Nutritional shortfalls, particularly in trace elements such as zinc, are common in aging populations and can exacerbate immune decline. In South Asia, where diets frequently emphasize plant-based staples high in phytates that inhibit zinc absorption, the prevalence of inadequate zinc intake remains substantial. National nutrition surveys in India have reported an increase in the proportion of individuals with inadequate absorbable zinc intake from approximately 17% in 1983 to 25% in 2011–12.^1.2 Studies in rural and tribal communities have documented prevalence levels exceeding 60–70% in certain population subgroups, including older adults.^2,3 Among elderly individuals (≥65 years), zinc inadequacy has been reported in around 50–60% of women in parts of central India, often occurring alongside broader micronutrient deficiencies^3,4. These deficiencies may contribute to sarcopenia, frailty, and cardiometabolic vulnerability in later life.

Globally, zinc deficiency has been identified as a meaningful and preventable contributor to morbidity and mortality in low- and middle-income countries, accounting for a measurable burden of infectious-disease outcomes in regions such as India, Nigeria, and Ethiopia^5,6. Zinc plays a central biological role in more than 300 enzymatic reactions, including those governing DNA synthesis, antioxidant defense, and immune signaling⁷. In older adults, zinc deficiency intersects with immunosenescence — the progressive decline in adaptive immunity characterized by thymic involution, reduced naïve T-cell output, and chronic low-grade inflammation⁸. Low serum zinc concentrations (<70 µg/dL) have been associated with reduced T-cell maturation, thymic atrophy, and dysregulated cytokine activity, which may increase susceptibility to respiratory infections, including pneumonia^9,10. Observational studies suggest that elders with marginal zinc status may experience higher rates of respiratory illness and slower recovery compared with zinc-replete peers^9,10. These vulnerabilities may be amplified in South Asian contexts due to socioeconomic constraints, limited access to fortified foods, and coexisting micronutrient deficits such as vitamin D and vitamin B12 insufficiency^3,4.

Restoring zinc status through supplementation has shown promise in older adults with low baseline zinc levels. Randomized controlled trials and meta-analyses have reported that zinc supplementation (≈15–50 mg/day) can improve immune biomarkers — including T-cell proliferation and natural-killer-cell activity — with greater effects observed among individuals who are zinc-deficient at baseline^11,12. More recent clinical protocols conducted in institutionalized and community-dwelling elders have explored moderate-dose regimens (30–60 mg/day) and reported improvements in serum zinc concentrations and inflammatory indices over short-term intervention periods^9-13. However, few studies have focused explicitly on older adults with confirmed low zinc status in resource-constrained South Asian settings, where dietary phytate exposure may further blunt absorption^2,4. Evidence on dose–response relationships and the feasibility of short-duration interventions adapted to local diets also remains limited. We therefore conducted this pilot study to examine whether 30 mg/day zinc for 3 months could improve serum zinc concentrations in older adults with marginal zinc status in a South Indian outpatient population and to explore associations with cellular immune function using mitogen-stimulated T-cell proliferation assays. By focusing on a real-world clinical setting, this study seeks to inform feasible, targeted supplementation strategies that could help reduce infection-related vulnerability and health-care burden among aging populations in South Asia.

“This single-center, randomized controlled pilot trial evaluated the short-term biochemical and immunological effects of zinc sulfate on adults aged ≥65 years with serum zinc <70 μg/dL at screening.” The study adhered to CONSORT guidelines for RCTs and was registered prospectively. After ethics approval from Khaja Banda Nawaz University (KBNU) Faculty of Medical Sciences’s Institutional Ethics Committee, we enrolled participants from local outpatient clinics in Gulbarga, Karnataka, South India, between January 2023 and June 2024 (overall study span: 18 months, including recruitment and follow-up). Recruitment involved screening 150 consecutive elders attending geriatric and general medicine clinics for routine check-ups; 45 (30%) met initial criteria based on medical history (no acute illness, stable chronic conditions). Serum zinc was then assayed in these 45, confirming 30 with deficiency (<70 μg/dL via flame atomic absorption spectrophotometry, calibrated against certified standards; inter-assay CV <5%). Inclusion required stable health (no acute illness or hospitalization in prior 4 weeks), no recent zinc or multimineral therapy (within 3 months), and ability to provide informed consent (or proxy from next-of-kin for participants with documented cognitive impairment). Exclusions encompassed severe organ failure (e.g., eGFR <30 mL/min), active malignancy, gastrointestinal disorders affecting absorption (e.g., untreated celiac), or medications known to alter zinc kinetics (e.g., proton pump inhibitors at high doses, chelators like deferoxamine). Eligible individuals gave written informed consent in local languages (Kannada/Urdu) and were randomized 1:1 to intervention (30 mg elemental zinc/day as zinc sulfate) or control (5 mg/day zinc sulfate, approximating minimal dietary needs to mimic placebo without full blinding challenges in a pilot) using computer-generated random blocks (block size 4; allocation concealed via sealed envelopes). Wording confirming envelopes were opaque and sequentially numbered. Both arms received identical-appearing gelatin capsules in weekly blister packs, taken with breakfast under clinic staff supervision during monthly visits to promote compliance (>90% verified via pill counts and self-reported logs). A standardized low-dose multivitamin (50% RDA for vitamins A, C, D, E, B-group, and minerals excluding zinc/copper; e.g., 400 IU vitamin D, 2 mg copper to mitigate antagonism) was provided to all, substituting prior supplements and issued monthly.

 Power and Allocation

Drawing from geriatric zinc trials anticipating a 15–20 μg/dL post-treatment rise (SD ~8 μg/dL; e.g., from 2020–2025 RCTs showing 10–25% gains in deficient cohorts), we calculated n=15/arm for 95% power (α=0.05, two-sided) using G*Power software (version 3.1.9.7; effect size d=1.0 based on prior deltas). This pilot prioritized feasibility over larger samples, with secondary outcomes exploratory (underpowered; the detectable effect size for proliferation outcomes was limited ~60% for proliferation).

 Assessments
At baseline (pre-dose) and 3 months (fasting, 8–10 AM), we collected 10 mL venous blood for comprehensive profiling. Serum zinc and copper were quantified by flame atomic absorption spectrophotometry (PerkinElmer Analyst 400; detection limit 0.5 μg/dL). Albumin and globulin used colorimetric assays (Beckman Coulter AU480); CRP via high-sensitivity ELISA (R&D Systems; range 0.1–10 mg/L, sensitivity 0.02 mg/L); LDH by enzymatic kinetic method (IFCC standard); metallothionein by commercial ELISA (Abcam; intra-assay CV <10%).

Full blood counts (CBC) and differentials were via automated analyzer (Sysmex XN-1000). Lymphocyte subsets employed flow cytometry (BD FACSCalibur): 100 μL EDTA-anticoagulated blood stained with fluorochrome-conjugated antibodies (CD3-FITC, CD4-PE, CD8-APC, CD19-PerCP, CD56-PE-Cy7; BD Biosciences), analyzed with FlowJo v10 (≥10,000 events/gate; absolute counts via Trucount beads).

T-cell proliferation isolated peripheral blood mononuclear cells (PBMCs) via Ficoll density gradient (1.077 g/mL), seeding 2×10⁵ cells/well. Normalization: cpm per 10³ T-cells/μL whole blood. Intra-assay variability <15%. Demographics, comorbidities… Safety: Adverse events graded per CTCAE v5.0, queried monthly. The primary outcome was 3-month serum zinc change (μg/dL). Secondaries encompassed proliferation metrics.

Analysis
We used SPSS v.27. Primary analysis: ANCOVA for changes (95% CIs), covarying baseline values and depression (as imbalanced).

Significance: P<0.05 (no multiplicity adjustment, exploratory). Intention-to-treat with last-observation-carried-forward for dropouts (no dropouts occurred during follow-up, verified through visit records); sensitivity: Per-protocol identical.

Thirty participants (mean age 72.5 years; 63% female) completed the trial, with no withdrawals. Baseline screening confirmed all <70 μg/dL; minor drifts occurred pre-randomization, but groups remained comparable (Table 1). Comorbidities (e.g., hypertension 60%, diabetes 40%) and supplement use (e.g., vitamin D 40%) were balanced. Depression prevalence was higher in controls (55% vs. 35%; P=0.04, chi-square), but other factors (e.g., NSAIDs 52%) did not differ. No zinc-impacting drugs were changed.

Biochemical Changes (Table 2)

Zinc rose robustly in the intervention arm (from 66.9 ± 11.6 to 85.0 ± 12.7 μg/dL) vs. decline in controls (63.2 ± 10.0 to 60.9 ± 9.3 μg/dL; adjusted β=20.91 ± 2.35 μg/dL, P<0.001; 31% relative gain). Copper, globulin, and metallothionein showed no group differences (all P>0.15). Albumin dipped slightly more in zinc recipients (β=−0.20 ± 0.06 g/dL, P=0.003), possibly nutritional. LDH increased notably in zinc (β=24.33 ± 5.55 IU/L, P<0.001), without clinical correlates. CRP was stable (P=0.520).

Immune Markers (Table 3)

Baseline lymphocyte subsets and proliferation were similar. Zinc enhanced anti-CD3/CD28 responses (β=8.73 ± 3.15 ×1000 cpm, P=0.010) and phytohemagglutinin (β=14.99 ± 3.70 ×1000 cpm, P<0.001). Total T-cells grew more in zinc (from ~1150 to ~1370/μL vs. minor control rise; β=219.75 ± 40.67/μL, P<0.001). However, per-cell proliferation (cpm/T-cell) lacked significance (P>0.10), implying number-driven effects. Other subsets (e.g., CD4+) trended up but not tested formally.

Adherence was 95%; mild GI upset occurred in 2 zinc participants (resolved).

TABLE-1: Baseline characteristics of subjects

Abbreviations: BMI = body mass index; NSAID = non-steroidal anti-inflammatory drug.

Note: Values are mean ± SD for continuous variables and % (n) for categorical variables.

Statistical tests: Welch’s t-test for continuous variables; Fisher’s exact test for categorical variables.

Dash (—) indicates p-value not applicable because values are identical across groups.

Table 2: ¹Effect of zinc supplementation on serum zinc and other biochemical measures in elderly with low serum zinc concentration

1t test for independent samples between zinc and placebo groups at baseline is not significant for all variables. CRP, C-reactive protein; LDH, lactate dehydrogenase.

2“Outcome measures” refers to the change in concentrations of measures of interest between baseline and month 3. β [difference in the mean outcome measures between treatment groups (zinc group = 1 and placebo group = 0)] ±SE and P value obtained with the use of ANCOVA with outcome measures of interest, controlling for their corresponding baseline concentrations. %Δ obtained as follows: [(outcome measure for zinc group/baseline measure for zinc group) X 100] - [(outcome measure for placebo group/baseline measure for placebo group) X 100].

3P values for this variable were obtained with data analyses done in the natural logarithmic scale to normalize distribution; however, unlogged data are presented in the table.

⁴95% CIs were derived as β ± 1.96 × SE.

Table 3: Effect of zinc supplementation on lymphocyte proliferation measures in elderly nursing home residents with low serum zinc concentration¹

Values are mean ± SD. Outcomes are reported in units of 1000 counts per minute (cpm ×10³).

Positive β indicates a greater increase in the zinc group versus control.

Adjusted effects represent the between-group difference in change from baseline to 3 months, estimated from a linear model adjusted for baseline value and T-cell count.

Serum zinc is widely used as the most practical biomarker for identifying population-level zinc deficiency, although individual variability remains a limitation. Consistent with prior trials in older adults, some studies have reported increases in circulating zinc following supplementation, whereas others show modest or no change; these differences likely reflect variation in study design, baseline status, and dose and duration of supplementation^14-16.

In our study, daily supplementation with 30 mg of zinc sulfate increased serum concentrations in participants with marginal deficiency, producing an adjusted net gain of approximately 21 μg/dL, while control participants showed small declines across follow-up. These findings are broadly consistent with earlier work reporting greater improvements at moderate-to-higher supplemental doses compared with low-dose regimens^16-22.

Participants with more severe deficiency at baseline (≤55–60 μg/dL) demonstrated a smaller rise in serum zinc, suggesting that longer supplementation or higher dosing may be required in this subgroup; however, the mechanism for this pattern cannot be inferred from our data and remains speculative.

The intervention was generally well tolerated, and estimated total zinc intake remained below the tolerable upper intake level. Copper concentrations were unchanged, and no hematologic abnormalities were observed. A rise in LDH was noted but occurred in the absence of clinical symptoms; this finding should be interpreted cautiously and monitored in future trials.

Zinc sufficiency is important for immune function across multiple cellular processes. In this cohort, supplementation was associated with increases in total T-cell counts and higher mitogen-stimulated proliferation responses. After adjusting for cell number, per-cell proliferation was not different between groups, indicating that the apparent functional gain may primarily reflect expansion of circulating T-cell populations rather than enhanced activity at the single-cell level. This interpretation is consistent with prior hypotheses that zinc status may influence survival or turnover of T-cell precursors, although the present study was not designed to test mechanistic pathways.

Key limitations include the small sample size, short duration, and absence of clinical outcomes such as infection events. Strengths include targeted recruitment of zinc-deficient older adults and standardized biochemical and immunologic assessments. Future studies should evaluate longer supplementation periods, explore tailored dosing for individuals with severe deficiency, and incorporate mechanistic and microbiome-related analyses where appropriate.

Zinc at 30 mg/day offers a safe, accessible means to improve the serum zinc levels and optimize status and T-cell pools in marginally deficient elders, with implications for immunity. Tailored protocols could curb age-linked vulnerabilities.

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