Introduction

Population ageing is an unavoidable demographic transition. A rapid increase in the number of elderly people (aged 60 years and above) has become a global phenomenon. By 2050, the world’s elderly population is expected to be 2 billion (22% of the total population), up from 0.9 billion (12% of the total population) in 2015, and 80% of them will live in low and middle-income countries.1 In India, there has been a sharp increase in the number of elderly people. It has been observed that the general population has grown by 12.4% during 2011–2021 in comparison with around 18% in 2001–2011, while the elderly population grew by 36% in each of the last two decades (2001–2011 and 2011–2021).2 India’s elderly population count is projected to reach 194 million in 2031 from 138 million in 2021.2

Anaemia is a condition in which the number of red blood cells and their oxygen-carrying capacity is insufficient to meet the body’s physiological needs.3 It is a common, multifactorial condition among the elderly. The prevalence of anaemia increases with age, representing an important health problem among older individuals. Worldwide, every fourth individual aged 60 years or above is anaemic,4 and in India, the prevalence ranges between 21% and 92%.5 6 Unfortunately, many symptoms of anaemia like weakness, fatigue and shortness of breath are often misdiagnosed as normal phenomena in elderly individuals. Anaemia has been reported to be associated independently with the decreased functional ability/physical function, increased dementia, and increased risks of falls, morbidity, and mortality in the elderly population.7–9 Altogether, anaemia among the elderly population grossly affects the health-related quality of life and also increases mortality considerably.

Rationale

In the elderly population, anaemia of chronic disease (ACD) and nutritional anaemia especially due to iron deficiency (IDA) are two of the most common causes of anaemia—each contributing around one-third of the total prevalence and both the conditions often coexist.10 This makes the treatment of anaemia in the elderly difficult, since response to oral iron is often slow, with a substantial fraction of patients showing refractoriness and requiring cumbersome intravenous administration.11 In the last decade, the discovery of the iron regulatory hormone hepcidin has revolutionised our understanding of iron metabolism. Hepcidin has been reported to be a systemic iron regulator and high hepcidin levels block intestinal iron absorption and macrophage iron recycling, causing iron-restricted erythropoiesis and anaemia.12 Vitamin D, which exists in two major forms (D3 and D2),13 has recently been linked in the stimulation of erythroid precursors and ultimately rate of erythropoiesis.14 An in vitro study carried out in human cell lines demonstrated that administration of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D for 6 hours led to a 50% reduction in hepcidin mRNA expression,15 supporting the postulated hypothesis that hepcidin suppression may facilitate iron status regulation by increasing iron uptake and storage. Evidence from a clinical trial supports the in vitro findings and showed that the intervention group had statistically significant increase in haemoglobin and haematocrit values over 8 weeks of follow-up.11 The study included 50 women aged 19–49 years who received a daily oral dose of vitamin D3 (1500 IU) supplementation with iron-fortified breakfast (containing approximately 9 mg of iron) as intervention. However, the sample size and duration were apparently small and short, respectively. It is known that for getting significant change in haemoglobin levels after oral supplementation, a minimum follow-up of 12 weeks is required.16–18 Moreover, no such study has been done so far among the elderly in India.

Most of the studies have reported that anaemia in the elderly is a significant public health problem with prevalence ranging from 23% to 92%.5 6 19–21 The severity of anaemia has been reported to increase with age, especially after 80 years of age. In most of the studies, significant association of anaemia has been reported with age, gender, socioeconomic status, types of diet, chronic disease and calorie intake.

Previous randomised controlled trials done to study the effect of vitamin D3 supplementation with iron on haematological indicators were not specifically done among the elderly population and were limited by inadequate sample size.11 22 23

ObjectivesPrimary objective

To estimate the impact of supplementation of a therapeutic package of iron and folic acid (IFA) and vitamin D3 on haemoglobin levels in the elderly with mild-to-moderate anaemia in comparison with IFA only.

Secondary objective

To estimate the changes in ferritin, hepcidin, 25-hydroxyvitamin D and parathyroid hormone (PTH) levels.

Methods and analysisTrial design

This study will be a community-based, placebo-controlled, double-blind, randomised trial. Groups will be parallel, allocation ratio will be 1:1 and framework will be superiority. The study will be done in the Kalyani municipality area. Kalyani is one of the earliest planned cities of India and is located 50 km away from the metropolitan city of Kolkata, West Bengal. The municipality area is divided into 21 ward/block areas—all similar in sociodemographic and economic characteristics and cater to a population of around one lakh. There are 2 urban primary health centres and 10 subcentres in the municipality.

Individuals aged 60 years and above and residing in the study setting will be the study population for the initial screening of anaemia and vitamin D3 levels. Elderly individuals with mild-to-moderate anaemia with normal vitamin D3 levels will be the study population for the trial. The study participants will be selected randomly.

Eligibility criteria

Individuals aged 60 years and above, permanent residents of the area, are expected to stay in the area for another 6 months, give consent to participate in the study, and diagnosed to have mild-to-moderate anaemia without vitamin D deficiency will be eligible for the study. Individuals with no anaemia (male: haemoglobin >130 g/L and female: haemoglobin >120 g/L), with severe anaemia (haemoglobin <80 g/L), with known history of gastrointestinal and metabolic disorders, with known history of chronic renal failure (>1.4 mg/dL creatinine level), who received blood donation in the last 6 months and regularly consumed nutritional supplements (iron, folic acid, vitamin B12, vitamin D and other multivitamins and minerals) will be excluded from the study. The screening form is provided as online supplemental file 1.

Operational definitionsAnaemia

Haemoglobin level <130 g/L in male and haemoglobin level <120 g/L in female will be considered as having anaemia. Haemoglobin level <80 g/L will be considered as severe anaemia in both sexes.

Iron-deficiency anaemia

Anaemia related to absolute iron deficiency (IDA) was characterised by a decreased serum ferritin level (<30 µg/mL) in combination with low serum C reactive protein (CRP) levels (<0.5 mg/dL).

Anaemia of chronic disease

ACD will be defined by high ferritin levels (>100 µg/mL) and increased CRP (≥0.5 mg/dL).

Iron-deficiency anaemia/anaemia of inflammation

Patients with ferritin levels between 30 µg/mL and 100 µg/mL and high CRP levels (≥0.5 mg/dL) will be classified as having mixed anaemia (IDA/anaemia of inflammation).

Compliance

Individuals who will consume ≥80% of both the tablets (IFA) and capsules (vitamin D3/olive oil) will be considered as compliant with the therapy.

Vitamin D3 deficiency

Serum vitamin D3 levels <20 ng/mL will be considered as vitamin D3 deficiency.

Intervention

In this study, the eligible individuals will be randomised into intervention and control group. In the intervention arm, the study participants will receive 60 mg of elemental oral iron as IFA tablets and 1500 IU of vitamin D3 as capsules daily for 3 months. In the control group, the study participants will receive 60 mg of elemental oral iron as IFA tablets and capsules containing olive oil for daily oral intake for 3 months. All the IFA tablets will be containing 500 µg folic acid. The tablets will be manufactured in certified laboratories.

The intervention and control groups will receive the same 60 mg of elemental oral iron as IFA tablets daily. The capsules for intervention and control arm will be different in their constituents (intervention group: vitamin D3 capsule 1500 IU; control group: olive oil capsule) but similar in all other aspects.

If any individual develops side effects associated with IFA supplementation like excessive vomiting even after consuming the medicine 1 hour after a major meal and without any other obvious reason, or side effects related to hypervitaminosis of D3 like tinnitus, palpitations, confusion or excessive fatigue, he/she will be advised to discontinue taking the medicine.

An individual will be considered lost to follow-up if he/she could not be contacted on two repeated visits with a minimal interval of 1 week, has transferred residency to another place permanently, died during the course of the trial or could not be available for blood collection at the end of the 3-month trial.

Participants requiring any other medical advice will be referred to All India Institute of Medical Sciences (AIIMS) Kalyani for further management.

OutcomesPrimary outcome

Change in haemoglobin levels from baseline to 12 weeks in both groups.

Secondary outcome

Change in serum ferritin, hepcidin, 25-hydroxyvitamin D and PTH levels from baseline to 12 weeks in both groups.

Participant timeline

Figure 1 shows the recruitment process and participant timeline.

Figure 1

Study flow chart. CRP, C reactive protein; Hb, haemoglobin; IFA, iron–folic acid; PTH, parathyroid hormone.

Sample size

Sample size was determined based on the primary outcome being change in haemoglobin levels from baseline to 12 weeks in both groups. Assuming a superiority margin of 0.4 g/dL, mean (SD) observed difference of 0.7 g/dL between the two groups before and after the intervention,11 power of 80%, alpha error of 5% and attrition rate of 10%, we need to recruit 150 participants in both the study arms. The sample size was calculated using nMaster V.2.0. Assuming prevalence of vitamin D3 deficiency was 56% or higher among the elderly,24 around 1000 individuals would have to be screened for their vitamin D3 levels.

Recruitment

The study setting being an urban field practice area of a medical college, the principal investigator has the list of households which have elderly people. Out of 22 100 households, currently, there are 2112 households which have elderly individuals. The first 1000 households will be selected randomly using computer-generated random numbers. If more than two elderly individuals reside in a single household, one of them will be selected by using the lottery method. The randomly selected individuals aged 60 years and above will be screened for their haemoglobin levels using the HemoCue 301 device. This device has very high sensitivity and specificity as compared with the gold-standard method.25 Individuals who will be diagnosed with mild-to-moderate anaemia and will be fulfilling other inclusion criteria will be requested for blood samples (5 mL) for the assessment of their vitamin D3 level. The blood sample will be drawn aseptically from the antecubital vein from each participant and will be stored in a plain vial. The sample will be handed over to the institute on the same day. Serum will be separated from the blood samples at the laboratory of the Department of Biochemistry using a centrifuge machine at 5000 rpm. The separated serum will be used to assess the vitamin D3 level and serum creatinine level. Individuals with vitamin D3 level >20 ng/mL and a normal serum creatinine level will be enrolled in the study. Written informed consent will be taken at the time of the blood sample collection and again during the enrolment in the study.

Randomisation

Before the start of the intervention, a third person not related to the study will do block randomisation dividing the intervention and control groups in equal numbers. He/she will sort and pack the similar-looking tablets and capsules into pouches which will be serially numbered according to the random sequence generated. The serial numbered opaque and sealed pouches will be subdivided into 30 tablets (IFA) and 30 capsules (vitamin D3/olive oil) each so that compliance can be measured on a monthly basis. The investigator will be handed over only serial numbered pouches containing IFA tablets and vitamin D3/olive oil capsules which will be unlabelled, and the original content inside each pouch will only be known to the third person who generated the random sequence and did the packaging to ensure allocation concealment. Since the tablets/capsules which will be used in the study will look similar in all aspects, both the investigator and the study participants will be blinded.

Treatment armsIntervention group

Study participants in this group will receive 60 mg of elemental oral iron therapy as IFA tablets and 1500 IU of vitamin D3 capsules daily for 3 months. The participant will be asked to take the tablets on an empty stomach and the capsules after lunch. The field investigators will visit each study participant on a weekly basis to record the side effects. Compliance will be calculated on a monthly basis. If the participant develops any side effect, the study participant will be asked to take the tablet 1 hour after a regular meal.

Control group

Study participants in this group will receive 60 mg of elemental oral iron therapy as IFA tablets and olive oil capsules for daily oral intake for 3 months. The participant will be asked to take the tablets on an empty stomach and the capsules after lunch. The field investigators will visit each of the study participants on a weekly basis to record the side effects. Compliance will be calculated on a monthly basis. If the participant develops any side effect, the study participant will be asked to take the tablet 1 hour after a regular meal.

Drugs

The drugs required for the study include two sets: (1) IFA tablets containing 60 mg of elemental iron and 500 µg folic acid; and (2) vitamin D3 capsules containing 1500 IU of vitamin D3. Placebo pills contain olive oil. The tablets will be manufactured in certified laboratories. Both the tablets and capsules will be similar in all aspects except for the vitamin D3 content.

Data collection

Baseline sociodemographic information (age, gender, area of residence, education, marital status, religion, caste, number of household members, gross family income, financial independence, etc) and personal information (diet type, presence of chewing disability, comorbidities, height, weight, etc) will be collected through a pretested semistructured interview from all included individuals (online supplemental file 2: case record form).

Five millilitres (5 mL) of blood will be drawn aseptically from the antecubital vein of each participant and will be stored in a plain vial. The sample will be handed over to the institute on the same day. Serum will be separated from the blood sample at the laboratory of the Department of Biochemistry using a centrifuge machine at 3000 rpm. The separated serum will be stored at −20°C. Initially, 25-hydroxyvitamin D (vitamin D3) and serum creatinine levels will be assessed. If the levels fulfil the inclusion criteria, the remaining serum sample will be used for measurement of serum hepcidin, ferritin, PTH and CRP. Ferritin will be measured using the chemiluminescence method. CRP and creatinine will be measured using a biochemical auto-analyser. Vitamin D and PTH will be measured using a chemiluminescence technique. All the tests will be done at the laboratory of the Department of Biochemistry. All the tools will be calibrated before and during estimation of haematological and biochemical parameters. The laboratory has their own internal and external quality control measures.

Field investigators will visit study participants on a weekly basis to collect data on side effects. They will also provide the drugs to the study participants on a monthly basis and collect the packets given during the earlier visit. They will calculate the participants’ compliance on a monthly basis. Each participant will be followed up for 12 weeks. The field investigators will be blinded to the group (intervention/control) of the study participants.

The haemoglobin level will be measured using HemoCue 301 after the completion of the intervention. Five millilitres (5 mL) of blood will be again drawn aseptically from the antecubital vein of each participant and will be stored in a plain vial, which will undergo similar transport, processing and finally storage as done for the baseline samples. The stored serum will be used for measurement of serum hepcidin, ferritin, 25-hydroxyvitamin D, PTH and CRP at the laboratory of the Department of Biochemistry. The technique for biomarker measurements (ferritin, CRP, hepcidin, 25-hydroxyvitamin D, PTH), calibration, and internal and external quality assessments will be similar as the one stated for the baseline samples. The team which will analyse the sample will be unaware of the group (intervention/control) of the study participants.

We will be assessing any side effects with routine visits every week (‘adverse drug reaction profile’ section in the case record form found in online supplemental file 2). Participants developing any adverse effect will be treated at AIIMS Kalyani, West Bengal, India.

Study tools

Haemoglobin will be measured using HemoCue 301 instrument, while hepcidin will be measured using the ELISA method. Moreover, CRP and creatinine will be measured in a biochemical auto-analyser, whereas ferritin, vitamin D and PTH will be measured using a chemiluminescence technique.

Study duration

The study will be completed in a duration of 12 months. The project will have three phases: (1) preparatory phase (2 months), (2) implementation phase (9 months), and (3) data analysis and final report writing phase (1 month).

Preparatory phase: recruitment of staff, project manual development, procurement of survey logistics, development of the study questionnaire, and training of field staff in data collection, quality control and blood sample collection will be done. The packaging of drugs and random sequence generation by a third person not related to study will also be done.

Implementation phase: screening, enrolment, randomisation, intervention and biochemical analysis of the samples will be done during this phase.

Recruitment of the participants started in September 2023, and the data collection is expected to be completed by March 2024.

Data analysis

The data will be collected in Epicollect V.5, a mobile-based app, and exported in a Microsoft Excel sheet. Data quality will be ensured during the data feeding on the mobile-based app, through usage of range checks and compulsory fields. The data entered will be rechecked for completeness and validity. The final data will be stored safely on an encrypted pen-drive which will be securely stored by the principal investigator.

The data will be analysed with the help of STATA V.14 statistical software. Categorical data will be presented as percentage (%). Pearson’s Χ2 test will be used to evaluate differences between groups for categorised variables. Continuous variables will be checked for normal distribution using Shapiro-Francia test. Normally distributed data will be presented as means and SD or 95% CI. Student’s t-test for independent samples will be used for comparison between groups with continuous variables and paired t-test will be used for comparison before and after changes. If the continuous variable is found to be non-normally distributed, median and IQR will be reported, and Wilcoxon signed-rank test will be applied to test for comparison for before and after changes. Both the per-protocol and intention-to-treat analyses will be done. A p value of <0.05 will be considered as statistically significant.

Patient and public involvement

None.

Ethics and dissemination

Ethical approval from the Institutional Ethics Committee of AIIMS Kalyani has been obtained (IEC/AIIMS/Kalyani/Meeting/2022/03). Guidelines pertaining to the participant information sheet (PIS) and participant informed consent form (PICF) will be followed. The PIS will be given to each study participant. The PIS will be in the local language (Bengali) and will be provided and read out to each study participant before their recruitment. Written informed consent will be obtained from each study participant willing to participate in the study, by the field investigator. The PICF is provided as online supplemental file 3. Participants developing adverse effects or requiring any other medical advice will be referred to AIIMS Kalyani for further management. Participants will be provided with the following information: (a) expected duration of participation in the study, (b) the benefits to be expected from the research, (c) any risk associated with the study, and (d) maintenance of confidentiality of records.

Data collected from individuals will be deidentified using only identity numbers and will be securely stored by the principal investigator.

Participants will have the freedom to participate or withdraw from the study at any time without penalty or loss of benefits to which the study participant would otherwise be entitled. The intervention used in the recent trial is safe as daily supplementation of 1500 IU of vitamin D3 is considered completely safe.26

The trial results will be reported through publication in a reputable journal and disseminated through health talks within the communities.

Any changes in the protocol during the trial will be informed in advance to the ethical committee of AIIMS Kalyani, and necessary permissions for the changes will be taken.

Any change in the protocol will be updated in the trial registry record and it will be ensured that all protocol amendments will be detailed in the journal manuscript where the trial results will be published.