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(Hypertension. 2002;39:777.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Oxidative Stress in Leukocytes Is a Possible Link Between Blood Pressure, Blood Glucose, and C-Reacting Protein

Kenichi Yasunari; Kensaku Maeda; Munehiro Nakamura; Junichi Yoshikawa

From the Department of Cardiology, Graduate School of Medicine, Osaka City University, Japan.

Correspondence to Kenichi Yasunari, MD, PhD, Department of Cardiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail yasunari{at}osaka.med.or.jp

	Abstract

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Because oxidative stress and inflammation are believed to play roles in the pathogenesis of cardiovascular diseases, oxidative stress in polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNCs) has been measured. A total of 529 subjects participated this study. Intracellular oxidative stress in PMNs and MNCs was measured by gated flow cytometry using carboxyfluorescin diacetate bis-acetoxymethyl ester. C-reacting protein (CRP), insulin action (homeostasis model assessment), and traditional risk factors such as age, gender, body mass index, triglycerides, LDL cholesterol, HDL cholesterol, hemoglobin A_1c, and mean blood pressure were also measured. Multiple regression analysis revealed a significant correlation between mean blood pressure and PMN oxidative stress (r=0.104, P=0.018). It also demonstrated a significant correlation between hemoglobin A_1c and PMN oxidative stress (r=0.112, P=0.021). A significant correlation was also found between CRP and MNC oxidative stress (r=0.116, P=0.008) by multiple regression analysis. In patients with both hypertension and diabetes, both PMN and MNC oxidative stress was increased (n=21, P=0.022 and P=0.006). These results suggest that both hypertension and diabetes lead to increased oxidative stress of PMNs and MNCs, and that CRP is related to MNC oxidative stress.

Key Words: leukocytes • arteries • diabetes mellitus • oxidative stress

	Introduction

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Although hypertension and diabetes are well-recognized risk factors for cardiovascular disease,^1,2 the links between hypertension or diabetes and oxidative stress are not fully understood. It has recently been shown that patients with hypertension or diabetes tend to have a higher body mass index and tend to be glucose-intolerant, hyperinsulinemic (insulin-resistant), and dyslipidemic, with high plasma triglyceride and low HDL cholesterol concentrations, which are recognized risk factors for cardiovascular disease.^3,4 Though these changes may account for the increased cardiovascular risk in patients with hypertension and those with diabetes, the mechanistic links between these syndrome and enhanced oxidative stress in vivo is not known.

Polymorphonuclear leukocytes (PMNs) are one of the main types of inflammatory cells. Once activated, PMNs release reactive oxygen species, including hydrogen peroxide, contributing to endothelial damage and cardiovascular disease.^5,6 Monocytes (MNCs) are crucial cells in the genesis of atherosclerotic lesions. MNCs, when stimulated, have been shown to induce adhesion to endothelium, which results in cardiovascular disease.⁷

In the present study, we have compared PMN and MNC oxidative stress in hypertensive and/or diabetic subjects and evaluated the relationship of each risk factor for cardiovascular diseases and PMN or MNC oxidative stress.

	Methods

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The study was performed a prospective study of 529 apparently healthy subjects who visited Osaka City University Hospital. The hypertensive group consisted of subjects who had not been treated or those who had stopped antihypertensive agents for 4 weeks with blood pressure 140/90 mm Hg, and the diabetes group consisted of untreated subjects with hemoglobin A _1C (HbA_1C) 6.5 mg/dL. Except for hypertension and diabetes, individuals enrolled in the study were determined to be healthy on the basis of medical history, physical examination, and results of routine laboratory tests. The study protocol was approved by the Osaka City University Institutional Review Board, and written informed consent was obtained from all subjects.

All studies were performed after an overnight fast. Degree of obesity was estimated by body mass index. Venous blood was used for measurement of plasma insulin, plasma glucose, plasma C-reacting protein (CRP), plasma cholesterol, triglyceride, and HDL cholesterol concentrations. Serum CRP was measured by a highly sensitive microparticle enzyme immunoassay. An estimate of insulin resistance by homeostasis model assessment (HOMA-IR) was calculated with the following formula: [fasting serum insulin (µU/mL)x fasting plasma glucose (mmol/L)/22.5].

Hydrogen peroxide production by PMNs and MNCs was measured using a gated flow cytometry derived from the technique described by Bass et al⁸ with some modifications.⁹ Fresh blood (1 mL) from participants was preincubated for 15 minutes with 2', 7'-carboxydichlorofluorescein diacetate bis-acetoxymethyl ester (CDCFH bis-AM ester; 100 µmol/l). CDCFH diacetate bis-AM ester is a nonpolar compound that is converted into a nonfluorescent polar derivative (CDCFH) by cellular esterases after incorporation into cells. CDCFH is membrane-impermeable and rapidly oxidized to the highly fluorescent carboxydichlorofluorescein (CDCF) in the presence of intracellular hydrogen peroxide and peroxidases. The oxidative stress in PMNs and MNCs was measured as fluorescence intensity by gated flow cytometry.

Valves are expressed as mean±SD, unless otherwise specified. Student’s unpaired t test was used to compare groups, preceded by 1-way ANOVA. The relationship between PMN or MNC oxidative stress and relevant covariates was examined by determination of standardized correlation coefficients and multiple regression analysis.

	Results

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Blood Pressure
All subjects were divided into hypertensive group (HT; BP 140/90 mm Hg, n=219) and normotensive group (NT; BP <140/90 mm Hg, n=310) Baseline characteristics of the 2 experimental groups are shown in Table 1. PMN oxidative stress was significantly increased in cell from the HT group. None of the other differences between measured variables in the 2 groups were significant. The relationship between MBP and oxidative stress in PMNs or MNCs is illustrated in Figure 1 and indicates that the higher the blood pressure, the greater the PMN oxidative stress.

View this table: [in this window] [in a new window]   Table 1. Baseline Characteristics of Normotensive and Hypertensive Subjects

View larger version (23K): [in this window] [in a new window]   Figure 1. Relationship between MBP and oxidative stress in PMNs (left) or MNCs (right). n=529

Blood Glucose (HbA_1C)
All subjects were divided into a diabetes group (DM; HbA_1C 6.5%, n=41) or a nondiabetic group (Non-DM; HbA_1C <6.5%, n=488). Baseline characteristics of the 2 experimental groups are shown in Table 2. MNC oxidative stress was significantly increased in the diabetes group. Significant difference was observed between the 2 groups in MNC oxidative stress, HDL cholesterol, HOMA-IR, and insulin (Table 2). The relationship between HbA_1C and PMN or MNC oxidative stress is shown in Figure 2 and indicates that the higher HbA_1C, the greater the PMN oxidative stress. This relationship was also observed between HbA_1C and MNC oxidative stress.

View this table: [in this window] [in a new window]   Table 2. Baseline Characteristics of Diabetic Subjects and Nondiabetic Subjects

View larger version (21K): [in this window] [in a new window]   Figure 2. Relationship between HbA1C and oxidative stress in PMNs (left) or MNCs (right). n=529

Hypertensive Diabetic Patients
All subjects were divided into 4 groups: NT and Non-DM (n=290), HT and Non-DM (n=198), HT and DM (n=20), and HT and Non-DM (n=21). Baseline characteristics of the 4 experimental groups are shown in Table 3. Only in HT and DM group, were both PMN and MNC oxidative stress significantly increased; there was also an increase in triglycerides and HOMA-IR, an indicator of insulin resistance, and a decrease in HDL cholesterol.

View this table: [in this window] [in a new window]   Table 3. Baseline Characteristics of Hypertensive Diabetic Subjects

Multiple Regression Analysis
Multiple regression analysis was used to quantify the impact of measured variables on PMN oxidative stress and MNC oxidative stress. The results shown in Table 4 indicate that MBP was significantly related to PMN oxidative stress, and that HbA_1C was significantly related to PMN oxidative stress. The results shown in Table 5 indicate that CRP is significantly related to MNC oxidative stress. The relationships between CRP and PMN or MNC oxidative stress are shown in Figure 3. Multiple regression analysis was also used to quantify the impact of measured variables on HOMA-IR, an indicator of insulin resistance. Body mass index (r=0.338, P<0.001, n=529), triglycerides (r=0.149, P=0.004, n=529), and HbA_1C (r=0.313, P<0.001, n=529) were significantly related to HOMA-IR. However, neither PMN oxidative stress (P=0.673) nor MNC oxidative stress (P=0.563) was related to HOMA-IR. Oxidative stress in PMNs and MNCs was related (r=0.153, P<0.001, n=529).

View this table: [in this window] [in a new window]   Table 4. Multiple Regression Analysis of the Relationship Between PMN Oxidative Stress and Other Associated Variables for the Entire Group

View this table: [in this window] [in a new window]   Table 5. Multiple Regression Analysis of the Relationship Between MNC Oxidative Stress and Other Associated Variables for the Entire Group

View larger version (18K): [in this window] [in a new window]   Figure 3. Relationship between CRP and oxidative stress in PMNs (left) or MNCs (right). n=529

	Discussion

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In the present study, we have demonstrated for the first time that only in hypertensive diabetic patients, both PMN and MNC oxidative stress were significantly increased (Table 3). Both hypertension and diabetes are concurrent,¹⁰ and the relationship with increased cardiovascular events is suggested.¹¹ Our findings may explain the mechanisms of increased cardiovascular events through the increase in PMN and MNC oxidative stress of hypertensive diabetic subjects.

We have shown that MBP or HbA_1C is related to PMN oxidative stress (Table 4), which is consistent with the previous observation.^12,13 PMNs contain the identical oxidative stress–generating system to endothelial cells. If they are activated, they would be an additional systemic source of oxidative stress.¹⁴

In the present study, we have also demonstrated for the first time that CRP is related to MNC oxidative stress (Tables 5). CRP has been extensively studied as a potential tool for the prediction of cardiovascular events.¹⁵ Thus, MNC oxidative stress may play a role as a potential tool for prediction of cardiovascular risk.

Risk factors for cardiovascular diseases such as hypertension, diabetes, and hyperlipidemia are related to both oxidative stress^9,16,17 and insulin resistance.^18,19 In the present study, neither PMN oxidative stress nor MNC oxidative stress was directly related to HOMA-IR, an indicator of insulin resistance. Multiple regression analysis showed that body mass index, triglycerides, and HbA_1C were related to HOMA-IR, suggesting that obesity-related metabolic abnormality was related to insulin resistance.

Possible mechanism of increase in oxidative stress in PMNs and MNCs in relation to hypertension, diabetes, and CRP remains to be elucidated. Pressure and high glucose are reported to activate protein kinase C activation in vitro,^9,18 and oxidative stress in PMNs is mediated by protein kinase C.²⁰ Protein kinase C may play a role in the increase in PMN oxidative stress induced by hypertension and diabetes.

In conclusion, we found a significant relationship between PMN oxidative stress and MBP or HbA_1C, in addition to a significant relationship between MNC oxidative stress and CRP. We have identified a possible cellular mechanism to explain why oxidative stress in hypertensive and diabetic individuals are increased.

	Acknowledgments

 
We would like to thank Junko Ohashi and Sayuri Takagi for excellent secretarial assistance. This study was supported by the Ministry of Education, Sports, Science and Technology; Kimura Memorial Heart Foundation; and Research Foundation of Community Medicine.

Received November 14, 2001; first decision December 3, 2001; accepted December 18, 2001.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yasunari, K. Articles by Yoshikawa, J. Search for Related Content PubMed PubMed Citation Articles by Yasunari, K. Articles by Yoshikawa, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB HYDROGEN PEROXIDE Related Collections Risk Factors Type 2 diabetes Clinical Studies