Definitions

Geriatric population. For the WHO, an older person is defined by a civil age over 65 years old. In high-income countries, most people above the age of 65 remain very active and healthy, making impossible to consider a definition of geriatrics solely based on chronological age. The concept of frailty is nowadays a cornerstone to define geriatrics.26 The cut-off in European hospitals for admission in geriatric units is usually 75 years old, and in most cases, patients admitted in geriatric units share criteria of frailty or even dependence.

Vitamin C and AA are used interchangeably in this study. However, on a biochemical basis the term vitamin C refers to the L-ascorbic acid enantiomer, which is the most encountered form. The D-ascorbic acid enantiomer has no significant antiscorbutic activity. Under physiological pH, AA is predominantly found under its anionic form called ascorbate.2 The vitamin C plasmatic level is known as ascorbemia, or plasmatic vitamin C. It is expressed in µmol/L.

Hypovitaminosis C is defined by an ascorbemia under the threshold of 23 µmol/L (figure 1). In some studies, the limit is set at 28 µmol/L; here we use the consensual threshold of 23 µmol/L.23 Depending on hypovitaminosis C depth, two subgroups are described: vitamin C deficiency and vitamin C depletion.

Vitamin C deficiency corresponds to a moderate hypovitaminosis C and is defined by a plasmatic asborbemia ranging between 11 and 23 µmol/L (figure 1). Symptoms are likely to be subclinical or absent.

Vitamin C depletion corresponds to a profound hypovitaminosis C with plasmatic asborbemia below 11 µmol/L (figure 1). This value defines the biological scurvy and is generally associated with clinical features of scurvy. Under 5 µmol/L, the depletion is severe.

Study design

To assess hypovitaminosis C prevalence in a population of hospitalised older patients, we designed a monocentric cross-sectional study with a prospective recruitment. Research objectives are summarised in table 2. Patients’ recruitment takes place within the department of gerontology of the Edouard Herriot Hospital (Hospices Civils de Lyon, France). Two geriatric acute care units (AGU) from this department participate to the enrolment (figure 2).

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Figure 2

Patient pathway in the study, from admission, to end of participation. GACU, geriatric acute care unit.

Three hundred and eighty-five patients are expected to be enrolled for a total of 7–9 months depending on the recruiting rate. The enrolment started on 9 January 2023.

All eligible patients will be offered participation in the study by the investigating physicians. If the written consent is obtained, an extra 4 mL blood sample dedicated to the determination of plasmatic ascorbemia will be collected. The requirements for this biological sampling are the following:

• The blood sampling will never be considered before the obtention of a written consent.

• The written consent will be obtained within a maximum of 2 days after admission in the AGU and the blood sampling will be done within a maximum of 3 days after admission.

• As often as possible, the blood sample will be taken concomitantly to another routine care blood test to avoid extra venipuncture. Exceptionally, if no routine care venipuncture is expected to meet the study timing, a sample dedicated to the determination of plasma ascorbemia may be taken with the patient’s agreement.

• After a fasting period of at least 6 hours to homogenise sampling conditions.

For patients with vitamin C deficiency, a dietary advice sheet aimed at normalising the intake of vitamin C intake through the diet will be given. For patients with a vitamin C depletion, an oral supplementation will be prescribed and a follow-up is organised to collect potential side effects of the oral supplementation.

Study population

The study will be presented to all men and women aged more than 75 years old and being admitted to the recruiting AGU from the Hospital Edouard Herriot. To take part of the study, an affiliation to the national social security system and a written consent are needed.

Participants will be excluded if they have been hospitalised for at least 7 days straight before their transfer to the AGU, or if a previous hospitalisation has ended within the 7 days before their admission to the AGU. The 7 days threshold has been arbitrarily set by the authors to avoid enrolment of patients with plasmatic ascorbemia not representative of the home level. Also, no blood sample will be taken if the admission in the geriatric unit is for a terminal palliative care; in this case the participation in the study will not be considered. For regulatory purpose, patients under legal protection (eg, guardianship and curatorship) will not be enrolled, likewise if a written consent is not obtained.

The different pathways of patients before their admission to the AGU are the following:

• After admission in the emergency department for an acute pathology such as a fall, an infection or an organ failure.

• After a transfer from another unit. Most likely within a few days after an orthopaedic surgery consecutive to a traumatic fall, but also after a stay in a critical care unit, or any other medical or surgery department.

• Straight from home at the request of the general practitioner for an acute pathology or a diagnostic assessment that cannot be done on an outpatient basis.

These characteristics define the targeted study population.

As this exploratory setting is a first step before a larger study, it has several limits we need to keep in mind. First, it is a monocentric study. Also, the recruited population concerns a significant proportion of patients with a heavy burden of comorbidities. Thus, the extrapolation of the results to the general geriatric population and particularly to patients living a successful ageing must be careful.

Finally, it is important to mention that geographical and socioeconomical aspects are likely to affect the generalisability of our results. Particularly in low-income populations and outside Europeanised countries where a heightened susceptibility to food-insecurity or difficulties accessing vitamin C rich food may exist. However, these remarks also suggest hypovitaminosis C could be even higher in these settings.

Consent

According to the French law, this study is considered as an excluding health products research involving human subjects with low risk and constraint. It implies the obtention of a written consent for patient’s participation (Jardé law, 2016).

Due to prevalence of acute confusion, neurocognitive disorders, or functional impairments among hospitalised older patients, different consent option must be considered:

• For patients able to consent, the study will be explained in appropriate terms and the written consent will be collected by investigating physicians after reflection time if wished by the participant.

• For patients who are not capable of giving their own written consent, the study will be explained to a trustworthy person, or failing that family, or failing that a close relative. The patient’s refusal will not be overridden, and a written consent will be collected from this referee.

As often as possible, the written consent will be sought the day of admission, so the ascorbemia blood test is done the next morning. To address specific situations, the consent can be obtained within a maximum of 2 days notably when the patient has been admitted to the AGU during the weekend, or when the written consent must be obtained from a third party. Any consent can be withdrawn at any time of the study by the signatory and for any reason; in this case, the patient will leave the study prematurely and his data collected up to the date of withdrawal will be analysed.

Data collection

Information collected from the initial geriatric evaluation and used by this research project are listed in table 1. Data from the geriatric and medical evaluation are variables of interest (secondary outcomes). Variables have been chosen to match with already known risk factors previously described, or to explore geriatric syndromes such as mobility and functional independence, falls, cognitive function, malnutrition and feeding issues, polypathology and polypharmacy. Social context information is also collected.

If the written consent is obtained, a unique inclusion number will be assigned to each patient. Data will be collected from the institutional electronic patient record, and anonymously reported on an electronical case report form (eCRF). The eCRF developed for this study uses the REDCAP software (V.10.8.5). REDCAP complies with the FDA recommendations on authentication and electronic signatures (21CFR part 11).

The result from the plasmatic ascorbemia will be implemented when obtained.

Patients are not allowed to participate more than once in the study.

Vitamin C assay

As AA is a strong antioxidant quickly degraded inside the sampling tube, the technical process has to be well protocolised.27 This implies a pretreatment phase consisting of a plasmatic deproteinisation and chelation. In the participating AGUs, biological samples are sent to the laboratory via a pneumatic tube transport system ensuring a rapid transfer. Samples in heparinised blood tubes must be sent at ambient temperature in a maximum of 2 hours after venipuncture.28 29

Immediately after samples arrive at the laboratory, the plasma is separated from blood formed elements with a 10 min centrifugation at 2500 g. Three hundred microlitres of plasma are pipetted and mixed with 600 µL of a stabilisation solution containing 6% sulfosalicilyc acid, EDTA 2 mM and N-ethylmaleimide 20 mM. The sample is vortexed for 10 s and kept frozen at −20°C.30 31

Samples are analysed by the reference method using high-performance liquid chromatography coupled to electrochemical detection with H-Class Waters chromatographic system (Waters, St Quentin en Yvelines, France) and Empower3_HF1_Enterprise software (V.7.30.00.00, Waters). After defrosting, stabilised plasma are centrifuged 10 min at 15000 g. The supernatant is vortexed in equal proportions with the vitamin C mobile phase buffer (Chromsystems, Gräfelfing, Germany) and injected into the chromatographic system. The separation is run at 37° over a reverse column using isocratic elution with vitamin C mobile phase (Chromsystems) containing KCl 3M (Merck KGaA, Darmstadt, Germany). AA is oxidised at a potential of +0.60V and quantity of electrons generated during this oxidation reaction is quantified. With this method, we only measure vitamin C in its reduced form.

Acidified supernatants are kept frozen for a maximum of 1 week at −18°C and batch vitamin C assay is performed at least once a week.

Because vitamin C is a powerful redox agent, there are some limits we need to be aware of. Preanalytical bias is well controlled and standardised in our setup as blood samples are quickly taken to the laboratory and stabilised. However, we cannot exclude an inter-individual variation of the timing between venipuncture and stabilisation. Concerning postanalytical limits, despite the commonly admitted value to define hypovitaminosis C,23 there is no strictly consensual threshold to affirm 23 µmol/L is the minimal sufficient ascorbemia to ensure vitamin C-related functions. Notably, a higher cut-off sometimes set at 28 by certain authors, would lead to a potentially overestimated prevalence. Thus, the estimated prevalence by this cross-sectional study must be considered as a minimal prevalence for hypovitaminosis C.

Also, the plasmatic vitamin C does not perfectly reflect the vitaminic pool of the human body; it is assumed it could be mostly representative of vitamin C intakes.2 Intra-leucocyte AA could be a better measure to assess vitamin C capital, but the technique is complex and not generalised within the scientific literature. Because of these limitations, ascorbemia assessment is not recommended in routine practice in France.

Follow-up

As the participation to the study may lead to the discovery of an unsuspected hypovitaminosis C and thus to an oral supplementation, a follow-up for concerned patients is organised after the obtention of ascorbemia within 7–14 days after admission in the AGU. Three cases are possible depending on the AA status (figure 2):

• Case number 1: normo-ascorbemia. No follow-up is required; the patient’s participation ends with the obtention of the plasmatic ascorbemia result.

• Case number 2: vitamin C deficiency (11–23 µmol/L). No follow-up is required. The patient’s participation ends with the obtention of the plasmatic ascorbemia result. However, a letter by post will be sent to the patient, including the vitamin C status notification together with a dietary advice summary. This document lists the most common vitamin C-riched fruits and vegetables, and the average daily amount required to insure a sufficient vitamin C intake. There is no national neither international recommendation to prescribe an oral supplementation for this group of patients, as the normalisation of food intakes is expected to restore the vitaminic status.

• Case number 3: vitamin C depletion (<11 µmol/L). For this specific group of patients, a letter by post will be sent including the vitamin C status notification and the dietary advice summary. Because of the depth of the hypovitaminosis C, an oral supplementation is required to prevent the evolution toward the potentially fatal scurvy. A prescription for an oral vitamin C supplementation will be attached together with a form to be reimbursed by the study sponsor (Hospices Civils de Lyon). The oral supplementation consists of 500 mg of oral vitamin C, two times a day, for 15 days. The normalisation of food intakes is then required to maintain a normal vitaminic status.

For participants eligible to an oral supplementation, a phone call will be given 1 month after sending the prescription, at the patient’s home, or failing that to the place where the patient is staying (eg, rehabilitation centre, retirement home). The purpose of this phone call is to ensure the oral supplementation has been well taken, and to ask for the occurrence of adverse events related to the treatment. Potential adverse events are headaches, gastrointestinal disorders, cutaneous rash and kidney stones formation. Oral vitamin C side effects are unlikely to occur if the 1 g/day posology is not exceeded,32 with a wide safety range even in case of overuse. Thus, vitamin C oral supplementation is considered as an easy and safe treatment. In case of adverse event, occurrences will be reported to relevant authorities according to the French laws.

Sample size

As hypovitaminosis C prevalence is poorly documented, we hypothesised it could concern half of the hospitalised geriatric patients in acute care units. Because of the greater uncertainty, this 50% estimated prevalence leads to the calculation of the greatest sample size.33 With an expected proportion of 50%, a precision of 5% and a bilateral alpha risk of 5%, the inclusion of 385 patients is needed.

Statistical analyses

Patient and public involvement

Patients or the public were not involved in the trial design or planning of the study. There are no plans to involve patients in dissemination of the results.