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disulfide redox status in hyperlipidemic subjects
Free Radical Biology & Medicine, Vol. 35, No. 10, pp. 1286 –1292, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter
doi:10.1016/j.freeradbiomed.2003.07.001
Original Contribution CHOLESTEROL LEVELS LINKED TO ABNORMAL PLASMA THIOL CONCENTRATIONS AND THIOL/DISULFIDE REDOX STATUS IN HYPERLIPIDEMIC SUBJECTS RALF KINSCHERF,* KONSTANTINOS CAFALTZIS,*1 FALK RO¨ DER,*1 WULF HILDEBRANDT,† LUTZ EDLER,† HANS-PETER DEIGNER,‡ RAOUL BREITKREUTZ,† GISO FEUSSNER,§ JO¨ RG KREUZER,㛳 EGON WERLE,¶ GERD MICHEL,# JU¨ RGEN METZ,* and WULF DRO¨ GE† *Department of Anatomy and Cell Biology III, Heidelberg University, Heidelberg, Germany; †German Cancer Research Center, Heidelberg, Germany; ‡SIRS Lab, Jena, Germany; §Department of Internal Medicine I, Heidelberg, Germany; 㛳Department of Internal Medicine III, Cardiology, Angiology und Pulmology, Heidelberg, Germany; and ¶Zentrallabor of the Medical University Hospital Heidelberg, Heidelberg, Germany; and #Abbott GmbH, Wiesbaden, Germany (Received 14 May 2003; Revised 29 July 2003; Accepted 31 July 2003)
Abstract—Treatment of hyperlipidemic patients with the thiol compound N-acetylcysteine (NAC) was previously shown to cause a significant dose-related increase in the high-density lipoprotein (HDL)-cholesterol serum level, suggesting the possibility that its disease-related decrease may result from a diminished thiol concentration and/or thiol/disulfide redox status (REDST) in the plasma. We therefore investigated plasma thiol levels and REDST in normo-/hyperlipidemic subjects with and without coronary heart disease (CHD). The thiol level, REDST, and amino acid concentrations in the plasma and intracellular REDST of peripheral blood mononuclear cells (PBMC) have been determined in 62 normo- and hyperlipidemic subjects. Thirty-three of these subjects underwent coronary angiography, because of clinical symptoms of CHD. All groups of hyperlipidemic patients under test and those normolipidemic individuals with documented coronary stenoses showed a marked decrease in plasma thiol concentrations, plasma and intracellular REDST of PBMCs, and a marked increase in plasma taurine levels. Individual plasma thiol concentrations and plasma REDST were strongly negatively correlated with the serum LDL-cholesterol and positively correlated with the serum HDL-cholesterol level. Together with the earlier report about the effect of NAC on the HDL-cholesterol serum level, our findings suggest strongly that lower HDL-cholesterol serum levels may result from a decrease in plasma thiol level and/or REDST possibly through an excessive cysteine catabolism into taurine. © 2003 Elsevier Inc. Keywords—Arteriosclerosis, Risk factors in hyperlipidemia, Glutathione in atherosclerosis, Redox status as a risk factor, Thiol level as a risk factor, Free radicals
INTRODUCTION
HDL serum cholesterol may be related to changes of the thiol level and/or the thiol/disulfide redox status (REDST) in the plasma. The present study was designed to test this hypothesis. The most important quantitatively low molecular weight thiol/disulfide redox couple in the plasma is the cysteine/ cystine couple. As the REDST of different thiol/disulfide couples are typically linked to one another by thiol/disulfide exchange reactions, changes in the cysteine/cystine REDST are typically associated with corresponding changes in the ratio of reduced versus oxidized form of plasma albumin, i.e., the major high molecular weight redox buffer in the plasma (reviewed in [3]). It should be noted, however, that different redox couples show different ratios due to differences in redox potentials. Whereas cystine, i.e., cysteine-
Abnormally high serum levels of low-density lipoprotein (LDL)-cholesterol and low serum levels of high-density lipoprotein (HDL)-cholesterol are associated with an increased risk for CHD [1]. The mechanisms responsible for these changes in serum lipid levels are not well understood. An earlier study showed, however, that HDL-cholesterol may be increased by N-acetylcysteine (NAC) [2], suggesting the possibility that a decrease in Address correspondence to: Dr. Ralf Kinscherf, Department of Anatomy and Cell Biology III, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany; Tel: ⫹49 (6221) 548306; Fax: ⫹49 (6221) 544912; E-Mail: [email protected]. 1 Both authors contributed equally. 1286
Cholesterol levels linked to plasma thiol levels and/or redox status
disulfide, is routinely determined by the amino acid analyzer, its reduced form cysteine can be determined in first approximation by a photometric thiol assay [4,5]. The analysis of plasma cystine by the amino acid analyzer as well as the determination of low molecular thiols requires that the high molecular (protein-bound) thiols are removed by acid precipitation. The remaining acid-soluble thiol is taken in first approximation as the cysteine concentration. Several redox-responsive physiological signaling pathways involve proteins with a redox-sensitive cysteine residue that controls the signaling function either by interaction with free radicals or in response to changes in the thiol/disulfide REDST (reviewed in [6]). We therefore focused in the present investigation on the analysis of plasma thiol levels and thiol/disulfide REDST rather than on the total antioxidant capacity of the plasma. Certain antioxidants such as gliclazide and red wine, which were previously shown to ameliorate lipid peroxidation, were also found to increase thiol concentrations [7,8], whereas quercetin, i.e., a major antioxidant from onions, was found to have no direct protective effect on LDL-oxidation [9]. MATERIALS AND METHODS
Subjects Twelve normolipidemic (NL; total serum cholesterol ⬍ 220 mg/dl; plasma triglyceride levels ⬍ 150 mg/dl) and 33 hyperlipidemic male patients [hypercholesterolemic (HC) total cholesterol levels ⬎ 220 mg/dl; hypertriglyceridemic (HT) plasma triglyceride levels ⬎150 mg/dl] without antihyperlipidemic treatment were randomly recruited from the Cardiologic and Lipid Outpatient Clinic of the Medical University Hospital of Heidelberg. From these 45 patients, 33 underwent coronary angiography because of clinical symptoms of CHD. Moreover, patient subgroups were installed, as indicated in Table 1, to identify subjects as diabetics and according to their smoking habit. Seventeen NL male individuals with a preventive lifestyle, regular physical exercise, without known risk factors/clinical symptoms of CHD, and any kind of regular medication served as control. The study was conducted according to the principles of the Declaration of Helsinki. Blood samples; determination of the plasma REDST Blood samples were routinely taken from the cubital vein. Total serum cholesterol, HDL- and LDL-cholesterol as well as triglyceride levels were measured by standard methods. Plasma amino acids were determined by high-performance liquid chromatography and acidsoluble thiol concentrations were analyzed within 30 min after drawing of the blood by a photometric assay as described previously [4,5]. In detail, plasma was depro-
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teinized with sulfosalicylic acid (SSA; final concentration 3.5%), vortexed and centrifuged at 10,000 ⫻ g (10 min). Clear supernatant (400 l) was mixed with buffer (400 l; 200 mM Na2HPO4, 10 mM ethylenediaminetetraacetic acid, pH 8), and the extinction was measured at 412 nm before and after adding of 20 l of 5,5⬘-Dithiobis(2-nitrobenzoic acid) (10 mM; Sigma-Aldrich, Taufkirchen, Germany). Several concentrations of L-cysteine (thiol) in 3.5% SSA were used as standards. Control experiments indicated that because of thiol oxidation, a significant decrease occurs within a few hours at room temperature or within a few days in frozen samples. The plasma REDST was defined as thiol2 cystine⫺1, because two thiol molecules yield one disulfide molecule upon oxidation. Homocysteine concentrations were measured in frozen plasma samples using the IMx homocysteine assay (Abbott Laboratories, Wiesbaden, Germany). Determination of total glutathione, glutathione disulfide (GSSG), and reduced glutathione (GSH) in PBMCs Human PBMCs were prepared from venous blood by Histopaque (Sigma, Munich, Germany) density gradient centrifugation, as described by the manufacturer’s instructions. The cells were washed three times with 10 mM phosphate-buffered saline (pH 7.4), mixed with 2.5% SSA, and subjected to sonication and centrifugation. The clear supernatants were finally used for determining total glutathione and GSSG by the method of Tietze [10] as described previously [11]. GSH was computed by subtraction. The pellets were subjected to protein determination [12]. The intracellular REDST of PBMCs was defined as GSH2GSSG⫺1. Statistical analysis Characteristics of patients subdivided into the four groups: hypercholesterolemics without CHD (group 2), normolipidemics with CHD (group 3), hypercholesterolemics with CHD (group 4), and normocholesterolemics/hypertriglyceridemics with CHD (group 5) and characteristics of controls (group 1) were presented as mean (⫾SEM). Differences of taurine and thiol levels and plasma and intracellular REDST levels between the five groups (1–5) were statistically analyzed by one-way analysis of variance using Scheffe´ contrasts (post-hoc testing) between the groups (2–5) and the control to adjust for multiple comparisons. Associations between HDL or LDL cholesterol levels and the cysteine-related parameters were illustrated for all groups and separately for patients by linear regression and were quantified by the Pearson correlation coefficient and the respective test for independence. Differences were considered as statistically significant if p ⬍ .05. The SPSS 11.5 program for
Table 1. Groups of Patients and Control Subjects
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Control of subjects HC w/o CHD No smokers/no diabetics Smokers and/or diabetics NL with CHD No smokers/no diabetics Smokers and/or diabetics HC with CHD No smokers/no diabetics Smokers and/or diabetics NC/HT with CHD No smokers/no diabetics Smokers and/or diabetics
n
Homocysteine ⬍15M (n)
Age (y)
Total cholesterol (mg/dl)
17
11
49.6 ⫾ 3.3
199.0 ⫾ 4.7
5 7
4 3
44.0 ⫾ 4.8 52.9 ⫾ 5.8
4 8
n.d. n.d.
6 7 5 3
HDL cholesterol (mg/dl)
LDL cholesterol (mg/dl)
TG (mg/dl)
Homocysteine (M)
GSH (nmol/mg)
GSSG (nmol/ mg)
78.6 ⫾ 3.9
103.0 ⫾ 5.5
88.4 ⫾ 8.6
12.0 ⫾ 0.7
23.2 ⫾ 2.9
1.7 ⫾ 0.4
254.6 ⫾ 10.4 270.3 ⫾ 28.0
37.2 ⫾ 2.9 42.1 ⫾ 2.0
196.4 ⫾ 8.2 190.3 ⫾ 27.1
140.6 ⫾ 17.7 209.6 ⫾ 52.1
13.2 ⫾ 1.9 19.1 ⫾ 3.8
20.1 ⫾ 5.4 17.1 ⫾ 1.7
2.2 ⫾ 0.6 1.3 ⫾ 0.1
67.5 ⫾ 3.6 66.1 ⫾ 2.7
162.0 ⫾ 8.8 167.3 ⫾ 9.7
33.5 ⫾ 5.0 41.7 ⫾ 3.1
103.0 ⫾ 6.2 113.3 ⫾ 7.9
127.5 ⫾ 9.6 87.4 ⫾ 9.71
n.d. n.d.
21.3 ⫾ 2.7 20.9 ⫾ 1.7
2.9 ⫾ 0.6 2.6 ⫾ 0.5
n.d. n.d.
66.7 ⫾ 0.9 62.9 ⫾ 2.4
241.5 ⫾ 7.6 231.0 ⫾ 6.4
40.8 ⫾ 4.2 42.6 ⫾ 5.1
163.8 ⫾ 10.3 154.4 ⫾ 1.6
210.7 ⫾ 36.1 158.7 ⫾ 22.4
n.d. n.d.
23.4 ⫾ 2.0 23.7 ⫾ 2.5
2.3 ⫾ 0.3 3.4 ⫾ 0.8
n.d. n.d.
64.2 ⫾ 2.7 64.7 ⫾ 3.2
184.2 ⫾ 10.7 201.0 ⫾ 7.0
35.8 ⫾ 2.6 46.3 ⫾ 11.3
106.1 ⫾ 11.1 117.3 ⫾ 6.5
211.2 ⫾ 26.0 187.0 ⫾ 4.6
n.d. n.d.
23.2 ⫾ 2.9 24.0 ⫾ 0.9
4.6 ⫾ 1.1 2.9 ⫾ 0.9
HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein; TG ⫽ triglycerides; GSH ⫽ reduced glutathione; GSSG ⫽ glutathione disulfide; HC ⫽ hypercholesterolemic; CHD ⫽ coronary heart disease; NL ⫽ normolipidemie; NC ⫽ normocholesterolemic; HT ⫽ hypertriglyceridemic; n.d. ⫽ not determined.
Cholesterol levels linked to plasma thiol levels and/or redox status
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Fig. 1. Abnormal plasma and intracellular redox state (REDST) in hypercholesterolemia and coronary heart disease (CHD). Plasma taurine and thiol concentrations, plasma REDST (defined as thiol2 cystine⫺1) and intracellular REDST (defined as GSH2 GSSG⫺1) in peripheral blood mononuclear cells of 17 healthy control subjects and the 8 patient groups described in Table 1. NL w/o CHD ⫽ normolipidemic without CHD (healthy controls); HC w/o CHD ⫽ hypercholesterolemic without CHD; NL⫹CHD ⫽ normolipidemic with CHD; HC⫹CHD ⫽ hypercholesterolemic with CHD; NC, HT⫹CHD ⫽ normocholesterolemic hypertriglyceridemic patients with CHD. Black symbols ⫽ smokers and diabetic patients; open symbols ⫽ patients without these risk factors. p-Values indicate the significance between the indicated group and the healthy control group.
Windows (SPSS Inc., Chicago, IL, USA) was used to calculate the statistics. RESULTS
All 45 patients under test (groups 2 to 5 in Table 1) showed, on the average, abnormally high plasma taurine
and low plasma thiol levels in comparison to the control group (Fig. 1). All patient groups revealed a pro-oxidative shift of the plasma REDST (thiol2 cystine⫺1), which in all hyperlipidemic groups was associated with a significant decrease of the plasma thiol (mainly cysteine) concentration (Fig. 1). In almost all patient groups this decrease in plasma thiol was accompanied by a signifi-
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R. KINSCHERF et al.
Fig. 2. Correlation between cholesterol levels and cysteine-related parameters The correlation between high-density lipoprotein (HDL) cholesterol level (upper panel) or low-density lipoprotein (LDL) cholesterol level (lower panel), and the corresponding individual plasma taurine and thiol concentrations and the plasma redox state (REDST) is indicated by the regression function (solid line, all groups; dotted line, patient groups), correlation coefficient (r), and significance level (p). The analysis was based on the total group of 45 patients with and without 17 healthy control subjects as indicated in Table 1. The symbols refer to the different subgroups of patients as described in legend to Fig. 1.
cant increase in plasma taurine concentration (Fig. 1). The decline in intracellular REDST (GSH2 GSSG⫺1) within the PBMCs was mostly due to increased GSSG concentrations, whereas the GSH levels often are not different among the groups (Table 1). It may be noted that all these changes were found in smokers and diabetic patients (black symbols, Fig. 1) as well as in patients without these risk factors and in hyperlipidemic patients with and without CHD as well as in CHD patients with cholesterol levels ⬍ 220 mg/dl (Fig. 1 and Table 1). In line with the reported effect of NAC on the HDLcholesterol level [2], our study revealed a significant positive correlation of the HDL-cholesterol level with the corresponding plasma thiol concentration and REDST and an inverse correlation with the plasma taurine level (Fig. 2, upper panel). In this context, analysis of the data of all patient groups separate from the control group revealed only marginal correlations of the HDLcholesterol level and the corresponding plasma thiol, taurine concentration, and REDST (Fig. 2, upper panel). These results seem to indicate a coincidence of two differences, which are possibly not causally determined. In addition, individual serum LDL-cholesterol levels were significantly inversely correlated with the corresponding plasma thiol concentration or the plasma REDST and strongly positively correlated with the plasma taurine concentration (Fig. 2, lower panel). Analysis of the data of all patient groups separate from the control group also revealed a significant inverse correla-
tion between serum LDL-cholesterol levels and the corresponding plasma thiol concentration or the plasma REDST and a positive correlation with the plasma taurine concentration (Fig. 2, lower panel). In this context, the regression function of the patient groups includes the control group and, thus, according to this is identical to the regression function of all groups (control plus patients). Furthermore, regression analyses without outliers do neither markedly alter the correlation coefficient nor the significance (not shown). Homocysteine, which was only analyzed in groups 1 and 2, showed a significant increase in smoking as well as diabetic hypercholesterolemic patients (Table 1). It should be noted that the widely used clinical parameter “homocysteine” refers to total homocysteine, which includes oxidized and proteinbound forms of homocysteine. An oxidative shift in the plasma REDST is typically associated with a profound increase in protein-bound homocyteine and a corresponding increase in total homocysteine (reviewed in [3]). DISCUSSION
Our study revealed that patients with abnormally high serum LDL-cholesterol levels and/or low serum HDLcholesterol levels had a markedly diminished acid soluble thiol level, and a significantly lower plasma and intracellular REDST within the PBMCs. These alterations were also found in patients belonging to the sub-
Cholesterol levels linked to plasma thiol levels and/or redox status
group of smokers and diabetics. In addition, the serum HDL-cholesterol level was strongly positively correlated with the individual plasma thiol level or plasma REDST, whereas the LDL-cholesterol serum level and total serum cholesterol level were inversely correlated. These findings together with the earlier report that the HDL-cholesterol concentration can be increased by treatment with NAC [2] strongly suggest that the low serum HDLcholesterol levels may be a direct consequence of the decrease in plasma cysteine concentration and/or plasma REDST. The mechanism of this linkage between HDLcholesterol level and plasma thiol level or REDST is still unknown. The conspicuous increase in the plasma taurine concentration, which is seen in all groups of patients under test, suggests that the decrease in plasma thiol (mainly cysteine) concentration might result, at least to some extent, from an increased catabolism of cysteine into taurine. The individual plasma taurine concentration was indeed significantly and inversely correlated with the corresponding plasma thiol concentration and the serum HDL-cholesterol level. The underlying pathomechanism and the cause of the increase in taurine concentration remain to be investigated. The oxidative shift in plasma REDST was associated with a corresponding shift of the intracellular REDST within PBMCs of all patient groups under test. This shift is believed to result partly from the decrease in plasma thiol concentration and partly from an increase in the plasma glutamate concentration, which was also found in all patient groups (not shown). The cysteine supply was shown to determine decisively the intracellular glutathione concentration of lymphocytes and macrophages [13], while elevated extracellular glutamate and vitamin C levels were found to inhibit competitively the membrane transport of cystine [14,15]. Increased extracellular glutamate concentrations were shown to decrease the intracellular glutathione level of macrophages [16]. The glutathione system is well known to play a key role in the cellular protection against oxidative stress. Low glutathione levels within human macrophages have been shown to facilitate LDL oxidation and to induce macrophage apoptosis [17–19]. Treatment with NAC prevented the oxLDL-mediated decrease of the intracellular glutathione and the induction of apoptosis [20]. Because various redox-sensitive signaling pathways respond not only to changes in concentration of reactive oxygen species but also to changes in REDST (reviewed in [3]), it is reasonable to assume that the change in REDST accounts also for the abnormal expression of redox-controlled gene products, such as vascular cell adhesion molecule-1, tumor necrosis factor-␣, and manganese superoxide dismutase, which were previously shown to play an important role in the pathogenesis of atherosclerosis. This hypothesis is supported by several
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studies with NAC. Treatment with NAC was shown to improve coronary and peripheral endothelial-dependent vasodilation [21]. Treatment of patients with CHD with the cysteine prodrug L-oxo-4-thiozolidinecarboxylate was found to reverse endothelial dysfunction in a placebo-controlled trial [22]. In vitro, LDL-oxidation was shown to be inhibited by NAC [23]. Treatment with NAC was also found to improve vascular stability by inhibiting the expression of matrix metalloproteinases in atherosclerotic lesions [24]. NAC also ameliorated the abnormal expression of the redox-regulated vascular cell adhesion molecule-1 in nonobese patients with non– insulin-dependent diabetes mellitus [25]. In conclusion, there is a strong possibility that the changes in plasma thiol level, plasma and intracellular REDST of PBMCs may play a causative role in the pathophysiology of the arteriosclerotic process and the development of CHD. This conclusion is in line with the fact that abnormally high “total homocysteine levels,” which are also typically associated with an oxidative shift in REDST [26] have been identified as an independent risk factor for CHD. The oxidative shift in REDST may therefore be a consensus risk factor common to several or all independent risk factors that have been identified previously. Our findings enforce the conclusions from earlier studies that NAC or other mechanistically related antioxidants may be useful for the treatment of cardiovascular diseases. Acknowledgements — We are grateful to Mrs. U. Traut, Mrs. U. Winter, and Mr. H. Lips for expert technical assistance and to the study participants for their cooperation and to Mrs. I. Fryson for her assistance in the preparation in this manuscript.
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ABBREVIATIONS
CHD— coronary heart disease GSH—reduced glutathione GSSG— glutathione disulfide HC— hypercholesterolemic HDL— high-density lipoprotein HT— hypertriglyceridemic LDL—low-density lipoprotein NAC—N-acetyl-cysteine NL—normolipidemic PBMC—peripheral blood mononuclear cells oxLDL— oxidized LDL REDST—thiol/disulfide redox state
doi:10.1016/j.freeradbiomed.2003.07.001
Original Contribution CHOLESTEROL LEVELS LINKED TO ABNORMAL PLASMA THIOL CONCENTRATIONS AND THIOL/DISULFIDE REDOX STATUS IN HYPERLIPIDEMIC SUBJECTS RALF KINSCHERF,* KONSTANTINOS CAFALTZIS,*1 FALK RO¨ DER,*1 WULF HILDEBRANDT,† LUTZ EDLER,† HANS-PETER DEIGNER,‡ RAOUL BREITKREUTZ,† GISO FEUSSNER,§ JO¨ RG KREUZER,㛳 EGON WERLE,¶ GERD MICHEL,# JU¨ RGEN METZ,* and WULF DRO¨ GE† *Department of Anatomy and Cell Biology III, Heidelberg University, Heidelberg, Germany; †German Cancer Research Center, Heidelberg, Germany; ‡SIRS Lab, Jena, Germany; §Department of Internal Medicine I, Heidelberg, Germany; 㛳Department of Internal Medicine III, Cardiology, Angiology und Pulmology, Heidelberg, Germany; and ¶Zentrallabor of the Medical University Hospital Heidelberg, Heidelberg, Germany; and #Abbott GmbH, Wiesbaden, Germany (Received 14 May 2003; Revised 29 July 2003; Accepted 31 July 2003)
Abstract—Treatment of hyperlipidemic patients with the thiol compound N-acetylcysteine (NAC) was previously shown to cause a significant dose-related increase in the high-density lipoprotein (HDL)-cholesterol serum level, suggesting the possibility that its disease-related decrease may result from a diminished thiol concentration and/or thiol/disulfide redox status (REDST) in the plasma. We therefore investigated plasma thiol levels and REDST in normo-/hyperlipidemic subjects with and without coronary heart disease (CHD). The thiol level, REDST, and amino acid concentrations in the plasma and intracellular REDST of peripheral blood mononuclear cells (PBMC) have been determined in 62 normo- and hyperlipidemic subjects. Thirty-three of these subjects underwent coronary angiography, because of clinical symptoms of CHD. All groups of hyperlipidemic patients under test and those normolipidemic individuals with documented coronary stenoses showed a marked decrease in plasma thiol concentrations, plasma and intracellular REDST of PBMCs, and a marked increase in plasma taurine levels. Individual plasma thiol concentrations and plasma REDST were strongly negatively correlated with the serum LDL-cholesterol and positively correlated with the serum HDL-cholesterol level. Together with the earlier report about the effect of NAC on the HDL-cholesterol serum level, our findings suggest strongly that lower HDL-cholesterol serum levels may result from a decrease in plasma thiol level and/or REDST possibly through an excessive cysteine catabolism into taurine. © 2003 Elsevier Inc. Keywords—Arteriosclerosis, Risk factors in hyperlipidemia, Glutathione in atherosclerosis, Redox status as a risk factor, Thiol level as a risk factor, Free radicals
INTRODUCTION
HDL serum cholesterol may be related to changes of the thiol level and/or the thiol/disulfide redox status (REDST) in the plasma. The present study was designed to test this hypothesis. The most important quantitatively low molecular weight thiol/disulfide redox couple in the plasma is the cysteine/ cystine couple. As the REDST of different thiol/disulfide couples are typically linked to one another by thiol/disulfide exchange reactions, changes in the cysteine/cystine REDST are typically associated with corresponding changes in the ratio of reduced versus oxidized form of plasma albumin, i.e., the major high molecular weight redox buffer in the plasma (reviewed in [3]). It should be noted, however, that different redox couples show different ratios due to differences in redox potentials. Whereas cystine, i.e., cysteine-
Abnormally high serum levels of low-density lipoprotein (LDL)-cholesterol and low serum levels of high-density lipoprotein (HDL)-cholesterol are associated with an increased risk for CHD [1]. The mechanisms responsible for these changes in serum lipid levels are not well understood. An earlier study showed, however, that HDL-cholesterol may be increased by N-acetylcysteine (NAC) [2], suggesting the possibility that a decrease in Address correspondence to: Dr. Ralf Kinscherf, Department of Anatomy and Cell Biology III, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany; Tel: ⫹49 (6221) 548306; Fax: ⫹49 (6221) 544912; E-Mail: [email protected]. 1 Both authors contributed equally. 1286
Cholesterol levels linked to plasma thiol levels and/or redox status
disulfide, is routinely determined by the amino acid analyzer, its reduced form cysteine can be determined in first approximation by a photometric thiol assay [4,5]. The analysis of plasma cystine by the amino acid analyzer as well as the determination of low molecular thiols requires that the high molecular (protein-bound) thiols are removed by acid precipitation. The remaining acid-soluble thiol is taken in first approximation as the cysteine concentration. Several redox-responsive physiological signaling pathways involve proteins with a redox-sensitive cysteine residue that controls the signaling function either by interaction with free radicals or in response to changes in the thiol/disulfide REDST (reviewed in [6]). We therefore focused in the present investigation on the analysis of plasma thiol levels and thiol/disulfide REDST rather than on the total antioxidant capacity of the plasma. Certain antioxidants such as gliclazide and red wine, which were previously shown to ameliorate lipid peroxidation, were also found to increase thiol concentrations [7,8], whereas quercetin, i.e., a major antioxidant from onions, was found to have no direct protective effect on LDL-oxidation [9]. MATERIALS AND METHODS
Subjects Twelve normolipidemic (NL; total serum cholesterol ⬍ 220 mg/dl; plasma triglyceride levels ⬍ 150 mg/dl) and 33 hyperlipidemic male patients [hypercholesterolemic (HC) total cholesterol levels ⬎ 220 mg/dl; hypertriglyceridemic (HT) plasma triglyceride levels ⬎150 mg/dl] without antihyperlipidemic treatment were randomly recruited from the Cardiologic and Lipid Outpatient Clinic of the Medical University Hospital of Heidelberg. From these 45 patients, 33 underwent coronary angiography because of clinical symptoms of CHD. Moreover, patient subgroups were installed, as indicated in Table 1, to identify subjects as diabetics and according to their smoking habit. Seventeen NL male individuals with a preventive lifestyle, regular physical exercise, without known risk factors/clinical symptoms of CHD, and any kind of regular medication served as control. The study was conducted according to the principles of the Declaration of Helsinki. Blood samples; determination of the plasma REDST Blood samples were routinely taken from the cubital vein. Total serum cholesterol, HDL- and LDL-cholesterol as well as triglyceride levels were measured by standard methods. Plasma amino acids were determined by high-performance liquid chromatography and acidsoluble thiol concentrations were analyzed within 30 min after drawing of the blood by a photometric assay as described previously [4,5]. In detail, plasma was depro-
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teinized with sulfosalicylic acid (SSA; final concentration 3.5%), vortexed and centrifuged at 10,000 ⫻ g (10 min). Clear supernatant (400 l) was mixed with buffer (400 l; 200 mM Na2HPO4, 10 mM ethylenediaminetetraacetic acid, pH 8), and the extinction was measured at 412 nm before and after adding of 20 l of 5,5⬘-Dithiobis(2-nitrobenzoic acid) (10 mM; Sigma-Aldrich, Taufkirchen, Germany). Several concentrations of L-cysteine (thiol) in 3.5% SSA were used as standards. Control experiments indicated that because of thiol oxidation, a significant decrease occurs within a few hours at room temperature or within a few days in frozen samples. The plasma REDST was defined as thiol2 cystine⫺1, because two thiol molecules yield one disulfide molecule upon oxidation. Homocysteine concentrations were measured in frozen plasma samples using the IMx homocysteine assay (Abbott Laboratories, Wiesbaden, Germany). Determination of total glutathione, glutathione disulfide (GSSG), and reduced glutathione (GSH) in PBMCs Human PBMCs were prepared from venous blood by Histopaque (Sigma, Munich, Germany) density gradient centrifugation, as described by the manufacturer’s instructions. The cells were washed three times with 10 mM phosphate-buffered saline (pH 7.4), mixed with 2.5% SSA, and subjected to sonication and centrifugation. The clear supernatants were finally used for determining total glutathione and GSSG by the method of Tietze [10] as described previously [11]. GSH was computed by subtraction. The pellets were subjected to protein determination [12]. The intracellular REDST of PBMCs was defined as GSH2GSSG⫺1. Statistical analysis Characteristics of patients subdivided into the four groups: hypercholesterolemics without CHD (group 2), normolipidemics with CHD (group 3), hypercholesterolemics with CHD (group 4), and normocholesterolemics/hypertriglyceridemics with CHD (group 5) and characteristics of controls (group 1) were presented as mean (⫾SEM). Differences of taurine and thiol levels and plasma and intracellular REDST levels between the five groups (1–5) were statistically analyzed by one-way analysis of variance using Scheffe´ contrasts (post-hoc testing) between the groups (2–5) and the control to adjust for multiple comparisons. Associations between HDL or LDL cholesterol levels and the cysteine-related parameters were illustrated for all groups and separately for patients by linear regression and were quantified by the Pearson correlation coefficient and the respective test for independence. Differences were considered as statistically significant if p ⬍ .05. The SPSS 11.5 program for
Table 1. Groups of Patients and Control Subjects
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Control of subjects HC w/o CHD No smokers/no diabetics Smokers and/or diabetics NL with CHD No smokers/no diabetics Smokers and/or diabetics HC with CHD No smokers/no diabetics Smokers and/or diabetics NC/HT with CHD No smokers/no diabetics Smokers and/or diabetics
n
Homocysteine ⬍15M (n)
Age (y)
Total cholesterol (mg/dl)
17
11
49.6 ⫾ 3.3
199.0 ⫾ 4.7
5 7
4 3
44.0 ⫾ 4.8 52.9 ⫾ 5.8
4 8
n.d. n.d.
6 7 5 3
HDL cholesterol (mg/dl)
LDL cholesterol (mg/dl)
TG (mg/dl)
Homocysteine (M)
GSH (nmol/mg)
GSSG (nmol/ mg)
78.6 ⫾ 3.9
103.0 ⫾ 5.5
88.4 ⫾ 8.6
12.0 ⫾ 0.7
23.2 ⫾ 2.9
1.7 ⫾ 0.4
254.6 ⫾ 10.4 270.3 ⫾ 28.0
37.2 ⫾ 2.9 42.1 ⫾ 2.0
196.4 ⫾ 8.2 190.3 ⫾ 27.1
140.6 ⫾ 17.7 209.6 ⫾ 52.1
13.2 ⫾ 1.9 19.1 ⫾ 3.8
20.1 ⫾ 5.4 17.1 ⫾ 1.7
2.2 ⫾ 0.6 1.3 ⫾ 0.1
67.5 ⫾ 3.6 66.1 ⫾ 2.7
162.0 ⫾ 8.8 167.3 ⫾ 9.7
33.5 ⫾ 5.0 41.7 ⫾ 3.1
103.0 ⫾ 6.2 113.3 ⫾ 7.9
127.5 ⫾ 9.6 87.4 ⫾ 9.71
n.d. n.d.
21.3 ⫾ 2.7 20.9 ⫾ 1.7
2.9 ⫾ 0.6 2.6 ⫾ 0.5
n.d. n.d.
66.7 ⫾ 0.9 62.9 ⫾ 2.4
241.5 ⫾ 7.6 231.0 ⫾ 6.4
40.8 ⫾ 4.2 42.6 ⫾ 5.1
163.8 ⫾ 10.3 154.4 ⫾ 1.6
210.7 ⫾ 36.1 158.7 ⫾ 22.4
n.d. n.d.
23.4 ⫾ 2.0 23.7 ⫾ 2.5
2.3 ⫾ 0.3 3.4 ⫾ 0.8
n.d. n.d.
64.2 ⫾ 2.7 64.7 ⫾ 3.2
184.2 ⫾ 10.7 201.0 ⫾ 7.0
35.8 ⫾ 2.6 46.3 ⫾ 11.3
106.1 ⫾ 11.1 117.3 ⫾ 6.5
211.2 ⫾ 26.0 187.0 ⫾ 4.6
n.d. n.d.
23.2 ⫾ 2.9 24.0 ⫾ 0.9
4.6 ⫾ 1.1 2.9 ⫾ 0.9
HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein; TG ⫽ triglycerides; GSH ⫽ reduced glutathione; GSSG ⫽ glutathione disulfide; HC ⫽ hypercholesterolemic; CHD ⫽ coronary heart disease; NL ⫽ normolipidemie; NC ⫽ normocholesterolemic; HT ⫽ hypertriglyceridemic; n.d. ⫽ not determined.
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Fig. 1. Abnormal plasma and intracellular redox state (REDST) in hypercholesterolemia and coronary heart disease (CHD). Plasma taurine and thiol concentrations, plasma REDST (defined as thiol2 cystine⫺1) and intracellular REDST (defined as GSH2 GSSG⫺1) in peripheral blood mononuclear cells of 17 healthy control subjects and the 8 patient groups described in Table 1. NL w/o CHD ⫽ normolipidemic without CHD (healthy controls); HC w/o CHD ⫽ hypercholesterolemic without CHD; NL⫹CHD ⫽ normolipidemic with CHD; HC⫹CHD ⫽ hypercholesterolemic with CHD; NC, HT⫹CHD ⫽ normocholesterolemic hypertriglyceridemic patients with CHD. Black symbols ⫽ smokers and diabetic patients; open symbols ⫽ patients without these risk factors. p-Values indicate the significance between the indicated group and the healthy control group.
Windows (SPSS Inc., Chicago, IL, USA) was used to calculate the statistics. RESULTS
All 45 patients under test (groups 2 to 5 in Table 1) showed, on the average, abnormally high plasma taurine
and low plasma thiol levels in comparison to the control group (Fig. 1). All patient groups revealed a pro-oxidative shift of the plasma REDST (thiol2 cystine⫺1), which in all hyperlipidemic groups was associated with a significant decrease of the plasma thiol (mainly cysteine) concentration (Fig. 1). In almost all patient groups this decrease in plasma thiol was accompanied by a signifi-
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Fig. 2. Correlation between cholesterol levels and cysteine-related parameters The correlation between high-density lipoprotein (HDL) cholesterol level (upper panel) or low-density lipoprotein (LDL) cholesterol level (lower panel), and the corresponding individual plasma taurine and thiol concentrations and the plasma redox state (REDST) is indicated by the regression function (solid line, all groups; dotted line, patient groups), correlation coefficient (r), and significance level (p). The analysis was based on the total group of 45 patients with and without 17 healthy control subjects as indicated in Table 1. The symbols refer to the different subgroups of patients as described in legend to Fig. 1.
cant increase in plasma taurine concentration (Fig. 1). The decline in intracellular REDST (GSH2 GSSG⫺1) within the PBMCs was mostly due to increased GSSG concentrations, whereas the GSH levels often are not different among the groups (Table 1). It may be noted that all these changes were found in smokers and diabetic patients (black symbols, Fig. 1) as well as in patients without these risk factors and in hyperlipidemic patients with and without CHD as well as in CHD patients with cholesterol levels ⬍ 220 mg/dl (Fig. 1 and Table 1). In line with the reported effect of NAC on the HDLcholesterol level [2], our study revealed a significant positive correlation of the HDL-cholesterol level with the corresponding plasma thiol concentration and REDST and an inverse correlation with the plasma taurine level (Fig. 2, upper panel). In this context, analysis of the data of all patient groups separate from the control group revealed only marginal correlations of the HDLcholesterol level and the corresponding plasma thiol, taurine concentration, and REDST (Fig. 2, upper panel). These results seem to indicate a coincidence of two differences, which are possibly not causally determined. In addition, individual serum LDL-cholesterol levels were significantly inversely correlated with the corresponding plasma thiol concentration or the plasma REDST and strongly positively correlated with the plasma taurine concentration (Fig. 2, lower panel). Analysis of the data of all patient groups separate from the control group also revealed a significant inverse correla-
tion between serum LDL-cholesterol levels and the corresponding plasma thiol concentration or the plasma REDST and a positive correlation with the plasma taurine concentration (Fig. 2, lower panel). In this context, the regression function of the patient groups includes the control group and, thus, according to this is identical to the regression function of all groups (control plus patients). Furthermore, regression analyses without outliers do neither markedly alter the correlation coefficient nor the significance (not shown). Homocysteine, which was only analyzed in groups 1 and 2, showed a significant increase in smoking as well as diabetic hypercholesterolemic patients (Table 1). It should be noted that the widely used clinical parameter “homocysteine” refers to total homocysteine, which includes oxidized and proteinbound forms of homocysteine. An oxidative shift in the plasma REDST is typically associated with a profound increase in protein-bound homocyteine and a corresponding increase in total homocysteine (reviewed in [3]). DISCUSSION
Our study revealed that patients with abnormally high serum LDL-cholesterol levels and/or low serum HDLcholesterol levels had a markedly diminished acid soluble thiol level, and a significantly lower plasma and intracellular REDST within the PBMCs. These alterations were also found in patients belonging to the sub-
Cholesterol levels linked to plasma thiol levels and/or redox status
group of smokers and diabetics. In addition, the serum HDL-cholesterol level was strongly positively correlated with the individual plasma thiol level or plasma REDST, whereas the LDL-cholesterol serum level and total serum cholesterol level were inversely correlated. These findings together with the earlier report that the HDL-cholesterol concentration can be increased by treatment with NAC [2] strongly suggest that the low serum HDLcholesterol levels may be a direct consequence of the decrease in plasma cysteine concentration and/or plasma REDST. The mechanism of this linkage between HDLcholesterol level and plasma thiol level or REDST is still unknown. The conspicuous increase in the plasma taurine concentration, which is seen in all groups of patients under test, suggests that the decrease in plasma thiol (mainly cysteine) concentration might result, at least to some extent, from an increased catabolism of cysteine into taurine. The individual plasma taurine concentration was indeed significantly and inversely correlated with the corresponding plasma thiol concentration and the serum HDL-cholesterol level. The underlying pathomechanism and the cause of the increase in taurine concentration remain to be investigated. The oxidative shift in plasma REDST was associated with a corresponding shift of the intracellular REDST within PBMCs of all patient groups under test. This shift is believed to result partly from the decrease in plasma thiol concentration and partly from an increase in the plasma glutamate concentration, which was also found in all patient groups (not shown). The cysteine supply was shown to determine decisively the intracellular glutathione concentration of lymphocytes and macrophages [13], while elevated extracellular glutamate and vitamin C levels were found to inhibit competitively the membrane transport of cystine [14,15]. Increased extracellular glutamate concentrations were shown to decrease the intracellular glutathione level of macrophages [16]. The glutathione system is well known to play a key role in the cellular protection against oxidative stress. Low glutathione levels within human macrophages have been shown to facilitate LDL oxidation and to induce macrophage apoptosis [17–19]. Treatment with NAC prevented the oxLDL-mediated decrease of the intracellular glutathione and the induction of apoptosis [20]. Because various redox-sensitive signaling pathways respond not only to changes in concentration of reactive oxygen species but also to changes in REDST (reviewed in [3]), it is reasonable to assume that the change in REDST accounts also for the abnormal expression of redox-controlled gene products, such as vascular cell adhesion molecule-1, tumor necrosis factor-␣, and manganese superoxide dismutase, which were previously shown to play an important role in the pathogenesis of atherosclerosis. This hypothesis is supported by several
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studies with NAC. Treatment with NAC was shown to improve coronary and peripheral endothelial-dependent vasodilation [21]. Treatment of patients with CHD with the cysteine prodrug L-oxo-4-thiozolidinecarboxylate was found to reverse endothelial dysfunction in a placebo-controlled trial [22]. In vitro, LDL-oxidation was shown to be inhibited by NAC [23]. Treatment with NAC was also found to improve vascular stability by inhibiting the expression of matrix metalloproteinases in atherosclerotic lesions [24]. NAC also ameliorated the abnormal expression of the redox-regulated vascular cell adhesion molecule-1 in nonobese patients with non– insulin-dependent diabetes mellitus [25]. In conclusion, there is a strong possibility that the changes in plasma thiol level, plasma and intracellular REDST of PBMCs may play a causative role in the pathophysiology of the arteriosclerotic process and the development of CHD. This conclusion is in line with the fact that abnormally high “total homocysteine levels,” which are also typically associated with an oxidative shift in REDST [26] have been identified as an independent risk factor for CHD. The oxidative shift in REDST may therefore be a consensus risk factor common to several or all independent risk factors that have been identified previously. Our findings enforce the conclusions from earlier studies that NAC or other mechanistically related antioxidants may be useful for the treatment of cardiovascular diseases. Acknowledgements — We are grateful to Mrs. U. Traut, Mrs. U. Winter, and Mr. H. Lips for expert technical assistance and to the study participants for their cooperation and to Mrs. I. Fryson for her assistance in the preparation in this manuscript.
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ABBREVIATIONS
CHD— coronary heart disease GSH—reduced glutathione GSSG— glutathione disulfide HC— hypercholesterolemic HDL— high-density lipoprotein HT— hypertriglyceridemic LDL—low-density lipoprotein NAC—N-acetyl-cysteine NL—normolipidemic PBMC—peripheral blood mononuclear cells oxLDL— oxidized LDL REDST—thiol/disulfide redox state