Impact of different severity hyperglycemia on erythrocyte rheological properties 1

Abstract

BACKGROUND:

The triad “insulin resistance, prediabetes, diabetes” is three independent neologies with characteristic features and development. In addition, each are characterized by progression and the possibility of transition from one form to other. Due to the fact that diabetes is one of the common diseases associated with high rates of disability, it is necessary to improve diagnostic methods and educational regimens for successful prevention and treatment of the disease.

OBJECTIVE:

We investigated Band 3 protein (B3p) level, osmotic resistance of erythrocytes, the total antioxidant activity (TAA) of blood serum, level of HbA1 in group patients with insulin resistance (IR), prediabetes, and Type 2 Diabetes Mellitus (T2DM) and comparative with health control group.

METHODS:

We used original, accurate research methods that measure the essence of the studied quantities.

RESULTS:

Disruptions of glucose and insulin homeostasis ay lead to the initiation of oxidative stress (in our study demonstrated by a decrease of TAA of blood serum) increased redox-sensitive PTP activity and aberrant band 3 phosphorylation, potentially leading to reduced erythrocyte deformability. At the same time glycation of Hb during T2DM may affect its cross-link with membrane proteins, in particular with B3p, and although appears to limit its cross-linking and decrease its clusterization ability, induces alterations in the cytoskeletal matrix, and thereby decrease erythrocytes’ osmotic resistance making them more susceptible to hemolysis.

CONCLUSIONS:

The osmotic resistance of the erythrocytes can be used as a sensitive marker for the detection of the early stages of hyperglycemia (prediabetes). This set of clinical trials will make it possible to identify diseases that make up the triad at an early stage. Early detection of disorders and continued research in this direction will help in the development of a diagnostic scheme for the prevention of such patients. Based on our data, research into anti-oxidation drugs is very important. With the help of the array of studies described in the article and antioxidant treatment, the likelihood of successful treatment will increase.

Keywords

Hyperglycemia erythrocyte band 3 protein oxidative stress osmotic resistance

List of abbreviations

insulin resistance

T2DM

Type 2 Diabetes Mellitus

B3p

Band 3 protein

TAA

Total antioxidant activity of blood serum

HbA1

Glycated hemoglobin

TAA

Total antioxidant activity

1 Introduction

The triad “insulin resistance, prediabetes, diabetes” is one of the most studied pathophysiological conditions, which, on the one hand, is a continuous pathological chain that promotes the outgrowth of one stage into another and also it is three independent nosology’s with characteristic features and development. The main feature of the constituents of the triad is manifested in the fact that each of them contributes to the development of diseases associated with impairment of macro- and microcirculation, leading to an unfavorable outcome, which in turn can be caused by the membrane properties and activity of red blood cells that form the bloodstream.

According to the data of the Centers for Disease Control in Europe insulin resistance (IR) is detected in every fourth practically healthy person; about 1 in 10 Europeans are suffered from prediabetes [1]. Globally about 9.3% of adults are living with diabetes. More than half of individuals with diabetes remain unaware of their diabetic status, thus untreated leading to complications (heart disease, stroke, peripheral vascular disease, and microvascular complications) [1, 2]. An increase in the risk of cardiovascular complications and mortality is observed already at the stage of early disorders of carbohydrate metabolism. T2DM remains one of the main causes of death and disability in the developed world.

Insulin resistance and prediabetes are considered as prerequisites for diabetes. It has not been proven that under any conditions insulin resistance always turns into prediabetes, and prediabetes turns into diabetes. However, it is important that insulin resistance is a favorable factor for prediabetes, and prediabetes is a risk factor for diabetes.

Several physiological conditions (old age, pregnancy, physical inactivity) contribute to the development of IR, however, it is more often caused by as pathological conditions as genetic defects, arterial hypertension, dyslipidemia, and a social problem such as passive lifestyle, unbalanced diet, sleep/wake disturbances, multiple environmental stresses. Other of the significant causes of IR is a mutation in the genes of the insulin receptor’s protein, glycogen synthetase, hormone-sensitive lipase, beta3 adrenergic receptors, TNF-α, and molecular defects in the insulin’s signals transmitting proteins [3]. Often against the background of chronic IR, there is hyperglycemia and gradual decrease in β-cell function. Against this background, prediabetes develops.

With the development of the metabolic response of the body to endogenous or exogenous insulin is disturbed, which leads to a relatively high, compared with physiological values, insulin concentration concerning the level of glucose in blood plasma. In this case, the effect of insulin might injure the vascular endothelium, or disbalance the protein and fat metabolism.

Already at the stages of prediabetes, the vascular wall, permeability and fragility of blood vessels change, which subsequently becomes an independent disease, and the likelihood of developing diabetes also increases.

Prediabetes is a fone for developing of the classic form of disease progression type 2 diabetes mellitus (T2DM) [4, 5] and increased the risk of developing T2DM.

There are cases of diabetes development without manifestation of prediabetes and the patient’s insulin-resistant state. We cannot say for sure whether the development of diabetes was primary, or whether it is the final link in the transition process of the triad “insulin resistance, prediabetes, diabetes”, because the patient consulted a doctor only when diabetes had already manifested itself clinical.

Such is due to the insufficiency of diagnostic and preventive means. Late access to a doctor, under examination and inadequate treatment are among the main factors that turn the disease into a so-called disease. malignant diabetes with complications. Registration of insulin resistance and prediabetes, as well as an adjusted treatment strategy, are very important for successful management of patients. Thus, identifying insulin resistance and prediabetes as prerequisites for type 2 diabetes is a very pressing modern problem.

Prevention of T2DM requires a modifying effect on insulin resistance and other pathological events leading to diabetes, slowing down, blocking, or reversing the β-cell dysfunction. Early diagnosis and prevention of T2DM is the most effective and economically feasible strategy to reduce chronic complications and mortality from T2DM [6 –8].

In this regard, it is especially important to monitor and study new parameters, and widespread introduction into clinical practice new methods that can be helpful for early diagnosis of glucose metabolism disorders complications. This will effectively affect the increase in the duration and improvement of the quality of life of patients, and also, which is important, will reduce the financial costs for the provision of all types of medical care in a cohort of the population with T2DM [7].

A distinctive feature of insulin resistance and prediabetes and diabetes is the deterioration of blood flow. Blood fluidity is directly related to the blood profile, which is formed by the flow of red blood cells. The state of erythrocyte membranes and their properties are key to the study of blood fluidity, which causes disruption of the functioning of organs and body systems in this triad of diseases [9, 10]. The purpose of our work was to study some properties of erythrocytes in insulin resistance and prediabetes as precursors of diabetes. For this we researched level of Band 3 protein, the osmotic resistance of erythrocytes, the total antioxidant activity (TAA) of blood serum, Level of HbA1, as properties, which make effect of erythrocytes on blood flow [11].

2 Materials and methods

2.1 Target group

Blood was obtained upon signed informed consent from healthy volunteers, and also patients with IR, prediabetes, and T2DM. The study protocol was proven by the Ethical Committee for Human Studies of the Multidisciplinary High School of NNLE Society of Rheology (N EC 18122101).

We followed three groups of patients: 1. Patients with IR (n = 20, average age – 45±3,4 years); 2. Patients with prediabetes (n = 20 average age 48±2,4 years) (no prior history of insulin resistance)); 3. Patients with T2DM (n = 20, average age 50±5,5 years) with an average age of the disease less than one year (without a previous record in the history of IR and prediabetes).

To confirm IR, we diagnosed in patients without carbohydrate metabolic disorders (glycated hemoglobin (HbA1c) <5.7%–6.4%; fasting plasma glucose <100–125 mg/dl, and plasma glucose 2-hour post 75 g oral glucose challenge <140–199 mg/dl), having two of three symptoms – overweightness/obesity (body mass index (BMI) – ≥27 kg/m²), or abdominal obesity (waist circumference ≥88 cm (for women), ≥94 cm (for men)), dyslipidemia (elevated plasma triglycerides (Tg) ≥150 mg/dL and/or decreased high-density lipoprotein cholesterol (HDL-Chol) <50 mg/dL), arterial hypertension (arterial pressure ≥135/85 mmHg or antihypertensive drug therapy), without carbohydrate metabolic disorders (glycated hemoglobin (HbA1c) <5.7%–6.4%; fasting plasma glucose <100–125 mg/dl, and plasma glucose 2-hour post 75 g oral glucose challenge <140–199 mg/dl).

To confirm a diagnosis of prediabetes, carbohydrate metabolic disorders tests were used: HbA1c test, fasting plasma glucose levels, and 2-hour post 75 g oral glucose challenge (HbA1c – 5.7%–6.4%; impaired fasting plasma glucose – 100–125 mg/dl, and plasma glucose 2-hour post 75 g oral glucose challenge – 140–199 mg/dl (impaired glucose tolerance)). If we observed having two metabolic disorders of three, we included this patient in the group with prediabetes.

To confirm the diagnosis of T2DM, carbohydrate metabolic disorders tests were used: HbA1c level, fasting plasma glucose, and an oral glucose tolerance test (HbA1c level of 6.5% or greater, fasting plasma glucose level of 126 mg/dl or greater, or a 2-hour post-load glucose level of 200 mg/dL or greater).

2.2 Determination of Bend 3 protein (B3p) level (Protein analytical electrophoresis under dissociated conditions)

The soluble and membrane-bound protein (solubilized in Laemmli buffer) content was determined using the DC (detergent-compatible) protein assay [12]. Protein analytical electrophoresis was performed under dissociated conditions in a 12.5% gradient polyacrylamide gel with 1 mm thick and 6 ml volume with 0.1% sodium dodecyl sulfate (SDS). The samples of membrane proteins were heated for 10 min at 100°C and loaded (20μg) on an 8% gel for protein staining by colloidal 0.2% Coomassie Blue G-250 [13]. A set of standard proteins (kDa) as electrophoresis markers was used.

The protein content in the erythrocyte membranes’ samples was determined by evaluating the area of the corresponding area on the electrophoretic picture by use of the special analytical device (TAS plus, Leitz, Germany).

2.3 The osmotic resistance of erythrocytes

The osmotic resistance of erythrocytes was studied based on the kinetics of their lysis, which was determined by a highly sensitive spectrophotometric differential method. After washing in an isotonic solution, erythrocytes were suspended at 0.5% hematocrit (isotonic solution) and then centrifuged (250C, 1200 g, 5 min) and resuspended in a 0.7% v/v NaCl solution at 0.05% hematocrit. Hemoglobin absorbance was measured at 405 nm wavelength at different time incubation intervals (5–200 min. of incubation) [14]. The main parameters of erythrocytes’ osmotic resistance (T – time of maximum hemolysis of erythrocytes (period of spherulation), and t – time elapsed from the introduction of hemolytic agent to the beginning of hemolysis (hemolysis onset time), 1/t – hemolysis rate) were determined by the erythrocyte lysis curve.

2.4 Determination of the total antioxidant activity (TAA) of blood serum

TAA was determined in deproteinized blood plasma by using the 2.2-diphenyl-1-picryl-hydrazine (DPPH)-scavenging assay, which was adapted from a study conducted by Chrzczanowicz et al. [15]. Plasma samples (1 ml) were deproteinized by adding 3 ml of acetonitrile and centrifuging them for 10 min (4°C, 9500 g). A supernatant was immediately collected and 1 ml was transferred to a tube, subsequently, 3 ml of DPPH was added, and the resultant solution’s absorbance was read at 515 nm A calibration curve was built with the use of gallic acid, wherein the absorbance values were interpolated and the results were expressed as equivalents of gallic acid (%).

2.5 Level of HbA1

Level of HbA1 messed by high-pressure liquid ion exchange chromatography with an automatic device GH-900Plus (Diavendor, Germany).

2.6 Statistical analysis

An analysis of variance (ANOVA) (SPSS-12 for Windows) was used for the comparative analysis of the data.

3 Results

3.1 Determination of the B3p expression level

The level of B3p in the IR group did not statistically significantly differ from its level in erythrocytes from healthy volunteers. In patients with prediabetes, its content decreased by 24% in comparison to the level in the healthy volunteers and patients with diabetes iB3p level accounted 89% of healthy volunteers’ level. B3p (100 kDa) expression levels in erythrocytes from healthy volunteers and patients with IR, prediabetes, and T2DM (see Table 1).

Table 1
B3p expression levels in erythrocytes from healthy volunteers and patients with IR, prediabetes, and T2DM

Parameter Healthy volunteers IR Prediabetes T2DM

B3p 1,47±0.04 1.38±0.07 1.13±0.04^* 1,31±0.02

Parameter	Healthy volunteers	IR	Prediabetes	T2DM
B3p	1,47±0.04	1.38±0.07	1.13±0.04^*	1,31±0.02

^*- statistically significant changes compared to the healthy volunteers (p < 0.001).

3.2 Osmotic resistance of erythrocytes

We investigated the resistance fragility in erythrocytes from healthy volunteers and patients with IR, prediabetes, and T2DM detected according to the absorbance of hemoglobin released during the incubation in a 0.7% v/v NaCl solution (5–200 min). As follows from the results of the study, the erythrocytes’ hemolysis rate (t) and spherulation time (T) of erythrocytes from patients in the IR group were not significantly different in comparison to the means of the healthy volunteers, the time, necessary for hemolysis (t) tends to decrease in patients from prediabetes group, the erythrocytes’ hemolysis rate (t) and spherulation time (T) importantly reduced in the patients with T2DM (see Fig. 1). Therefore, the osmotic resistance of erythrocytes decreases compared to the control level with an increasing degree of impaired glucose metabolism.

Fig. 1

Erythrocyte’s hemolysis rate in the control group, and the patients’ group with IR, prediabetes, T2DM. M±m.

3.3 TAA of patients’ blood serum

Our results showed, alterations in blood redox status in in patients’ blood serum with IR, prediabetes, and T2DM TAA statistically significantly decreased in comparison to the control level; TAA was especially low in patients with prediabetes (see Fig. 2).

Fig. 2

Mean of total antioxidant activity (TAA) in blood serum in the control group, and the patients’ group with IR, prediabetes, T2DM. M±m.

3.4 Level of HbA1c in patients’ blood

Our results showed in patients’ blood with IR and prediabetes HbA1c statistically significantly did not change in comparison to the control level; in patients with T2DM level of HbA1c statistically significantly increased in comparison to the control level (see Fig. 3).

Fig. 3

Mean of HbA1 in the control group, and the patients’ group with IR, prediabetes, T2DM. M±m.

4 Discussion

Chronic hyperglycemia is a major factor in diabetic complications development [16]. Usually, chronic IR leads to hyperglycemia a gradual decrease in β-cell function (prediabetes), and the development of T2DM. An elevated level of glucose affects the hemorheological parameters (hematocrit, plasma proteins, plasma and whole blood viscosity, erythrocyte deformability and aggregation, etc.) of blood accompanied by generalized microcirculation disturbance [17 –20], impaired tissue perfusion and the development of severe chronic leg ischemia, leading to foot ulceration, diabetic retinopathy, nephropathy (retinal and renal failure) [21].

Erythrocytes are the most glucose-consuming cells; in the presence of long-lasting hyperglycemia, the morphology, metabolism, and function of erythrocytes undergo a series of changes that can affect hemorheology and microcirculation [22].

Investigating of changes occur in erythrocytes, and links between that cinching with diabetes progression is very important and actuality fundamental question and significant for clinical endocrinology.

In this research, we aimed to elaborate on the changes observed in erythrocytes as hyperglycemia progresses and identify erythrocyte-related indicators that can be used to monitor disease progression.

Glycosylated hemoglobin (HbA1c) (a fraction of Hb, in vivo, produced by non-enzymatic binding of glucose to N-terminal amino acids of Hb A beta chains [23]) is one of the nonenzymatic glycosylation product of Hb and reflects the average concentration of blood glucose during the past 2–3 months. Clinically, HbA1c is usually used as an important diagnostic indicator of diabetes [24]. The level of HBA1 was significantly higher in the patients with T2DM in comparison to patients from IR and prediabetes groups, studied by us.

When glucose concentration increases in the blood, it binds to the erythrocytes’ Hb; once HbA1c is formed, it does not easily decompose. HbA1c has an enhanced affinity toward O2, therefore, high HbA1c concentrations lead to difficulties in releasing oxygen to cells and reduced oxygen-transporting function of erythrocytes [25]. Local hypoxia in patients with diabetes is one of the causes of diabetic retinopathy, leading to the thickening of the glomerular basement membrane and thus inducing diabetic nephropathy, increasing the risk of periodontitis. Prolonged systemic hypoxia [23] and hyperglycemia [26] can promote reactive oxygen species (ROS) accumulation [27] and decrease antioxidant levels in tissues, such as vitamin E, GSH, catalase, SOD, and glutathione (GSH) [28], thereby altering redox balance in humans.

Clinical evidence and experimental data testify that the production of ROS is increased in T2DM and that diabetes development is strictly related to oxidative stress [11]. We revealed a decrease in blood serum TAA in patients with IR, prediabetes, and T2DM (Fig. 3).

Erythrocytes are highly susceptible to oxidative damage due to the high cellular concentration of transition metal ions, molecular oxygen, and oxyhemoglobin – potentially powerful promoters for the oxidative processes [29]. During their 120 days life span, human erythrocytes are constantly exposed to oxidant compounds (glucose and other molecules) present in the blood. In chronic hyperglycemia and oxidative stress conditions, several biochemical modifications are triggered in the erythrocytes’ membranes – glycation of membrane proteins with a subsequent decrease in their activity, lipids peroxidation, and changes in lipid-protein interactions, causing the subsequent structural and functional disruption of the membranes, alterations their shape and deformability, reducing rheological properties of erythrocytes, the appearance of phosphatidylserine (PS) on the external surface of their membranes and consequent eryptosis. Accordingly, in diabetic patients structural and functional differences in erythrocytes’ membrane in comparison to healthy individuals were detected [11]. The topographical nanostructure of the erythrocyte’s membrane can be classified as independent morphological characteristics responsible for their functional status [30], further playing an important role in the pathogenesis of diabetes and its complications.

As follows from the results of the study, in patients with impaired glucose metabolism (IR, prediabetes, and T2DM), the osmotic resistance of erythrocytes is reduced compared to the control, correlates with an increase in the degree of carbohydrate metabolism disorders, and is especially low in patients with T2DM. It was demonstrated that erythrocytes with low osmotic resistance are characterized by low deformability of their membranes [31]. Because the size of erythrocytes is typically approximately 8μm (while the lumen of capillary vessels – is 4–8μm) [16, 32], and for perfusion, it is essential that the red blood cells pass through the capillaries and supply oxygen to the surrounding tissues [33], the deformability of the erythrocytes have an important impact on microcirculation. It has been suggested that the impaired perfusion at the tissue level, as a complication of diabetes mellitus, is primarily due to reduced erythrocyte deformability [34].

In the RBC membrane, a basic triangular complex of α- and β-spectrin molecules connected to band 3, ankyrin, and protein 4.1 provides the RBC with a certain stability and simultaneously the ability to deform [35]. The shape of erythrocytes and their deformability are critically related to the Band 3 protein (B3p) [36] (90–100kDa) which is the most abundant integral protein of erythrocytes’ membrane (composed of approximately 25% of proteins) and the major linkage between the cytoskeleton and lipid bilayer. The B3p is an anion exchanger, responsible for ion balance and gas exchange (Cl−/HCO₃) through erythrocyte membranes and thus regulates erythrocytes’ as well as whole-body homeostasis.

As follows from the results of our studies, in patients with disorders of carbohydrate metabolism the expression of B3p (90–100 kDa) is reduced in comparison to the control level and appears the oligomerized form of B3p (oB3p, 180 kDa). The level of B3p in the erythrocytes from the IR patients group statistically significantly did not differ from its level in healthy volunteers, in patients with prediabetes, its content decreased by 24%, and in patients with T2DM – by 11% in comparison to the levels in healthy volunteers, while expression of the oligomerized form of B3p (oB3p) is maximal in patients with prediabetes.

Normally junctional complexes linking B3p to the cytoskeleton prevent the formation of large aggregates of B3p. Clusterization of B3p and formation of the high-molecular-mass aggregates through disulfide cross-linking dimerization are initiated by oxidative damage of the erythrocyte’s membrane [37]. The post-translational modifications of B3p, modulating its clusterization capability regulated by tyrosine phosphorylation via phosphotyrosine kinases (PTKs) or phospho-tyrosine phosphatase (PTP) which appears to be facilitating oxidatively modified B3p clusterization [37, 38].

It was suggested that B3p as a redox sensor, is regulated by phosphorylation: in oxidative stress conditions, rapid intense Tyr-phosphorylation of B3p affects its interactions with the cytoskeletal proteins triggering a cascade of events inducing alteration of deformability and resistance of erythrocytes membrane, its destabilization and finally leading their hemolysis [39]. Hyperphosphorylation of band 3 was observed during the prooxidant hemolytic disorders, malaria, diabetes types I and II, and intermediate thalassemia, and this phenomenon is closely related to the formation of hemichromes [40].

Disruptions of glucose and insulin homeostasis are leading to initiation of oxidative stress (in our study revealed by a decrease of TAA of blood serum) increased redox-sensitive PTP activity and aberrant band 3 phosphorylation, potentially leading to reduced erythrocyte deformability. At the same time glycation of Hb during T2DM may affect its cross-link with membrane proteins, in particular with B3p, and although appears to limit its cross-linking and decrease its clusterization ability, induces alterations in the cytoskeletal matrix [11, 30], and thereby decrease erythrocytes’ osmotic resistance making them more susceptible to hemolysis.

Therefore, it seems that in the process of modification of erythrocyte membranes of patients with disorders of carbohydrate metabolism both mechanisms – hyperglycemia and oxidative stress, are involved. In patients with prediabetes, the especially low TAA of blood serum (relatively high intensity of oxidative stress) and high content of the oligomerized form of B3p in the erythrocyte’s membranes indicate on the prevalence of the oxidative mechanism. During T2DM especially high levels of glycemia and HGA1C cause prevalence of glycosylation mechanisms, which limit of clusterization of B3p and decrease oB3p content, but affect its linking to cytoskeletal proteins and induce a sharp decrease of osmotic resistance compared to the levels in patients with IR, prediabetes, and healthy volunteers.

5 Conclusions

We have already planned and are carrying out pilot studies of patients using anti-oxidation drugs. The preliminary findings that our international research team will present in future publications are encouraging. Further improvement of treatment tactics has great prospects, which will have a positive impact on the quality of life of both patients with diabetes and conditions that can develop into diabetes (IR, prediabetes), as well as for healthcare in general.

Footnotes

Acknowledgments

We are grateful to the administration of Tbilisi State Medical University and Beritashvili Center of Experimental Biomedicine for technical support.

Conflict of interest

The authors have no conflict of interest to declare.

Funding

This work was performed in the frames of PhD programs at Javakhishvili State University.

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Impact of different severity hyperglycemia on erythrocyte rheological properties 1

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

List of abbreviations

1 Introduction

2 Materials and methods

2.1 Target group

2.2 Determination of Bend 3 protein (B3p) level (Protein analytical electrophoresis under dissociated conditions)

2.3 The osmotic resistance of erythrocytes

2.4 Determination of the total antioxidant activity (TAA) of blood serum

2.5 Level of HbA1

2.6 Statistical analysis

3 Results

3.1 Determination of the B3p expression level

Table 1 B3p expression levels in erythrocytes from healthy volunteers and patients with IR, prediabetes, and T2DM Parameter Healthy volunteers IR Prediabetes T2DM B3p 1,47±0.04 1.38±0.07 1.13±0.04* 1,31±0.02

5 Conclusions

Footnotes

Acknowledgments

Conflict of interest

Funding

References

Table 1
B3p expression levels in erythrocytes from healthy volunteers and patients with IR, prediabetes, and T2DM

Parameter Healthy volunteers IR Prediabetes T2DM

B3p 1,47±0.04 1.38±0.07 1.13±0.04^* 1,31±0.02