Nasir Abdelrafie Hamad^1*, Lienda Bashier Eltayeb²and Habab Merghani Yassin³

¹Department of Biochemistry, College of Medicine and Health Sciences, National University of Sciences and Technology, Sohar, Oman. College of Medicine, El-Neelain University, Sudan.

²Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, Al-Kharj 11942, Riyadh, Saudi Arabia.

³Department of Biology, Faculty of Science, King Khalid University, Abha 62529, Saudi Arabia.

Corresponding Author E-mail:nasircosha@gmail.com.

DOI : https://dx.doi.org/10.13005/bpj/3137

Abstract

Background: Diabetic foot ulcer (DFU) has emerged as a leading cause of morbidity and mortality among diabetic patients, with the numbers of amputation and death are significantly increasing. DFU has a complex multifactorial etiology with several pathophysiological mechanisms and risk factors implicated. Finding reliable indicators to predict the prognosis of diabetic foot has become an urgent and critical need. The current study aimed to investigate the association between levels of serum calcium, vitamin D and C-reactive protein and the development of DF. Methods: One hundred and twenty diabetic patients with a confirmed diagnosis of DFU were included in the study. The ratio of cases and control subjects was 1:1.  We measured serum calcium, vitamin D, CRP, HbA1C, blood urea nitrogen (BUN), creatinine, total cholesterol, low-density lipoproteins (LDL) and triglycerides in both cases and control subjects. Results: The study found significantly low levels of serum calcium and vitamin D as well as high levels of HbA1C, CRP, serum creatinine, LDL, and TAG among cases. Conclusion: Serum calcium, vitamin D and CRP were significantly associated with DFU. As the treatment of DFU is challenging, these parameters might predict the development of DFU in diabetic patient and might therefore help reduce the morbidity and mortality associated with DFU.

Keywords

Calcium; Diabetic; DFU; Diabetes; Foot; Predictors;  Ulcer; Vitamin D

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Copy the following to cite this article: Hamad N. A, Eltayeb L. B, Yassin H. M. Serum Calcium, Vitamin D and C-Reactive Protein as Early Predictors for Diabetic Foot Ulcer. Biomed Pharmacol J 2025;18(1).

Copy the following to cite this URL: Hamad N. A, Eltayeb L. B, Yassin H. M. Serum Calcium, Vitamin D and C-Reactive Protein as Early Predictors for Diabetic Foot Ulcer. Biomed Pharmacol J 2025;18(1). Available from: https://bit.ly/4kI4Z6e

Introduction

Diabetic foot ulcer (DFU) is a common and devastating complication of long standing, poorly controlled diabetes mellitus,¹ with a significant morbidity and mortality,² in addition to the psychological and financial burden on the patient and his family³. Globally, 19-34% of all diabetic patients will develop DFU.⁴Around 20% of patients with DFU eventually necessitate amputation.⁴ Additionally, around 10% of DFU patients will die within one year after the diagnosis has been made.⁵  DFU is defined as a break through the epidermis and part of the dermis in a diabetic patient that fails to heal quickly⁶. The literature suggested that DFU has a multifactorial origin.⁷ The most important factors in the development of DFU are the existence of peripheral neuropathy, peripheral artery disease (PAD) and secondary bacterial infaction.² Other contributing factors include poor glycemic control, the presence of foot deformities and calluses, as well as trauma.⁷ However, peripheral artery disease remains the most significant contributing factor.²  Around 66% of diabetic patients develop peripheral neuropathy.⁸ According to the presence or absence of peripheral neuropathy, DFU can be classified into purely neuropathic, purely ischemic, and mixed neuroischemic.⁹ Around 50% of all cases of DFU have a neuroischemic origin.⁹

Peripheral neuropathy causes loss of pain and temperature sensation, resulting in an insensitive foot.¹⁰ In this case, even a minor trauma can precipitate foot ulceration. In addition to the sensory deficit, motor dysfunction can also precipitate the foot ulceration.¹¹ Autonomic neuropathy can also decrease sweating leading to skin dryness and increased predisposition to infection.¹¹ The end result is a progressive foot ulceration that is difficult to heal.¹²

PAD usually presents with intermittent claudication and rest pain.¹³ Intermittent claudication aggravates the disability already present.¹⁴ Risk factors for PAD include hypertension, smoking and dyslipidemia¹⁴. Together, these factors precipitates the ischemia and worsen the ulceration.¹⁵  In addition to the direct effect of PAD on the ulceration process, it also contributes to the ongoing neuropathy.¹⁵ The hypoglycemia accompanying diabetes augments to the ongoing neuropathy and ulceration via the formation of advanced glycation end-products (AGEs).¹⁶

Both neuropathy and peripheral artery disease precipitate secondary infection. Staphylococcus aureus accounts for 30% of all cases.¹⁷ Chronic recurrent infections are mostly caused by impaired immune system,¹⁷ which is aggravated by the interplay of production of AGE and reactive oxygen species secondary to hyperglycemia.  The interplay of these two factors slows down the process of wound healing.¹⁸ Several classification systems for DFU were developed, including Wagner-Meggitt classification,¹⁹  University of Texas Classification,²⁰ and the SINBAD²¹ (site, ischemia, neuropathy, bacterial infection, area, depth) system. The SINBAD system is straightforward and fast to use, necessitating no specialized equipment beyond clinical examination alone. The SINBAD system assesses six elements (site, ischemia, neuropathy, infection, area, and depth) with scores ranging from 0 to 1, creating a severity scale from 0 to 6. It contains all the essential information required by specialized team.²¹ Furthermore, this classification has been rigorously validated for both ulcer healing and amputation prediction, demonstrating exceptional results and undeniable reliability.²¹ The SINBAD system scores 0 or 1 for the site, extent and depth of the ulcer, presence or absence of ischemia, neuropathy, or infection. The total score for each category is one, and the overall score is six.²¹

Table 1: SINBAD classification system

Category	Explanation	Score
Ulcer site	Anterior footBack and middle foot	01
Ulcer extent	< 1 cm2> 1 cm2	01
Ulcer deepness	Confined to skinExtends beyond skin	01
Ischemia	Normal blood flowReduced blood flow	01
Neuropathy	Intact sensationLost sensation	01
Bacterial infection	NoPresent	01
Total score		6

 Risk factors for DFU

Risk factors for DFU can be categorized as patient-related and foot-related²². One of the most significant patient-related factors is age.²³ Moreover, literature stated that DFU is significantly associated with males rather than females.²⁴ Other factors include race and ethnicity,²⁵ low socioeconomic class,²⁶ smoking,²⁷ high body mass index²⁸ (BMI) and obesity,²⁸ as well as poor glycemic control²⁹. Comorbidities associated with high risk and severe outcome of DFU include cardiovascular disordersو³⁰ end-stage renal disease,³¹ as well as retinopathy.³²

Literature has investigated the biochemical and radiological findings as predictors for DFU in diabetic patients. For an instance, Caruso³³ concluded that parathyroid hormone and high alkaline phosphatase levels are independent risk factors for DFU. Potential biomarkers that can be helpful in early diagnosis of DFU included procalcitonin, C-reactive protein (CRP), interleukins, TNF-α, arginine, leucine and isoleucine.³⁴ Moreover, a study found low lymphocyte absolute value and high platelet count to be associated with DFU.³⁴ Interestingly, a study by Vijaya³⁵ concluded that LDL cholesterol and cell-surface expression of CD63 on monocytes are predictors of DFU. Furthermore, a study by Katya³⁶ showed significantly higher levels of blood urea and serum creatinine among DFU patients. In his review, Fujita³⁷ finalized that methyl glyoxal, adiponectin, semaphorin, and nerve growth factor are biomarkers for diabetic neuropathy that can predict the development of DFU. In addition, Guttikonda³⁸ stated that  high levels of total cholesterol, LDL, and triglycerides were associated with a higher risk of DFU. Interestingly, Xu showed that neutrophil-to-lymphocyte ratio (NLR), serum calcium and albumin levels can be reliably used as predictors for the development and prognosis of DFU.³⁹  In a meta-analysis-study investigating the association between vitamin D levels and DFU, lower levels of vitamin D were significantly associated with DFU.⁴⁰

With the global increase in the prevalence of DFU together with the associated morbidity and mortality and the doubtful effectiveness of treatment modalities for DFU, finding a biomarker that can reliably predict DFU as early as possible becomes a must. In the present study, we aimed to investigate the association between serum calcium, vitamin D, and parathyroid hormones and DFU to assess their predictive and prognostic roles in DFU.

Materials and methods

Study type and population

The current observational case-control study was conducted among diabetic patients attending different primary healthcare facilities in Khartoum, Sudan during the period from February-June 2023. Cases included diabetic patients with an established diagnosis of DFU. DFU was graded using the SINBAD system²¹. Scores ≥ 3 signify a severe ulceration. Control individuals were selected from diabetic patients without DFU.

Sample size

Sample size was calculated based on a 95% confidence interval, a 5% margin of error, an estimated 5% population proportion, assuming a normal sample distribution⁴¹. The population proportion was taken as 18% based on previous study in Sudan⁴². The following formula⁴³ was used to calculate the sample size:

n = (tα2 x p x q x N)/((N-1)  X e2+tα2 x p x q)

Given that: n = sample size, N = population size, p = expected percentage of the variable, q = 1-p, e = accepted margin of error, tα = 1.96 for 95% confidence interval. Accordingly, A total of 120 diabetic patients with DFU were recruited for the current study. The ratio of the case to control participants was 1:1.

Data collection

Sociodemographic and medical data related to diabetes and DFU were obtained from hospital medical records and by using a validated self-administered questionnaire. The questionnaire was composed of two parts and 10 closed-ended questions. The first part included questions covering the patient’s sociodemographic data (age, gender, and smoking). The second part included questions inquiring about the medical history of diabetes and DFU, as well as associated comorbidities. All patients signed informed consent before participating.

Biochemical investigations

About 10 ml of fasting venous blood was collected in an EDTA tube and centrifuged for 10 minutes at 1,000-2,000 x g for plasma and serum extractions. Serum calcium was measured using RayBio® Calcium Colorimetric Assay Kit. The normal range is 8.8 -10.4 mg/dL.⁴⁴ Additionally, vitamin D3 level was measured using Elabscience® VitD ELISA kit. The normal range is given as 40-60 ng/mL.⁴⁵ Regarding renal profile, BUN was measured using DetectX® BUN kit, with a normal range of 5-20 mg/dl.⁴⁶ For serum creatinine, it was measured using DetectX® Serum Creatinine Kit. The normal range for creatinine is 0.6-1.2 mg/dl.⁴⁶ Moreover, total cholesterol, triglyceride, and low-density lipoproteins were measured using Qucare® Multi Meter kit. The normal value for total cholesterol is less than 200 mg/dl.⁴⁷ (TAG is less than 150 mg/dl⁴⁷) and LDL is less than 100 mg/dl.⁴⁷ Furthermore, CRP was measured using Human CRP Instant ELISA™ Kit (normal value is lower than 0.3 mg/dl⁴⁸). Finally, HbA1C levels were measured using Getein HbA1c fast test kit with the normal value lower than 5.7%.⁴⁹ BMI was obtained from the patient’s medical records.

Data presentation and analysis

Descriptive data were presented as means and standard deviations for quantitative variables and frequencies for qualitative variables. Results were statistically analysed using the SPSS (18th version). Unpaired T-test was used to compare the mean age, BMI as well as the various biochemical parameters among cases and control subjects. Chi-Square test was used to compare distribution of gender and comorbidities between cases and control subjects. Analysis of variances (NOVA) was used to compare means of serum calcium, vitamin D, and C-reactive protein among groups of patients having mild, moderate and severe DFU. P-value was considered significant when ≤ 0.05.

Ethical approval

All participants signed an informed consent before recruitment. Ethical approval was obtained from the ethical committee at College of Medicine, Neelain University, Sudan and Ministry of Health, Sudan.

Results

Sociodemographic findings

The current study included 120 diabetic patients with DFU. The ratio of the case to control participants was 1:1. The mean age was 53.54 years for cases and 46.71 years for control subjects (table 1). As shown in table (2), mean BMI was 29.55kg/m² for cases and 25.41 kg/m² for control subjects.

Table 2: Mean age and BMI  among cases and control subjects

Age (years)	Cases	Controls	Analysis
Count, N:Sum, Σx: Mean, x̄: SD SEM	1206425 53.54 2.3005144961027 0.21000728056385	1205606 46.72 6.8134608900118 0.62198103735644	Two-tailed p value is less than 0.0001 t = 10.3964 df = 238 Standard error of difference = 0.656
BMI	Cases	Controls	The two-tailed P value is less than 0.0001 t = 18.8859 df = 238 Standard error of difference = 0.219
Count, N:Sum, Σx: Mean, x̄: Variance, s2: SD SEM	1203547 29.55 3.0890056022409 1.757556713805 0.16044224304094	1203050 25.41 2.6820728291316 1.6377035229649 0.14950119367226

The cases included 89 males and 31 females whereas the control subjects included 56 males and 64 females (table 3).

Table 3: Gender distribution among cases and control subjects

Males	Observed Expected Chi Square distribution	Cases	Controls	Row totals	Analysis
		89 72.5 3.7552	56 72.5 3.7552	145 95 240	Chi-Square Statistic: 18.9735df: 1 p-value: 0.000
Females	ObservedExpected Chi Square distribution	3147.5 5.7316 120	6447.5 5.7316 120
Column totals

Table 4 shows the distribution of smoking among cases and control subjects. As shown in the table, smokers represented 61.7%  of cases and 45.8% of control subjects.

Table 4: Distribution of smoking among cases and control subjects

Smoking	Observed Expected Chi Square distribution	Cases	Controls	Row totals	Analysis
		74 64.5 1.3992	55 64.5 1.3992	129 111 240	Chi-Square Statistic: 6.0507 df: 1 p-value: 0.0139
No smoking	ObservedExpected Chi Square distribution	4655.5 1.6261 120	6555.5 1.6261 120
Column totals

The frequency and percentage of DFU among patients with DFU was shown in chart 1. All cases had SINBAD scores of either 4, 5 or 6 which were considered to be severe ulceration with variable degrees (4 the less severe and 6 the most severe). As shown in the chart, approximately 43% of patients had a score  of 6, 29% of patients had a score of 5, and 28% had a score of 4.

Chart 1: Distribution of DFU grades among casesClick here to view Figue

Comorbidities

The frequency of retinopathy, hypertension, cardiovascular disease (CVD), and renal diseases among cases and control subjects are shown in table 5. As shown in the table, the most common comorbidity associated with DFU was renal disease and retinopathy.

Table 5: Distribution of comorbidities among cases and control subjects

Comorbidity	Cases	Controls	df	Chi Square statistic	p-value
RetinopathyHypertension CVD Renal disease	4533 7 56	2824 2 33	1	5.68941.8637 2.886 9.4471	0.01710.1722 0.0894 0.0021

 Biochemical findings

The mean values for serum calcium, vitamin D3, BUN, creatinine, total cholesterol, LDL, TAG, HbA1C and CRP were shown in table 6.

Table 6: Mean values for different biochemical findings among cases and control subjects

Parameter	Cases	Controls	p-value
Serum calciumHBA1C Vitamin D3 BUN Creatinine Total cholesterol LDL TAG CRP	6.178.07 35.99 17.28 2.67 159.05 122.20 125.30 6.700	8.635.10 55.73 16.83 1.16 158.50 89.15 115.65 0.975	0.00010.0001 0.0001 0.5424 0.0001 0.9373 0.0001 0.0107 0.0001

We have also compared the mean vitamin D, serum calcium and CRP levels among groups of patients having grades 4, 5, and 5 of DFU as shown in table 7.

Table 7: Mean vitamin D level among different grades of DFU

Grade	n	Mean VD	F-statistic	SD	p-value
45 6	3533 52	35.060635.1818 33.2115	4.6754	5.63992.0533 1.7415	0.0112
Grade	n	Mean calcium	F-statistic	SD	p-value
45 6	3533 52	6.08576.2424 6.4528	3.1016	0.81790.7084 0.574	0.0487
Grade	n	Mean calcium	F-statistic	SD	p-value
45 6	3533 52	5.73535.7188 6.0943	3.1241	0.75110.7719 0.8381	0.0477

Discussion

The global mortality of DFU is as high as 50% within five years,⁵⁰ with ischemic heart disease being the leading cause of mortality associated with DFU.⁵¹ Morbidity associated with DFU is high too, with an approximate rate of recurrence of 65% within 3-5 years⁵¹ and around eight-fold increase in the risk of amputation than non-diabetics.⁵² Additionally, around 50% of amputated patients will die within 5 years.⁵² The main focus of the present study is to identify sociodemographic and biochemical findings that can predict the incidence of DFU in diabetic patients.

The study revealed that DFU was more prominent among older patients. Middle aged group in the study was not an exclusion.⁵³ The effect of age on the incidence of DFU has been already established.²³ The effect of age on DFU can be attributed to the long-term cumulative effects of hyperglycemia and advanced end-glycation products (AGEs) with the subsequent modulation of gene expression, production of ROS, and improper functioning of nitric oxide and growth factors.⁵⁴  Moreover, the study found that severe and advanced DFU was significantly associated with middle-aged group. Accordingly, we agreed with the conclusion that middle aged patients have more advanced ulceration and a higher rate of hospitalization than elderly patients.⁵⁵ DFU is associated with advanced age. However, Shi⁵⁶ found that the prognosis of DFU is independent of age. Moreover, elderly patients with DFU still have some probability of healing despite the poor outcome.⁵⁷

Our study found a significant association between DFU and male gender. In this context, we agreed with the previous studies which confirmed a higher risk of DFU in males than in females.^23,58 It’s important to consider that sex differences in health outcomes are often shaped by a range of factors such as access to care, screening procedures, and the commitment to following treatment plans. Moreover, women were more likely to follow guidelines related to foot care.⁵⁹ Furthermore, it’s crucial to recognize that men with diabetes face a significantly elevated risk of developing peripheral neuropathy (PN), peripheral artery disease (PAD), and cardiovascular disease. This underscores the urgent need for increased awareness and proactive management of these associated health challenges.⁶⁰

Our study suggested an increased risk of DFU with higher BMI. This suggestion was also made by other studies.^28,61 Excessive body fat can affect important biological processes involved in wound healing. For instance, having more fat can lead to reduced blood vessel formation and poor circulation, which can hinder the healing of ulcers. This poor circulation can also result in decreased oxygen delivery, creating an environment that promotes the growth of certain bacteria and fungi.⁶²

In the present study, we concluded that smoking is associated with the development of DFU. The study was in an agreement with Conte.²⁷ This association can be explained based on the fact that smoking negatively impacts glycemic control and increases the production of AGEs and ROS.⁶³

The most common comorbidity associated with DFU in the present study was chronic kidney disease. Chronic kidney disease is unequivocally associated with a significantly heightened risk of diabetic foot ulcers (DFU), prolonged healing times, increased recurrence rates, and increased rates of lower extremity amputation.³¹ Even the minimal impairment in kidney function was linked to DFU.⁶⁴ This finding is quite explainable as peripheral artery disease and chronic kidney disease were considered as one disease from a pathophysiological point of view. In other words, The histopathophysiological processes that link oxidative stress to PAD from one side, and oxidative stress to CKD from the other side, are similar.⁶⁴ Proteinuria, lower extremity edema, malnutrition and renal dialysis are key factors that directly contribute to the occurrence and exacerbation of DFU, significantly increasing the complexity of condition65. The second most common morbidity associated with DFU in our study was retinopathy. Again, our study was consistent with other studies in this regard.²² DFU and retinopathy serve as strong indicators of advanced microvascular disease, offering compelling evidence that may help elucidate this association.²² In fact, the oxidative stress and endothelial dysfunction responsible for retinopathy also contributes to DFU.⁶⁶ In contrast to our study, Hwang⁶⁷ did not find a significant association with retinopathy. Unlike other studies that found a strong association linking DFU to hypertension⁶⁸ and cardiovascular diseases,³⁰ the present did not a significant association.

The present study supported a strong association between DFU and abnormally high levels of HBA1C, suggesting an association with poor glycemic control. Accordingly, the study was consistent with previous studies in this regard.²⁹ The cumulative effects of hyperglycemia and the related microvascular complications are cornerstones in the firm establishment of this connection.²⁹

Our study revealed a significant association between CRP and DFU. Furthermore, we reported the highest levels of CRP among the group of patients with grade 6 OF DFU. In fact, CRP was considered as the most significant inflammatory marker to be associated with DFU.⁶⁹ The value of CRP in predicting, diagnosing, as well as determining the prognosis of DFU relies on being a highly sensitive acute phase protein inflammatory marker.⁶⁹ Moreover, Sharma⁷⁰ considered CRP and procalcitonin to be the best markers for DFU.

With regards to lipid profile, our study concluded that high levels of LDL and TAG were significantly associated with DFU. This finding supported Guttikonda³⁸ who stated that high levels of total cholesterol, LDL, and triglycerides were associated with a higher risk of DFU. These lipids have a direct link to the development of atherosclerosis, which significantly impedes blood flow to the lower limbs and substantially raises the risk of developing foot ulcers.⁷¹

With regards to renal function profile, our study found a significant association between DFU and elevated serum creatinine, supporting Fujita³⁷ and other studies. According to one study,⁷² nephropathy was considered to be a contributing factor in impaired microcirculation accompanying DFU. In fact, high serum creatinine signifies a chronic kidney disease, which was already proved to predict the development of DFU.^31,64

Interestingly, the present study has significantly and successfully linked low levels of vitamin D to DFU. Moreover, we found that the lowest levels of vitamin D were significantly associated with grade greater severity of  DFU.  In his meta-analysis study, Dai¹ studies reported low serum vitamin D levels among patients with DFU. The exact mechanism behind the link between low vitamin D level and DFU is not fully understood. Vitamin D deficiency was linked to several chronic disorders including cardiovascular diseases, metabolic disorders, autoimmune and infectious diseases, cancer and diabetes mellitus.⁵³ The active form of vitamin D, vitamin D3  or dihydroxycholecalciferol is thought to affect the expression of several genes involved in regulation of immune function and promoting insulin sensitivity and pancreatic function.⁵³ The vitamin inhibits the release of inflammatory mediators and proinflammatory cytokines, thereby minimizing inflammation. Additionally, it modifies both cellular and humoral immunity.⁷³ In his meta-analysis study, Li⁴⁰ has proposed several theories explaining the negative effect of low vitamin D levels on DFU. These theories highlighted the positive effect of vitamin D on the glycemic control , endothelial system, and immune functioning.

Several studies reported low serum calcium levels among patients with DFU. In the present study, patients with grade 6 of DFU displayed lower levels of serum calcium than groups with grade 1 or 2. In his study, Xu³⁹ related low serum calcium levels to DFU, amputation and death. The study considered serum calcium as an inflammatory marker that decreases as the inflammation progresses. As a micronutrient, calcium is essential for promoting effective wound healing via its powerful antioxidant and anti-inflammatory properties, coupled with its role in stabilizing collagen and regulating cell growth and differentiation.⁷⁴

Limitations

The study has several limitations. First, the sample size was relatively small. Second, we could not assess other important molecules like other minerals and ptoteins that might have important roles in the process of wound healing . Finally, the study was conducted in local health care facilities in Khartoum City, which renders generalization to the entire population illogical.

Conclusion

In conclusion, the current study found a significant association between low levels of serum calcium and vitamin D with DFU. Moreover, the study reported significantly higher levels of HbA1C, CRP, serum creatinine, LDL, and TAG among patients with DFU. patients with severe DFU have the lowest levels of serum calcium and vitamin D, and the highest level of CRP.

Acknowledgement

This paper and the research behind it would not have been possible without the exceptional help from the support and paramedic staffs of Khartoum Teaching Hospital and Diabetic Foot Care Center. I am also thankful to individuals who agreed to participate in this study.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

Ethical approval was obtained from the ethical committee at College of Medicine, Neelain University, Sudan and Ministry of Health, Sudan (NU/COMHS/EBC0013/2023).

Informed Consent Statement–

All participants signed an informed consent before recruitment.

Clinical Trial Registration

This research does not involve any clinical trials.

Author contribution

The authors confirm contribution to the paper as follows:

• Nasir Abdelrafie Hamad: Study conception,  design and Data collection
• Habab Merghani Yassin: Analysis and interpretation of results
• Lienda Bashier Eltayeb: Draft manuscript preparation.

References

1. Dai J, Jiang C, Chen H, Chai Y. Vitamin D and diabetic foot ulcer: A systematic review and meta-analysis. Nutrition & Diabetes. 2019;9(1).
   CrossRef
2. Akkus G, Sert M. Diabetic foot ulcers: A devastating complication of diabetes mellitus continues non-stop in spite of new medical treatment modalities. World Journal of Diabetes. 2022;13(12):1106-1121.
   CrossRef
3. Schaper NC, Netten JJ, Apelqvist J, Bus SA, Hinchliffe RJ, Lipsky BA. Practical Guidelines on the prevention and management of diabetic foot disease. Diabetes/Metabolism Research and Reviews. 2020;36(1).
   CrossRef
4. Armstrong DG, Boulton AJM, Bus SA. Diabetic Foot Ulcers and Their Recurrence. New England Journal of Medicine. 2017;376(24):2367-2375.
   CrossRef
5. Hoffstad O, Mitra N, Walsh J, Margolis DJ. Diabetes, lower-extremity amputation, and death. Diabetes care. 2015;38(10):1852-1857.
   CrossRef
6. McDermott K, Fang M, Boulton AJM, Selvin E, Hicks CW. Etiology, epidemiology, and disparities in the burden of diabetic foot ulcers. Diabetes Care. 2022;46(1):209-221.
   CrossRef
7. Sa BC, Maskan Bermudez N, Shimon SV, Kirsner RS. Diabetic foot ulcers: A review of debridement techniques. Surg Technol Int. 2024;15(44).
   CrossRef
8. G Pallavicini, Cetta G, F Sinigaglia, R Badiello, Tamba M. Radiolysis of a pig gastric glycopeptide. British Journal of Cancer. 1975;32(6):761-762.
   CrossRef
9. Game F. Treatment strategies for neuroischemic diabetic foot ulcers. Lancet Diabetes Endocrinol. 2018;6(3):159-160.
   CrossRef
10. ‌Hicks CW, Selvin E. Epidemiology of peripheral neuropathy and lower extremity disease in diabetes. Current Diabetes Reports. 2019;19(10):1-8.
    CrossRef
11. ‌ Fealey RD, Low PA, Thomas JE. thermoregulatory sweating abnormalities in diabetes mellitus. Mayo Clinic Proceedings. 1989;64(6):617-628.
    CrossRef
12. Dayya D, O’Neill O, Huedo-Medina T, Habib N, Moore J, Iyer K. Debridement of diabetic foot ulcers. Advances in Wound Care. 2021;11(12).
    CrossRef
13. Soyoye DO, Abiodun OO, Ikem RT, Kolawole BA, Akintomide AO. Diabetes and peripheral artery disease: A review. World Journal of Diabetes. 2021;12(6):827-838.
    CrossRef
14. Liao F, An R, Pu F, Burn’s S, Shen S, Jan YK. Effect of exercise on risk factors of diabetic foot ulcers. American Journal of Physical Medicine & Rehabilitation. 2019;98(2):103-116.
    CrossRef
15. Wang X, Yuan CX, Xu B, Yu Z. Diabetic foot ulcers: Classification, risk factors and management. World Journal of Diabetes. 2022;13(12):1049-1065.
    CrossRef
16. Brings S, Fleming T, Freichel M, Muckenthaler M, Herzig S, Nawroth P. Dicarbonyls and advanced glycation end-products in the development of diabetic complications and targets for intervention. International Journal of Molecular Sciences. 2017;18(5):984.
    CrossRef
17. Stacey HJ, Clements CS, Welburn SC, Jones JD. The prevalence of methicillin-resistant Staphylococcus aureus among diabetic patients: A meta-analysis. Acta Diabetologica. 2019;56(8):907-921.
    CrossRef
18. Kim J. The pathophysiology of diabetic foot: a narrative review. Journal of Yeungnam Medical Science. 2023;40(4).
    CrossRef
19. Shah P, Inturi R, Anne D, Jadhav D, Viswambharan V, Khadilkar R, Dnyanmote A, Shivangi Shahi1. Wagner’s Classification as a tool for treating diabetic foot ulcers: our observations at a suburban teaching hospital. Cureus.
20. PN VC, PP P, LJM T, AH T. Comparison of WIFi, University of Texas and Wagner classification systems as major amputation predictors for admitted diabetic foot patients: A prospective cohort study. Malaysian Orthopaedic Journal. 2020;14(3):114-123.
    CrossRef
21. Monteiro‐Soares M, Russell D, Boyko EJ, Jeffcoate W, Mills Joseph L, M Stephan, G Fran. Guidelines on the classification of diabetic foot ulcers (IWGDF 2019). Diabetes/Metabolism Research and Reviews. 2020;36(1).
    CrossRef
22. McDermott K, Fang M, Boulton AJM, Selvin E, Hicks CW. Etiology, epidemiology, and disparities in the burden of diabetic foot ulcers. Diabetes Care. 2022;46(1):209-221.
    CrossRef
23. Zhang P, Lu J, Jing Y, Tang S, Zhu D, Bi Y. Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis. Annals of Medicine. 2016;49(2):106-116.
    CrossRef
24. Rossboth S, Lechleitner M, Oberaigner W. Risk factors for diabetic foot complications in type 2 diabetes: A systematic review. Endocrinology, Diabetes & Metabolism. 2020;4(1).
    CrossRef
25. Canedo JR, Miller ST, Schlundt D, Fadden MK, Sanderson M. Racial/ethnic disparities in diabetes quality of care: the role of healthcare access and socioeconomic status. Journal of Racial and Ethnic Health Disparities. 2017;5(1):7-14.
    CrossRef
26. Fanaroff AC, Yang L, Nathan AS, Khatana Sameed Ahmed M, Julien Howard, Wang Tracy Y, Armstrong Ehrin J, Treat-Jacobson Diane , Glaser Julia D, Wang Grace, Damrauer Scott M, Giri Jay, Groeneveld Peter W. Geographic and socioeconomic disparities in major lower extremity amputation rates in Metropolitan areas. Journal of the American Heart Association. 2021;10(17).
    CrossRef
27. Xia N, Morteza A, Yang F, Cao H, Wang A. Review of the role of cigarette smoking in diabetic foot. J Diabetes Investig. 2019;10(2):202-215.
    CrossRef
28. ‌Chamberlain RC, Fleetwood K, Wild SH, Colhoun Helen M, Lindsay Robert S, Petrie John R, McCrimmon Rory J, Gibb Fraser, Philip Sam, Sattar Naveed, Kennon Brian, Leese Graham P. Foot ulcer and risk of lower limb amputation or death in people with diabetes: A national population-based retrospective cohort study. Diabetes Care. 2021;45(1):dc211596.
    CrossRef
29. Goldman MP, Clark CJ, Craven TE, Davis R P, Williams T K, VR Gabriela, Hurie J B, Edwards M S. Effect of intensive glycemic control on risk of lower extremity amputation. J Am Coll Surg. 2018;227(6):596-604.
    CrossRef
30. ‌Fang M, Hu J, Matsushita K, Selvin E, Hicks CW. Long-term consequences of diabetic foot infection: Atherosclerosis risk in communities (ARIC) study. Diabetes. 2022;71(1):126.
    CrossRef
31. Ndip A, Lavery LA, Boulton AJM. Diabetic foot disease in people with advanced nephropathy and those on renal dialysis. Current Diabetes Reports. 2010;10(4):283-290.
    CrossRef
32. Serban D, Papanas N, Dascalu AM, Stana D, Nicolae V A, Vancea G, Badiu C D, Tanasescu D, Tudor C, Balasescu S A, PS Anca. Diabetic retinopathy in patients with diabetic foot ulcer: A systematic review. Int J Low Extrem Wounds.
33. Caruso P, Maria Ida Maiorino, Scappaticcio L, Porcellini C, Matrone R, Cirillo P, Macera M, Gicchino M, Vietri M T, Bellastella G, Coppola N, Esposito K. Biochemical predictors of diabetic foot osteomyelitis: A potential diagnostic role for parathormone. Diabetes Metab Res Rev. 2023;39(1):e3590.
    CrossRef
34. Wang Y, Shao T, Wang J, Huang X, Deng X, Cao Y, Zhou M, Zhao C. An update on potential biomarkers for diagnosing diabetic foot ulcer at early stage. Biomed Pharmacother. 2021;133:110991.
    CrossRef
35. Raghavan JV, Sagar SK, Dorai VK, Samuel R, Arunachalam P, Chaluvanarayana H C, Belahalli P, Kalpana S R, Jhunjhunwala S. Cholesterol levels and monocyte phenotype are predictors of nonhealing in individuals with low-grade diabetic foot ulcers: A Prospective cohort study. Adv Wound Care (New Rochelle). 2023;12(6):316-326.
    CrossRef
36. Katya WE, Salman AN, Mohammed AG. Assessment of biochemical markers in diabetic foot ulcers patients At Thi-Qar province. International journal of health sciences. Published online September 9, 2022:3614-3620
    CrossRef.
37. Fujita Y, Murakami T, Nakamura A. Recent advances in biomarkers and regenerative medicine for diabetic neuropathy. International Journal of Molecular Sciences. 2021;22(5):2301.
    CrossRef
38. ‌Guttikonda Rajendra, Kiran Role of biochemical parameters in prediction of diabetic peripheral neuropathy. Journal of Research in Applied and Basic Medical Sciences. 2024, 10(2): 169-177.
    CrossRef
39. Xu S, Wang Y, Hu Z, Ma L, Zhang F, Liu P. Effects of neutrophil‐to‐lymphocyte ratio, serum calcium, and serum albumin on prognosis in patients with diabetic foot. International Wound Journal. 2022;20(5):1638-1646.
    CrossRef
40. Li X, Kou S, Chen G, Zhao B, Xue J, Ding R, Zhao X, Ye M, Yang Y, Yue R, Li F. The relationship between vitamin D deficiency and diabetic foot ulcer: A meta‐analysis. International Wound Journal. 2023;20(8):3015-3022.
    CrossRef
41. Althubaiti A. Sample Size determination: a Practical guide for health researchers. Journal of General and Family Medicine. 2022;24(2):72-78.
    CrossRef
42. Almobarak AO, Awadalla H, Osman M, Ahmed MH. Prevalence of diabetic foot ulceration and associated risk factors: an old and still major public health problem in Khartoum, Sudan. Annals of Translational Medicine. 2017;5(17):1-1.
    CrossRef
43. Rodríguez del Águila M, González-Ramírez A. Sample size calculation. Allergologia et Immunopathologia. 2014;42(5):485-492.
    CrossRef
44. Walker MD, Shane E. Hypercalcemia: A Review. JAMA. 2022;328(16):1624-1636.
    CrossRef
45. Dundar ZD, Kucukceran K, Ayranci MK. Blood urea nitrogen to albumin ratio is a predictor of in-hospital mortality in older emergency department patients. Am J Emerg Med. 2021;46:349-354.
    CrossRef
46. Stark J. Interpretation of BUN and Serum Creatinine: An interactive exercise. Critical Care Nursing Clinics of North America. 1998;10(4):491-496.
    CrossRef
47. Fernández-Friera L, Fuster V, López-Melgar B, Oliva Belén, García-Ruiz José M, Mendiguren José, Bueno Héctor, Pocock Stuart, Ibáñez Borja, Fernández-Ortiz Antonio, Sanz Normal LDL-cholesterol levels are associated with subclinical atherosclerosis in the absence of risk factors. J Am Coll Cardiol. 2017;70(24):2979-2991.
    CrossRef
48. Sproston NR, Ashworth JJ. Role of C-reactive protein at sites of inflammation and infection. Front Immunol. 2018;9:754.
    CrossRef
49. Sherwani SI, Khan HA, Ekhzaimy A, Masood A, Sakharkar MK. Significance of HbA1c test in diagnosis and prognosis of diabetic patients. Biomark Insights. 2016;11:95-104.
    CrossRef
50. Chen L, Sun S, Gao Y, Ran X. Global mortality of diabetic foot ulcer: A systematic review and meta‐analysis of observational studies. Diabetes, Obesity and Metabolism. 2022;25(1).
    CrossRef
51. Brian Zhaojie Chin, Lee P, Sia CH, Choon Chiet Hong. Diabetic foot ulcer is associated with cardiovascular-related mortality and morbidity: A systematic review and meta-analysis of 8062 patients. Endocrine. Published online January 27, 2024.
52. Mansoor Z, Modaweb A. Predicting amputation in patients with diabetic foot ulcers: A systematic review. Cureus. 2022;14(7).
    CrossRef
53. Tong T, Yang C, Tian W, Liu Zhiping, Liu Bo, Cheng Jun, Cheng Qingfeng, Zhou Bo. Phenotypes and outcomes in middle-aged patients with diabetic foot ulcers: A retrospective cohort study. Journal of Foot and Ankle Research. 2020;13(1).
    CrossRef
54. Huijberts MSP, Schaper NC, Schalkwijk CG. Advanced glycation end products and diabetic foot disease. Diabetes/Metabolism Research and Reviews.
55. Hicks CW, Canner JK, Mathioudakis N, Lippincott C, Sherman RL, Abularrage CJ. Incidence and risk factors associated with ulcer recurrence among patients with diabetic foot ulcers treated in a multidisciplinary setting. Journal of Surgical Research. 2020;246:243-250.
    CrossRef
56. Shi L, Xue J, Zhao W, Wei X, Zhang M, Li Lijun, Xu Z, Wang A. The prognosis of diabetic foot ulcer is independent of age: A comparative analysis of the characteristics of patients with diabetic foot ulcer in different age groups: A cross-sectional study from China. Int J Low Extrem Wounds. 2022, 10 (1177).
    CrossRef
57. Gershater MA, Apelqvist J. Elderly individuals with diabetes and foot ulcer have a probability for healing despite extensive comorbidity and dependency. Expert Review of Pharmacoeconomics & Outcomes Research. 2020;21(2):1-8.
    CrossRef
58. Lin C, Liu J, Sun H. Risk factors for lower extremity amputation in patients with diabetic foot ulcers: A meta-analysis. PLoS One. 2020;15(9).
    CrossRef
59. Jarl G, Alnemo J, Tranberg R, Lundqvist LO. Gender differences in attitudes and attributes of people using therapeutic shoes for diabetic foot complications. Journal of Foot and Ankle Research. 2019;12(1).
    CrossRef
60. ‌Sorber R, Abularrage CJ. Diabetic Foot Ulcers: Epidemiology and the role of multidisciplinary care teams. Seminars in Vascular Surgery. 2021;34(1):47-53.
    CrossRef
61. Bekele F, Chelkeba L, Fekadu G, Bekele K. Risk factors and outcomes of diabetic foot ulcer among diabetes mellitus patients admitted to Nekemte Referral Hospital, Western Ethiopia: Prospective observational study. Annals of Medicine and Surgery. 2020;51:17-23.
    CrossRef
62. Kalan L, Loesche M, Hodkinson BP, Heilmann Kristopher, Ruthel Gordon, Gardner Sue E, Grice Elizabith A. Redefining the chronic-wound microbiome: fungal communities are prevalent, dynamic, and associated with delayed healing. mBio. 2016;7(5).
    CrossRef
63. Bechara N, Tien-Ming Hng, Gunton JE. The association between tobacco smoking and systolic toe pressures in active foot ulceration. Scientific reports. 2024;14(1).
    CrossRef
64. Bonnet JB, Sultan A. Narrative review of the relationship between CKD and diabetic foot ulcer. Kidney International Reports. Published online December 2021.
    CrossRef
65. Yan S, Yao D, Wang Y, Zhang J. Risk factors of foot ulcers in patients with end‐stage renal disease on dialysis: A meta‐analysis. International Wound Journal. 2023;21(1).
    CrossRef
66. Zafar S, Rahim K, Inayat Ullah Khan, Yasin M, Dawood M, Shamim Saleha. Prevalence and association of diabetic retinopathy with diabetic foot ulcer: A cross-sectional observational study. Frontiers in Ophthalmology and Ocular Imaging. 2019.
    CrossRef
67. Hwang DJ, Lee KM, Park MS, Choi Sung Hee, Park Ji In, Cho Joon Hee, Park Kyu Hyung, Woo Se Jun. Association between diabetic foot ulcer and diabetic retinopathy. PLoS One. 2017;12(4):e0175270.
    CrossRef
68. Khan MIH, Azhar U, Zubair F, Khan ZA. Can we link foot ulcer with risk factors in diabetics? A study in a tertiary care hospital. Pakistan Journal of Medical Sciences. 2018;34(6):1375-1380.
    CrossRef
69. Zhang WQ, Tang W, Hu SQ, Fu XL, Wu H,  Shen WQ, Chen H L. C-reactive protein and diabetic foot ulcer infections: A meta-analysis. J Tissue Viability. 2022;31(3):537-543.
    CrossRef
70. Sharma H, Sharma S, Krishnan A, Yuan D, Vangaveti V N, Malabu U H, Haleagrahara N. The efficacy of inflammatory markers in diagnosing infected diabetic foot ulcers and diabetic foot osteomyelitis: Systematic review and meta-analysis. Singh K, ed. PLOS ONE. 2022;17(4):e0267412.
    CrossRef
71. Ardelean A, Neamtu AA, Balta DF, Neamtu C, Goldis D, Rosu M, Nesiu A, Moldovan S, Tarta C, Totoliki B D. Lipid profile paradox: investigating improved lipid levels in diabetic mellitus patients with foot ulcer infections-a prospective descriptive study. Diagnostics (Basel). 2023;13(23):3531. Published 2023 Nov 25.
    CrossRef
72. ‌Jin L, Xu W. Renal function as risk factor for diabetic foot ulcers: A meta‐analysis. International wound journal. Published online November 22, 2023.
    CrossRef
73. Wang H, Chen W, Li D, Yin X, Zhang X, Olsen N, Zheng S G. Vitamin D and chronic diseases. Aging and disease. 2017;8(3):346-353.
    CrossRef
74. Kurian SJ, Baral T, Unnikrishnan MK, Benson R, Munisamy M, Saravu K, Rodrigues GS, Rao M, Kumar A, Miraj SS. The association between micronutrient levels and diabetic foot ulcer: A systematic review with meta-analysis. Frontiers in Endocrinology. 2023;14. ‌
    CrossRef