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Clinical Science

Brief Report: Zinc Supplementation and Inflammation in Treated HIV

Dirajlal-Fargo, Sahera MS, DOa,b; Yu, Jiao BSb; Kulkarni, Manjusha PhDc; Sattar, Abdus PhDb; Funderburg, Nicholas PhDc; Barkoukis, Hope PhDb; Mccomsey, Grace A. MD, FIDSAa,d

Author Information

aDepartment of Pediatrics, Division of Pediatric Infectious Diseases, Rainbow Babies and Children's Hospital, Cleveland, OH;

bDepartment of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH;

cDivision of Medical Laboratory Science, Ohio State University School of Health and Rehabilitation Sciences, Columbus, OH; and

dDepartment of Nutrition, School of Medicine, University Hospitals Cleveland Medical Center, Cleveland, OH.

Correspondence to: Grace McComsey, MD, FIDSA, Medicine University Hospitals Cleveland Medical Center, Case School of Medicine, 2061 Cornell Road, Mail Stop: 5083, Cleveland, OH 44106 (e-mail: [email protected]).

Supported by a grant from NCCIH (AT009153) to G.A.M. Additional support was provided by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) to S.D.-F (K23HD088295-01A1).

Presented in part at the Conference for Retroviruses and Opportunistic Infections, Seattle, WA, March 2019.

G.A.M. served as a consultant for Gilead, BMS, GSK/Viiv, and Merck, and has received research funding from Gilead, Merck, GSK/Viiv, and BMS. N.F. serves as a consultant for Gilead. The remaining authors have no conflicts of interest to disclose.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

G.A.M. designed the study. A.S. and J.Y. provided statistical support. M.K. and N.F. assisted with the biomarker assays. S.D.-F wrote the first draft of the manuscript. All authors contributed to data analysis and reviewed the manuscript for intellectual content.

JAIDS Journal of Acquired Immune Deficiency Syndromes: November 1, 2019 - Volume 82 - Issue 3 - p 275-280
doi: 10.1097/QAI.0000000000002129
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Abstract

INTRODUCTION

The WHO estimates that nearly 2 billion individuals may be zinc-deficient in resource-limited settings. Before effective antiretroviral therapy (ART), zinc deficiency was prevalent in adults living with HIV (PLHIV).1–3 Low plasma zinc levels have been linked with disease progression.4 Unlike what is observed with most nutrients, the prevalence of zinc deficiency seems to be high, even after participants are treated with ART.3

Studies of PLHIV show that although ART decreases inflammation,5 inflammatory markers remain elevated and increasing evidence suggests that chronic inflammation plays an important role in cardiovascular disease (CVD).

Zinc deficiency may be associated with diseases, similarly to HIV, that are associated with ongoing inflammation such as rheumatoid arthritis, diabetes, and atherosclerosis.6–8 Zinc homeostasis is important for several aspects of the immune system; however, there is a lack of data related to the effect of zinc status on the heightened inflammation and monocyte activation state in HIV.

The current study is an open-labeled randomized double-arm study, assessing the efficacy and safety of zinc supplementation for 16 weeks in PLHIV, who were on stable ART, and had documented zinc deficiency. Our primary objective was to examine the effect of zinc on inflammation markers in this population; secondary objectives were to assess the effect of the intervention more specifically on monocyte activation, gut epithelial barrier dysfunction and microbial translocation that have been associated with comorbidities and increased mortality in HIV.9–12

METHODS

Study Design

This is a pilot open-labeled randomized double-arm study. Participants were prospectively enrolled at University Hospitals Case Medical Center, Cleveland, OH. The study was approved by the local institutional review board, and written informed consent was provided by all participants (ClinicalTrials.gov Identifier: NCT02856269). All participants were aged ≥18 years, with HIV-1 infection on stable ART for at least 3 months with cumulative ART duration of at least 6 months, HIV-1 RNA <400 copies/mL in the last 4 months before study entry. Participants were excluded if they were pregnant, lactating, or had an active infectious or inflammatory condition. Uncontrolled diabetes and known CVDs were excluded. We focused on participants with documented zinc deficiency, as defined by participants having serum zinc levels ≤75 µg/dL in the last 60 days.

Zinc Supplementation

The recommended dietary allowance for zinc is an intake of 11 mg zinc/d for men and 8 mg zinc/d for women.13 Normal serum zinc levels range between 75 and 150 µg/dL. We have used 45 and 90 mg elemental zinc/d as oral supplementation, because it has been used safely in nonHIV studies for a 1-year duration, and at this concentration, with no serious adverse events.14 We used zinc gluconate as the form most commonly used and with the most available safety data in clinical trials outside of HIV.14–20

Randomization and Blinding

The randomization schedule was performed by a statistician using SAS software to create a list based on permuted variably sized block randomization. Participants were randomized by the study pharmacist to gluconate capsules at a dose of 45 mg (low-dose) or 90 mg (high-dose) of elemental zinc daily.

Patients either took 1 (45 mg) or 2 (90 mg) zinc gluconate capsules. The research assistant, the principle investigator, and all laboratory personnel performing the study assays were blinded to treatment assignment.

Primary and Secondary Outcomes

At entry and week 16, fasting (for at least 8 hours) blood draws were obtained and plasma and serum stored for measurement of a marker of monocyte activation soluble CD14 (sCD14), of systemic inflammation [high sensitivity C reactive protein and soluble tumor necrosis alpha receptor I and II (sTNFR-I and II)], which were measured by ELISA (R &D Systems, Minneapolis, MN and ALPCO, Salem, NH and Mercodia, Uppsala, Sweden). Lipopolysaccharide binding protein (LBP), a marker of microbial translocation (Hycult Biotech Inc., Plymouth Meeting, PA) and a marker of gut integrity, intestinal fatty acid binding protein (IFABP; R &D Systems) were measured by ELISA.21–23 The intra-assay and interassay variability were less than 10% for all markers. All assays were done at Dr Funderburg's laboratory at Ohio State University, Columbus, OH.

At entry, weeks 4, 10 and 16, zinc adherence and adverse events were assessed.

The DSMB met every 6 months to review safety data and adverse events.

Statistical Analysis

The primary objective of this pilot study was to assess the safety and tolerability of zinc in PLHIV. The secondary outcomes were to demonstrate the impact on the biological signatures in PLHIV (markers of inflammation, monocyte activation, coagulation, and microbial translocation).

First, we presented summary statistics for most of the relevant biomarkers and demographic characteristics measured at the baseline. We compared various markers and characteristics for the 2 groups of participants using the Wilcoxon rank sum tests, χ2 tests, and Fisher exact tests, as appropriate. Next, we presented the changes of the zinc levels over the study period using Box plots for the 2 arms of the study. We calculated the percentages of participants who experienced a biomarker reduction to evaluate the impact of zinc supplementation. To assess the meaningful change in biological signatures, we performed significance tests for the shift of distributions of the selected biomarkers after the zinc supplementation using the Kolmogorov-Smironow (K-S) test. The amount of shift or effect size was measured using Cohen D value. All statistical analyses for this study were conducted using the statistical software Stata 15.024 and R 3.4.0.

RESULTS

Baseline Characteristics

A total of 52 participants were enrolled (25 participants in the low-dose arm and 27 participants in the high-dose arm). Baseline characteristics are shown in Table 1. Median (interquartile range) age was 49 (38, 60) years, 77% were men and 73% were African Americans. Participants in the low-dose arm were significantly older and had longer duration of ART. At baseline, median zinc levels were 73 (64, 86) µg/dL.

T1
TABLE 1.:
Baseline Characteristics

Zinc Safety and Changes Over Time

After 16 weeks, loss to follow-up was minimal with 94% retention. There was 1 loss to follow-up in the low-dose arm and 3 participants lost to follow-up in the high-dose arm. There was one possible study-related grade 3 adverse event: a patient who developed nausea and abdominal cramps 6 days after initiating high-dose zinc supplement.

Median circulating zinc levels increased from 74 to 91 µg/dL in the low-dose arm and from 73 to 100 µg/dL in the high-dose arm.

In addition, 88% of participants in the low-dose arm and 96% in the high-dose arm reached zinc levels >75 µg/dL.

Impact on Biological Signature

Overall, 48%–60% of participants experienced a reduction in biomarkers in the combined arms (Fig. 1). There was a larger proportion of participants with reduction in sCD14 and IFABP in the participants in the low-dose arm; however more participants had decreases in LBP and sTNF-RI in the high-dose arm. Biomarker reductions were meaningful with Cohen D ranging from 5.25 to 19.44.

F1
FIGURE 1.:
The proportion of participants with biomarker changes.

DISCUSSION

In this pilot study, we found that in PLHIV on effective ART, zinc supplementation is safe, effective at increasing zinc levels and decreasing biomarkers known to be elevated in this population and associated with clinical comorbidities.

Zinc Deficiency

Zinc is an essential nutrient and is involved in vital pathways including protein synthesis and gene transcription.25 Zinc deficiency is the fifth leading cause for loss of healthy life years.26 In the Nutrition for Healthy Living study, 40% of men and 36% of women on ART had low zinc levels, and participants in the upper quartiles of zinc had lower HIV viral load levels than those in the lowest quartile.3 Mean zinc levels in PLHIV from different cohorts range between 60 and 75 µg/dL.3,27,28 In previous zinc supplementation trials performed in PLHIV, the participants were either not on ART, or not virally suppressed.27–32 In this study, we assessed for the first time in HIV, the effects of zinc supplementation in participants virally suppressed on effective ART with low baseline zinc levels. We found that zinc supplementation at both high- and low-doses is well-tolerated with no serious toxicity. Importantly, we also found that 45 mg and 90 mg of elemental zinc daily are enough in PLHIV to raise serum zinc levels above 75 µg/dL in most.

Zinc and Inflammation

Zinc deficiency may be associated with diseases, similarly to HIV, that are associated with ongoing inflammation such as rheumatoid arthritis, diabetes, and atherosclerosis.6–8 Zinc has known antioxidative and anti-inflammatory properties33 and is involved in immune regulation including cell maturation, cell differentiation, and apoptosis.34 Zinc homeostasis is important for several aspects of the immune system. Zinc deficiency results in an increased sensitivity to the effects of oxidative stress. As previously noted, no studies have investigated the relationship between zinc status and systemic inflammation in ART-treated PLHIV. We measured soluble CD14, a marker of monocyte activation in response to microbial translocation, because it has been linked to mortality in HIV10 and by our group, to coronary calcification and subclinical vascular disease.35 The other markers of systemic inflammation were chosen because they have predicted HIV comorbidities,36–38 and are relevant to CVD research.

In our study, we found that zinc supplementation in PLHIV with zinc deficiency can decrease markers of systemic inflammation, monocyte activation, and microbial translocation in some cases in more than half of the participants. Although the margin of reduction in the biomarkers after zinc supplementation did not surpass 21%, the quantitative measure of the magnitude of change represented by Cohen's D(the difference of the means divided by the SD) is large for all biomarkers. We hypothesize that the size of the reduction is likely secondary to the short period of supplementation in our study.

Zinc and Intestinal Barrier

In PLHIV, the causes of the heightened immune activation remain unclear, but one of the key contributors is likely gut epithelial barrier dysfunction and microbial translocation.39 The benefits of zinc supplementation during diarrheal episodes, in areas with high prevalence of zinc deficiency, are well-documented.40 Dietary zinc deficiency causes impairment of gut architecture, such as shorter and narrower jejunal villi, reduced absorptive surface area, decreased number of mitochondria, swelling of the endoplasmic reticulum, and atrophic Golgi apparatus, accompanied by increased membrane permeability and declined cell mobility.41 In this study, we measured IFABP, a marker of enterocyte damage, as IFABP leaks out of damaged small intestine epithelial cells.42 We also measured LPB, which is produced by hepatocytes in response to the presence lipopolysaccharide (LPS) and other bacterial products in the portal venous system. The differences seen in the levels of LBP and IFABP between the 2 arms were unexpected. We suspect, however, that the regulation of the intestinal barrier is likely a complex process, and immune and dietary factors both modulate the barrier function. Our findings support our hypothesis that zinc supplementation in ART-treated HIV infection has the potential to be an effective agent in decreasing microbial translocation and improving gut epithelial barrier dysfunction. Further studies are needed to determine zinc's role on the intestinal barrier in HIV, and whether its effect is dose-dependent.

Our study has several limitations. Because of the small sample size and short study period, we cannot establish causation. We did not include a placebo control group for comparison. Our study focused on participants with treated HIV and documented zinc deficiency in the United States; therefore, our findings cannot be generalized to different populations or to resource-limited settings. We did not obtain dietary or physical activity patterns. Finally, the participants in the high-dose arm were significantly younger than the participants in the low-dose arm and with shorter ART duration, which may have affected our findings.

This is the first study to investigate zinc supplementation in PLHIV with viral suppression and zinc deficiency. Studying the relationship between zinc and inflammation in PLHIV is an innovative approach to prevention of comorbidities as opposed to only treatment of established disease. In addition, we offer potential mechanistic pathways for the relationship between zinc and systemic inflammation by demonstrating a potential role of zinc on gut epithelial barrier dysfunction and result microbial translocation.

Larger studies are crucial to investigate the role of zinc supplementation in HIV comorbidities linked to inflammation.

ACKNOWLEDGMENTS

The authors would like to thank the patients who participated in this research.

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Keywords:

HIV; zinc; inflammation; immune activation; morbidity; gut integrity

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