Hyperglycemia associated blood viscosity can be a nexus stimuli

Abstract

BACKGROUND:

The viscosity of a fluid is a measure of its resistance. It is the thickness and stickiness of blood, and a direct measure of the resistance of blood to flow through the vessels. Various factors in the blood have direct or indirect impact on blood viscosity. These hemorheological factors play an important role in the pathogenesis of many diseases. Glucose is one such factor, which, when increased in the blood, causes resistance in the blood flow.

OBJECTIVE:

The present study is aimed to assess the changes in blood viscosity associated with hyperglycemia in rodents.

METHODS:

Diabetic patients were grouped, depending on the duration of their diabetic status assessed by their increased HbA1c. Similarly rodents were subjected to acute or chronic hyperglycemic conditions in various experiments. In vivo, perfusion study was performed using micro probe in diabetic mice. Flow cytometry was used to assess the expression of VCAM-1 on endothelial surface.

RESULTS:

An approximate 40% increase in blood viscosity is observed in individual who were diabetic for the past 15 years than those who were diagnosed just one year back. Similarly such increase in blood viscosity was evident in different experiments of rodents. Our in vivo perfusion study did not showed conclusive finding however long term hyperglycemia can have deleterious effect on flow rate. Vascular pathology which was evident from the data of flow cytometry, where increase in VCAM-1 expression on the endothelial surface was observed in response to glucose and in diabetic mice.

CONCLUSIONS:

Hyperglycemia implicates the blood viscosity which in turn can have tedious effect on metabolic syndromes thus causing the serious effect in the tissue perfusion of an organs.

Keywords

Glucose viscosity HbA1c metabolic syndrome vascular pathology diabetic hyperglyecemia

1 Introduction

Viscosity of blood [non-Newtonian fluid] is an intrinsic property of blood and is determined by the internal friction experienced by blood during its flow. These internal friction are in turn determined by the composition of the blood and on the shear rate which is determined by various factors.

The non-Newtonian behavior of blood is due to the tendency of erythrocytes to aggregate at low shear rates. Furthermore, when shear rate is high, the erythrocytes are deformed to optimally adapt to flow conditions. In normal circumstances, in capillaries, high shear rates occur when blood viscosity is low [1, 2].

The notions of shear rate should be clearly comprehended to understand the phenomena of viscosity. It is defined as the rate at which adjacent layers of fluid move with respect to each other and usually expressed as reciprocal seconds. Hence there is non-linear relation between blood viscosity and blood flow. Specifically, blood viscosity is higher at low shear rates and is reduced as shear rate increases [3]. Viscosity of blood has been shown as an important risk factor associated vascular pathology [4].

Change in blood viscosity that could lead to changes in organ perfusion is offset by autoregulation. But in diseased conditions as in ischemia, structural or physiological dysfunction, these auto-regulatory mechanisms are inoperative [5]. Hence identifying changes in blood viscosity and its implications in management will help us devise methods to increase the organ perfusion.

In addition to vascular changes in diabetes, blood flow plays a crucial role in organ perfusion. If blood flow is impaired then the diabetic micro and macrovascular complications are likely to occur. Studies have shown that increased blood viscosity owing to hyperglycemia is a decisive risk factor for flow-related insulin resistance; type II Diabetes Mellitus [DM] [6]. There is a negative relationship between whole-blood viscosity and insulin sensitivity [7]. Such a relationship exists even among young healthy men [8]. Blood viscosity predicted the development of diabetes in both men and women [9]. Increase in blood viscosity has been positively correlated with blood glucose levels and negatively correlated with blood flow rate [10].

Increased blood viscosity plays a role in small vessel diseases and has been implicated in stroke patients, being more common in small artery occlusion and in large artery atherosclerotic lesions [11 –14]. Hemorheological factors play an important role in the pathogenesis of severe complications of diabetes and cardiovascular events. Fowkes, et al. in their landmark study published in the European heart journal, that a strong and independent association exists between blood viscosity and arterial pressure [15].

Our study intends to measure the blood viscosity in rodents and correlate the blood glucose values with blood viscosity. This will help deepen the understanding of pathophysiology of type II DM and its vascular pathology. Our aim is to study the relationship between hyperglycemia and blood viscosity in various simulating experiments of hyperglycemia in rodents and compare them with controls to ascertain the direct correlation between the two parameters.

2 Materials and methods

Major part of this study was on animal, however to produce better understanding of the concept, diabetic patients were also included who were randomly picked up from the general hospital record and were followed, requesting them to donate a sample of blood for our study. They were grouped into four groups irrespective of socioeconomic status, marital status, age or gender. This work in support of animal study via and ethical committee was approved under, 2015-000-20135.

2.1 Exclusion DM

Individual with known diagnosis of DM but suffering from any hemtological disorder, cancer, renal failure, plasma protein disorder, liver failure and those who were on aspirin were dropped from the study.

2.2 Inclusion DM

Individual with known diagnosis of DM but had uncontrolled DM which was evident by measuring HbA1c, were included in the study and they were grouped as;

Individual who had confirmed onset of diabetes for 01 year

Individual who had confirmed onset of diabetes for 05 year

Individual who had confirmed onset of diabetes for 10 year

Individual who had confirmed onset of diabetes for 15 year

Mice were bred and kept in animal housing facilities at Chonbuk National University Medical School under specific pathogen-free conditions. All experimental animals were used under a protocol approved by the institutional animal care of the Chonbuk National University Medical School. Standard guidelines for laboratory animal care were followed. The procedure was conducted on mouse having weight of approximately 25–30 gm.

2.3 Blood collection

Essential protocol described earlier were followed for collecting the blood sample from DM individual. After consent from all individual, Phlebotomy method was employed to take blood sample in standard tube containing EDTA. However from the mice blood was drawn by cardiac puncture into tube containing EDTA.

2.4 Blood glucose measurement

Commercially available device “ACCU-CHECK Active Glucose Monitoring” was used to assess the blood sugar levels.

2.5 HbA1c measurement

The quantitative detection of HbA1cwas performed using commercially available kit of Crystal Chem; catalog #80099, essentially manufacturer’s instructions were followed.

2.6 Induction of diabetes in mice

Mice weighing 25–30 gm were injected inter peritoneum with single moderate size dose of 100 mg/kg streptozotocin (STZ). The mice are observed daily until 05–07 days post injection, when they are weighed and their blood glucose levels determined, which was elevated suggesting the dysfunction of glucose metabolic pathway. Control group of mice were injected with same volume of buffer solution. Streptozotocin makes pancreas swell and at last causes degeneration in Langerhans islet beta cells and induces experimental diabetes in the animal model.

2.7 Viscosity measurement

Hematocrit adjustments is necessary protocol for whole blood viscosity measurement. This is done by adding or removing the calculated amounts of autologous plasma to or from the samples. Plasma is separated by centrifugation of whole blood at 1500 g, 5 minutes at room temperature.

Brookfield digital rheometer Model DV-III was used to assess the viscosity of whole blood. Device is sensitive it can measure the viscosity of 0.5 ml of blood at moderate to high shear rates. Instrument is pre-warmed by its in-let and out-let tubes using standard water bath. Temperature control is necessary since plasma viscosity changes by about 2% per degree Celsius [7]. It is measured in milli-pascal-second (mPa-s).

2.8 Assessment of in vivo perfusion

Mice which were earlier made diabetic using STZ were subjected to assess the perfusion of abdominal viscera like intestine. Mice were anesthetized using ketamine by intra-peritoneal injection. Mice were placed on a pre-warmed surface. Bilateral peritoneal incisions were made and the abdominal contents were exposed. Exposed intestine were carefully examined and the superior mesenteric artery (SMA) was located. Feces-free area which was directly supplied by SMA was located. Saline soaked filter paper was placed and the segment of intestine was placed on top of it which was studied for perfusion. The intestine was sprayed with pre-warmed saline after every 2 min. Blood perfusion was recorded using a small animal Laser Doppler flow meter (Model BLF 21, Transonic Systems Inc) for up to 30 min. Arterial flow; perfusion was recorded for data analysis and was expressed as; perfusion –AU (Arbitrary Units).

2.9 Endothelial Cell (EC) preparation

Fresh thoracic and abdominal aorta as one piece was separated from the anesthetized mouse. Four to five mouse were used at a time. Aorta from STZ mice was also processed with same protocol. For the separation of endothelial cell, rest of the procedure was the same as previously described [16].

2.10 Flow cytometry

Washed EC were stimulated with 5, 10 or 50 mM/ml of glucose for 2 min at 37°C in EP tube. However the EC of STZ mice was processed as such without any stimulation. After the appropriate time, the reaction was stopped by adding 1 ml of 3% ice cold formalin diluted in PBS. After 15 min of fixation on ice, the tubes were spun at 3,000 RPM for 3 min at 4°C and washed once with ice cold PBS. After washing; 0.5 ml of PBS containing 3% BSA was added and left on a rotator in a cold room for 60 min. The tubes were centrifuged again at 3,000 RPM for 3 min at 4°C and washed once with ice cold PBS. After washing, pellets were re-suspended in 0.5 ml PBS containing 1% BSA and were incubated with VCAM-1 Ab conjugated with FITC for 120 min in dark cold room. After incubation EC were washed once and re-suspended in appropriate volume and the sample was subjected to FACS along with the control which did not have the FITC antibody. A total of 10,000 events per sample were collected with a BD FACS Calibur using the Cell-Quest program. Color and light sensitive substance was run with control for calibration purposes.

2.11 Statistics

Statistical analysis was carried on raw data using Sigma Plot 9 by using the unpaired Student t test, P < 0.05 was considered statistically significant. Values are expressed as mean of three to four independent experiment on rodents.

3 Results and discussion

Perpetual hyperglycemia is evident finding in individual suffering from diabetes mellitus, especially if it is un-controlled. HbA1c measurement gives the indirect estimation of blood sugar levels for the previous three months. Viscosity measurement is slightly increased in those who were diabetic for the past 05 years than those who were diagnosed just one year back. Similarly very significant increase in viscosity measurement is noted in those who were having DM for 10 or more years when compared with those suffering from DM for the past one year. Hence uncontrolled diabetes can progress with an increase in viscosity: Table 1. Our data is consistent with an earlier study where they confirmed that patients suffering from metabolic syndrome show elevated level of viscosity measurements [17]. Likewise it has been concluded that elevated blood viscosity deserve attention as an emerging risk factor for insulin resistance type II DM [6]. Furthermore increase in the viscosity is much obvious when one is suffering various other metabolic disorders at the same time. In an interesting study it has been summarized that each species has its own rheological fingerprints [18]. The physiological significance of these variations in different mammalian species is not entirely clear at present. However microcirculatory dysfunction in cardiac syndrome [19] and other metabolic syndrome is a well-established fact as documented in literature, arising due to abnormal blood rheology [7 , 21].

Table 1
Comparison of viscosity measurement among the diabetic groups, exhibiting uncontrolled DM, as indicated by HbA1c measurement

Diabetic Since Average Average Viscosity at Shear rate

HbA1c % mg/dl (1/s) 400 –mPa-s

A. More than 01 Year 08.4±0.1 2.03±0.4

B. More than 05 Year 09.6±0.3 2.21±0.2

C. More than 10 Year 10.1±0.2 2.68±0.1

D. More than 15 Year 10.8±0.3 2.81±0.2

Diabetic Since	Average	Average Viscosity at Shear rate
A. More than 01 Year	08.4±0.1	2.03±0.4
B. More than 05 Year	09.6±0.3	2.21±0.2
C. More than 10 Year	10.1±0.2	2.68±0.1
D. More than 15 Year	10.8±0.3	2.81±0.2

To further strengthen our study, we used an animal model. Mice were placed in two groups and their blood glucose level were assessed before the administration of any material. Mice were then injected with glucose 0.001 gm/gm body weight or saline solution of equal volume in the peritoneum. After 10 min of the injection, blood was drawn and viscosity was measured. Significant differences was observed in the two groups at both shear rate: Table 2.

Table 2

Comparison of viscosity measurement in rodents, injected with appropriate amount of glucose in the peritoneum. Following is the result of three independent experiment

Parameter	Saline 0.9%	Glucose 0.001 gm/gm body wt.
Blood Glucose mg/dl
Before	137	134
After	161	286
Viscosity –mPa-s
Shear rate (1/s) 100	3.02 * ±0.2	3.73 * ±0.1
Shear rate (1/s) 400	2.69 ** ±0.4	3.17 ** ±0.2

^*p = 0.03 and

^**p = 0.02, measured by t test.

It has evidently been proven that acute effects of ingesting glucose solutions on blood and plasma volume results in a decrease in plasma volume, most likely, due to a temporary net secretion of water from the blood into the intestinal lumen. This may result in a transient hypo-hydration, but the resulting hypertonicity and a fall in plasma volume may prevent the initiation of the diuresis and together these critical events can substantially make the blood more viscous and hence decrease in perfusion of vital organ is expected [22]. This data is backed up by other segmental perfusion studies [23]. Perfusion of hypertonic solutions can result in net secretion of water into the lumen. Therefore avoidance of ingestion of strongly hyperosmolar carbohydrate meal is advisable, especially in those individual who are already going through the episodes of different metabolic syndrome components.

In another experiment two groups of mice, one of the group were injected with appropriate amount of STZ to make them diabetic by destroying the normal function of pancreas. After 05–07 days sugar level of mice were assessed, STZ injected mice showed elevated level of blood glucose. Next day both group were sacrificed and viscosity was measured. STZ injected mice showed highly increased viscosity measurements as compared to controlled group at both share rate: Table 3.

Table 3

Comparison of viscosity measurement in rodents which were induce diabetic. Following is the result of two independent experiment

Parameter	Controlled	Diabetic - STZ
Blood Glucose mg/dl
Before	131	133
After	156	271
Viscosity –mPa-s
Shear rate (1/s) 100	3.11 * ±0.4	3.81 * ±0.2
Shear rate (1/s) 400	2.57 ** ±0.2	3.09 ** ±0.2

^*p = 0.04 and

^**p = 0.02, measured by t test.

Hallmark of uncontrolled DM is persistent elevated level of glucose level accompanied by increase in HbA1c. Increased level of HbA1c has long been attributed as a risk factor in the out-come of various metabolic syndrome. However its confounder, glucose, has wide range of vulnerability in damaging of various metabolic pathways. Furthermore its increase level also contribute in the viciousness of blood thus impeding the perfusion of various vital body parts. Our study is congruent with statement that increased blood viscosity has been considered a major cardiovascular risk factor and play a role in the vascular complications of diabetes [18]. Furthermore such an increase of glucose level in poorly controlled diabetic patients, blood viscosity may be very useful marker of diabetic complications [24] both microvascular and macrovascular level.

Yet in another experiment two group of mice were subjected to metabolic cage for 3 days. One group of mice were given normal water to drink and the other group were given glucose water which was prepared by dissolving 1 gm of glucose in 50 ml of normal drinking water. Pre-measurements, like body weight and blood sugar were assessed. However viscosity measurements were increased in the group which were given glucose water: Table 4.

Table 4

Comparison of viscosity measurement in rodents, subjected in the metabolic cage and given glucose water. Following is the result of three independent experiment

Parameter	Normal Water	Glucose 1 gm/50 ml
Blood Glucose mg/dl
Before	134	129
After	169	241
Viscosity –mPa-s
Shear rate (1/s) 100	3.08 * ±0.2	3.63 * ±0.3
Shear rate (1/s) 400	2.61 ** ±0.1	2.91 ** ±0.2

^*, p = 0.03 and

^**, p = 0.04, measured by t test.

Therefore any large reduction in blood or plasma volume following ingestion of hypertonic solutions may lead to an increase in blood viscosity. Because of this, individuals suffering from cardiac insufficiency or hypertension should perhaps avoid sudden or large changes in blood volume that may threaten cardiovascular stability. Such a perpetual hyperglycemia and increase in blood viscosity in an otherwise healthy individual can be alarming indication for the occurrence of Type II DM [12, 18].

It was imperative to demonstrate this in vivo tissue perfusion to see if hyperglycemia could impede the blood flow to vital organs. Diabetic mouse was used for this purpose and they were compared with non-diabetic mouse in term of perfusion of intestine. Though not very significant and conclusive data could be obtained from this experiment: Fig. 1, however we believe that long term standing of hyperglycemia in diabetes would eventually have deleterious effects, which are evident as peripheral vascular complications, in the advanced stage of the this disease.

Fig. 1

In vivo perfusion of rodent intestine. The perfusion of intestine and time are shown on the y axis and x axis, respectively. Data represent two individual experiments. AU, arbitrary units.

Blood viscosity modulates tissue perfusion; at times in few places, each organ possesses specific properties for controlling microvascular perfusion. Such specificity provides an opportunity to design transfusion fluids that target thrombo-embolic or vasospasm induced ischemia in a particular organ or that optimize overall perfusion from systemic shock. Yet documented in another study, low hematocrit per blood viscosity ratio is a mortality risk factor in coronary heart disease [14]. Furthermore as Blood viscosity is inversely related to flow, this might thereby contribute to flow-related insulin resistance, because of inability of substrates to reach the target organ [6, 25].

Finally, how hyperglycemia has its toxic effect on the endothelia surface was demonstrated by isolating the EC cell from the aorta of normal and STZ mice. Expression of VCAM-1 were assessed on the surface; Fig. 2. As the concentration of glucose increase, expression of VCAM-1 increases and moderate rise is observed in the EC separated from the STZ mice. Expression of VCAM-1 mediates the signal transduction resulting the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium and hence playing a role in the development of vascular pathology; atherosclerosis. Hence the upregulation of VCAM-1 in response to increase blood glucose, on endothelial cells exacerbate vascular pathology [26 –28].

Fig. 2

Washed EC were stimulated with appropriate amount of glucose (STZ not stimulated). VCAM-1 fluorescence intensity measured in flow-cytometry.

Increased blood viscosity is associated with microvascular dysfunction and has been shown to predict ischemic heart disease. Fibrinogen is also one of the major contributors to plasma viscosity and important in the complex mechanisms of coronary microvascular dysfunction [29, 30]. However hyperglycemia, transient or persistent does modulate the cardiovascular pathology by increasing blood viscosity [31, 32]. Taken together, fibrinogen-induced mechanisms or episodes of increase glucose does orchestrate the microcirculation of vital organs.

Elevated whole blood viscosity is also associated with higher body mass index, fasting glucose, fibrinogen, red blood cells, white blood cells, and triglyceride levels [6 , 33–35]. Most of these dynamics elements have been credited to the sedentary life style. It is hence one of the major modifiable risk factor for cardiovascular disease. Performing regular physical activity is related to reduction of cardiovascular mortality.

The mechanisms associated with physical activity includes beneficial effect on endothelial function [36, 37] and hence probably reduces blood viscosity. Indeed, physical exercise has been demonstrated to increase coronary microvascular function in healthy volunteers as well as in patients with stable coronary artery disease and has beneficial effects on cardiovascular disease incidence as well as other components of metabolic syndrome.

4 Conclusion

Our findings conclude that hyperglycaemia, transient or perpetual, implicates a direct effect on blood viscosity. Viciousness of blood in turn can impede the perfusion of vital organs thus causing deleterious effects on health of the individual. Though most of our study was on rodent, however it is less likely that different pathophysiological parameters would be involved in augmenting the vascular pathology.

Conflict of interest disclosure

The authors declares that there is no conflict of interest of what so ever.

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