The impact of vitamin D supplementation on peripheral neuropathy in a sample of Egyptian prediabetic individuals

Background: Vitamin D deficiency is seen more frequently in diabetic patients with distal symmetrical polyneuropathy. Unfortunately, there is a shortage of data concerning prediabetic individuals with peripheral neuropathy (PN). Therefore, we aimed to study the association of vitamin D deficiency with PN severity and to determine the effect of vitamin D supplementation on PN in prediabetics. Methods: A case-control study was conducted consisting of 89 prediabetic individuals with PN and a control group of prediabetics without PN, recruited from the outpatient department of the National Institute of Diabetes and Endocrinology, Cairo, Egypt. All patients were screened for PN using clinical examination and Douleur Neuropathique 4 diagnostic questionnaire (DN4). Group A (with PN) was assessed for neuropathic severity using the Short-Form McGill Pain Questionnaire (SF-MPQ). In addition, 25-hydroxyvitamin D, ionized calcium, phosphorus, parathyroid hormone (PTH), glycated hemoglobin (HbA1c), fasting blood glucose (FBG), 2-hour post 75g glucose (2h-PPBG) and lipid profile were measured for both groups. Prediabetic patients with PN were given vitamin D3 200.000 IU IM monthly for three months. After three months, clinical assessment, DN4, SF-MPQ and all laboratory measures were repeated. Results: Vitamin D was not associated with the severity of PN patients. However, supplementation of vitamin D resulted in a highly significant improvement in glycemic parameters , p≤0.001. Interestingly, neuropathy score and severity before vitamin D supplementation were (6.4±1.6 and 28.3±7.2) and after became (2.5±0.9 and 17±6.3, p≤0.001). Conclusion: Correction of vitamin D deficiency in prediabetics with PN as well as hypovitaminosis D, improves glycemic parameters, PN score and severity.

Amendments from Version 1 1. Further clarification of the study design: An analytic casecontrol followed by an interventional one arm clinical trial design (quasi experiment) were performed to fulfill the preset objectives. The case-control study included 89 prediabetic patients with peripheral neuropathy (Group A) and 89 prediabetics without peripheral neuropathy (Group B). Patients of group A were invited to participate in a quasi-experiment by administration of therapeutic dose of vitamin D. None of the 89 patients participating in the case-control study refused to be enrolled in the interventional study 2. Laboratory studies included: ionized calcium, phosphorous, PTH, and 25(OH)vitamin D, FBG, 2h-PPBG, glycated hemoglobin and total lipid profile (total cholesterol, low density lipoprotein (LDL) and triglycerides) 3. During the study, we found few patients with ionized calcium 0.8 mmol/L. they were 11 out of 178 prediabetic patients. They were asymptomatic (mostly due to chronic hypocalcemia) and once they were recognized to be hypocalcemic, they were given the proper management.

Introduction
Diabetes mellitus (DM), a significant world health problem, is a metabolic disease, which occurs due to a defect in insulin release and or insulin resistance 1 . Globally, the prevalence of type 2 diabetes (T2DM) is high and rising across all regions 2 .
There is a higher frequency of idiopathic polyneuropathy, small fiber neuropathy and painful sensory neuropathy among prediabetics. These findings suggest an involvement of the small unmyelinated nerve fibers that carry pain, temperature, and regulate autonomic function during prediabetes, before the development of diabetes 3 .
Vitamin D, which is a fat-soluble hormone, has multiple physiological roles, which extends far beyond calcium metabolism 4 . Vitamin D deficiency is a worldwide health problem, patients with prediabetes, T2DM, gestational diabetes and obesity represent a high-risk group 5 .
Recently, a lot of studies have been done to assess the association between vitamin D level and the diabetic peripheral neuropathy in patients with diabetes mellitus and to study the effect of vitamin D on painful neuropathy, but there is a lack of data concerning prediabetic individuals 1 .
The aim of this work was to determine the association of vitamin D deficiency with peripheral neuropathy severity and evaluate the effect of vitamin D supplementation on peripheral neuropathy in prediabetics with hypovitaminosis D.

Methods
An analytic case-control followed by an interventional one arm clinical trial design (quasi experiment) were performed to fulfill the preset objectives. The case-control study included 89 prediabetic patients with peripheral neuropathy (Group A) and 89 prediabetics without peripheral neuropathy (Group B).

Study Participants and Case Definition:
Prediabetic individuals were diagnosed, according to the American Diabetes Association 2019, with impaired fasting (100-125 mg/dl) and/or impaired glucose tolerance (140-199 mg/dl), and/or glycated haemoglobin (5.7-6.4%) 6 (they were all ages between 18-60 years old). Participants were recruited from the National Institute of Diabetes and Endocrinology (NIDE), Cairo, Egypt, in the period from September 2018 to March 2019 after proven informed written consent.
Ethical Considerations: A written informed consent was obtained from all patients. All data were made confidential and an ethical approval on study conduction was obtained from the Local Research Ethical Committee (REC) of the Faculty of Medicine, Ain Shams University. FWA 000017858.
All participants were subjected to full medical history including smoking habits, alcohol consumption, drug history, thorough clinical examination including blood pressure, weight, height and BMI.
Screening for peripheral neuropathy All participants were screened for peripheral neuropathy by 10 g monofilament for assessing the loss of protective sensation, tuning fork (vibration sense testing using a 128-Hz tuning fork), ankle reflex, pinprick (for perception of pain) and Douleur Neuropathic 4 diagnostic questionnaire (DN4) 7 that assesses symptoms reflecting pain in the form of burning, painful, cold, electric shocks, tingling, pins and needles. If the patients score is ≥4 the patient likely suffers from neuropathic pain. Patients found to have peripheral neuropathy were given the Short-Form McGill Pain Questionnaire (SF-MPQ) 8 that assesses the severity of pain; an increase in the score indicates increasing severity.

Laboratory studies
Laboratory studies included: ionized calcium, phosphorous, PTH, and 25(OH)vitamin D, FBG, 2h-PPBG, glycated hemoglobin and total lipid profile (total cholesterol, low density lipoprotein (LDL) and triglycerides) Participants were first instructed to fast for eight hours (overnight fasting), 10 ml of venous blood were then collected by venipuncture without tourniquet. 2 ml of the collected blood were taken in an EDTA containing tube for the assay of the glycated hemoglobin and it was stored at 4°C to be carried out within one week. 2 ml were taken in a fluoride containing tube and then separated by centrifugation and the sample was used for measurement of FBG, serum Ca, phosphorus, PTH and 25(OH)vitamin D. 2 ml sample were collected two hours after 75 g oral glucose load for the measurement of the 2h-PPBG. On a separate day, 2 ml of venous blood were collected by venipuncture (after an overnight 12 hour fast), the sample was collected in a fluoride containing tube and then separated by centrifugation and used for measurement of total lipid profile (total cholesterol, low density lipoprotein (LDL), triglycerides (TG)) by enzyme colorimetric assay.

Exclusion criteria
Patients with renal impairment, hypo or hyperthyroidism, patients on vitamin D supplementation or antiepileptic or any medication affecting calcium and vitamin D level, pregnant or breast-feeding females were excluded from the study.
Based on the results of previous studies, the majority of patients with and without peripheral neuropathy were found to be either insufficient or deficient as regard their level of vitamin D. The current analytical study has also demonstrated that all prediabetics with and without peripheral neuropathy were also either insufficient or deficient as regard their level of vitamin D.
Interventional Study: Patients of group A were invited to participate in a quasi-experiment by administration of therapeutic dose of vitamin D. None of the 89 patients participating in the case-control study refused to be enrolled in the interventional study. 200.000 IU of Vitamin D (cholecalciferol) were intramuscularly administered every month for three successive months to all patient with peripheral neuropathy.
Clinical assessments were repeated in the last visit after three months to assess the improvement in peripheral neuropathy in those patients. Retesting is advised after three months, as suppression of parathyroid hormone after supplementation with cholecalciferol takes at least three months and the response differs between individuals. So, most guidelines recommend repeat testing after three months 10 .
All laboratory tests were conducted at the beginning of the study and after three months of vitamin D supplementation.

Statistical analysis
Collected data were analyzed using SPSS (version 17, 2012, IBM Corporation, USA) (An open-access alternative that can perform an equivalent function is the R Stats package). The continuous quantitative variables included Age (Years), BMI (kg/m2), Systolic BP (mmHg), Diastolic BP (mmHg), HbA1c (%), 2h-PPBG (mg/dl), T. cholesterol (mg/dl), LDL (mg/dl), HDL (mg/dl), TG (mg/dl), 25 (OH) Vit D (ng/ml), Ionized Ca (mg/dl), Phosphorus (mg/dl), PTH (pg/ml) and they were described as mean and standard deviation. Student's T-test was used to compare two independent groups (group A and group B) for quantitative data. Continuous variables before and after vitamin D intake were assessed using paired t-test. Regarding categorical/qualitative data, we measured vitamin D status (Sufficient, Insufficient, Deficient) and they were presented as numbers and percentages and the Chi-Square test was used to compare two independent groups (group A and group B) with qualitative data. Additional qualitative data that were measured were clinical examination for peripheral neuropathy using ankle reflex, tuning fork (vibration) and 10 g monofilament and they were presented as numbers and percentages and McNemar and McNemar Bowker tests were used to compared group A before and after vitamin D supplementation.

Results
Comparison between the two studied groups regarding clinical and laboratory characteristics is shown in

After vitamin D supplementation
There was a highly significant improvement in vitamin D level in group (A) after intramuscular injection of vitamin D, from which 42 (47%) prediabetic patients became sufficient, 38 (42.7%) became insufficient and only 9 (10.1%) remained deficient (P≤0.001). There was a highly significant improvement of glycemic profile as shown in (Table 3).
Neuropathic pain score and its severity A statistically significant improvement in neuropathic pain severity was observed by the SF-MPQ (28.3±7.2 before and 17±6.3 after vitamin D supplementation, P≤0.001). There was also a statistically significant reduction in the DN4 questionnaire score from 6.39 ±1.64 to 2.5 ± 0.9, ≤0.001 (≥4 denote neuropathic pain) with an improvement of neuropathic pain in 82% of patients (73 out of 89) (P≤0.001) (Figure 1).

Clinical examination for peripheral neuropathy
We found a highly significant improvement in vibration sense by tuning fork and protective sense measured by the 10 g monofilament test (P≤0.001), while there was no improvement regarding ankle reflex (P>0.05) ( Table 4).

Discussion
Diabetic peripheral neuropathy in recently diagnosed diabetic patients may reach about 8% and more than 50% in patients with long-standing diabetes 11 . Recently, the American Diabetes Association stated that there is no strong evidence that supports the lifestyle management or efficacy of glycemic control in the treatment of neuropathic pain, which means that pharmaceutical interventions such as pregabalin, duloxetine, or tapentadol are the only way of treatment 12 . Accordingly, we aimed to demonstrate the association of vitamin D status with peripheral neuropathy and determine the effect of vitamin D supplementation on painful neuropathy in prediabetics.
Even with our sunny country, none of our patients (0%) had sufficient vitamin D level.  On the other hand, administration of vitamin D causes increase in serum calcium, decrease in circulating free fatty acid levels, increase in insulin release and improvement in glucose levels 17 .  Reduced 0 (0.0) 3 (7.9) 6 (13.3) On the other hand, Alkhatatbeh et al., (2019) showed that the only significant predictor for neuropathic pain was female gender, while vitamin D level, BMI, age, FBG, duration of T2DM, DBP and SBP were not 23 . The divergence in the results of previous studies may be due to the use of different methods to assess neuropathy and because the studies were directed on different populations.

MNB=McNemar Bowker test between patients with peripheral neuropathy before and after vitamin D injection
Injection of vitamin D 200.000 IU intramuscular every month for three successive months is in accordance with the guidelines for vitamin D supplementation and treatment of deficiency in Central Europe individuals with proved vitamin D deficiency which require higher doses of vitamin D than doses recommended for the general population. The therapeutic dose in severe deficiency should be 1.000-10.000 IU/day (~50.000 IU/week), depending on the patient's body weight and age. The duration of the treatment varies from 1-3 months, depending on the degree of vitamin D deficiency 24 . Our patients showed significant improvement and reduction in neuropathy severity score and also showed clinical improvement by monofilament and tuning fork. This is in line with Bell (2012) who found great improvement in neuropathic symptoms after supplementation with 50.000 IU of vitamin D 2 every week in a case report of a patient suffering from diabetic peripheral neuropathy. The patient had been refractory to different types of treatment like tricyclic's, gabapentin, oxycodone and pregabalin 25 . As well, Shehab et al., (2015) in their study applied vitamin D replacement therapy as a single intramuscular vitamin D dose of 300.000 IU and this application significantly enhanced the DN4 questionnaire scores of the patients with diabetic neuropathy 26 . Correspondingly, Lee and Chen (2008) showed that oral cholecalciferol resulted in an approximate 50% reduction in painful neuropathic symptoms and a significant reduction in SF-MPQ score from 32.
who considered vitamin D as a neurotrophic substance, which modulates neuronal growth and differentiation, and neuromuscular functions 28,29 . Its exact role in diabetic neuropathic pain is uncertain; insufficiency of vitamin D may increase damage of diabetic nerve and may affect the function of nociceptors leading to pain at a higher threshold of serum 25 (OH) vitamin D concentration higher than that in the non-diabetic individuals 27 .
Therefore, the results of previous studies corroborate our findings that vitamin D supplementation improves peripheral neuropathy and can be used as a safe treatment for peripheral neuropathy in prediabetic patients with hypovitaminosis D.
Opposing previous results, a study by Alam et al., (2016) reported no significant decrease in neuropathic pain scores after vitamin D administration 30 . This study was based on all or none values instead of assessing the quantity of pain score, which may have led to a failure of observing a reduction in pain scoring.
Glycemic parameters of our patients showed significant improvement after the administration of 200.000 IU of vitamin D every four weeks for 12 weeks, which was the same result found by Kuchay et al., (2015) who revealed that correcting vitamin D deficiency in people with prediabetes significantly reduces FBG, two hours plasma glucose and A1C levels in 12 months 13 . However, contrary to our findings, He et al., (2018) proclaimed in their meta-analysis that vitamin D supplementation did not improve fasting glucose levels or insulin resistance, nor did it prevent T2DM in non-diabetics 31 . Furthermore, Moreira-Lucas et al., (2017) confirmed that vitamin D supplementation did not improve fasting or post challenge measures of insulin sensitivity, β-cell function or HbA1c 32 .
Among the limitations of the study were a small sample size compared to previous studies. Our study is the first to discuss the effect of vitamin D supplementation on peripheral neuropathy in prediabetic individuals whereas other studies have discussed the effect on diabetic patients. Finding prediabetic participants with peripheral neuropathy to include in the study was challenging.

Conclusion
This study found that vitamin D supplementation in prediabetics with peripheral neuropathy and hypovitaminosis D improves neuropathy in those patients as assessed by McGill and DN4 scores, as well as glycemic parameter namely HbA1c, FBG and 2h-PPG.

Consent statement
Written informed consent was obtained from all individual participants included in our study.

Open Peer Review
The The methodology was perfectly planned, well written, and well-arranged so that the reader can understand the steps of the study and how the authors managed to find prediabetic individuals with peripheral neuropathy. Also, the number of patients is very suitable to the study design, helping to get more valid data on the effect of vitamin D supplementation on peripheral neuropathy as previous studies were lacking evidence due to either small sample size or defect in follow-up.
The statistics are well prepared, the sample size is quite enough for the study and the data is analyzed by SPSS. The statistical analysis and tests are properly described and appropriate for the study, used correctly which helped to get the results that are interpreted in a clear way. The tables and figures are well presented and have a crucial role in the study and writing of the manuscript and helped to finalize the results of the study. Finally, data files are uploaded which will help produce more results further on it, but it would be much better to present the raw data in a simplified way.
The results were nicely presented; the authors first presented that vitamin D deficiency was highly prevalent in the studied group. Second, the authors compared clinical and laboratory data between the two studied groups. Third, they presented the effect of vitamin D supplementation over the arm with the peripheral neuropathy and clarified the benefit on the vitamin D levels and neuropathy score and severity and clinically by peripheral neuropathy indicators (ankle reflex, tuning fork vibration, and 10 g monofilament).
The discussion was organized and updated, citing each part of the study with other papers, discussing with cons, and clarifying the cause of each result.
The conclusion was short, to the point, summarizing all the aims in the study design.
Despite this, I would be thankful if the authors could clarify the following points: The main results are not sufficiently presented in the abstract (e.g. the mean and SD of the vitamin D level in the study groups, the mean of HbA1c results, and the other glucose parameters, etc).
Yes find patients with such inclusion criteria given my expertise. The authors declared that one limitation was collecting patients compatible with the inclusion criteria, it's really challenging to find a prediabetic patient accept, confirming the diagnosis by fasting and doing OGTT test then assessment of neuropathic pain, so this effort from the authors is really appreciated in my opinion.
The conclusion was short but I think it is enough to support the results since it is mentioning the primary outcome for this paper which is that replacement of Vit D in neuropathic prediabetes patients is useful.
○ Finally, I find this version accepted and well written.