Keywords
Diabetic foot - fibrinogen - high-sensitive C-reactive protein - ischemia-modified
albumin
Introduction
Diabetic foot ulcer (DFU) is one of the major complications in patients with diabetes,
predominantly seen in elderly male patients above 60 years of age. The incidence rate
of DFU over the lifespan of a patient with diabetes is 15%–25%.[[1]] Regardless of extensive efforts, DFUs remain to be predisposed for a large number
of amputations of the lower limb and are related to diminished quality of life in
addition to higher risk of morbidity and mortality.[[2]],[[3]] Glycemic status plays a very important role in DFUs. The long-term persistent hyperglycemia
creates an environment wherein the cells are exposed to the reducing sugars and amino
groups of biomolecules leading to the production of advanced glycation-end products.
This reaction is known as the Maillard reaction, which has been hypothesized to be
an important mechanism in the pathophysiology of diabetes complications.[[4]],[[5]]
The abnormal metabolic state in type 2 diabetes mellitus (T2DM) involves chronic hyperglycemia,
dyslipidemia, and insulin resistance. This abnormal metabolic condition affects the
function of cells of endothelium, smooth muscle, and inflammatory cells. In normal
individuals, there is a balance between reactive oxygen species and antioxidant levels.
However, in T2DM, this balance is disturbed leading to a condition of increased oxidative
stress, due to rise in the oxidant contents and fall in antioxidant level in the serum.
There is hypercoagulability of blood in addition to the oxidative stress.[[6]] Patients with T2DM have diminished fibrinolysis and heightened production of procoagulants.
Thus, the oxidative stress, enhanced levels of procoagulants, and reduced fibrinolysis
favor the development of atherosclerosis that commonly progress to DFU.[[7]] The etiology of DFUs is multifactorial, the risk factors being peripheral neuropathy,
vascular disease, deformities of the foot, limited joint mobility, minor trauma, impaired
visual acuity, etc.[[8]]
The ischemic pathophysiological events, together with hypoxia and free radicals, lead
to a characteristic conformational change of N-terminal end of albumin, which results
in the development of ischemia-modified albumin (IMA) with decreased affinity binding
to transition metals.[[8]] Currently, IMA is being explored as a general marker for ischemic tissues, and
its role in DFU is one of the recent areas of research.[[9]] C-reactive protein (CRP) and fibrinogen are two of the best known acute-phase proteins
that are synthesized primarily by the hepatic tissue in response to a variety of inflammatory
cytokines and in the setting of acute inflammation, their concentration may increase
several hundred fold.[[10]]
Low-grade immune activation implicated in the development of T2DM is also responsible
for several microvascular and macrovascular complications associated with DM.[[11]] These patients have diminished fibrinolysis and a higher production of procoagulants,
leading to an inflammatory state, playing a chief role in the development of atherosclerosis
and its complications.[[11]],[[12]] Fibrinogen, a simple and effective inflammatory marker, has been estimated to be
elevated in the presence of DFU.[[13]],[[14]] The present study is taken up to explore the role of IMA and fibrinogen levels
in relation to high sensitive CRP (hsCRP) in patients with DFU when compared to patients
with diabetes without foot ulcers.
Methods
This study is a hospital-based, cross-sectional study conducted on the participants
admitted with DFU in the general surgery department of a tertiary care hospital. The
study was carried out over a period of 2 months, i.e., from June 01, 2017, to July
31, 2017. The sample size for this study was determined to be 30 cases with 95% confidence
interval at 90% power based on the works of Muhtaroğlu S. et al.[[8]] The study was approved by the Institutional Ethics Committee. All T2DM patients
presenting with foot ulcers who were admitted in the general surgery department of
a tertiary care hospital were considered for this study. However, patients with chronic
kidney disease with estimated glomerular filtration rate <30 ml/min/1.73 m2, liver
failure, cardiovascular disorders, venous ulcer, pregnancy, and malignancy were excluded
from the study. The history of the patients was obtained, following a clear explanation
and understanding of the purpose of the study through an informed consent, which was
signed by the patients. A thorough physical examination of the patients was done which
included recording the height, weight, blood pressure, pulse rate, and a general physical
examination followed by the inspection of the ulcer site in the DFU patients. Data
relevant to biochemical parameters such as glycated hemoglobin, plasma sugars, renal
function test, and liver function tests were obtained from the patient's medical records
at the time of admission.
After obtaining informed consent from the patients, 5 ml blood was collected in a
sterile plain vacutainer and 3 ml blood in citrated vacutainer for plasma. The serum
and plasma were separated by centrifugation at 2500 × g for 15 min, and stored at
minus 80°C until further analysis. Serum hsCRP was measured by immunoturbidimetry
method using commercially available kits by Abbott on ARCHITECT ci4100 (USA) analyzer.
Plasma fibrinogen was estimated on a semi-automated coagulation analyzer using commercially
available kits based on Clauss method.[[15]] Serum IMA was measured by colorimetric assay based on the biochemical properties
of albumin to bind exogenous cobalt, as previously described by Bar-Or et al.[[16]] IMA was reported in absorbance units; 1 ABSU = 100 × ΔOD (Optical Density of Test
minus Blank). To adjust for the interference of the serum albumin, albumin adjusted
IMA was calculated based on the following formula and was expressed in ABSU.[[17]]
Statistical analysis
Data analysis was done using (trial version IBM SPSS Statistics for Windows, Version
20.0. Armonk, NY: IBM Corp.). The data were tested for normality by using the Kolmogorov-Smirnov
test; further data were compared using student's independent sample t-test for parametric
data and Mann–Whitney U-test for nonparametric data. Pearson's correlation was done
to assess the relation of biomarkers with hsCRP. P values were considered statistically
significant at < 0.05.
Results
Following the inclusion criteria, a total of sixty patients were enrolled in the study
after obtaining informed consent from them. The control group comprised of patients
with diabetes without foot ulcer and the cases were the patients with DFU, based on
the inclusion criteria. Among them, 20 were females and 40 were men, with more percentage
of men (90%) suffering with DFU. In this study, the cases and controls were matched
for age and duration of having diabetes [[Table 1]]. The biochemical parameters of the two study groups are given in [[Table 2]]. Based on the HbA1c status, 66.7% of cases with DFU were found to have very poor
glycemic control.
Table 1: Clinical characteristics of the study subjects
Table 2: Biochemical parameters of the study subjects (Mean±SD)
The IMA varied from the lowest 43.00 ABSU to a maximum of 95.00 ABSU in the cases
with DFU with a mean ± standard deviation of 67.1 ± 14 ABSU. On comparing the levels
of glycemic groups with IMA levels, it showed an increased trend, implying raise in
glycated hemoglobin level is associated with increase in the serum IMA level [[Figure 1]]., Linear regression analysis between IMA and HbA1c revealed a significant moderate
positive correlation coefficient, r2 = 0.30 (P < 0.05).
Figure 1: Serum ischemia-modified albumin levels correlated with HbA1c in the study subjects
When glycated Hb was correlated with hsCRP, IMA, and AAIMA, all the three parameters
correlated positively with the HbA1c (P < 0.05) [[Table 3]]. Further, on correlating the values of serum albumin with hsCRP, IMA, and AAIMA,
it revealed that all these parameters had an inverse correlation with serum albumin
[[Table 3]]. Based on the inverse correlation between hsCRP and serum albumin, it is suggested
that high hsCRP levels may accompany the decrease in serum albumin [[Figure 2]]. Similar is the scenario with serum fibrinogen and serum albumin. Furthermore,
plasma fibrinogen levels correlated positively with serum IMA indicating rise in fibrinogen
levels is associated with rise in IMA levels in the serum [[Figure 3]].
Figure 2: Correlation of serum albumin with high sensitive-C-reactive protein in cases with
diabetic foot ulcer
Figure 3: Correlation of plasma fibrinogen with serum ischemia-modified albumin levels in cases
with diabetic foot ulcer
Table 3: Correlation of glycated hemoglobin and serum albumin with high-sensitive C reactive
protein, ischemia modified albumin, albumin adjusted ischemia modified albumin and
fibrinogen among patients with diabetic foot ulcer
Discussion
The prevalence of diabetes mellitus (DM) shows an increased propensity worldwide,
the most important complication being the lower-extremity vascular disease associated
with diabetes.[[18]],[[19]] Patients often ignore the initial signs and symptoms of the peripheral vascular
disease. The lower extremity injury either due to trauma, infection, or both in the
presence of diabetic neuropathy often presents as foot ulcers of varied degree. One
of the major causes of hospitalization and amputation among patients with diabetes
are the complications of the foot ulcers leading to significant health-care costs.
This is evidenced by the fact that around 20%–40% of health-care resources are spent
on diabetes-related diabetic foot.[[20]]
When the cases were grouped according to American Diabetes Association[[21]] into good glycemic control (HbA1c ≤7.0%) and poor glycemic control (HbA1c >7.0%),
about 25 patients with DFU (83.3%) were found to be having poor glycemic control.
Long-term testing and maintenance of glycemic targets especially HbA1c in the normal
range is of utmost importance for delaying and preventing the onset of vascular complications
of DM.[[22]]
The IMA levels were not correlated with the duration of diabetes implying that it
could be more of an acute phenomenon rather than a chronic one. The serum IMA levels
correlated with the HbA1c levels showing an increased trend of serum IMA levels with
increase in the HbA1c levels. Similar observations have been reported by other studies.[[7]],[[23]],[[24]] The serum IMA levels correlated negatively with serum albumin levels (P < 0.001).
Hs-CRP an acute-phase protein has been used in the diagnosis and monitoring of acute
inflammation and infection and DFU is invariably such a condition, where inflammation
is prone to occur and thus hsCRP levels are expected to be elevated. From this study,
it is evident that hsCRP levels are significantly on a higher side in DFU when compared
to patients with DM without DFU. Elevated levels in DFU could be due to various reasons
such as atherosclerosis due to endothelial damage, underlying inflammation, peripheral
arterial disease that has led to DFU.[[25]] It is also noted that hsCRP levels are elevated in the controls, i.e., diabetics
without foot ulcer which could be due to underlying inflammatory process.
Fibrinogen is an acute-phase protein, in addition to being a major blood coagulation
protein and a prime determinant of blood viscosity and platelet aggregation.[[26]] This protein is expected to be elevated in the inflammatory settings and is an
important risk factor in the vascular events that occur in DFU.[[27]] Kunutsor et al.[[28]] have shown that fibrinogen is an independent risk factor for sudden cardiac death.
Fibrinogen also plays a significant role in endothelial injury, formation of fibrin
clot which has low permeability, thrombosis, hyperactivity of platelets, and blood
flow abnormalities. All these contribute to subclinical atherosclerosis.[[29]] Based on the current study, it is noteworthy that fibrinogen levels are considerably
much elevated in patients with diabetes with DFU when compared to the values in patients
with diabetes without DFU, which is similar to the findings in other studies.[[13]],[[14]]. In controls, the elevated levels could be due to the initial stages of progression
to vascular diseases. Therefore, this could be another good marker in DFU as DFU is
generally associated with inflammation.
The present study was done at a single tertiary care center, with a very limited sample
size in a limited time frame. Furthermore, the IMA should have been estimated on follow-up
in the same set of patients but could not be executed due to the lack of time and
appropriate funds. As, this study was done with a small sample size, it is warranted
that further studies with larger sample size comprising of patients presenting with
various stages of DFU would be required to optimize the fibrinogen and IMA cut-off
levels in estimating the disease severity at initial evaluation as well as during
follow-up.
Conclusions
The present study has demonstrated that IMA levels and fibrinogen levels are significantly
elevated in patients with DFU and had significant correlation with serum albumin and
hs-CRP. Higher IMA levels were associated with elevated HbA1c levels, in patients
with DFU. In addition to IMA, fibrinogen levels also play an important role in estimating
the disease severity. The elevated fibrinogen levels and the serum IMA levels can
give a significant clue to the clinician for estimating the disease severity and decide
on initiation of the treatment earlier. These two markers can serve as potential prognostic
markers in T2DM patients with DFU, and early intervention can prevent lower limb amputation,
thereby decreasing the morbidity and mortality of patients with diabetes.
Acknowledgments
We would like to thank the participants of this study and for providing samples and
consent. We thank the Indian Council of Medical Research (ICMR), Government of India
for research support to the undergraduate student HA under the ICMR-STS program.
Financial support and sponsorship
This project was sponsored under the Indian Council of Medical Research (ICMR) - short
term studentship programme.
