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Brief Reports

Lack of benefit with omega-3 fatty acid supplementation in HIV patients: A randomized pilot study

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Pages 99-105 | Received 07 Feb 2019, Accepted 02 Aug 2019, Published online: 03 Sep 2019

Abstract

Objective: To assess the effect of omega-3 polyunsaturated fatty acids (n-3 PUFA) supplementation on bone metabolism in HIV-infected patients presenting with hypertriglyceridemia.

Methods: Patients were randomized 1:1 to receive 2 g of n-3 PUFA or fenofibrate (FF). The primary endpoint was % change in bone mineral density (BMD) from baseline to month 24 in lumbar spine (LS) and femoral neck (FN). Secondary endpoints were changes in Z-score, calcitriol, calcitonin, parathyroid hormone, osteocalcin, and C-terminal telopeptide of type I collagen (CTX-I) at 12 and 24 months. Differences in continuous variables were evaluated using the t test or Mann-Whitney U-test for independent samples and differences in means of intra- and inter-subject continuous variables using a general linear model. Categorical variables were compared by the chi-squared or Fisher’s exact test.

Results: 30 patients were included in each arm; 23 in the n-3 PUFA arm and 22 in the FF arm completed follow-up. No significant differences between arms were observed after 24 months in either region (FN: −12.51% ± 7.89 in the n-3 PUFA arm and -8.18% ± 7.72 in the FF arm, p = .07; LS: 2.94% ± 6.63 in the n-3 PUFA arm, −3.07% ± 16.85 in the FF arm, p = .07), although the BMD reduction in the FN region after 24 months was noticeable in both arms (n-3 PUFA: −12.51% ± 7.89%, p =< .001; FF: −8.183% ± 7.72%, p =< .001). There was a significant difference in calcitriol changes between arms after 96 weeks. No differences in Z-score or bone turnover markers were observed between the two arms.

Conclusions: Omega-3 fatty acid supplementation resulted in no beneficial changes in BMD or bone turnover markers. n-3 PUFA supplementation achieved similar reductions in triglyceride levels as FF.

Background

Life expectancy for people living with HIV is likely to be similar to the uninfected population. However, age-related co-morbidities are now the main contributors to mortality, particularly in stable, treated HIV-infected patients.

Osteoporosis is highly prevalent in this populationCitation1 and accounts for a higher incidence of fractures.Citation2,Citation3 In addition to traditional risk factors, other causes have been observed to increase bone disorders. Metabolic abnormalities, antiretroviral therapy (ART) including protease inhibitors (PI) or tenofovir disoproxil fumarate (TDF), and residual and persistent inflammatory response with high concentrations of TNF-α, interleukin-1, interleukin-6 and interleukin-11 can promote significant loss of bone mineral density (BMD).Citation4 HIV viral proteins and the host immune response contribute to changes in the balance of bone formation and resorption. In vitro, HIV proteins Tat and Nef reduce the number of bone marrow mesenchymal stem cells available to differentiate into osteoblasts by inducing senescence of these cells through NF-kB activation and inhibition of autophagy, respectively. HIV proteins Tat and Rev increase monocyte differentiation into osteoclasts and also boost osteoclast resorption function by increasing reactive oxygen species and TNF-α production in osteoclast precursors. B-cell dysfunction associated with HIV infection has also been suggested as a possible mechanism for bone loss in HIV. It has been shown that there is a higher frequency of RANKL-expressing B cells (resting memory and exhausted tissue-like memory B cells) and a lower frequency of OPG-expressing B cells (resting memory B cells) in HIV-infected patients compared with HIV-uninfected individuals. RANKL/OPG ratio has also been correlated with total hip BMD and with T and Z-scores in HIV-infected participants.Citation4 A decrease in osteoblast function and vitamin D function as well as osteoclast activation may explain some of these changes.Citation5–9

Omega-3 polyunsaturated fatty acids (n-3 PUFA) has shown lipid-lowering effects in subjects with hypertriglyceridemia, it is recommended for the treatment of this abnormality in the HIV population.Citation10–12 Long-term n-3 PUFA supplementation in HIV-uninfected individuals resulted in BMD increase as well as improvement in bone markers. Two different mechanisms have been postulated: 1) a reduction in pro-inflammatory cytokines, critical to turnover regulation and calcium balance modulation, and 2) upregulation of intestinal calcium absorption, making n-3 PUFA crucial for maximal vitamin D-dependent calcium absorption.Citation13,Citation14

The aim of our study was to assess the effect of n-3 PUFA supplementation on bone metabolism in HIV-infected patients presenting with hypertriglyceridemia.

Design and methods

A randomized, open-label pilot clinical trial was conducted at the HIV Unit of a teaching hospital in Barcelona. HIV-infected persons aged 40 years or older with a CD4 count >200 cells/µL and serum triglycerides >2.2 mmol/L were eligible for recruitment. All subjects were on stable ART and had undetectable viral load for the previous 3 months. Exclusion criteria included use of glucocorticoids and/or oral anticoagulants. Vitamin D supplementation was not allowed during follow-up. The study was approved by the hospital ethics committee, and written consent for participation was signed by all participants (EudraCT number: 2010-023071-25).

Patients were randomized 1:1 to receive either n-3 PUFA, 2 g daily (460 mg eicosapentaenoic acid and 540 mg docosahexaenoic acid per capsule; Omacor®, Laboratorios Ferrer, Sant Just Desvern, Spain) or fenofibrate (FF), 145 mg daily. Arms were stratified by sex (male vs female), age (40-59 y vs 60 y or older), and ART (non-nucleoside reverse-transcriptase inhibitors [NNRTIs] vs PI).

All subjects underwent a 24-month follow-up. The primary endpoint was percent change from baseline to month 24 in lumbar spine (LS) and femoral neck (FN) density assessed by QDR 4500 DX (Hologic Iberia, Barcelona, Spain). Secondary endpoints were changes from baseline in calcitriol, calcitonin, parathyroid hormone, osteocalcin (N-Mid™ Osteocalcin), and C-terminal telopeptide of type I collagen (CTX-I) (Serum CrossLaps® BioVendor, Immunodiagnostic Systems, Tyne & Wear, United Kingdom) at 12 and 24 months. We retrospectively assessed calcidiol levels at the baseline visit. Adherence was assessed by remaining pill counts at each study visit and self-reported adherence.

Statistical analysis

The sample size was calculated using ene v3.0 software (Servei d’Estadística Aplicada, Universitat Autònoma de Barcelona, Cerdanyola, Spain), obtaining a power of 90% to detect differences in the contrast null hypothesis by a two-tail Student t test for two independent samples. In order to calculate the sample size, we took data from a study published by Jeannie Huang et al.Citation15 In this clinical trial, authors evaluated changes in BMD in HIV-positive subjects after receiving a single dose of intravenous zoledronate. Mean ± SD BMD increase in the experimental group was 3.7%. Significance was set at a p value ≤ 5%, and a mean variation of 0.00% and 3.70% in BMD values was assumed for the control and experimental groups, respectively. Because SD was 4.10 units for each group, in our study we included 27 patients in each arm and increased to 30 participants, assuming a 10% loss to follow-up, giving a total of 60 participants.

Continuous variables were compared between arms by the t test for independent samples or by the Mann-Whitney U-test. Categorical variables were compared by the chi-squared or Fisher test. We used the t test for independent measurements adjusted by Bonferroni for multiple comparisons to evaluate the differences in means of intra- and inter-subject continuous variables using a general linear model. Continuous variables are expressed as the mean ± SD or as the median (interquartile range), depending on the normality of the data, allowing intra- and inter-subject changes to be assessed over the course of the study. For all statistical analyses performed, a p value less than .05 was considered significant. All analyses were performed using SPSS, version 19.0 (IBM SPSS, Chicago, IL, USA).

Results

Between June 2011 and September 2012, 60 patients were included in the study: 53% of participants were on a PI-based regimen, 43% were receiving a NNRTI-based regimen, and the rest were receiving integrase strand transfer inhibitors (INSTIs). No significant differences were found between the two arms at baseline (). A total of 23 subjects in the n-3 PUFA arm and 22 in the FF arm completed follow-up. In the n-3 PUFA arm, 6 subjects withdrew consent, 1 patient died due to recreational drug intoxication, and 1 patient was excluded from the final analysis due to missing data. In the FF arm, 7 patients withdrew consent and 1 patient was lost to follow-up.

Table 1 Baseline characteristics

We observed a significant decrease in BMD from baseline to month 24 in FN in both arms. Subjects receiving n-3 PUFA had a noticeable reduction in BMD in FN (−12.51% ± 7.89%; p < .001) (). Participants in the FF arm also had a significant decrease in BMD (−8.18% ± 7.72%; p < .001). However, this difference between arms was not statistically significant (n-3 PUFA arm: −12.51% ± 7.89%, FF arm: −8.183% ± 7.72%, p = .07). No significant changes in LS were observed between the arms at month 24 (n-3 PUFA arm: 2.94% ± 6.63%; FF arm: −3.07% ± 16.85%, p = .079). We also observed a significant decrease in Z-score from baseline to month 24 in FN in both groups. The n-3 PUFA arm had a significant reduction in Z-score in the FN region (−0.26 ± 0.41; p = .01), and similar results were observed in the FF arm (−0.33 ± 0.38; p = .01) with no differences observed between arms. No significant changes in LS were observed between groups at month 24.

Table 2 Changes from baseline to month 24

Despite the FN changes observed, the decrease observed in BMD in both arms showed no increase in the proportion of new diagnoses of osteoporosis after 24 months (n-3 PUFA arm: FN osteoporosis baseline = 3.8%, 24 months = 4.2%, p = 1; LS osteoporosis baseline = 14.3%, 24 months = 12.5%, p = 0.235; FF arm: FN osteoporosis baseline = 0%, FN at 24 months = 0%, p = 1; LS at baseline = 4.5%; LS at 24 months = 0%).

At month 24, we observed a significant decrease in calcitriol levels in the n-3 PUFA arm (mean change, −34.15 ± 41.52 pmol/L; p = .01). In the FF arm, a nonsignificant reduction was observed between baseline and month 24 (−6.22 ± 44.51 pmol/L; p = 1). A comparison between arms showed significant differences in calcitriol changes (n-3 PUFA arm: −34.15 ± 41.52 pmol/L; FF arm: −6.22 ± 44.51 pmol/L; p = .04).

No significant differences were found between arms in calcidiol levels at baseline (n-3 PUFA arm: 50.14 ± 26.13 nmol/L; FF arm: 60.76 ± 23.24 nmol/L; p = .1). Although we did not observe altered parathyroid hormone levels, mean calcidiol at baseline in the n-3 PUFA arm was near the lower end of the normal range for vitamin D.

No changes were observed in parathyroid hormone, osteocalcin, or CTX-I levels in either arm after month 24.

After 24 months of treatment, plasma triglycerides decreased in both arms (n-3 PUFA arm: −1.32 mmol/L (IQR = −1.82—0.65), p < .001; FF group: −1 mmol/L (IQR = −2.14—0.33), p = .002). Changes between arms from baseline were statistically significant but clinically irrelevant.

We found no statistical association between BMD changes and ART drugs (PI or TDF) after 24 months of follow-up. Age (<50 y or ≥50 y) and immunologic status (CD4 count at baseline stratified by <500 or ≥500 cells/m3) did not influence the changes observed.

Discussion

We performed a randomized, controlled pilot trial of n-3 PUFA supplementation in patients with well-controlled HIV infection and hypertriglyceridemia. Although several studies have suggested that BMD may be reduced with the use of TDF, especially when given with PIs, we did not expect the BMD reductions within arms to be so large. There are at least five different randomized clinical trials in the literature showing no significant effects of n-3 PUFACitation13 supplementation in HIV-uninfected individuals. However, other studies have investigated the role of a variety of n-3 PUFA preparations in subjects with bone disorders, and it has been suggested that the potential effects on bone-related outcomes might be enhanced by including n-3 PUFA fatty acids in foods high in calcium, vitamins, and minerals or in concentrated oil mixtures with other polyunsaturated fatty acids and calcium.Citation13 During our literature review, we found no relevant studies in HIV-infected subjects.

There are several potential mechanisms whereby n-3 PUFA may impact bone; two of the most well recognized involve decreasing proinflammatory cytokines crucial to the regulation of bone turnover and calcium balance. These cytokines are key regulators of the osteoprotegerin/receptor activator of NFKB ligand (OPG/RANKL) ratio in bone.Citation7 RANKL is expressed in osteoblasts and activates its receptor, RANK, which is expressed on osteoclasts. These changes promote osteoclast formation and activation, as well as suppress apoptosis of osteoclasts. Osteoprotegerin (OPG) is a secretory glycoprotein expressed by osteoblasts which blocks RANKL from activating RANK. Although the OPG/RANKL ratio seems to be critical in the pathogenesis of bone disease, with a higher ratio indicating less bone resorption,Citation7 our study did not determine the OPG/RANKL ratio.

Higher n-3 PUFA doses seemed to have no impact on the trend of BMD.Citation16,Citation17 In such case, the lack of effect in our trial could be attributed to an imbalance between the proportion of n-3 PUFA and n-6 PUFA in patient diet. Orchard et al.Citation13 reported that the increase in daily intake of n-3 PUFA was not enough by itself to observe positive effects and that the ratio between n-6 PUFA and n-3 PUFA should be adequate, with the optimal ratio being 1:3.

Baseline calcidiol levels were balanced between arms, but the n-3 PUFA arm had lower values than the FF arm, and half the subjects in this arm had values close to the lower limit to diagnose calcidiol deficiency. Deficiency may lead to a further reduction in calcitriol. The European AIDS Clinical Society guidelinesCitation18 recommend calcidiol supplementation for this situation. Unfortunately, we assessed these results retrospectively, and vitamin D was not supplemented. This may have contributed to the significant loss in BMD in the n-3 PUFA arm.

Our study has a number of limitations. The small sample size (30 patients per arm) with many patients lost to follow-up resulted in insufficient power to confirm that supplementation had no effect. Additionally, the dose administered was probably enough to reduce hypertriglyceridemia, but might not have been sufficient to induce anti-inflammatory changes. Other significant limitations were low levels of 25-(OH)D3 left untreated, lack of monitoring of inflammation biomarkers and OPG/RANKL, and the lack of data on smoking status and androgen/testosterone use. In a study designed to evaluate the beneficial effects of n-3 PUFA on BMD, we observed considerable bone loss in HIV-infected patients with good initial bone mass; hence, its effect on the proportion of osteoporosis patients was low. This bone loss cannot be explained by increased bone turnover, impaired immunologic status, or pharmacologic treatment. Bone loss was lower in n-3 PUFA-treated patients than in FF-treated patients.

In conclusion, n-3 PUFA supplementation yielded no beneficial changes in BMD or bone turnover markers but did help reduce triglyceride levels similarly to FF.

Acknowledgments

We thank the CERCA Program of the Generalitat de Catalunya for institutional support. We also thank the Instituto de Salud Carlos III for funding this study. We dedicate this paper to our colleague and friend Elena Ferrer, MD, who has recently passed away. She largely contributed to the design and implementation of this study. Thanks, Helen, for all your important contributions to our HIV Unit over more than two decades. We will greatly miss you.

Additional information

Notes on contributors

Antonio Navarro-Alcaraz

Antonio Navarro-Alcaraz MSc, He obtained a Nutrition and Dietetics Degree from the University of Barcelona, Spain in 2009 and a MSc degree in Nutrition and Metabolism by the University of Barcelona, Spain in 2011. His clinical practice and research areas include cardiovascular disease, bone metabolism, lipodystrophy and gut microbiota alterations. He is working at the HIV and STD Unit, Bellvitge University Hospital since 2009.

Juan Tiraboschi

Dr Juan Manuel Tiraboschi He obtained a Medical Degree from the National University of Córdoba, Argentina in 1999. He trained in Medicine and Infectious Diseases in Argentina and France (2000–2007). He obtained a PhD degree in 2015 by the University of Barcelona investigating on “Penetration and antiviral activity of antiretroviral drugs in the Central Nervous System”. Between 2013 and 2016, Dr. Tiraboschi completed a fellowship as Clinical Research Doctor in HIV at St Thomas Hospital in London, UK. He joined the HIV and STD Unid at Bellvitge University Hospital in 2016. His clinical and research interests include the effects of HIV and antiretroviral therapy on Central Nervous System and viral reservoirs.

Carmen Gómez

Dr Carmen Gómez She obtained a Medical Degree from the University of Barcelona, Spain in 1991. She completed her Reumathology Medical training in The Hospital Universitari de Bellvitge (1991–1995). She obtained a PhD degree in 2000 by the University of Barcelona.

Beatriz Candas-Estébanez

Dr Beatriz Candas-Estébanez Specialist in Clinical Biochemistry (Hospital Universitari de Bellvitge, 2010). PhD by the University of Barcelona in the Human Molecular Genetics Program with Cum laude qualification (2011). Between 2011 and 2013 associate biochemistry in Servei Laboratori UDIAT of the Sanitary Corporation of the Parc Taulí Hospital (Sabadell) in Core of Biochemistry and serology and genetics of hepatitis. From 2013-present, associate biochemistry in Servei Laboratori Hospital de Bellvitge in the area of special biochemistry and molecular biology responsible for the biochemical aspects related to hormonal biochemistry and fertility. Director of 3 doctoral theses in progress, one of which ends in 2020. Member of the commission of lipidology and cardiovascular risk of the Spanish Society of Laboratory Medicine and the Commission of genetics of the Catalan Association of Clinical Laboratory Sciences. Author of more than 15 scientific publications of national and international scope. Awarded for the best communication in congress of national scope in 2 occasions (first author) and in other three (collaborating author or director of the work). Principal investigator in three calls for competitive public bodies (two completed and one in progress), and collaborator of another of the FIS Carlos III.

María Saumoy

Dr María Saumoy I completed Specialized Medical Training in Internal Medicine in 2003. Since then I have been combining the hospital care task with research in the field of HIV infection. In 2004, through a contract from the Instituto de Salud Carlos III, a post-MIR study carried out at Joan XXIII Hospital in Tarragona, I carried out research on the pathogenesis of metabolic complications related to antiretroviral treatment and the genetic role of these complications. I completed this line of research with a stay at the Institute of Microbiology of Lausanne, with the team of Dr. Amalio Telente. These studies allowed me to elaborate my doctoral thesis. In 2007 I joined the HIV Unit of the Infectious Diseases Service of the Hospital Universitario de Bellvitge where I have continued to this day. I have combined the care activity, basically to patients with HIV infection, with the research. Within the research I have participated in multiple randomized studies of antiretroviral drugs both nationally and internationally. On the other hand, I participated in studies promoted from our Unit with a line of research focused on the study of the pathogenesis of metabolic alterations related to HIV infection and antiretroviral treatment, as well as a study of cardiovascular risk associated with HIV. Some of these studies have been funded by competitive calls from public and private administrations. The result of these studies are the publications in which I appear as the first signer. For the past year, I have been consulting on Sexually Transmitted Diseases and Screening for Anal Cancer, in which we also carry out a research task.

Arkaitz Imaz

Dr Arkaitz Imaz received his MD from University of Navarra in 2002 and completed his Internal Medicine training at Vall d'Hebron Hospital (Barcelona) between 2003 and 2008. In 2010 he joined the Department of Infectious Diseases of Bellvitge University Hospital where he works as a physician in the HIV and STI Unit. Since 2008 he has been involved in clinical research in the field of HIV infections participating in research projects and clinical trials. He obtained his PhD from the Autonomous University of Barcelona in 2012. His main areas of interest are penetration, pharmacokinetics and activity of antiretroviral drugs in reservoirs (especially genital tract), drug-resistance and salvage therapy, sexually transmitted infections and HCV co-infection.

Daniel Podzamczer

Dr Daniel Podzamczer He qualified from the University of Buenos Aires and undertook training in Internal Medicine at Bellvitge University Hospital (Barcelona, Spain). He obtained a PhD degree in 1991 investigating on “predictors of progression to AIDS” (University of Barcelona, Spain). He started to see HIV-infected patients in the first years of the epidemic, that is in the mid of 80'. Thus he has lived very different periods in the history of AIDS. Beginning when AIDS was a fatal disease and patients died every day until now when HIV has become a chronic disease with a prolonged survival and an excellent life quality of patients receiving antiretroviral therapy. He is currently the head of an HIV and STD Unit within an Infectious Disease Service in a University teaching hospital in Barcelona. In his position he has contributed to clinical research, participating and organizing clinical trials related to opportunistic infections, HIV complications and antiretroviral therapy. He is a co-author of more than 250 scientific papers published in national and international Journals such as New Engl. J Med, Lancet, Ann Intern Med, Clin. Infect Dis., J Infect Dis., AIDS and JAIDS among others. He has also participated in expert panels for Guidelines for the management of opportunistic infections (NIH/CDC/IDSA), antiretroviral therapy, opportunistic infections and neurocognitive disorders (GESIDA). He is also a peer reviewer for a number of international Journals.

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