The independent relationship between hemoglobin A_1c and homeostasis model assessment of insulin resistance in non-diabetic subjects

Introduction

Insulin resistance (IR) is defined as the impaired ability of target tissues of fat, liver, and muscle to show various metabolic effects of insulin, including glucose uptake [1]. Insulin resistance plays an important patho-physiological role in the development of diabetes, dyslipidemia, hypertension, and cardiovascular disease [2], [3]. Prospective studies have shown that it is a powerful predictor of the likelihood of an individual developing diabetes or cardiovascular disease [4]. Accurate measurement of IR requires complex techniques that are expensive and time-consuming. A number of surrogate indices of IR had been developed. The homeostatic model of assessment-insulin resistance (HOMA-IR), which uses fasting insulin and glucose levels to calculate IR, is the most widely used [5]. However, HOMA-IR results are reasonably correlated with those of clamping studies (the “gold standard”).

Glycated hemoglobin (HbA_1c) is well recognized and widely used as a measure of glycemic control reflecting the mean blood glucose level over the preceding weeks to months. Until recently, hemoglobin A_1c has only been used to monitor people already diagnosed with diabetes, serving as the gold-standard measure of glycemic levels over a 3-month period. More recently, as with measures of glucose, HbA_1c levels have been used to describe a continuum of risk for the development of diabetes and associated conditions. Because it can be measured regardless of food intake, A_1c is simpler than fasting plasma glucose (FPG) or oral glucose tolerance tests [6].

Recently, the International Expert Committee recommended that HbA_1c be added to the diagnosis of diabetes mellitus (DM); the 2010 American Diabetes Association (ADA) clinical practice recommendation defining HbA_1c levels of over 6.5% as DM, and an HbA_1c between 5.7 and 6.4% as pre-diabetes [7], [8]. It was shown that pre-diabetes is associated with insulin resistance which is a risk factor for development of type 2 diabetes. How well a HbA_1c level in the pre-diabetic range (5.7%–6.4%) predicts insulin resistance is not clear. Publications associated with the relationship between HbA_1c and insulin resistance are limited. Therefore, the aim of the current study was to clarify the relationship between insulin resistance and HbA_1c.

Materials and methods

We retrospectively analyzed serum fasting glucose, fasting insulin, 2 h glucose and HbA_1c records of 18–85 years-old outpatients who underwent a 75 g OGTT in Department of Biochemistry, Tepecik Teaching and Research Hospital during the period January 2010–May 2014. The samples for all parameters were collected in the same day. Patients with a serum creatinine level above reference range and patients with DM according to ADA criteria (fasting glucose>125 mg/dL or 2 h glucose >199 mg/dL or HbA_1c>6.4%) were excluded. The present study included a total of 365 patients of whom 281 were women and 84 were men. The study was conducted with the approval of the Local Hospital’s Ethics Committee.

Glucose tolerance status was assessed with the 75-g OGTT and serum fasting glucose defined according to the 2010 ADA criteria [8]. Our study used the HOMA-IR as the diagnostic criteria for insulin resistance and HOMA-IR≥2.5 was identified as an indicator of insulin resistance. HOMA-IR was calculated using the following formula: HOMA-IR=fasting serum glucose (mg/dL)×fasting serum insulin value (μU/mL)/405 [5]. Serum glucose levels were measured by the hexokinase method using the Olympus AU 2700 analyzer (Olympus Diagnostics. GmbH, Hamburg, Germany).

Serum insulin levels were measured using direct chemiluminescence technology on Siemens Immulite 2000 XPI analyzer (Siemens Healthcare Diagnostics, Deerfield, IL, USA). HbA_1c analysis was performed by affinity chromatography HPLC on Primus Ultra2 Analyzer (Primus Corporation, Kansas City, Kansas, USA).

Results

A total of 365 non-diabetic subjects were included in the study. Mean age of the subjects were 40±11 and 281 (77%) of the subjects were female, while 84 (23%) of the subjects were male. There were 191 individuals with normal glucose tolerance and 174 individuals with pre-diabetes (impaired fasting glucose, impaired glucose tolerance, or both according to ADA criteria). Insulin resistance (HOMA-IR>2.5) were detected in 147 (40%) of the subjects. Table 1 shows the characteristics of the study population.

The subjects were divided into two groups according to their HOMA-IR values: group 1, insulin sensitive (HOMA IR≤2.5) and group 2, insulin resistant (HOMA-IR>2.5). HbA_1c and other biochemical parameters of insulin resistant and insulin sensitive subjects were compared. Analysis showed that insulin resistant subjects with normal glucose tolerance had significantly higher HbA_1c and fasting glucose levels compared to insulin sensitive subjects with normal glucose tolerance (p=0.002, p<0.001, respectively). Similarly, insulin resistant subjects with pre-diabetes had significantly higher HbA_1c and 2-h glucose levels compared to insulin sensitive subjects with pre-diabetes (p=0.016, p=0.013, respectively) (Table 2).

To estimate the independent relationship between HbA_1c and HOMA-IR, multiple linear regression analysis was used. Age, gender, fasting glucose, 2-h glucose and HOMA-IR were entered in the regression model as independent variables. Since HOMA-IR values had skewed distribution, log transformed HOMA-IR was used in analysis. Regression analysis showed that age, fasting glucose, 2-h glucose, and log transformed HOMA-IR values are significant predictors of HbA_1c. Higher HOMA-IR values are associated with higher HbA_1c levels in non-diabetic subjects. Standardized β coefficients demonstrate the relative importance of predictor variables in regression model. In the regression model age showed the highest standardized β coefficient. In non-diabetic subjects age was the most important factor affecting HbA_1c values (Table 3). HbA_1c values were also presented in different age groups (Table 4).

Dividing the study population as normal and pre-diabetic subjects and re-performing the regression analysis did not change the results fundamentally. Age was still most effective factor in predicting HbA_1c values in both normal group and pre-diabetic group. Log HOMA was still a significant variable (data not shown).

To visualize the data a regression plot showing the relation between HbA_1c and HOMA-IR was generated. The relation between HbA_1c and HOMA-IR was similar in different sample groups (group 1: fasting glucose <96 mg/dL, group 2: fasting glucose >96 mg/dL) (Figure 1).

Figure 1:

Scatterplot showing correlation between HOMA-IR and HbA_1c in different sample groups. Group 1: fasting glucose <96 mg/dL. Group 2: fasting glucose >96 mg/dL.

Discussion

It was clearly reported that lower HbA_1c values were associated with reduced microvascular and macrovascular complications in diabetic patients [9]. In type 2 diabetes a HbA_1c target level of <7% is currently recommended by ADA and HbA_1c is the basis guiding diabetes therapy. Recently ADA recommended using HbA_1c also for the diabetes diagnosis and consolidated its importance in diabetes mellitus. The use of HbA_1c as a diagnostic criterion necessitates a more accurate measurement and careful interpretation. Several factors other than plasma glucose levels were reported to affect glycated hemoglobin levels. Any condition affecting turnover of red blood cells like hemolytic anemia and iron deficiency affect HbA_1c levels. Previous studies showed that genetic-ethnic factors, sex hormones and age are associated with HbA_1c values [10], [11], [12], [13], [14]. In the present study we report that HOMA-IR is a factor affecting HbA_1c values independent of fasting and 2-h post-load glucose concentrations. The relationship between glycated hemoglobin levels and glucose levels are higher in diabetic patients than in non-diabetic patients therefore it can be concluded that non-glycemic factors affecting HbA_1c levels are more important in non-diabetic patients [15].

Previous studies investigated the relationship between insulin resistance and HbA_1c. Gallwitz et al. showed that with increasing HbA_1c levels, there was a statistically significant increase in HOMA-IR in patients with type 2 DM [16]. Heianza et al. reported that in subjects without a history of diabetes a HbA_1c level of >5.9% were significantly associated with higher HOMA-IR values [17]. In another study Borai et al. showed that the correlation between HbA_1c and insulin resistance were higher in subjects with normal glucose tolerance than in patients with pre-diabetes and diabetes [18]. None of the aforementioned studies reported corrected results independent of glycemic status. Venkataraman et al. showed that in a multivariate regression model with HbA_1c as dependent variable HOMA-IR(log) were significantly associated with HbA_1c independent of fasting glucose but the effect of post-load glucose on HbA_1c was lacking [19]. One recent study indicated that HbA_1c was associated with HOMA-IR independent of 0- and 120-min glucose in pregnant women with gestational diabetes mellitus [20]. The results of our study reveal that HbA_1c is associated with HOMA-IR independent of fasting and post-load glucose status also in non-diabetic subjects.

Glycation is the nonenzymatic attachment of free aldehyde groups of carbohydrates to the unprotonated free amino groups of proteins [21]. The binding of glucose molecules to potential glycation sites in hemoglobin molecule leads to formation of HbA_1c. Via condensation with glucose, hemoglobin A first forms a labile intermediate adduct, which is thereafter rearranged to the more stable ketoamine adduct (HbA_1c) form [22]. Physiological factors like pH, inorganic phosphate, oxidative stress, deglycation, and Schiff base inhibitors can affect the rate of HbA_1c formation [23], [24], [25], [26], [27]. Another point to be considered is that glycation of hemoglobin occurs in the intracellular compartment. Previously, it was demonstrated that the erythrocyte glucose–to–plasma glucose concentration ratio may affect hemoglobin glycation and contributes to the variation in HbA_1c levels [28].

Oxidative stress is a process that was proposed to be associated with the multifactorial etiology of insulin resistance. It was shown that plasma markers of oxidative stress were correlated with the degree of insulin resistance [29], [30]. Oxidative stress which is a factor co-existing with insulin resistance may also be responsible for the increased hemoglobin glycation. Oxidative stress biomarkers as lipid peroxides were reported to be associated with hemoglobin glycation [31], [32]. LDL oxidation was also suggested to increase HbA_1c values [33]. Furthermore there is evidence that antioxidants can partially inhibit the formation of HbA_1c [31].

Consistent with previous studies, age was a significant factor affecting HbA_1c levels independent of glycaemia [13], [34]. However the mechanisms involved in the age related HbA_1c increase remain to be established. Since our model showed that age was the most effective factor contributing to the variation of HbA_1c levels in non-diabetic subjects it should not be ignored when interpreting an HbA_1c result. The question whether age-specific diagnostic and treatment criteria would be appropriate was previously mentioned [13].

Due to its retrospective design the current study has some limitations. Firstly the information of some possible confounding variables (e.g. BMI) could not be gathered; therefore confounding factors may exist. Second, HOMA-IR is not the gold standard method for measuring insulin sensitivity. The euglycemic hyperinsulinemic clamp technique is the gold standard for quantifying insulin sensitivity; however, this technique requires insulin infusion and repeated blood sampling. HOMA-IR is a relatively simple method to determine insulin sensitivity. It can be calculated from a single blood sample and it was reported to have a linear correlation with glucose clamp technique [35].

In conclusion, our data showed that HOMA-IR is associated with glycated hemoglobin values independent of glycaemia and age is a very important factor affecting HbA_1c values in non-diabetic subjects.