Effects of Monthly Intramuscular High-Dose Vitamin D2 on Serum 25-Hydroxyvitamin D and Immune Parameters in Very Elderly Chinese Patients with Vitamin D Deficiency

Purpose Vitamin D deficiency is highly prevalent among the very elderly and is associated with a wide variety of clinical conditions other than musculoskeletal diseases. This study aims to ascertain the efficacy and safety of high-dose intramuscular vitamin D2 in very elderly Chinese patients with vitamin D deficiency. Methods Very elderly (aged 80 years or over) Chinese patients with vitamin D deficiency were recruited to receive monthly intramuscular injections of 600,000 IU vitamin D2 until their serum 25-hydroxyvitamin D (25(OH)D) reached ≥30 ng/mL. The serum levels of 25(OH)D2, 25(OH)D3, iPTH, BTMs, immune parameters, and other biochemical parameters were measured at baseline and one month after each dose. Results Of the 30 very elderly Chinese patients who had been recruited into the study, 27 (90.0%) had their vitamin D deficiency corrected, and 26 (86.7%) reached vitamin D sufficiency. The mean time (±SD) was 3.1 (±1.3) months for vitamin D deficiency to be corrected, and 6.1 (±0.8) months for vitamin D sufficiency to be reached. The mean (±SD) serum level of 25(OH)D2 increased from 0.69 (±1.51) ng/mL to 29.07 (±5.68) ng/mL, while the mean (±SD) serum level of 25(OH)D3 decreased from 9.82 (±2.75) ng/mL to 5.30 (±3.09) ng/mL (both P < 0.001). The total T cells in serum remained unchanged (P > 0.05), and the CD4 and B cells (CD19+) were increased significantly (both P < 0.05). In addition, no significant change was observed in the serum levels of iPTH and BTMs. Conclusion Monthly intramuscular injection of 600,000 IU vitamin D2 is an effective and safe dosing regimen to reach vitamin D sufficiency and enhances immune function in the very elderly Chinese patients with vitamin D deficiency.


Introduction
Vitamin D deficiency, which is defined as a serum level of 25-hydroxyvitamin D (25(OH)D) below 20 ng/mL [1], is an ever-increasing health concern globally. In addition to musculoskeletal diseases such as muscle weakness, osteomalacia, osteoporosis, and fractures, it has been supposed to associate with an increased risk for a wide variety of clinically relevant conditions in adults such as chronic obstructive pulmonary disease (COPD) [2], acute respiratory distress syndrome (ARDS) [3], COVID-19 [4], infectious diseases [5,6], diabetes mellitus [7,8], and autoimmune diseases [9]. On the other hand, vitamin D supplementation has been reported to accelerate inflammation resolution in hospitalized AECOPD patients [10], reduce mortality in mechanically ventilated patients [11], prevent acute respiratory tract infections [12], etc. Because of insufficient sunlight exposure, decreased function of the skin to synthesize vitamin D diminished intestinal absorption and/or dietary intake of vitamin D, and vitamin D deficiency is widespread in the elderly [13]. As for the very elderly (aged 80 years and over), the prevalence of vitamin D deficiency was 65.7% among 367 Belgian [14], 75.8% among 1324 Chinese [15], and even as high as 89.5% among 153 Lithuanians [16].
A serum level of 25(OH)D ≥30 ng/ml is generally considered vitamin D sufficiency in adults [1]. In addition to increasing sun exposure and dietary intake of vitamin D-rich foods, oral intake and intramuscular injection of vitamin D pharmaceutical preparations are two major routes to reach this target [17]. Daily intake of low-dose vitamin D often needs a long period to reach target vitamin D status, while maintaining long-term adherence is not easy [18]. As a result, dosing regimens with high vitamin D doses at less frequent intervals have been proposed to improve patient compliance and obtain desired intervention outcomes [19]. Some studies demonstrated that intramuscular high-dose vitamin D is an effective approach to vitamin D sufficiency [20,21], and that intramuscular administration is more effective than oral intake in the long term [22].
As we all know, there are two forms of vitamin D in humans. Vitamin D2, also known as ergocalciferol, is entirely obtained from dietary sources such as mushrooms. Vitamin D3, also known as cholecalciferol, is the major source of the human body's requirements for vitamin D [23]. It is mainly derived from dermal synthesis by the action of ultraviolet B (UVB) radiation on 7dehydrocholesterol (7DHC) to generate previtamin D3, which further undergoes a slow thermal isomerization to form vitamin D3 [24]. Vitamin D is hydroxylated at C-25 in the liver to produce 25(OH)D, with the latter being further hydroxylated at C-1 in the kidney to form 1,25(OH)2D, the functional, hormonally active forms of vitamin D. Both vitamin D2 and vitamin D3 injections are suggested for the treatment and prevention of vitamin D deficiency [1]. Vitamin D3 injection, however, is not always commercially available as a pharmaceutical preparation [25].
us far, studies regarding intramuscular high-dose vitamin D supplementation have focused more on vitamin D3 [20,21] than on vitamin D2 [26,27], with the latter being evaluated only for the outcome of a single dose. Furthermore, there has been no common agreement worldwide to define a standard dose of vitamin D2 injection for the treatment and prevention of vitamin D deficiency. Especially, the efficacy and safety of monthly intramuscular injection of high-dose vitamin D2 to reach vitamin D sufficiency in very elderly Chinese patients have not been elucidated.
is study was designed to ascertain the time needed to correct vitamin D deficiency and to reach vitamin D sufficiency in very elderly Chinese patients with monthly intramuscular injections of 600,000 IU vitamin D2. e effects of this intervention on serum levels of iPTH, BTMs, immune parameters, and other biomedical parameters were also examined.

Study Design.
All qualified patients were given monthly intramuscular injections of 600,000 IU vitamin D2 (vitamin D2 injection, 200,000 IU/1 mL; Jiangxi Gannan Pharmaceutical, Ganzhou, China) into the deltoid muscle of the upper arm. ey were subjected to measurement of serum levels of 25(OH)D2, 25(OH)D3, iPTH, BTMs, immune parameters, and other biomedical parameters at baseline and one month after each shot. All participants were asked to have adequate fluid intake, take regular outdoor activities as usual, and receive about 20 minutes of sunlight exposure per day if weather permits during the study.

Subjects.
From December 2020 to June 2021, very elderly Chinese patients with vitamin D deficiency, who were hospitalized in three 70-bed geriatrics wards for older cadres, Wangjiangshan Branch, Zhejiang Provincial People's Hospital, were recruited into the study. Exclusion criteria: (i) patients with hypoparathyroidism, hypercalcaemia, or hypercalciuria; (ii) with severe hepatic or renal dysfunction, moderate to severe cognitive dysfunction, or advanced malignancy; (iii) on antiosteoporosis agents such as bisphosphonates and teriparaptide; (iv) receiving potent enzyme inducers of vitamin D metabolism such as phenytoin, phenobarbital, carbamazepine, and rifampin; (v) taking calcium and/or vitamin D supplements, active forms of vitamin D or analogues. Each participant was asked to complete a questionnaire for collecting information on age, gender, height, weight, medical history, medication history, and status of vitamin D supplementation. e comorbidities they had were verified in accordance with the guidelines for specific diseases.

Sample Collection.
All participants fasted overnight before scheduled sample collection. Venous blood was drawn right before the first dose of injection (baseline) and one month after each dose. Blood samples were centrifuged at room temperature to separate serum, assayed immediately, or stored at −80°C until assay.

Efficacy Observation.
e primary efficacy endpoint was the time needed to reach serum levels of 25(OH)D ≥30 ng/ mL in the very elderly Chinese patients with monthly intramuscular injections of 600,000 IU vitamin D2. e secondary efficacy endpoint was the effects of this dosing regimen on the serum levels of iPTH, BTMs, and immune function parameters.

Safety Observation.
e safety profile of this dosing regimen was assessed in terms of changes in biochemical parameters and adverse drug reactions (ADRs) reported by the subjects. All participants were asked to report potential ADRs such as anorexia, diarrhea, constipation, nausea, vomiting, bone pain, drowsiness, continuous headache, irregular heartbeat, loss of appetite, muscle and joint pain, frequent urination, excessive thirst, weakness, nervousness, and itching.

Ethical Approval.
e study was performed in accordance with the principles outlined in the Declaration of Helsinki and was approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital. All the participants provided their written informed consent for the study and were free to withdraw from the study at any time.
2.10. Statistical Analysis. Sample size was calculated with an expected parameter estimate based on a pilot study conducted in our department, and the minimum sample size required was 31 patients in each study group within a 95% confidence and 80% power. All statistical analyses were performed by using SPSS 17.0 for Windows (SPSS Inc., IBM Company, Chicago, USA). Continuous variables were expressed as mean ± standard deviation (SD) and tested for normality before comparisons. Paired t-test or Wilcoxon rank sum test was performed for between-time comparisons depending on whether normality assumption was met. A P value less than 0.05 was considered of statistical significance. Continuous variables were tested for normality.

Baseline Demographic and Clinical Characteristics of the Participants.
A total of 30 very elderly Chinese patients were recruited into the study. Out of them, 27 (90.0%) patients had their vitamin D deficiency corrected, 26 (86.7%) patients reached vitamin sufficiency and completed the study. e data from these 26 patients were used to analyze the effects of the intervention. All variables compared were normally distributed. e baseline characteristics of the subjects are presented in Table 1. e mean (±SD) serum level of 25(OH) D at baseline was 10.42 (±2.79) ng/mL in the participants who completed the study.  (Figure 1). ere was no significant change observed in serum levels of iPTH, beta-CTx, P1NP, OC, calcium, phosphorus, urea nitrogen, creatinine, total bilirubin, AST, ALT, and GGT.

Changes in Serum Levels of Immune Function Parameters.
As shown in Table 3, after intervention, the serum level (mean ± SD) of total T cells (CD3+) remained unchanged (P > 0.05), and the levels (mean ± SD) of CD4 and B cells (CD19+) were increased significantly (both P < 0.05), while the level (mean ± SD) of CD8 decreased as the result of increased CD4 (P < 0.05).

Time Needed to Correct Vitamin D Deficiency and Reach
Vitamin D Sufficiency. With monthly intramuscular injections of 600,000 IU vitamin D2, we found the time (mean ± SD, range) was 3.1 ± 1.3 (1-6) months to correct vitamin D deficiency and 6.1 ± 0.8 (5-8) months to reach vitamin D sufficiency in the very elderly Chinese patients with vitamin D deficiency. e number of patients and the time they spent to reach vitamin D sufficiency are detailed in Figure 2.    International Journal of Endocrinology

Adverse Drug Reactions.
e changes in biochemical parameters after the intervention are shown in Table 1. e highest serum level of 25(OH)D was 44.71 ng/mL, which was far less than the concentration (100 ng/mL) considered perfectly safe [1].
ere were no hypercalcaemia, hyperphosphatemia, and no hypoparathyroidism observed in all participants. In addition to one case of swelling and lump at the injection site, which resolved after hot compress therapy, there was no abovementioned adverse drug reaction reported by the participants.

Discussion
Vitamin D, in its most hormonally active form of 1α, 25dihydroxyvitamin D (1α, 25(OH)2D), has been associated with a myriad of functions other than maintaining musculoskeletal health through regulation of calcium and phosphorus homeostasis. Some studies have demonstrated that most peripheral tissues have the capacity to convert 25(OH)D to 1α,25(OH)2D to meet the local needs in a paracrine way [28], which largely depends on the availability of circulating 25(OHD). erefore, to sustain sufficient serum 25(OH)D has important clinical relevance. Solar exposure becomes increasingly limited among the very elderly people, which is the direct result of lifestyle changes such as increased clothing and decreased outdoor activities. Even worse, the capacity of the skin to synthesize vitamin D3 decreases as the human body ages, leading to insufficient endogenous vitamin D production [29]. It was reported that the amount of dermal-origin vitamin D in a 70-year-old person is only one-fourth of that synthesized in a 20-year-old person [30]. In addition, the ability of the intestine to absorb dietary vitamin D in the very elderly was also presumably diminished [31]. As a result, supplementation of exogenous vitamin D by intramuscular injection would be a pragmatic option for the treatment and prevention of vitamin deficiency among the very elderly.
In spite of the continued controversy regarding the relative effectiveness of vitamin D2 and D3 on raising serum levels of 25(OH)D [32,33], vitamin D2 has been used for more than 50 years for the treatment and prevention of vitamin D deficiency [25]. 25(OH)D2, one of the intermediates of the most bioactive vitamin D metabolites (1,25(OH) 2D), was found not to be bound as tightly to vitamin D binding protein (DBP) as 25(OH)D3 [34], leading to a higher free level of 25(OH)D2 compared to that of 25(OH) D3 [35]. From this point of view, vitamin D2 would be theoretically more biologically effective than vitamin D3 because the free type of 25(OH)D is thought to have the most physiologic effects [25].
Xu and colleagues found that, after a single high dose of intramuscular vitamin D2, the level of 25(OH)D2 increased slowly and plateaued for 12 weeks [27]. In general, a single high-dose of oral vitamin D2 is initially more effective than the equivalent intramuscular one in increasing serum 25(OH)D, while the latter can produce a slower but more long-lasting increase [22,33]. A single intramuscular dose of 300,000 IU vitamin D2 was shown not to effectively correct vitamin D deficiency [26,33]. Moreover, researchers found that a single intramuscular dose of 600,000 IU vitamin D2 could correct vitamin D deficiency, but it could not reach vitamin D sufficiency [27]. Taking all this into account, we adopted a monthly intramuscular 600,000 IU vitamin D2 to reach vitamin D sufficiency in the very elderly Chinese people with vitamin D deficiency. We found in our current study that the mean (±SD) serum level of 25(OH)D was elevated from 10.42 (±2.79) ng/mL at baseline to 34.36 (±4.63) ng/mL after the intervention. e mean (±SD) time was 3.1 (±1.3) months to correct vitamin D deficiency and 6.1 (±0.8) months to reach vitamin D sufficiency in the very elderly Chinese patients with vitamin D deficiency.
Vitamin D2 treatment has been associated with a decrease in the serum level of 25(OH)D3 [27]. It was postulated to be the response to increased total 25(OH)D following vitamin D2 supplementation because a decrease in the serum level of 25(OH)D2 was also observed after vitamin D3 treatment [36]. We found in our present study that, in parallel with the rapidly increasing of serum 25(OH)D2, serum 25(OH)D3 decreased gradually, which partially offsets the net increase in total 25(OH)D. Intriguingly, the changes in serum levels of 25(OH)D2 after vitamin D3 intervention and 25(OH)D3 after vitamin D2 intervention were found to be similar in magnitude, signifying there would be a common regulatory mechanism [37].
iPTH and vitamin D are both involved in bone metabolism and have an inverse correlation [38]. iPTH   International Journal of Endocrinology concentrations are regarded as a functional parameter of vitamin D status [39] and are directly modulated and suppressed by 25(OH)D concentrations [32]. When vitamin D levels are low, a compensatory mechanism, which is known as secondary hyperparathyroidism, is trigged to stimulate the release of iPTH through an action on calciumsensing receptors located on parathyroid cells. High levels of serum iPTH are often observed in patients with insufficient vitamin D, while vitamin D supplementation can decrease serum iPTH concentrations [38]. In contrast to previous reports that vitamin D at doses of 50,000 IU orally, 200,000 IU or above intramuscularly reduced serum levels of iPTH [17,27], we observed no significant change in serum levels of iPTH in very elderly Chinese patients after highdose intramuscular vitamin D2 intervention. is could be explained at least in part by the lower baseline iPTH serum levels observed in our present study [22]. A higher target 25(OH)D level may be needed to suppress the serum concentrations of iPTH in the very elderly patients. 25(OH)D is converted to 1,25(OH)2D by the enzyme 1alpha-hydroxylase in the kidneys, with the latter in turn increasing the intestinal absorption of calcium and phosphorous, stimulating bone resorption, and promoting reabsorption of calcium and phosphorous by renal tubules.
is leads to a concern about elevated levels of calcium and BTMs when serum levels of 25(OH)D are enhanced. Despite vitamin D intoxication is extremely rare [25], supplementation of high-dose vitamin D is deemed to have the potential to cause toxicity, which is characterized by elevated serum levels of calcium and phosphorus [25]. A single intramuscular dose of 300,000 IU vitamin D has been considered well tolerated and safe [26]. Some studies also showed that a single dose of 300,000 IU or 600,000 IU intramuscular vitamin D2 would not lead to hypercalcaemia [26,27,33] and hyperphosphatemia [22]. Xu et al. found that a single dose of intramuscular vitamin D2 up to 600,000 IU did not alter the serum levels of BTMs [27]. In our present study, the serum levels of calcium, phosphorus, and BTMs in the very elderly Chinese patients remained unchanged after they had received monthly intramuscular 600,000 IU vitamin D2 up to 8 months. Furthermore, during the whole study, none of the participants had reached a serum 25(OH) D concentration of 100 ng/mL, which was considered perfectly safe [1].
Vitamin D receptor has been found on almost all cells of the immune system, which links vitamin D to immune function. Low circulating vitamin D was shown to be associated with impaired innate immune function [40]. We demonstrated in the current study that, with monthly intramuscular 600,000 IU vitamin D2, CD4, one of the main immunoactive factors, was increased significantly in serum in the very elderly Chinese people. In addition, the serum level of B cells (CD19+) was also lifted significantly.
ese results suggest that vitamin D supplementation can improve cellular and humoral immune function in the very elderly people.
ere are some limitations in our study. One is the relatively small sample size, which makes it a little less representative to depict the changes in serum levels of 25(OH)D and iPTH resulted from the intervention. e other one is the same dose of vitamin D2 for each single patient, which ignored the impact of body weight on increasing circulating 25(OH)D levels [33]. e last one is that the results were not adjusted for sun exposure and seasonal factor despite that they play a crucial role in the variation of serum 25(OH)D levels. Further welldesigned studies, which consider as many influential factors as possible, are warranted to identify an optimal and specific approach to correct vitamin D deficiency and reach vitamin D sufficiency in the very elderly people.
To draw a conclusion, monthly intramuscular 600,000 IU vitamin D2 is an effective and safe strategy to achieve vitamin D sufficiency and enhance immune function in the very elderly Chinese patients with vitamin D deficiency.
Data Availability e data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethical Approval e study was approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital.

Consent
All participants provided written informed consent.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Pingda Bian and Xue Jin contributed equally to the manuscript. Zhangxuan Shou conceptualized the study and contributed to the reviewing, editing, and finalizing the manuscript. Pinda Bian contributed to methodology and investigation; Xue Jin contributed to data collection and analysis and writing of the original draft. All authors reviewed and approved the final manuscript. 6 International Journal of Endocrinology