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Exhaled Carbon Monoxide Levels Elevated in Diabetes and Correlated With Glucose Concentration in Blood
Exhaled Carbon Monoxide Levels Elevated in Diabetes and Correlated With Glucose Concentration in Blood* A New Test for Monitoring the Disease? Paolo Paredi, MD; Wojciech Biernacki, MD; Giovanni Invernizzi, MD; Sergei A. Kharitonov, MD, PhD; and Peter J. Barnes, MA, DM, DSc
Purpose: In diabetes, the interaction of glycated proteins with their cell-surface binding sites leads to oxidative stress and induction of the stress protein heme oxygenase (HO)-1. Considering that carbon monoxide (CO) is a product of HO activity, we studied the level of exhaled CO as a marker of oxidative stress in diabetes. Methods: Eight patients with insulin-dependent diabetes mellitus (type 1) (4 men, 4 women; [mean 6 SEM] age, 50 6 8 years) were studied, of whom 2 had peripheral neuropathy and 1 had renal failure. Sixteen patients with non–insulin-dependent diabetes mellitus (type 2) (5 men, 11 women; age 63 6 8 years) were studied, of whom 2 had peripheral neuropathy. Glycosylated hemoglobin (HbA1c) levels were higher (7.4 6 0.3%) in patients with type 1 (mean duration of the disease, 20 6 5 years) than in type 2 (4.9 6 0.4%; p < 0.05; mean duration of the disease, 11 6 2 years). All of the patients were lifelong nonsmokers. Results: Levels of exhaled CO were higher in patients with diabetes (type 1, 4.0 6 0.7 ppm; type 2, 5.0 6 0.4 ppm) when compared to 37 nonsmoking healthy subjects (20 men, 17 women; age, 33 6 3 years) (2.9 6 0.2 ppm; p < 0.05). There was a positive correlation between exhaled CO levels and the incidence of glycemia in all subjects (r 5 0.52, p < 0.05) and the duration of diabetes (r 5 0.48, p < 0.05), but there was not a strong correlation with concentrations of HbA1c (r 5 0.06, p 5 0.8). In addition, an oral glucose tolerance test was performed in five healthy nonsmoking volunteers (three men; age, 33 6 4 years). The maximal glucose increase (from 3.9 6 0.2 to 5.5 6 0.1 mmol/L at 15 min; p < 0.05) was associated with a significant increase in exhaled CO concentration (from 3.0 6 0.5 to 6.3 6 1.0 ppm; p < 0.05). Both parameters returned to the baseline at 40 min after glucose administration. Conclusions: Elevated levels of exhaled CO in diabetes may reflect HO-1 induction and oxidative stress. The measurement of CO may be a new tool for disease monitoring. (CHEST 1999; 116:1007–1011) Key words: carbon monoxide; glucose tolerance test; oxidative stress Abbreviations: AGE 5 advanced glycation end product; CO 5 carbon monoxide; HbA1c 5 glycosylated hemoglobin; HO 5 heme oxygenase; OGTT 5 oral glucose tolerance test
the interaction of glycated proteins I nwithdiabetes, their cell-surface binding sites leads to oxidative stress, manifested by the appearance of malondialdehyde determinants in the vessel wall, thio*From the Department of Thoracic Medicine (Drs. Barnes, Paredi, Biernacki, and Kharitonov), Imperial College, School of Medicine, National Heart and Lung Institute, London, England; and General Practice (Dr. Invernizzi), Chiavenna, Italy. Supported by grants from University of Milan and the British Lung Foundation (UK). Manuscript received November 10, 1998; revision accepted April 2, 1999. Correspondence to: P. J. Barnes, MA, DM, DSc, Department of Thoracic Medicine, National heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK; e-mail: [email protected]
barbituric acid-reactive substances in the tissues, and induction of the transcription factor nuclear factor-kB and of heme oxygenase (HO)-1.1 This may explain the previously reported finding of elevated exhaled carbon monoxide (CO) levels.2 Oxidative stress also plays an important role in the development of diabetes-related complications; superoxide anions and elevated levels of plasma peroxide have been implicated in retinal damage,3 and lipid peroxides in the severity of coronary atherosclerosis4 in patients with type 1 and type 2 diabetes. CO is a product of heme degradation by HO. Two isoforms of HO have been described: the constitutive HO-2, which is highly expressed in the brain and CHEST / 116 / 4 / OCTOBER, 1999
1007
testes; and the inducible HO-1, which is ubiquitous. The latter is activated by a variety of pro-inflammatory cytokines,5,6 nitric oxide,7,8 H2O2, endotoxin,9 and oxidants,10 and is an important part of a protective response to oxidative stress.11,12 Glucose directly modifies HO activity and CO production, and high levels of CO promote insulin secretion.13 This has been suggested as a novel regulatory system for the stimulation of insulin release. Recent studies showing an activation of HO-1 by agents that cause oxidative stress10 have generated interest in the study of the level of CO as a marker of oxidation. In view of the evidence for increased oxidative stress in diabetes and of the direct activation of HO by glucose we investigated the relation between exhaled CO levels and disease control in patients with insulin-dependent diabetes (type 1) and patients with non–insulin-dependent diabetes (type 2).
Materials and Methods Subjects We have compared exhaled CO levels in 8 patients with type 1 disease (4 men, 4 women; [mean 6 SEM] age, 50 6 8 years), in 16 patients with type 2 disease (5 men, 11 women; age, 63 6 8 years), and in 37 healthy subjects (20 men, 17 women; age, 33 6 3 years). All subjects were lifelong nonsmokers. The mean duration of disease for patients with type 1 diabetes was 20 6 5 years and, for type 2 patients,11 6 2 years. Patients who had a history of respiratory symptoms or who exhibited clinical or radiologic abnormalities of the cardiorespiratory system were excluded from the study. None of the patients had a history of allergic disorders. Each patient was clinically assessed for the presence of cardiovascular, renal, retinal, and neurologic alterations of diabetes. Of the patients with type 1 diabetes, two had peripheral neuropathy and one had established nephropathy; of the patients with type 2 diabetes, two had peripheral neuropathy. Glycosylated hemoglobin (HbA1c) concentration was used as an index of diabetes control over the 3-month period before the study. HbA1c levels were significantly higher in patients with type 1 diabetes (7.5 6 0.3%) than in those with type 2 diabetes (4.9 6 0.4%; p , 0.05), showing a poorer metabolic control in the type 1 group. The mean duration of type 1 diabetes was 20 years (SEM, 5 years; range, 6 months to 40 years), and for type 2, 11 years (SEM, 2 years; range, 1 to 35 years). In addition, the concentration of exhaled CO was measured during an oral glucose tolerance test (OGTT) in five healthy nonsmoking volunteers (3 men, 2 women; [mean 6 SEM] age, 33 6 4 years). The study was approved by the Research Ethics Committee of the Royal Brompton Hospital.
Blood Glucose Level Measurement Blood glucose levels were measured with a portable blood glucose meter (Reflolux S; Boehringer Mannheim; Mannheim, Germany), which was previously shown to provide glucose levels comparable to those of the hexokinase method and to have a low coefficient of variation.14 The device was calibrated before every series of measurements. OGTT An OGTT was performed in the morning in five fasting healthy volunteers, with the administration of 75 g glucose. Monitoring of glucose levels was performed every 10 min for 2 h after glucose administration. The concentration of exhaled CO was monitored at the same time points. The OGTT response was normal (exhaled CO, , 200 mg [11.1 mmol] over 2 h) in all subjects evaluated. Exhaled CO Level Exhaled CO concentration was measured by a modified analyzer (EM50 MICRO Smokerlyser; Bedfont Scientific Ltd; Upchurch, Essex, England) that was sensitive to CO concentrations from 1 to 500 ppm (by volume), adapted for online recording of CO concentration. The subjects exhaled slowly from functional vital capacity with a constant flow (5 to 6 L/min) against resistance (3 6 0.4 mm Hg) over 20 to 30 s into the analyzer. Two successive recordings were made, and maximal values were used in all calculations. Ambient CO levels were recorded before each measurement. Statistical Analysis Analysis of variance tests were used for comparisons among groups. Linear regression analysis was used to assess the relationship between exhaled CO level and blood glucose concentration. All data were expressed as mean 6 SEM. Significance was set at p , 0.05.
Results Exhaled CO Concentration Exhaled CO concentration was higher in patients with diabetes (type 1 patients, 4.0 6 0.7 ppm; type 2 patients, 5.0 6 0.4 ppm; p . 0.05) than in nonsmoking healthy subjects (2.9 6 0.2 ppm; p , 0.05) (Fig 1, left, A). There was a positive correlation between exhaled CO level and blood glucose level (r 5 0.52; p , 0.001, Fig 1, right, B) and duration of diabetes (r 5 0.48; p , 0.05). There was no correlation between exhaled CO level and HbA1c concentration (r 5 0.06; p 5 0.8). Results indicated that exhaled CO levels were not influenced by gender (men, 4.0 6 0.3 ppm; women, 5.0 6 0.2 ppm; p . 0.05).
Pulmonary Function Pulmonary function tests were performed within 2 weeks of the measurement of exhaled CO levels. Curves for FEV1, FVC, and flow volume were obtained using a spirometer (Vitalograph; Buckingham, UK). The results of pulmonary function tests performed on all subjects were normal. 1008
Exhaled CO Concentration During OGTT Maximal increase in glucose concentration (from 3.9 6 0.2 to 5.5 6 0.1 mmol/L, at 15 min; p , 0.05) was associated with a significant increase in the level Clinical Investigations
Figure 1. Levels of exhaled CO in healthy subjects and patients with diabetes (left, A). Correlation between exhaled CO concentration and blood glucose level in patients with diabetes (right, B).
of exhaled CO (from 3.0 6 0.5 to 6.3 6 1.0 ppm; p , 0.05). Both parameters returned to the baseline 40 min after glucose administration (Fig 2). Discussion Exhaled CO levels are elevated in patients with diabetes, correlate with blood glucose levels and duration of the disease, and may reflect the amount of HO-1 induction caused by increased oxidative stress. In diabetes, a consequence of hyperglycemia is the glycation of both circulating proteins and the immobilized proteins within the vascular wall. When proteins or lipids are exposed to aldose sugars, they undergo nonenzymatic glycation and oxidation to form advanced glycation end products (AGEs),1,15 From this reaction, reversible products such as
Figure 2. Changes in exhaled CO and blood glucose levels after OGTT in healthy subjects.
HbA1c are formed, and complex molecular rearrangements result in the irreversible formation of the AGEs. AGEs can produce reactive oxygen intermediates1,16 by interacting with their surface binding sites and generating malondialdehyde determinants in the vessel wall and thiobarbituric acid-reactive substances in the tissues, and by inducing the nuclear factor-kB.1,17 The plasma and tissues of diabetic patients show increased evidence of oxidative stress,16 which plays a role in the development of diabetic complications.16,18 Increased oxidative stress induced by AGE activity and autoxidation associated with compromised mechanisms designed to scavenge free radicals19 is a common pathway for the pathogenesis of complications in diabetes.16 HO plays a role in the protective response to oxidative stress. The activation of HO-1 in response to AGEs induced by oxidative stress1 may explain the finding of elevated exhaled CO levels in patients with diabetes.2 Competitive inhibitors of HO such as Sn-mesoporphyrin and zinc protoporphyrin effectively reduce hyperbilirubinemia20 and CO production in newborns,21 but their effects have not been evaluated in adults. HO activators in asthmatic patients have been investigated in a previous study where the inhalation of hemin was shown to increase the levels of exhaled CO, whereas such levels were reduced in steroid-treated patients.22 Furthermore, in the same study we showed that the high levels of exhaled CO found in asthmatic patients were associated with an CHEST / 116 / 4 / OCTOBER, 1999
1009
increased expression of HO-1. Taken together, these data indicate that the exhaled CO concentration may reflect HO activity. We found a correlation between exhaled CO concentration and blood glucose level. The level of CO was also increased by acute elevations of blood glucose level after OGTT. This may be explained by the activation of HO by glucose23 and the positive modulation of CO on insulin secretion,24 whereby acute CO level increases may be part of a counterregulatory mechanism activated in response to changes in glucose levels. High CO levels during OGTT may also be a reflection of HO activation in response to the induction of the lipid peroxidation cascade as proved by elevated levels 8-isoprostane in the plasma of patients with diabetes and reduction of this marker of oxidation after metabolic control.25 Other potential sources of rapidly induced oxidative stress by hyperglycemia include the presence of free radicals generated by autoxidation and nonenzymatic glycation of unsaturated lipids and membrane proteins, and the reduced antioxidant properties of albumin.26 The lack of correlation between levels of exhaled CO and HbA1c may be due to the rapid fluctuations of CO production, as shown in the OGTT study. Furthermore, the concentration of HbA1c is an expression of the average blood glucose levels in the 2 to 3 months before the test and does not reflect the total oxidative burden associated with the duration of the disease. Although epinephrine is known to induce HO activity, 27steroids are the only nonmetalloporphyrin compounds known to inhibit the induction of HO1.28 The effect of different medications on HO activity has not been systematically investigated. Furthermore, the treatment of diabetes by modifying the levels of glucose and the production of AGEs may modulate the level of oxidative stress15 and indirectly change HO-1 activity and CO production.12 Therefore, the effect of treatment on CO levels should also be investigated in future studies. In this study, only five patients had complications of diabetes. Disease severity may be associated with different levels of oxidative stress; therefore, further studies are needed to investigate whether CO levels are influenced by disease severity. Although oxidative stress is now widely accepted as playing an important role in the aging process,29 the levels of exhaled markers of oxidation such as pentane and ethane are similar in healthy children and adults.30 Furthermore, preliminary results indicate that the levels of exhaled CO are not influenced by sex and age in healthy subjects. Therefore, the level of exhaled CO should not be adjusted for these parameters. 1010
In patients with diabetes, exhaled CO levels may indicate the level of oxidative stress in the manner in which the glycation of plasma proteins and hemoglobin reflects glycemic stress. Eventually, these studies may lead to the development of effective strategies for limiting the damage from oxidation of proteins or for complementing other therapeutic approaches to the treatment of complications in diabetes. Further studies are necessary to investigate the possible role of exhaled CO level as a prognostic factor in the development of systemic complications and in monitoring of the disease. References 1 Yan SD, Schmidt AM, Anderson GM, et al. Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem 1993; 269:9889 –9897 2 Nikberg II, Murashko VA, Leonenko IN, et al. Carbon monoxide concentration in the air exhaled by the healthy and the ill. Vrach Delo 1972; 12:112–114 3 Satto Y, Hotta N, Sakamoto N, et al. Lipid peroxide level in plasma of diabetic patients. Biochem Med 1979; 21:104 –107 4 Tamura M, Tanaka A, Yui K, et al. Oxidation of remnant-like particles from serum of diabetic patients, patients with ischemic heart disease and normal subjects. Horm Metab Res 1997; 29:398 – 402 5 Cantoni L, Rossi C, Rizzardini M, et al. Interleukin 1 and tumor necrosis factor induce hepatic heme oxygenase feedback regulation by glucocorticoids. Biochem J 1991; 279:891– 894 6 Lavrovsky Y, Drummond GS, Abraham NG. Downregulation of the human heme oxygenase gene by glucocorticoids and identification of 56b regulatory elements. Biochem Biophys Res Commun 1996; 218:759 –765 7 Kim YM, Bergonia HA, Muller C, et al. Loss of degradation of enzyme-bound heme induced by cellular nitric oxide synthesis. J Biol Chem 1995; 270:5710 –5713 8 Durante W, Kroll MH, Christodoulides N, et al. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res 1997; 80:557–564 9 Otterbein L, Sylvester SL, Choi A. Hemoglobin provides protection against lethal endotoxemia in rats: the role of heme oxigenase-1. Am J Respir Cell Mol Biol 1995; 13:595– 601 10 Applegate LA, Luscher P, Tyrrel RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res 1991; 51:974 –978 11 Nath KA, Balla G, Vercellotti GM, et al. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest 1992; 90:267–270 12 Augustine MK, Choi K, Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 1996; 15:9 –19 13 Henningsson R, Alm P, Elkstrom P, et al. Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release; a biochemical, immunohistochemical, and confocal microscopic study. Diabetes 1999; 48:66 –76 14 Devreese K, Leroux, RJ. Laboratory assessment of five glucose meters designed for self-monitoring of blood glucose concentration. Eur J Clin Chem Clin Biochem 1993; 31:829 – 837 Clinical Investigations
15 Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetes complications. N Engl J Med 1988; 318:1315–1321 16 Baynes JW. Role of oxidative stress in the development of complications in diabetes. Diabetes 1991; 40:405– 412 17 Hunt JV, Smith CC, Wolff SP. Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes 1990; 39:1420 –1424 18 Schmidt AM, Weidman E, Lalla EB et al. Advanced glycation endproducts (AGEs) induce oxidant stress in the gingiva: a potential mechanism underlying accelerated periodontal disease associated with diabetes. J Periodont Res 1996; 31:508 – 515 19 Godin D, Wohajeb S, Garnett M, et al. Antioxidant enzyme alterations in experimental clinical diabetes. Mol Cell Biochem 1988; 84:223–231 20 Martinez JC, Garcia HO, Otheguy LE, et al. Control of severe hyperbilirubinemia in full-term newborns with the inhibitor of bilirubin production Sn-mesoporphyrin. Pediatrics 1999; 103:1–5 21 Vreman HJ, Rodgers PA, Stevenson DK. Zinc protoporphyrin administration for suppression of increased bilirubin production by iatrogenic hemolysis in rhesus neonates. J Pediatr 1990; 117:292–297 22 Horvath I, Donelly LE, Paredi P, et al. Elevated levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of chronic inflammation. Thorax 1998; 53:668 – 672
23 Henningsson R, Alm P, Elkstrom P, et al. Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release; a biochemical, immunohistochemical, and confocal microscopic study. Diabetes 1999; 48:66 –76 24 Henningsson R, Alm P, Lundquist I. Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets. Am J Physiol 1997; 273(suppl 2 pt 1):C703–C709 25 Davı` G, Ciabattoni G, Consoli A, et al. In vivo formation of 8-iso-prostaglandin F2 alpha and platelet activation in diabetes mellitus: effect of improved metabolic control. Circulation 1999; 99:224 –229 26 Bourdon E, Loreau N, Blache D. Glucose and free radicals impair the antioxidant properties of serum albumin. FASEB J 1999; 13:233–244 27 Bakken AF, Thaler MM, Schmid R. Metabolic regulation of heme catabolism and bilirubin production: hormonal control of hepatic heme oxygenase activity. J Clin Invest 1972; 51:530 –536 28 Lutton JD, da Silva J-L, Moqattash S, et al. Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3B) by environmental agents. J Cell Biochem 1992; 49:259 –265 29 Knight JA. Free radicals: their history and current status in aging and diseased. Clin Lab Sci 1999; 28:331–346 30 Kneepkens CM, Lepage G, Roy CC. The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Radic Biol Med 1994; 17:127–160
CHEST / 116 / 4 / OCTOBER, 1999
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Purpose: In diabetes, the interaction of glycated proteins with their cell-surface binding sites leads to oxidative stress and induction of the stress protein heme oxygenase (HO)-1. Considering that carbon monoxide (CO) is a product of HO activity, we studied the level of exhaled CO as a marker of oxidative stress in diabetes. Methods: Eight patients with insulin-dependent diabetes mellitus (type 1) (4 men, 4 women; [mean 6 SEM] age, 50 6 8 years) were studied, of whom 2 had peripheral neuropathy and 1 had renal failure. Sixteen patients with non–insulin-dependent diabetes mellitus (type 2) (5 men, 11 women; age 63 6 8 years) were studied, of whom 2 had peripheral neuropathy. Glycosylated hemoglobin (HbA1c) levels were higher (7.4 6 0.3%) in patients with type 1 (mean duration of the disease, 20 6 5 years) than in type 2 (4.9 6 0.4%; p < 0.05; mean duration of the disease, 11 6 2 years). All of the patients were lifelong nonsmokers. Results: Levels of exhaled CO were higher in patients with diabetes (type 1, 4.0 6 0.7 ppm; type 2, 5.0 6 0.4 ppm) when compared to 37 nonsmoking healthy subjects (20 men, 17 women; age, 33 6 3 years) (2.9 6 0.2 ppm; p < 0.05). There was a positive correlation between exhaled CO levels and the incidence of glycemia in all subjects (r 5 0.52, p < 0.05) and the duration of diabetes (r 5 0.48, p < 0.05), but there was not a strong correlation with concentrations of HbA1c (r 5 0.06, p 5 0.8). In addition, an oral glucose tolerance test was performed in five healthy nonsmoking volunteers (three men; age, 33 6 4 years). The maximal glucose increase (from 3.9 6 0.2 to 5.5 6 0.1 mmol/L at 15 min; p < 0.05) was associated with a significant increase in exhaled CO concentration (from 3.0 6 0.5 to 6.3 6 1.0 ppm; p < 0.05). Both parameters returned to the baseline at 40 min after glucose administration. Conclusions: Elevated levels of exhaled CO in diabetes may reflect HO-1 induction and oxidative stress. The measurement of CO may be a new tool for disease monitoring. (CHEST 1999; 116:1007–1011) Key words: carbon monoxide; glucose tolerance test; oxidative stress Abbreviations: AGE 5 advanced glycation end product; CO 5 carbon monoxide; HbA1c 5 glycosylated hemoglobin; HO 5 heme oxygenase; OGTT 5 oral glucose tolerance test
the interaction of glycated proteins I nwithdiabetes, their cell-surface binding sites leads to oxidative stress, manifested by the appearance of malondialdehyde determinants in the vessel wall, thio*From the Department of Thoracic Medicine (Drs. Barnes, Paredi, Biernacki, and Kharitonov), Imperial College, School of Medicine, National Heart and Lung Institute, London, England; and General Practice (Dr. Invernizzi), Chiavenna, Italy. Supported by grants from University of Milan and the British Lung Foundation (UK). Manuscript received November 10, 1998; revision accepted April 2, 1999. Correspondence to: P. J. Barnes, MA, DM, DSc, Department of Thoracic Medicine, National heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK; e-mail: [email protected]
barbituric acid-reactive substances in the tissues, and induction of the transcription factor nuclear factor-kB and of heme oxygenase (HO)-1.1 This may explain the previously reported finding of elevated exhaled carbon monoxide (CO) levels.2 Oxidative stress also plays an important role in the development of diabetes-related complications; superoxide anions and elevated levels of plasma peroxide have been implicated in retinal damage,3 and lipid peroxides in the severity of coronary atherosclerosis4 in patients with type 1 and type 2 diabetes. CO is a product of heme degradation by HO. Two isoforms of HO have been described: the constitutive HO-2, which is highly expressed in the brain and CHEST / 116 / 4 / OCTOBER, 1999
1007
testes; and the inducible HO-1, which is ubiquitous. The latter is activated by a variety of pro-inflammatory cytokines,5,6 nitric oxide,7,8 H2O2, endotoxin,9 and oxidants,10 and is an important part of a protective response to oxidative stress.11,12 Glucose directly modifies HO activity and CO production, and high levels of CO promote insulin secretion.13 This has been suggested as a novel regulatory system for the stimulation of insulin release. Recent studies showing an activation of HO-1 by agents that cause oxidative stress10 have generated interest in the study of the level of CO as a marker of oxidation. In view of the evidence for increased oxidative stress in diabetes and of the direct activation of HO by glucose we investigated the relation between exhaled CO levels and disease control in patients with insulin-dependent diabetes (type 1) and patients with non–insulin-dependent diabetes (type 2).
Materials and Methods Subjects We have compared exhaled CO levels in 8 patients with type 1 disease (4 men, 4 women; [mean 6 SEM] age, 50 6 8 years), in 16 patients with type 2 disease (5 men, 11 women; age, 63 6 8 years), and in 37 healthy subjects (20 men, 17 women; age, 33 6 3 years). All subjects were lifelong nonsmokers. The mean duration of disease for patients with type 1 diabetes was 20 6 5 years and, for type 2 patients,11 6 2 years. Patients who had a history of respiratory symptoms or who exhibited clinical or radiologic abnormalities of the cardiorespiratory system were excluded from the study. None of the patients had a history of allergic disorders. Each patient was clinically assessed for the presence of cardiovascular, renal, retinal, and neurologic alterations of diabetes. Of the patients with type 1 diabetes, two had peripheral neuropathy and one had established nephropathy; of the patients with type 2 diabetes, two had peripheral neuropathy. Glycosylated hemoglobin (HbA1c) concentration was used as an index of diabetes control over the 3-month period before the study. HbA1c levels were significantly higher in patients with type 1 diabetes (7.5 6 0.3%) than in those with type 2 diabetes (4.9 6 0.4%; p , 0.05), showing a poorer metabolic control in the type 1 group. The mean duration of type 1 diabetes was 20 years (SEM, 5 years; range, 6 months to 40 years), and for type 2, 11 years (SEM, 2 years; range, 1 to 35 years). In addition, the concentration of exhaled CO was measured during an oral glucose tolerance test (OGTT) in five healthy nonsmoking volunteers (3 men, 2 women; [mean 6 SEM] age, 33 6 4 years). The study was approved by the Research Ethics Committee of the Royal Brompton Hospital.
Blood Glucose Level Measurement Blood glucose levels were measured with a portable blood glucose meter (Reflolux S; Boehringer Mannheim; Mannheim, Germany), which was previously shown to provide glucose levels comparable to those of the hexokinase method and to have a low coefficient of variation.14 The device was calibrated before every series of measurements. OGTT An OGTT was performed in the morning in five fasting healthy volunteers, with the administration of 75 g glucose. Monitoring of glucose levels was performed every 10 min for 2 h after glucose administration. The concentration of exhaled CO was monitored at the same time points. The OGTT response was normal (exhaled CO, , 200 mg [11.1 mmol] over 2 h) in all subjects evaluated. Exhaled CO Level Exhaled CO concentration was measured by a modified analyzer (EM50 MICRO Smokerlyser; Bedfont Scientific Ltd; Upchurch, Essex, England) that was sensitive to CO concentrations from 1 to 500 ppm (by volume), adapted for online recording of CO concentration. The subjects exhaled slowly from functional vital capacity with a constant flow (5 to 6 L/min) against resistance (3 6 0.4 mm Hg) over 20 to 30 s into the analyzer. Two successive recordings were made, and maximal values were used in all calculations. Ambient CO levels were recorded before each measurement. Statistical Analysis Analysis of variance tests were used for comparisons among groups. Linear regression analysis was used to assess the relationship between exhaled CO level and blood glucose concentration. All data were expressed as mean 6 SEM. Significance was set at p , 0.05.
Results Exhaled CO Concentration Exhaled CO concentration was higher in patients with diabetes (type 1 patients, 4.0 6 0.7 ppm; type 2 patients, 5.0 6 0.4 ppm; p . 0.05) than in nonsmoking healthy subjects (2.9 6 0.2 ppm; p , 0.05) (Fig 1, left, A). There was a positive correlation between exhaled CO level and blood glucose level (r 5 0.52; p , 0.001, Fig 1, right, B) and duration of diabetes (r 5 0.48; p , 0.05). There was no correlation between exhaled CO level and HbA1c concentration (r 5 0.06; p 5 0.8). Results indicated that exhaled CO levels were not influenced by gender (men, 4.0 6 0.3 ppm; women, 5.0 6 0.2 ppm; p . 0.05).
Pulmonary Function Pulmonary function tests were performed within 2 weeks of the measurement of exhaled CO levels. Curves for FEV1, FVC, and flow volume were obtained using a spirometer (Vitalograph; Buckingham, UK). The results of pulmonary function tests performed on all subjects were normal. 1008
Exhaled CO Concentration During OGTT Maximal increase in glucose concentration (from 3.9 6 0.2 to 5.5 6 0.1 mmol/L, at 15 min; p , 0.05) was associated with a significant increase in the level Clinical Investigations
Figure 1. Levels of exhaled CO in healthy subjects and patients with diabetes (left, A). Correlation between exhaled CO concentration and blood glucose level in patients with diabetes (right, B).
of exhaled CO (from 3.0 6 0.5 to 6.3 6 1.0 ppm; p , 0.05). Both parameters returned to the baseline 40 min after glucose administration (Fig 2). Discussion Exhaled CO levels are elevated in patients with diabetes, correlate with blood glucose levels and duration of the disease, and may reflect the amount of HO-1 induction caused by increased oxidative stress. In diabetes, a consequence of hyperglycemia is the glycation of both circulating proteins and the immobilized proteins within the vascular wall. When proteins or lipids are exposed to aldose sugars, they undergo nonenzymatic glycation and oxidation to form advanced glycation end products (AGEs),1,15 From this reaction, reversible products such as
Figure 2. Changes in exhaled CO and blood glucose levels after OGTT in healthy subjects.
HbA1c are formed, and complex molecular rearrangements result in the irreversible formation of the AGEs. AGEs can produce reactive oxygen intermediates1,16 by interacting with their surface binding sites and generating malondialdehyde determinants in the vessel wall and thiobarbituric acid-reactive substances in the tissues, and by inducing the nuclear factor-kB.1,17 The plasma and tissues of diabetic patients show increased evidence of oxidative stress,16 which plays a role in the development of diabetic complications.16,18 Increased oxidative stress induced by AGE activity and autoxidation associated with compromised mechanisms designed to scavenge free radicals19 is a common pathway for the pathogenesis of complications in diabetes.16 HO plays a role in the protective response to oxidative stress. The activation of HO-1 in response to AGEs induced by oxidative stress1 may explain the finding of elevated exhaled CO levels in patients with diabetes.2 Competitive inhibitors of HO such as Sn-mesoporphyrin and zinc protoporphyrin effectively reduce hyperbilirubinemia20 and CO production in newborns,21 but their effects have not been evaluated in adults. HO activators in asthmatic patients have been investigated in a previous study where the inhalation of hemin was shown to increase the levels of exhaled CO, whereas such levels were reduced in steroid-treated patients.22 Furthermore, in the same study we showed that the high levels of exhaled CO found in asthmatic patients were associated with an CHEST / 116 / 4 / OCTOBER, 1999
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increased expression of HO-1. Taken together, these data indicate that the exhaled CO concentration may reflect HO activity. We found a correlation between exhaled CO concentration and blood glucose level. The level of CO was also increased by acute elevations of blood glucose level after OGTT. This may be explained by the activation of HO by glucose23 and the positive modulation of CO on insulin secretion,24 whereby acute CO level increases may be part of a counterregulatory mechanism activated in response to changes in glucose levels. High CO levels during OGTT may also be a reflection of HO activation in response to the induction of the lipid peroxidation cascade as proved by elevated levels 8-isoprostane in the plasma of patients with diabetes and reduction of this marker of oxidation after metabolic control.25 Other potential sources of rapidly induced oxidative stress by hyperglycemia include the presence of free radicals generated by autoxidation and nonenzymatic glycation of unsaturated lipids and membrane proteins, and the reduced antioxidant properties of albumin.26 The lack of correlation between levels of exhaled CO and HbA1c may be due to the rapid fluctuations of CO production, as shown in the OGTT study. Furthermore, the concentration of HbA1c is an expression of the average blood glucose levels in the 2 to 3 months before the test and does not reflect the total oxidative burden associated with the duration of the disease. Although epinephrine is known to induce HO activity, 27steroids are the only nonmetalloporphyrin compounds known to inhibit the induction of HO1.28 The effect of different medications on HO activity has not been systematically investigated. Furthermore, the treatment of diabetes by modifying the levels of glucose and the production of AGEs may modulate the level of oxidative stress15 and indirectly change HO-1 activity and CO production.12 Therefore, the effect of treatment on CO levels should also be investigated in future studies. In this study, only five patients had complications of diabetes. Disease severity may be associated with different levels of oxidative stress; therefore, further studies are needed to investigate whether CO levels are influenced by disease severity. Although oxidative stress is now widely accepted as playing an important role in the aging process,29 the levels of exhaled markers of oxidation such as pentane and ethane are similar in healthy children and adults.30 Furthermore, preliminary results indicate that the levels of exhaled CO are not influenced by sex and age in healthy subjects. Therefore, the level of exhaled CO should not be adjusted for these parameters. 1010
In patients with diabetes, exhaled CO levels may indicate the level of oxidative stress in the manner in which the glycation of plasma proteins and hemoglobin reflects glycemic stress. Eventually, these studies may lead to the development of effective strategies for limiting the damage from oxidation of proteins or for complementing other therapeutic approaches to the treatment of complications in diabetes. Further studies are necessary to investigate the possible role of exhaled CO level as a prognostic factor in the development of systemic complications and in monitoring of the disease. References 1 Yan SD, Schmidt AM, Anderson GM, et al. Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem 1993; 269:9889 –9897 2 Nikberg II, Murashko VA, Leonenko IN, et al. Carbon monoxide concentration in the air exhaled by the healthy and the ill. Vrach Delo 1972; 12:112–114 3 Satto Y, Hotta N, Sakamoto N, et al. Lipid peroxide level in plasma of diabetic patients. Biochem Med 1979; 21:104 –107 4 Tamura M, Tanaka A, Yui K, et al. Oxidation of remnant-like particles from serum of diabetic patients, patients with ischemic heart disease and normal subjects. Horm Metab Res 1997; 29:398 – 402 5 Cantoni L, Rossi C, Rizzardini M, et al. Interleukin 1 and tumor necrosis factor induce hepatic heme oxygenase feedback regulation by glucocorticoids. Biochem J 1991; 279:891– 894 6 Lavrovsky Y, Drummond GS, Abraham NG. Downregulation of the human heme oxygenase gene by glucocorticoids and identification of 56b regulatory elements. Biochem Biophys Res Commun 1996; 218:759 –765 7 Kim YM, Bergonia HA, Muller C, et al. Loss of degradation of enzyme-bound heme induced by cellular nitric oxide synthesis. J Biol Chem 1995; 270:5710 –5713 8 Durante W, Kroll MH, Christodoulides N, et al. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res 1997; 80:557–564 9 Otterbein L, Sylvester SL, Choi A. Hemoglobin provides protection against lethal endotoxemia in rats: the role of heme oxigenase-1. Am J Respir Cell Mol Biol 1995; 13:595– 601 10 Applegate LA, Luscher P, Tyrrel RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res 1991; 51:974 –978 11 Nath KA, Balla G, Vercellotti GM, et al. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest 1992; 90:267–270 12 Augustine MK, Choi K, Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 1996; 15:9 –19 13 Henningsson R, Alm P, Elkstrom P, et al. Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release; a biochemical, immunohistochemical, and confocal microscopic study. Diabetes 1999; 48:66 –76 14 Devreese K, Leroux, RJ. Laboratory assessment of five glucose meters designed for self-monitoring of blood glucose concentration. Eur J Clin Chem Clin Biochem 1993; 31:829 – 837 Clinical Investigations
15 Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetes complications. N Engl J Med 1988; 318:1315–1321 16 Baynes JW. Role of oxidative stress in the development of complications in diabetes. Diabetes 1991; 40:405– 412 17 Hunt JV, Smith CC, Wolff SP. Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes 1990; 39:1420 –1424 18 Schmidt AM, Weidman E, Lalla EB et al. Advanced glycation endproducts (AGEs) induce oxidant stress in the gingiva: a potential mechanism underlying accelerated periodontal disease associated with diabetes. J Periodont Res 1996; 31:508 – 515 19 Godin D, Wohajeb S, Garnett M, et al. Antioxidant enzyme alterations in experimental clinical diabetes. Mol Cell Biochem 1988; 84:223–231 20 Martinez JC, Garcia HO, Otheguy LE, et al. Control of severe hyperbilirubinemia in full-term newborns with the inhibitor of bilirubin production Sn-mesoporphyrin. Pediatrics 1999; 103:1–5 21 Vreman HJ, Rodgers PA, Stevenson DK. Zinc protoporphyrin administration for suppression of increased bilirubin production by iatrogenic hemolysis in rhesus neonates. J Pediatr 1990; 117:292–297 22 Horvath I, Donelly LE, Paredi P, et al. Elevated levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of chronic inflammation. Thorax 1998; 53:668 – 672
23 Henningsson R, Alm P, Elkstrom P, et al. Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release; a biochemical, immunohistochemical, and confocal microscopic study. Diabetes 1999; 48:66 –76 24 Henningsson R, Alm P, Lundquist I. Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets. Am J Physiol 1997; 273(suppl 2 pt 1):C703–C709 25 Davı` G, Ciabattoni G, Consoli A, et al. In vivo formation of 8-iso-prostaglandin F2 alpha and platelet activation in diabetes mellitus: effect of improved metabolic control. Circulation 1999; 99:224 –229 26 Bourdon E, Loreau N, Blache D. Glucose and free radicals impair the antioxidant properties of serum albumin. FASEB J 1999; 13:233–244 27 Bakken AF, Thaler MM, Schmid R. Metabolic regulation of heme catabolism and bilirubin production: hormonal control of hepatic heme oxygenase activity. J Clin Invest 1972; 51:530 –536 28 Lutton JD, da Silva J-L, Moqattash S, et al. Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3B) by environmental agents. J Cell Biochem 1992; 49:259 –265 29 Knight JA. Free radicals: their history and current status in aging and diseased. Clin Lab Sci 1999; 28:331–346 30 Kneepkens CM, Lepage G, Roy CC. The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Radic Biol Med 1994; 17:127–160
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