Blood sampling: sampling was done for fasting plasma glucose (FPG) in the morning (8.00 – 10.00 AM, after 8 hours of overnight fasting); 2-hour postprandial plasma glucose (2h PPPG), glycated hemoglobin (HbA1c)22 ; complete blood count (CBC); serum aspartate transaminase (AST) and alanine transaminase (ALT); serum creatinine; fasting lipid profile (after 12 – 14 hours of overnight fasting), including serum total cholesterol (TC) and triglycerides (TG), serum high-density lipoprotein cholesterol (HDL-C), and serum low-density lipoprotein cholesterol (LDL-C); PAI-1 (using ELISA)23; high-sensitivity C-reactive protein (hs-CRP); and 1^st hour erythrocyte sedimentation rate (ESR). The estimated glomerular filtration rate (eGFR) was calculated using the MDRD formula for staging of kidney disease. Early morning urine samples were obtained to assess urine albumin-to-creatinine ratio (UACR). Dilated fundus examination was performed for assessment and staging of the presence of diabetic retinopathy24.

Data were input into the computer and analyzed using IBM SPSS software package version 20.0 (IBM Corp, Armonk, NY, USA). The Kolmogorov–Smirnov test was used to verify the normality of distribution. Statistical significance of the obtained results was set at the 5% level. Correlations between quantitative continuous variables and parameters were evaluated by multivariate regression analysis.

There was no statistically significant difference between the three groups regarding gender distribution (p = 0.067) or age (p = 0.166). Smokers constituted 97% of group I patients, compared to 63% for group II (p = 0.001). Patients with T2DM with DFD had longer duration of diabetes (p < 0.001). Regarding DFD, 29.5% of cases were mild, 32.0% were moderate, and 38.5% were severe. Foot ulcers were found in 39.5% of patients, and below knee amputations in 4%. Foot deformities were more prevalent in group I, where 9.5% had hammer/claw toe and 5.5% had Charcot foot. Diabetic retinopathy was more prevalent in group I, where 58% had NPR and 15.5% had PR. In T2DM patients with DFD, 48.5% were smokers (p = 0.001), 81.5% were hypertensive (p < 0.001), 17.5% had CHD (p = 0.001), 70% had claudication (p < 0.001), and 14.5% had a history of laser photocoagulation (p < 0.001). Patients with T2DM had higher BMI and WC values compared to the control group (p < 0.001 and p < 0.001, respectively). Systolic and diastolic blood pressure levels were also higher in patients with T2DM compared to the control group (p < 0.001, p < 0.001, and p < 0.001, respectively). No significant differences were observed when comparing patients with T2DM and DFD to those with T2DM without DFD regarding BMI, WC, and systolic and diastolic blood pressure (p = 0.480, p = 0.317, p = 0.376, and p = 0.325, respectively). Regarding ABI, there was a significant difference between T2DM patients with DFD compared to T2DM patients without DFD (p < 0.001), as shown in Table 1.

Table 1

	Cases with T2DM with DFD	Cases with T2DM without DFD	Control group	p1	p2	p3
Duration of diabetes
Mean ± SD.	8.05 ± 4.18	3.69 ± 1.47	–	<0.001*	–	–
Waist circumference
Mean ± SD.	93.80 ± 8.65	92.55 ± 8.98	89.66 ± 7.67	0.317	<0.001*	0.017*
BMI (kg/m2)
Mean ± SD.	29.29 ± 2.94	28.96 ± 3.0	27.24 ± 2.33	0.480	<0.001*	<0.001*
Ankle brachial index (ABI)
Mean ± SD.	0.83 ± 0.14	1.01 ± 0.04	1.03 ± 0.05	<0.001*	<0.001*	0.235
Systolic blood pressure (mmHg)
Mean ± SD.	134.1 ± 10.93	132.6 ± 12.20	119.3 ± 8.34	0.376	<0.001*	<0.001*
Diastolic blood pressure (mmHg)
Mean ± SD.	79.70 ± 9.18	81.0 ± 9.81	71.95 ± 7.15	0.325	<0.001*	<0.001*

p₁: p value for comparing between Cases with T2DM with DFD and Cases with T2DM without DFD, p₂: p value for comparing between Cases with T2DM with DFD and Control group, p₃: p value for comparing between Cases with T2DM without DFD and Control group, *: Statistically significant at p ≤ 0.05

There was no significant difference regarding ALT, serum creatinine, and eGFR between the three groups (p = 0.322, p = 0.680, and p = 0.419, respectively). However, UACR was significantly different between the groups (p < 0.001). There was no significant difference between group I and group II regarding TG and HDL (p = 0.250 and p = 0.067, respectively). However, group I showed significantly higher mean TC and LDL compared to group II (p < 0.001). There was a significant difference regarding FPG, 2h PPPG, and HbA1C between the groups; group I showed the highest mean and group III showed the lowest mean (p < 0.001), as shown in Table 2.

Table 2

	Cases with T2DM with DFD	Cases with T2DM without DFD	Control group	p1	p2	p3
Fasting blood glucose (FBG)
Mean ± SD.	193.2 ± 50.97	178.5 ± 53.98	88.99 ± 6.52	0.005*	<0.001*	<0.001*
2 hours post prandial blood glucose (2H PPBG)
Mean ± SD.	250.6 ± 56.63	234.0 ± 66.90	123.4 ± 9.99	0.009*	<0.001*	<0.001*
HbA1c
Mean ± SD.	8.15 ± 0.68	7.93 ± 0.75	5.21 ± 0.21	0.002*	<0.001*	<0.001*
TC
Mean ± SD.	203.1 ± 32.31	192.9 ± 35.68	151.3 ± 19.12	0.004*	<0.001*	<0.001*
TG
Mean ± SD.	156.4 ± 28.58	152.0 ± 31.40	118.5 ± 15.41	0.250	<0.001*	<0.001*
HDL-C
Mean ± SD.	42.32 ± 5.29	43.74 ± 5.86	46.17 ± 4.88	0.067	<0.001*	<0.001*
LDL-C
Mean ± SD.	114.3 ± 37.39	94.15 ± 28.90	79.80 ± 10.80	<0.001*	<0.001*	<0.001*
Serum Creatinine
Mean ± SD.	0.72 ± 0.09	0.73 ± 0.09	0.73 ± 0.11	>0.05	>0.05	>0.05
eGFR
Mean ± SD.	95.99 ± 4.07	95.17 ± 5.19	96.40 ± 8.88	>0.05	>0.05	>0.05
Urinary albumin/ Creatinine ratio (UACR)
Mean ± SD.	69.18 ± 50.72	33.08 ± 27.78	15.91 ± 4.81	<0.001*	<0.001*	<0.001*
ALT
Mean ± SD.	27.12 ± 8.87	26.13 ± 9.13	25.71 ± 6.52	>0.05	>0.05	>0.05

The mean PAI-1 was 29.76 ± 6.31 for group I, 25.78 ± 8.58 for group II, and 22.07 ± 6.12 for group III. Group I and group II showed significantly higher mean PAI-1 values than group III, with group I showing the highest mean PAI-1 (p < 0.001), as shown in Figure 1.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

There was a significant positive correlation between PAI-1 and metabolic control parameters (including FPG, 2h PPPG and HbA1c), as well as duration of diabetes in group I and group II (p < 0.001). A significant positive correlation was found between PAI-1 and severity of DFD in group I (p < 0.001), as shown in Figure 2.

The mean hs-CRP was 2.29 ± 0.82 mg/dl for group I, 1.21 ± 0.57mg/dl for group II, and 0.69 ± 0.12 mg/dl for group III. Group I and group II showed significantly higher mean hs-CRP values than group III, with group I showing the highest mean hs-CRP (p < 0.001), as shown in Figure 3.

There was a significant positive correlation between hs-CRP and metabolic control parameters (including FPG, 2h PPPG and HbA1c), as well as duration of diabetes in group I and group II (p < 0.001). A significant positive correlation was found between hs-CRP and severity of DFD in group I (p < 0.001), as shown in Figure 2.

The mean 1^st hour ESR was 39.18 ± 16.85 mm/h for group I, 24.33 ± 11.59 mm/h for group II, and 9.87 ± 2.81 mm/h for group III. Group I and group II showed significantly higher mean 1^st hour ESR values than group III, with group I showing the highest mean 1^st hour ESR (p < 0.001), as shown in Figure 4.

There was a significant positive correlation between 1^st hour ESR and metabolic control parameters (including FPG, 2h PPPG, and HbA1c), as well as duration of diabetes in group I and group II (p < 0.001). A significant positive correlation was found between 1^st hour ESR and severity of DFD in group I (p < 0.001), as shown in Figure 2.

ROC curve analysis for PAI-1, hs-CRP, and 1^st hour ESR was performed to discriminate DFD from non-DFD. It was found that hs-CRP has greater diagnostic value in the detection of DFD; the area under the curve (AUC) of hs-CRP was 0.856, which was significant (p < 0.001) and revealed its better performance. The best hs-CRP cut-off point in this regard was more than 1.9 for the detection of DFD (with a sensitivity of 70.5%) and non-DFD (with a specificity of 87.0%). The positive and negative predictive values were 84.4 and 74.7, respectively.

The AUC of PAI-1 was 0.629, which was significant (p < 0.001). The cut-off point was more than 29.75 for the detection of DFD (with a sensitivity of 59.0%) and non-DFD (with a specificity of 67.50%). The positive and negative predictive values were 64.5 and 62.2, respectively.

The AUC of 1^st hour ESR was 0.754, which was significant (p < 0.001). The cut-off point was more than 29 for the detection of DFD (with a sensitivity of 70.0%) and non-DFD (with a specificity of 65.0%). The positive and negative predictive values were 66.7 and 68.4, respectively, as shown in Figure 5.

DM is a worldwide problem, and its prevalence is increasing25. In particular, T2DM is currently considered to be one of the major challenges to the healthcare system.

Chronic uncontrolled hyperglycemia is associated with microvascular and macrovascular complications, which both play an important role in DFD2. DFD is a major problem and is considered to be the most common cause of non-traumatic amputation in developing countries26.

In this study, we evaluated the link between inflammatory markers (including PAI-1, hs-CRP, and 1^st hour ESR), metabolic control parameters (including FPG, 2h PPPG, and HbA1c), and DFD. The results showed that both groups of patients with T2DM had significantly higher mean serum levels of inflammatory markers (including PAI-1, hs-CRP, and 1^st hour ESR) compared to the control group, and the mean level of inflammatory markers was higher in T2DM patients with DFD compared to T2DM patients without DFD, indicating that inflammatory markers play a role in DFD. In contrast to our findings, Kulwas et al.27 found that the difference in PAI-1 levels between diabetic patients with foot ulcers, those without foot ulcers, and the control group was not statistically significant. In agreement with our results, Weigelt et al.28 found that diabetic individuals with an acute foot ulcer had greater CRP levels than those without foot ulcers (p < 0.01), and Abolfotouh et al.29 observed 1^st hour ESR levels greater than 60mm/hr in 70% of DFD patients, but in only 30% of non-DFD patients, which was statistically significant. (p = 0.002). In the present study, the mean serum levels of FPG, 2h PPPG, and HbA1C were significantly higher in T2DM patients with DFD compared to T2DM patients without DFD, indicating that poor metabolic control plays an important role in DFD. In agreement with our results, Kulwas et al.27 observed that HbA1C was >7% in diabetic patients with foot ulcers and <6.5% in those without foot ulcers group, and Hwang et al.30 found that HbA1c was 8% in diabetic patients with foot ulcers and 7.4% in those without foot ulcers. In our study, there was a significant positive correlation between inflammatory markers (including PAI-1, hs-CRP, and 1^st hour ESR) and metabolic control parameters (including FPG, 2h PPPG, and HbA1C) in both groups of diabetic patients. In agreement with our study, PAI-1 and HbA1c—as well as RBG—were found to have substantial positive relationships in the study conducted by Adly et al.31. In another study, Romuk et al.32 found that PAI-1 expression was strongly and positively linked with HbA1c concentration in both diabetic groups (T1DM and T2DM), showing the impact of glycemic management on PAI-1 molecule expression. Increased hs-CRP in diabetic patients with complications or high PAI-1 was found by Adly et al.,31 which is in agreement with Coulon et al.,33 who discovered that CRP levels were three-fold higher in uncomplicated diabetic patients compared to controls and five-fold higher in diabetic patients with subclinical complications. Abolfotouh et al.29 used logistic regression analysis with the presence of DFD as the dependent variable to evaluate the risks of DFD in diabetic patients, after adjusting for age and gender, and found that peripheral neuropathic pain (PNP), duration of diabetes, and ESR level were significant predictors of DFD in diabetic patients.

Some limitations should be considered in our study. For example, it lacks generalizability in that it was only conducted at the Diabetes Clinic at Alexandria Main University Hospital. In addition, the duration of the study was relatively short (only 14 months). Furthermore, the study only included 500 patients, which is a relatively small sample size.

Inflammation is a potential therapeutic target and may be both a cause and an effect of DM and its complications. DFD is associated with elevated indicators of systemic and vascular inflammation. There was a high incidence of early chronic problems, which was linked to HbA1c and increased inflammatory biomarkers. We recommend that PAI-1, hs-CRP, and 1^st hour ESR be considered as early predictors for vascular tissue damage which can be used for the early detection of complications in diabetic patients.

ABI: Ankle brachial index, ACR: Albumin creatinine ratio, BMI: Body mass index , CHD: Coronary heart disease, DFS: Diabetic foot syndrome, DFD: Diabetic foot disease, DM:Diabetes mellitus, HSCRP: High sensitive CRP, Ist ESR: First hour ESR, IR: Insuline resistance, PAD: Peripheral arterial disease, PAI 1: Plasminogen activator inhibitor 1, T2DM: Type 2 diabetes mellitus, WC: Waist circumference

This work was supported by Internal Medicine Department (Diabetes Unit), Faculty of Medicine, Alexandria University, Egypt.

Study design (Fathi Z. Elsewy and Magdy H. Megalaa), Acquisition of data (Nader F Tadros), Laboratory tests supervision (Neveen L.Michael), Analysis and interpretation of data (Magdy H. Megalaa), drafting the article (Neveen L.Michael) and critical revision of the manuscript (Fathi Z. Elsewy and Magdy H. Megalaa). All authors read and approved the final manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

This study was conducted in accordance with the amended Declaration of Helsinki. The institutional review board approved the study, and all participants provided written informed consent.

Not applicable.

The authors declare that they have no competing interests.