, bone loss caused by ART is evident by significant increase in the mean level of PTH in HIV infected patients taking ART compared HIV patients not taking ART.This finding is in agreement with the work done by Zhao et al. [35] that indicated increased plasma PTH level caused an increase in bone resorption.Study done by Bonofiglio et al. [36] also stated that PTH is inversely related to bone mineral density after adjustment for calcium intake and bone age.
According to Kota et al. [37] people with hyperparathyroidism produce too much parathyroid hormone for too long.This can cause calcium to be dissolved from the bones.Over time, if the hyperparathyroidism is not treated, loss of calcium from the bones can lead to decreased bone density and weakening of the bones.Eventually, osteoporosis may develop.
According to our finding significant increase in the mean level of Osteocalcin was also observed in HIV infected patients taking ART compared to HIV patients not taking ART.This is in consistent with the work done by Deeks et al. [38] that indicated ART is accompanied by increases in bone biomarker activation, which may lead to mechanisms underlying bone loss in HIV infected individuals.
Our study observed that the mean levels of Vitamin D was <30 ng/ml in all groups.According to Sahota et al. [39], the lower levels of Vitamin D (<30 ng/ml) and increasing PTH levels were associated with lower bone mineral density.This

Statistical analysis
Statistical analysis was performed using statistical package for windows (SPSS v16).The data were entered into the SPSS format and the continuous variables were summarized by using the mean and standard error of the mean.The data were analyzed by one way ANOVA followed by Tukey-Kramer post hoc test.P values less than 0.05 were considered to be statistically significant.Pearson's correlation test was also performed to compare the bone metabolism biomarkers with CD4 cell count and viral load.

Determination of bone biomarkers
The mean level of iPTH and Osteocalcin increased significantly in HIV infected patients taking ART compared to HIV patients not taking ART (p value<0.05).There were no significant differences in plasma levels of phosphorous and Vitamin D in HIV patients taking ART and not taking ART (p value>0.05)(Table 1).
There was no significant variation between male and females with HIV before and after treatment in plasma levels of iPTH, Osteocalcin and phosphate.However; there is a significant difference in plasma level of Vitamin D (Table 2).
The results of the Pearson's correlation test showed CD4 cell count was positively correlated with Osteocalcin and negatively correlated with iPTH, Vitamin D and phosphate (Table 3).Viral load was negatively correlated with plasma levels of iPTH, Osteocalcin, Vitamin D and phosphate (Table 4).This study showed significant decrease in mean levels of Vitamin D in female when compared to male.This finding is in agreement with study done by Verdoia et al. [40], where female gender was associated with lower Vitamin D levels.

Conclusions
As the duration of treatment with ART extended, further increase of the higher rate of bone turnover in HIV-infected patients was observed.

Recommendation
Assessment, screening, diagnosis and follow-up of bone turnover should be conducted in all HIV patients commencing ART; throughout the course of ART therapy.Moreover; ART drug for osteoporosis should be identified and remedial action should be taken accordingly.

Limitation of the Study
This study didn't show Calcium pattern because blood Plasma samples taken in EDTA tubes were used that is Calcium can be masked by plasma EDTA.

Table 1 :
Comparison of the Mean ± SEM of the bone biomarker parameters

Table 3 :
Bivariate correlation between CD4 and variables

Table 4 :
Bivariate correlation between viral load and variables