Association of altered hemorheology with oxidative stress and inflammation in metabolic syndrome

Abstract

Objective

We have shown increased whole blood viscosity (WBV), decreased erythrocyte deformability, and increased erythrocyte aggregation in metabolic syndrome (MetS) in our previous study. The objective of the study was to find out if the altered hemorheology shown in MetS in our previous study is associated with chronic inflammation and oxidative stress in the same subjects.

Methods

One hundred recruited participants were classified into three groups based on the number of the MetS components present following National Cholesterol Education Program, Adult Treatment Panel III definitions. WBV, erythrocyte aggregation, erythrocyte deformability, oxidative stress markers (erythrocyte reduced glutathione (GSH), superoxide dismutase (SOD), and urinary isoprostanes), inflammatory markers high-sensitivity C-reactive protein (hsCRP), and thrombotic marker D-dimer were measured. Data were analyzed by IBM SPSS 20 software.

Results

We found a significant association of altered hemorheology with chronic inflammation and oxidative stress in MetS. There was a linear increase in the level of hsCRP and a linear decrease in the level of SOD and GSH across the quartiles of erythrocyte aggregation. Similarly, the thrombotic marker D-dimer showed a linear increase and oxidative stress marker GSH showed a linear decrease trend across the quartiles of WBV.

Discussion

Alterations of hemorheology in MetS are probably due to the effect of chronic inflammation and oxidative stress. The negative effects of chronic inflammation and oxidative stress on the cardiovascular system could be due to the resulting altered hemorheology.

Keywords:

• Erythrocyte aggregation
• Erythrocyte deformability
• Glutathione
• Metabolic syndrome
• Whole blood viscosity

Introduction

Chronic low-grade inflammation has been hypothesized to play a role in the development of metabolic syndrome (MetS).¹ The American Heart Association has indicated that high-sensitivity C-reactive protein (hsCRP) measurements might provide information for a global risk assessment for coronary heart disease beyond that obtained from the established risk factors.² hsCRP has also been mooted as a marker for the primary prevention of cardiovascular diseases.³ Oxidative stress, due to chronic inflammation, also contributes to the development of MetS.^4,5 Oxidative stress and inflammation play a major role in vascular disorders and circulation defects.^6,7 Alterations in macrovascular circulation and microcirculation are common in MetS,⁸ and we have recently reviewed the effect of various hemorheological components on MetS.^9–11 In our recent study, we have shown altered hemorheology¹² among subjects with MetS when compared to healthy controls. Hemorheology is one of the major factors that could possibly alter the microcirculation.¹³ The objective of the study, in this context, was to find out if the altered hemorheology shown in MetS in our previous study¹² is associated with chronic inflammation and oxidative stress in the same subjects.

Materials and methods

The detailed methodology and the principles of the instruments used in the study have been described elsewhere.¹² Briefly, 100 participants were recruited from a rural town of Australia from June–Dec 2013. Pregnant women, non-ambulatory patients, and children under 18 years of age were excluded from the study. Recruited participants were divided into three groups on the basis of absence or presence of MetS and its components. A modified National Cholesterol Education program Adult Treatment Panel-III (NCEP ATP-III) guideline was used to define MetS.¹⁴ According to NCEP ATP-III guidelines, the individual is said to be in the state of MetS if he/she fulfills three criteria from the following five criteria: waist circumference >102 cm in male and >88 cm in female; triglyceride (TG) ≥1.7 mmol/l or specific treatment; high-density lipoprotein cholesterol (HDL-C) <1.0 mmol/l in male and <1.3 mmol/l in female or specific treatment; blood pressure ≥130/85 mm Hg or previously diagnosed hypertension; and fasting glucose ≥5.6 mmol/l or previously diagnosed DM. Group I consists of the participants without any positive components of MetS (healthy controls); group II consists of the participants with one or two positive components; and group III consists of participants with three or more positive components. Participants in groups I and II are non-MetS whereas participants of group III are with MetS. The study was approved by the university human research ethics committee (2012/131). Anthropometric measurements (height, weight, and waist circumference) were obtained from the participants.

Twenty milliliters of blood was taken from the participants for the analysis of erythrocyte aggregation (critical time and critical stress), erythrocyte deformability (EI_max and SS_1/2), whole blood viscosity (WBV) (shear stress 150/second), oxidative stress markers (erythrocyte superoxide dismutase (SOD) and erythrocyte-reduced glutathione (GSH)), inflammatory marker hsCRP, and thrombotic marker D-dimer. Erythrocyte deformability and aggregation measurements were carried out using a RheoScan-AnD 300 system (RheoMeditech Inc., Korea). Two indices were used to define erythrocyte aggregation: critical time and critical stress. The lesser the critical time, the faster the erythrocyte aggregation process and the higher the critical stress, the faster the erythrocyte aggregation.¹⁵ Two measurement indices were used to define erythrocyte deformability: EI_max and SS_1/2.^16,17 EI_max is the maximum erythrocyte elongation index at infinite shear stress and SS_1/2 is the shear stress required for half of maximal deformation.

WBV measurement was carried out using a Brookfield DV-II + programmable viscometer (MA, USA), using a CP40 spindle at 37 °C. All the rheological measurements were performed within 2 hours of blood collection after adjusting EDTA anticoagulated whole blood to the hematocrit of 40%.

Inflammatory marker hsCRP and thrombotic marker D-dimer were measured on the day of collection in a commercial clinical pathology laboratory. GSH was measured by the 5,5′-di-thiobis-(2-nitrobenzoic acid) method on the same day of blood collection from the erythrocyte lysate (from the washed cells) after protein precipitation by metaphosphoric acids.¹⁸ SOD was measured from the hemolysate using a commercially available kit (Cayman Chemical Company) using xanthine oxidase and tetrazolium salts. Hemolysate was prepared from packed red blood cells. One milliliter of packed red blood cells were mixed with 4 ml of ice-cold water and centrifuged at 1260 g for 9 minutes to obtain hemolysate. The hemolysate was stored in 1.5 ml Eppendorf tubes at −80°C in an ultra low-temperature freezer until tests were performed. Urinary 15-isoprostanes F_2t were measured using a commercially available kit (NWLSS™) and were expressed as ng of isoprostanes per mmol of urinary creatinine (Cayman Chemical Company). The urine sample was stored in 1.5 ml Eppendorf tubes at −80°C in an ultra low-temperature freezer until tests were performed.

Statistical analysis

Data were analyzed by IBM SPSS Statistics version 20. Normality of the data was tested by Shapiro–Wilk test. Kruskal–Wallis test was used to compare the median between three groups for non-normal data. Spearman's correlation was performed between hemorheological parameters and oxidative stress/inflammatory markers. Regression analysis was performed to find an association between MetS and hemorheology. Linear regression analysis was performed for prediction of hemorheology factors by inflammatory and oxidative stress parameters. Jonckheere trend test was used to analyze the trend of oxidative stress and inflammatory markers across the quartiles of critical stress, critical time, EI_max, and WBV. All the P-values were two-tailed, and those <0.05 were considered statistically significant.

Results

Baseline characteristics of the participants with and without MetS have been published in our previous report.¹² Demographic characteristics of the participants and mean values of MetS components in three different groups (groups I, II, and III) are shown in Table 1. hsCRP, D-dimer, SOD, glutathione, and isoprostanes were not normally distributed across the three different groups as assessed by Shapiro–Wilk test (P < 0.05). Hence, non-parametric tests were used. There were significant differences in the median value of hsCRP, D-dimer, SOD, glutathione, and isoprostanes across the three different studied groups (Table 2). Correlation of oxidative stress and inflammatory markers with the hemorheological parameters are shown in Table 3.

Table 1. Demographic characteristic of the participants and mean values (±SD) of MetS components in three different groups

Parameters	Group I (n = 31)	Group II (n = 33)	Group III (n = 36)	P-value
Age (years)	45.4 ± 8.9	56.52 ± 12.9	63.0 ± 12.1	<0.0005
Sex (male/female)	10/21	19/14	24/12	—
Waist circumference (cm)	81.16 ± 8.4	95.79 ± 13.23	110.25 ± 15.46	<0.0005
Height (cm)	166.0 ± 9.0	167.9 ± 6.2	170.8 ± 8.2	0.050
Weight (kg)	67.63 ± 9.27	76.25 ± 13.32	90.14 ± 16.74	<0.0005
BMI (kg/m^2)	24.49 ± 2.29	27.02 ± 4.66	30.89 ± 5.50	<0.0005
Systolic blood pressure (mm Hg)	116.2 ± 9.8	127.5 ± 15.1	142.5 ± 19.1	<0.0005
Fasting blood glucose (mmol/l)	6.92 ± 2.64	6.06 ± 1.97	4.93 ± 0.41	<0.0005
HDL-C (mmol/l)	1.45 ± 0.27	1.43 ± 0.43	1.09 ± 0.22	<0.0005
Triglyceride (mmol/l)	1.09 ± 0.34	1.37 ± 0.62	2.06 ± 0.85	<0.0005
Current smokers (n)	1	4	7	—
Antidiabetic medications	—	11	22	—
Antihypertensive medications	—	13	29	—
Antilipidemic medications	—	3	12	—

Table 2. Median values (interquartile range) of oxidative stress and inflammatory markers in three different groups

Parameters	Group I	Group II	Group III	P-value
hs-CRP (mg/l)	0.44 (0.96)	1.25 (3.78)	2.33 (5.27)	0.001
D-dimer (µg/ml)	0.36 (0.23)	0.39 (0.17)	0.53 (0.33)	0.003
SOD (U/ml)	630.05 (206.8)	587.69 (145.78)	524.79 (147.18)	0.021
Glutathione (mg/dl)	65.1 (9.0)	61.80 (13.0)	52.0 (10.0)	<0.0005
Urinary isoprostanes (ng/mmol)	65.18 (88.94)	144.09 (167.21)	165.71 (195.21)	0.001

Table 3. Spearman's correlation of hemorheological parameters with inflammatory and oxidative stress markers

Parameters	Critical time (seconds)	Critical stress (Pa)	EImax	SS1/2	WBV (mPa/second)
hsCRP (mg/l)
r	−0.517	0.450	−0.126	0.039	0.120
P-value	0.000	0.000	0.211	0.701	0.233
D-dimer (µg/ml)
r	−0.262	0.314	0.011	−0.175	0.171
P-value	0.009	0.002	0.914	0.083	0.092
SOD (U/ml)
r	0.241	−0.198	−0.075	0.240	−0.093
P-value	0.016	0.048	0.458	0.016	0.355
Glutathione (mg/dl)
r	0.336	−0.234	0.167	0.018	−0.251
P-value	0.001	0.019	0.097	0.860	0.012
Urinary isoprostanes (ng/mmol)
r	−0.288	0.257	−0.280	0.105	0.225
P-value	0.004	0.010	0.005	0.299	0.024

Post-hoc analysis of median comparison among three groups

Pairwise comparisons were performed using Dunn's (1964) procedure with a Bonferroni correction for multiple comparisons. Post-hoc pairwise comparisons revealed that hsCRP level was significantly different between groups I and III (P = 0.001) and between groups II and III (P = 0.015). Glutathione level was significantly different between groups I and III (P < 0.0005) and between groups I and II (P = 0.001). In the case of urinary isoprostanes level (P = 0.001), SOD level (P = 0.018), and D-dimer level (P = 0.003), post-hoc pairwise statistics revealed the significant difference between groups I and III only.

The WBV, EI_max, critical stress, and critical time were divided into four groups (quartiles) using twenty-fifth, fiftieth, and seventy-fifth percentiles. Since, in our previous study, we could not demonstrate significant difference in the value of SS_1/2 among three different groups,¹² this marker was not analyzed by the Jonckheere trend test. The Jonckheere trend test was used to analyze the trend of oxidative stress and inflammatory markers across the quartiles. There was a linear increase in the level of hsCRP (P trend < 0.0005), D-dimer (P trend = 0.002) and urinary isoprostanes (P trend = 0.007) and a linear decrease in the level of SOD (P trend = 0.035) and glutathione (P trend = 0.004) across the quartiles of critical stress. On the contrary, there was a linear decrease in the level of hsCRP (P trend < 0.0005), D-dimer (P trend = 0.021), and urinary isoprostanes (P trend = 0.016) and a linear increase in the level of SOD (P trend = 0.036) and glutathione (P trend = 0.001) across the quartiles of critical time. Similarly, D-dimer (P trend = 0.049) and urinary isoprostanes (P trend = 0.015) showed a linear increase, and glutathione showed a linear decrease (P trend = 0.005) across the quartiles of WBV whereas hsCRP (P trend = 0.147) and SOD (P trend = 0.542) did not follow any significant trend across the quartiles. Among the markers analyzed, only urinary isoprostanes showed a significant decreasing trend across the quartiles of EI_max (P trend = 0.005). Similarly, regression analysis showed that oxidative stress and inflammatory markers significantly predicted erythrocyte aggregation (Table 4).

Discussion

Oxidative stress is defined as an imbalance between prooxidant and antioxidant factors in favor of the prooxidants.¹⁹ In order to look for evidence of oxidative damage in vivo, erythrocytes from patients with MetS, with only one or two components of MetS and healthy control subjects were assessed for erythrocyte GSH, SOD, and urinary isoprostanes in the present study. This study shows the increased oxidative stress in MetS groups (group III) when compared to healthy controls (group I) (Table 2), suggesting that the erythrocyte antioxidant defense mechanism is exhausted in MetS due to overproduction of reactive oxygen species. These observations are consistent with studies that have highlighted oxidative stress as a pathophysiologic component of MetS.^4,5Increased oxidative stress has been suggested as a prior event in the development of MetS,²⁰ and it is likely to be a common second-level abnormality in MetS.²¹

Table 4. P-values (regression coefficient R^2*) of linear regression analysis for prediction of hemorheological factors by inflammatory and oxidative stress parameters

Predictors (continuous variable)	Outcome variables
	Critical time	Critical stress	EImax	SS1/2	WBV
hs-CRP (mg/l)	<0.0005 (0.199)	<0.0005 (0.187)	0.110	0.381	0.682
D-dimer (µg/ml)	0.358	0.226	0.943	0.201	0.577
SOD (U/ml)	0.009 (0.068)	0.078 (0.031)	0.720	0.016 (0.058)	0.273
Glutathione (mg/dl)	0.004 (0.080)	0.003(0.086)	0.106	0.516	0.043(0.041)
Urinary isoprostanes (ng/mmol)	0.005 (0.076)	0.018 (0.056)	0.003 (0.086)	0.354	0.005 (0.077)

*Regression coefficient is shown only for the parameters with P-value <0.05.

Erythrocyte GSH and SOD levels were decreased in MetS group when compared to healthy controls. The ubiquitous nature of the erythrocyte and the fact that it is an expendable cell (replaced on average every 120 days) make it an ideal cell to protect other body tissues from free radical damage even at the expense of their own structure and function.²² In carrying out their role of free radical scavenging, erythrocytes become damaged by oxidation, which consumes endogenous-reducing substances thereby decreasing the level of erythrocyte antioxidant GSH and SOD. Reactive oxygen species superoxide anion crosses the erythrocyte membrane through specific superoxide channel and is neutralized by the antioxidant SOD inside the erythrocyte.^22,23 SOD is inactivated by its own product hydrogen peroxide.²⁴ Hydrogen peroxide is broken down to water and oxygen by the enzyme catalase and glutathione peroxidase, the latter of which consumes GSH. Similar to our study, Koziróg et al. showed lower erythrocyte activity of SOD, GSH, and catalase in MetS subjects when compared to healthy controls.²⁵

Oxidative stress causes damage to critical biomolecules including DNA, lipids, and proteins.²⁶ The increased concentration of urinary isoprostanes seen in the MetS group in the present study is due to the damage caused by free radicals. This observation is consistent with that of Tsai et al. who reported elevated urinary isoprostanes level in MetS group when compared to healthy controls.²⁷ Similarly, the present study shows an increased inflammation level demonstrated by increased hsCRP level in the MetS group compared to healthy controls. Adipocytes in obese patients with MetS release high amounts of tumor necrosis factor-alpha and interleukin-6 into the circulation, which stimulate the production of hsCRP by the liver and induce insulin resistance.²⁸ Several studies in the past have confirmed this association of hsCRP with MetS^3,29,30 and altered hemorheology.³¹

The present study provides the evidence that oxidative stress and chronic inflammation is present in MetS. This study further investigated to what extent these two parameters affect hemorheology. The altered hemorheology in MetS that was demonstrated in our previous study¹² seems to be associated with the chronic inflammation and oxidative stress. This study was conducted among patients that take anti-inflammatory drugs. Nevertheless, hsCRP showed a linear increase across the quartiles of critical stress, linear decrease across the quartiles of critical time, and significantly predicted critical stress and critical time. It seems likely that increased erythrocyte aggregation is due to the presence of increased adhesive proteins (such as inflammatory cytokines and acute phase proteins) in their milieu in MetS.

Erythrocyte GSH and SOD showed negative correlation with erythrocyte aggregation and WBV whereas erythrocyte SOD showed negative correlation with erythrocyte aggregation (Table 3). The data appear to support the proposition that the increased erythrocyte aggregation is also due to the oxidative damage of erythrocytes. The labile groups in the proteins and lipids of the erythrocyte's cytoskeleton are oxidized due to the decreased antioxidant level, and this oxidative modification of membrane proteins and lipids possibly increases the tendency of ‘damaged’ erythrocytes to adhere with other erythrocytes thereby increasing erythrocyte aggregation. Supporting this, Richards and Nwose³² observed increased erythrocyte oxidative stress with increased WBV in different stages of diabetes pathogenesis. Abnormal WBV was linked to oxidative stress in 76% of the apparently healthy controls.³² It was shown that intravenous infusion of reduced glutathione significantly reduced blood viscosity and erythrocyte aggregation in atherosclerotic patients³³ supporting the observations of the present study that the depletion of the antioxidant is associated with alterations in hemorheological profile.

The present study demonstrated a positive correlation of urinary isoprostanes with WBV and erythrocyte aggregation, and a negative correlation with erythrocyte deformability (Table 3). Unfavorable alteration in erythrocyte lipid membrane fluidity in MetS participants has been reported by Kowalczyk et al.³⁴ MDA concentration in erythrocyte of MetS was shown to be higher when compared to healthy controls. Supporting the findings of the present study, membrane lipid peroxidation was considered a possibility for altered membrane properties.³⁴

Regression analysis in the present study showed that all of the oxidative stress markers and hsCRP significantly predicted erythrocyte aggregation parameters and urinary isoprostanes predicted all of the hemorheological parameters except SS_1/2. This reveals that oxidative stress and chronic inflammation influence blood rheological profile. However, regression coefficient indicates that the influence is not big. Though the present study suggests some association between rheological parameters and oxidative stress/chronic inflammation, large studies should be undertaken to see how big the effect is.

Yedgar et al. have suggested that the inflammation and oxidative stress may alter the erythrocyte flow due to altered rheological properties and may lead to vascular occlusion.³⁵ In the present study, D-dimer is positively correlated with the erythrocyte aggregation. The possible vascular occlusion and slow flow due to the aggregates could enhance endothelial dysfunction and activate the coagulation system. Increased concentration of D-dimer has been associated with coronary heart diseases.³⁶ Another plausible mechanism suggested by studies demonstrating an effect by free radicals on fibrinogen molecules is that a shift in the oxidant/antioxidant dynamic balance could lead to increased in vivo oxidation of the fibrinogen molecule promoting its conversion to fibrin and ultimately D-dimer.^37,38

Recently, Toth et al. have shown the effect of hemorheological factors in cardiovascular medicine.³⁹ The findings of the present study highlight the cause that leads to alterations in hemorheology and the effect of these alterations in the cardiovascular system through increased D-dimer. Moreover, recently, we have shown the positive correlation of increased WBV and erythrocyte aggregation with peripheral arterial atherosclerotic marker: toe brachial pressure index.¹² The possible effect of hemorheological factors in cardiovascular medicine has begun to be noticed for several years now.^13,39,40 Taken together, the findings of the present study and our previous study would seem to suggest that the negative effect of inflammatory and oxidant molecules seen in the cardiovascular system could be due to the resulting altered hemorheology.

Conclusion

Inflammation is increased and antioxidant capacity is decreased in MetS. Erythrocyte aggregation, WBV, and erythrocyte deformability showed significant association with one or more of the markers analyzed. Alterations of hemorheology in MetS are probably due to the effect of chronic inflammation and oxidative stress. The negative effect of chronic inflammation and oxidative stress in the cardiovascular system could be due to the resulting altered hemorheology.

Disclaimer statements

Contributors All authors contributed equally.

Funding None.

Conflicts of interest None.

Ethics approval Obtained from HREC, CSU.