A total of 200 diabetic patients with mean age of 59.00 ± 9.11 years were included in this study. Most of the participants were females (71.0%), have had diabetes for 13.92 ± 6.19 years and had been on metformin therapy for 12.88 ± 6.15 years. Furthermore, 32.5% and 6.5% presented with neuropathy based on MNSI-Q and MNSI-PE, respectively. Other clinical, immunological, metabolic and hematological parameters are shown in Table 1.
Table 1
Demographic, clinical and immune-metabolic profile of the entire study participants.
Variables
|
Mean ± SD
|
Age (years)
|
59.00 ± 9.11
|
BMI (kg/m2)
|
26.10 ± 3.70
|
SBP (mmHg)
|
132.21 ± 17.60
|
DBP (mmHg)
|
77.35 ± 9.93
|
DM duration (years)
|
13.92 ± 6.19
|
Metformin duration (years)
|
12.88 ± 6.15
|
Sex
|
Frequency (%)
|
Female
|
142 (71.0)
|
Male
|
58 (29.0)
|
Metformin dosage (mg/day)
|
|
1000
|
32 (16.0)
|
2000
|
130 (65.0)
|
3000
|
38 (19.0)
|
MNSI-Q
|
|
Neuropathy absent
|
135 (67.5)
|
Neuropathy present
|
65 (32.5)
|
MNSI PE
|
|
Neuropathy absent
|
187 (93.5)
|
Neuropathy present
|
13 (6.5)
|
Circulation impairment
|
|
Absent
|
112 (56.0)
|
Present
|
88 (44.0)
|
General asthenia
|
|
Absent
|
110 (55.0)
|
Present
|
90 (45.0)
|
Immunological parameters
|
|
IFA (AU/mL)
|
1.10 (1.0-1.21)
|
GPA (pmol/l)
|
5.20 (3.71–6.80)
|
TNF-α (pg/ml)
|
42.67 (30.00-50.92)
|
IL-6 (pg/ml)
|
66.67 (58.17-85.00)
|
Metabolic parameters
|
Median (IQR)
|
FPG (mmol/L)
|
6.90 (6.00-9.05)
|
HbA1c (%)
|
6.90 (6.00-8.20)
|
TC (mmol/L)
|
5.10 (4.20–6.30)
|
TG (mmol/L)
|
1.60 (1.20–1.90)
|
HDL (mmol/L)
|
1.30 (0.90–1.90)
|
LDL (mmol/L)
|
3.02 (2.23–4.32)
|
Serum albumin (g/dl)
|
4.10 (4.05–4.20)
|
Corrected calcium (mmol/L)
|
9.04 (8.82–9.44)
|
Hematological parameters
|
|
TWBC (103/𝜇L)
|
5.12 ± 1.71
|
RBC (106/𝜇L)
|
4.02 ± 0.60
|
Hemoglobin (g/dL)
|
12.05 ± 1.54
|
HCT (%)
|
34.12 ± 4.79
|
MCV (fL)
|
83.39 ± 7.94
|
MCH (pg)
|
30.82 ± 2.44
|
MCHC (g/dL)
|
36.56 ± 1.87
|
PLT (103/𝜇L)
|
187.0 (155.0-210.0)
|
MNSI-Q: Michigan Neuropathy Screening Instrument Questionnaire; Michigan Neuropathy Screening Instrument Physical Examination (MNSI-PE); IFA: Intrinsic factor antibody; GPA: Gastric parietal cell antibody; TNF-α: Tumor necrosis factor-alpha; IL-6: Interleukin 6; FPG: Fasting plasma glucose; HbA1c: Glycated hemoglobin; TC: Total cholesterol; TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TWBC: Total White Blood Cells; RBC: Red Blood Cells; HCT: Haematocrit; MCV: Mean Cell Volume; MCH: Mean Cell Haemoglobin; MCHC: Mean Cell Haemoglobin Concentration; PLT: Platelet count. |
The average sB12, hTC, MMA and Hcy were 142.10 (59.00-178.90) pmol/L, 27.02 (15.03–45.66) pmol/L, 0.17 (0.14–0.20) µmol/L and 6.50 (4.35–13.80) µmol/L, respectively. The median Fedosov’s wellness score (ω) was − 0.18 (-0.97-0.32) (Fig. 1A). Using the combined indicator (4cB12), the prevalence of vitamin B12 deficiency was 40.5% (Fig. 1B).
IFA (1.18 (1.00-1.30) vs 1.00 (0.98–1.20), AU/ML, p = 0.003), GPA (5.80 (4.10–7.90) vs 4.80 (3.60–6.50) pmol/l, p = 0.005) and TNF-α (45.00 (33.47–56.67) vs 41.67 (28.33–48.33), pg/ml p < 0.0001) levels were significantly higher among the participants with vitamin B12 deficiency compared to those with normal vitamin B12 levels. The levels of IL-6 did not differ significantly between the deficient and non-deficient group (Fig. 2).
Participants with vitamin B12 deficiency presented with significantly higher TC (5.60 (4.60–6.80) vs 5.10 (4.10-6.00) mmol/L, p = 0.01), LDL (3.08 (2.46–4.84) vs 2.86 (2.15–3.85) mmol/L, p = 0.005) and albumin (4.20 (4.10–4.30) vs 4.10 (4.00-4.20) g/l, p = 0.021) levels compared to those with normal vitamin B12 levels (Fig. 3).
Although vitamin B12 deficient participants had marginally higher mean cell volume (macrocytosis) and platelet count, there was no statistically significant association between hematological parameters and vitamin B12 status (Table S1).
Correlation analysis revealed a statistically significant negative association between 4cB12 and the immunological markers [IFA (rs= -0.301, AU/ml, p < 0.0001), GPA (rs= -0.244, pmol/l, p = 0.001), TNF-α (rs= -0.242, pg/ml, p = 0.001) and IL-6 (rs= -0.145, pg/ml, p = 0.041)]. Likewise, 4cB12 was negatively associated with TC (rs= -0.203 mmol/l, p = 0.004) and LDL (rs= -0.222, mmol/l, p = 0.002) but positively correlated with HDL (rs = 0.196, mmol/l, p = 0.005). A negative correlation was also observed between sB12 and IFA (rs=-0.341, p < 0.0001), GPA (rs= -0.234, p = 0.001) and TNF-α (rs= -0.169, p = 0.016) whereas hTC correlated negatively with only TNF-α (rs= -0.22, p = 0.002). Conversely, MMA correlated positively with AIF (rs = 0.189, p = 0.007), GPA (rs = 0.262, p < 0.0001), IL-6 (rs = 0.185, p = 0.009) and calcium (rs = 0.145, p = 0.041) but negatively with HDL (rs= -0.19, p = 0.007). Correspondingly, Hcy showed a positive correlation with the immunological markers [AIF (rs = 0.278, p < 0.0001), GPA (rs = 0.342, p < 0.0001), TNF-α (rs = 0.174, p = 0.014), IL-6 (rs = 0.186, p = 0.008) as well as TC (rs = 0.211, p = 0.003) and LDL (rs = 0.217, p = 0.002) but a negative correlation with HDL (rs= -0.18, p = 0.011) (Fig. 4).
Among the 32.5% and 6.5% presented with neuropathy based on MNSI-Q and MNSI-PE, respectively, a higher proportion also had vitamin B12 deficiency (20.0% and 4.0% for MNSI-Q and MNIS-PE, respectively). Furthermore, vitamin B12 deficiency was significantly associated with over 3-fold increase in the odds of neuropathy compared to the non-deficient counterparts by MNSI-Q (COR = 3.67, 95% CI (1.97–6.82), p < 0.0001). Although vitamin B12 deficiency was also associated with higher odds of neuropathy by MNSI-PE, the association was not statistically significant (Table 2).
Table 2
Association between vitamin B12 status and diabetic neuropathy
Vitamin B12 status
|
MNSI-Q
|
p-value
|
|
Neuropathy absent
|
Neuropathy present
|
COR (95% CI)
|
|
Non-deficient
|
94 (47.0)
|
25 (12.5)
|
1
|
|
Deficient
|
41 (20.5)
|
40 (20.0)
|
3.67 (1.97–6.82)
|
< 0.0001
|
|
MNSI-PE
|
|
Non-deficient
|
114 (57.0)
|
5 (2.5)
|
1
|
|
Deficient
|
73 (36.5)
|
8 (4.0)
|
2.50 (0.79–7.93)
|
0.12
|