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Beaudet AL, Sly WS, Valle D, eds. Metabolic and molecular bases of inherited disease, 7th ed. New York: McGraw-Hill, 1995:791– 801. Tanaka K, Orr JC, Isselbacher KJ. The identification of -hydroxyisovaleric acid in the urine of a patient with isovaleric acidemia. Biochem Biophys Acta 1968;152:638 – 41. Tanaka K, Ikeda Y, Matsubara Y, Hyman D. Molecular basis of isovaleric acidemia in the study of the acyl-CoA dehydrogenase family. Adv Neurol 1988;48:107–31. Truscott RJ, Malegan D, McCairns E, Burke D, Hick L, Sims P, et al. New metabolites in isovaleric acidemia. Clin Chim Acta 1981;110:187–203. Hine DG, Tanaka K. The identification and the excretion pattern of isovaleryl glucoronide in the urine of patients with isovaleric acidemia. Pediatr Res 1984;18:508 –13. Lehnert W. Excretion of N-isovalerylglutamic acid in isovaleric acidemia. Clin Chim Acta 1981;116:249 –53. Lehnert W. N-Isovalerylalanine and N-isovalerylsarcosine: two new metabolites in isovaleric acidemia. Clin Chim Acta 1983;134:207–12. Millington DS, Roe CR, Maltby DA, Inoue F. Endogenous catabolism is the major source of toxic metabolites in isovaleric acidemia. J Pediatr 1987; 110:56 – 60. Lehnert W. 3-Hydroxyisoheptanoic acid: a new metabolite in isovaleric acidemia. Clin Chim Acta 1981;113:101–3. Chalmers RA, Lawson AM. Organic acids in man: analytical chemistry, biochemistry and diagnosis of the organic acidurias. New York: Chapman and Hall, 1982:523pp. Duran M, Bruinvis L, Ketting D, Kamerling JP, Wadman SK, Schutgens RBH. The identification of E-2-methylgutaconic acid, a new isoleucine metabolite, in the urine of patients with -ketothiolase deficiency, propionic acidemia and methylmalonic acidemia. Biomed Mass Spectrom 1982;9:1–5. Hagenfeldt L, Naglo AS. New conjugated urinary metabolites in intermediatetype maple syrup urine disease. Clin Chim Acta 1987;169:77– 83. Liebich HM, Fo¨rst C. Urinary excretion of N-acetylamino acids. J Chromatogr 1985;338:187–91. Loots DT, Mienie LJ, Bergh JJ, van der Schyf CJ. Acetyl-L-carnitine prevents total body hydroxyl free radical and uric acid production induced by 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the rat. Life Sci 2004;75: 1243–53. Van der Westhuizen FH, Pretorius PJ, Erasmus E. The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase. J Biochem Mol Toxicol 2000;14:102–9. DOI: 10.1373/clinchem.2005.048421
Relationship of Plasma Homocysteine with the Severity of Chronic Heart Failure, Markus Herrmann,1 Ingrid Kindermann,2 Stephanie Mu¨ller,1 Thomas Georg,3 Michael Kindermann,2 Michael Bo¨hm,2 and Wolfgang Herrmann1* (1 Abteilung fu¨r Klinische Chemie und Laboratoriumsmedizin/ Zentrallabor, 2Klinik fu¨r Innere Medizin III, and 3 Institut fu¨r Medizinische Biometrie, Epidemiologie und Medizinische Informatik, Universita¨tsklinikum des Saarland, Homburg/Saar, Germany; * address correspondence to this author at: Abteilung fu¨r Klinische Chemie und Laboratoriumsmedizin/Zentrallabor, Universita¨tsklinikum des Saarland, D-66421 Homburg/Saar, Germany; fax 49-68411630703, e-mail
[email protected]) Chronic heart failure (CHF) is a major public health problem causing considerable morbidity and mortality (1–3 ). Prevention of CHF by identifying risk factors is therefore a major issue. Previous studies found that hypertension, smoking, diabetes mellitus, obesity, and advancing age are the most important risk factors for CHF (4 ). Recently, plasma homocysteine (Hcy) has been suggested as a newly recognized risk factor (5, 6 ). However, there are no data regarding the association between Hcy
and various objective as well as subjective measures of CHF. The demonstration of such relationships would help to clarify the role of hyperhomocysteinemia in CHF. We hypothesized that plasma Hcy is associated with clinical and echocardiographic signs of CHF as well as with N-terminal pro-brain natriuretic peptide (NT-proBNP), suggesting a relationship between Hcy and the severity of CHF. Accordingly, we investigated the relationships of plasma Hcy with serum NT-proBNP and clinical and echocardiographic indices of CHF in patients and in controls. For this study, 95 patients with systolic CHF and 12 healthy persons without cardiac diseases were interviewed and examined by the same 2 experienced cardiologists, who were blinded to the study. All participants had a medical history, physical examination, venous blood sampling, 6-min walking test (6-MWT), electrocardiography, and echocardiography. Eighty-two patients underwent a cardiac catheterization according to the American Heart Association guidelines (7 ). Additionally, 37 patients performed a symptom-limited bicycle exercise test (Ergoline cardio-systems) with gas-exchange analysis (MedGraphics CPX/D spiroergometry system; Medical Graphics Corporation) to determine maximum oxygen uptake (Vo2max). Informed consent was obtained from all participants, and the study protocol was approved by the Institutional Review Board. Nonfasting venous blood samples (plasma and serum) were drawn during the office visits and centrifuged within 45 min. Total Hcy was measured in EDTA-plasma by an HPLC application (Immundiagnostik) according to the method of Araki and Sato (8 ). Inter- and intraassay CVs were ⬍5.1%. Serum NT-proBNP and cardiac troponin T were measured with commercial chemiluminescence assays on an Elecsys 2010 analyzer (Roche Diagnostics). Intra- and interassay CVs were ⬍2.7% and ⬍3.2%, respectively, at concentrations ⱖ175 ng/L. Anthropometric data are provided as the mean (SD) and were compared by a Student t-test. Because Hcy and NT-proBNP were not normally distributed, we performed a logarithmic transformation before further data exploration. We then performed a Pearson correlation analysis. Hcy and NT-proBNP are influenced by renal function and age; we therefore also calculated a partial correlation controlling for age and creatinine. Calculations were done with the software package SPSS 11.0 (SPSS Inc.). Most of the patients investigated were classified as New York Heart Association (NYHA) classes II (n ⫽ 27) and III (n ⫽ 39; Table 1). The mean age increased (P ⫽ 0.03) with increasing NYHA class [controls, 44 (10) years; NYHA class I, 51 (16) years; NYHA class II, 53 (11) years; NYHA class III, 55 (12) years; NYHA class IV, 61 (15) years]. Weight and height did not differ among the NYHA classes. As expected, physical performance decreased with increasing NYHA classes. The 6-MWT decreased from 530 (70) m in controls to 26 (82) m in NYHA class IV patients (P ⬍0.001). Additionally, Vo2max decreased from 25 (13) mL 䡠 min⫺1 䡠 (kg body weight)⫺1 in NYHA class I patients to 16 (4) mL 䡠 min⫺1 䡠 (kg body weight)⫺1 in
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NYHA class III patients (P ⫽ 0.026). Because symptoms were present at rest, NYHA class IV patients were excluded from spiroergometry. Serum Hcy was lowest in controls and increased with increasing NYHA class (P ⫽ 0.002; Fig. 1A). Median Hcy concentrations in NYHA class II–IV patients were ⱖ12 mol/L, which represents the recommended cutoff of the German, Austrian, and Swiss consensus conference (9 ). Moreover, we observed significant negative correlations between serum Hcy and the 6-MWT (r ⫽ ⫺0.266; P ⫽ 0.014) and Vo2max (r ⫽ ⫺0.528; P ⬍0.001). In addition to the associations of Hcy with clinical measures, we also observed correlations between serum Hcy and echocardiographic measures of CHF (Fig. 1, C
Table 1. Characteristics of patients and controls. Patients (n ⴝ 95)
Anthropometric data Mean (SD) age, years Mean (SD) weight, kg Mean (SD) height, cm M/F, n NYHA classification, n I II III IV Mean (SD) blood pressure, mmHg RRsysb RRdia Echocardiography/Catheterization EF, % FS, % LVDD, mm Mean (SD) laboratory values Hcy, mol/L NT-proBNP, ng/L Troponin T, g/L Creatinine, mg/L Cardiac risk factors, % Current/previous/nonsmoker Hypertension Coronary artery disease Myocardial infarction Stroke Diabetes mellitus Medication, % Beta blockers Diuretics ACE inhibitors Digoxin/digitoxin Antiarrhythmics Aspirin Marcumar
54 (13) 84 (20) 173 (9) 74/21
Controls (n ⴝ 18)
44 (10)a 76 (11) 177 (10) 12/6
16 27 39 13
0 0 0 0
75 (13) 120 (20)
77 (5) 121 (9)
38 (20) 19 (9) 64 (10)
67 (8)a 37 (6)a 50 (4)a
17.1 (20) 2630 (4290) 0.013 (0.009) 11 (3)
9.6 (2.6)a 38 (38)a ⬍0.01 (0.00) 9.4 (1.2)
25/54/21 62 35 17 5 15
13/7/80 0 0 0 0 0
88 51 81 14 18 25 32
0 0 0 0 0 0 0
P ⬍0.01 vs patients. RRsys, Riva-Rocci systolic; RRdia, Riva-Rocci diastolic; EF, ejection fraction; FS, fractional shortening; ACE, angiotensin I-converting enzyme. a b
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and D). As serum Hcy increased, left ventricular end diastolic diameter (LVDD) increased and ejection fraction decreased. Partial correlation analysis controlling for age and creatinine confirmed the relationship between Hcy and LVDD (P ⫽ 0.041). NT-proBNP increased with increasing NYHA class (P ⬍0.001). Correlation analysis revealed a significant relationship between Hcy and NT-proBNP (Fig. 1B). Partial correlation analysis controlling for age and creatinine confirmed this association (P ⫽ 0.002). The main finding of this study is a consistent association of plasma Hcy with clinical and echocardiographic measures of CHF as well as with NT-proBNP, indicating a relationship between Hcy and the severity of CHF. Although NYHA classification, 6-MWT, and Vo2max describe different domains of clinical status and each of these tests has its limitations (10 –18 ), the overall results suggest a relevant association between Hcy and clinical status in CHF patients. Our data show that this relationship can be observed even at moderately increased Hcy concentrations. Only controls and NYHA class I patients had a median Hcy below the 12 mol/L cutoff (9 ). These results are supported by the study by Vasan et al. (5 ), who reported an increased incidence of CHF in individuals with Hcy concentrations ⱖ11–12 mol/L. Both studies suggest that an Hcy concentration of 12 mol/L is a reliable cutoff for an increased risk of CHF. NT-proBNP is known to have a high negative predictive value (19 –21 ) and might be useful for differential diagnosis of dyspnea in CHF and pulmonary diseases (22, 23 ). In the persons investigated, we found a positive association of Hcy and NT-proBNP. However, Hcy and NT-proBNP are influenced by age and renal function (9, 24, 25 ). We therefore performed a partial correlation analysis controlling for age and creatinine. This analysis confirmed the relationship between Hcy and NT-proBNP. Sex and body mass index are other potential confounders of NT-proBNP. However, because of the limited number of women among our participants, separate statistics for men and women were not appropriate. Additionally, we also observed a relationship between Hcy and LVDD, which remained significant after correction for age and creatinine. Animal studies support the existence of morphologic changes in the presence of increased Hcy. Joseph and coworkers observed ventricular hypertrophy (26 ) and adverse cardiac remodeling (27, 28 ) in the presence of high Hcy concentrations. Because Hcy depends strongly from folate, vitamin B6, and vitamin B12 status, it would be of particular interest to consider these data in our statistics. Unfortunately, these data were not available for our patients. Therefore, future studies are needed to clarify the role of these vitamins in CHF. Our data and the results from animal studies (26 –29 ) suggest adverse effects of Hcy on the myocardium mediated by 2 potential mechanisms: direct actions of Hcy on cardiac myocytes or Hcy-derived vascular damage leading to reduced perfusion. Direct effects of Hcy on cardiac myocytes could be attributable to the diminished Hcy degradation capacity of these cells (30 ) and the oxidative
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Technical Briefs
Fig. 1. Relationship between Hcy concentrations and other indices of CHF. (A), boxplot (25th–75th percentiles) showing the relationship between Hcy and NYHA classification. Co, controls. (B), Pearson correlation analysis of log(Hcy) and log(NT-proBNP). (C), Pearson correlation analysis of log(Hcy) and LVDD. (D), Pearson correlation analysis of log(Hcy) and ejection fraction (EF).
capacity of Hcy (31, 32 ). The vascular hypothesis is extended by data from Kennedy et al. (29 ), who reported a negative inotropic effect of Hcy that is mediated by a “coronary endothelium-derived agent”. However, more data are needed to understand the mechanistic role of Hcy in CHF. The clinical impact of our results is not fully clear at the moment. Further studies are needed to analyze whether increased Hcy concentrations in CHF patients are associated with faster progression of the disease and a worse clinical outcome. If Hcy is associated with outcome in CHF, Hcy-lowering therapy with folate, vitamin B12, and vitamin B6 supplementation might help to reduce progression of the disease and improve the clinical outcome. Because Hcy-lowering therapy has almost no side effects,
it might be a promising basic therapy comparable to anticoagulants in thrombotic diseases. Data from intervention trials are not currently available, however; therefore, an evidence-based recommendation to supplement B vitamins in CHF patients is not justified at the moment.
Financial support was provided by the “CompetenceNetwork Heart Failure” of the National Ministry of Education and Research. References 1. O’Connell JB, Bristow MR. Economic impact of heart failure in the United States: time for a different approach. J Heart Lung Transplant 1994;13: 107–12.
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2. Kannel WB, Belanger AJ. Epidemiology of heart failure. Am Heart J 1991; 121:951–7. 3. Kannel WB. Epidemiology and prevention of cardiac failure: Framingham Study insights. Eur Heart J 1987;8:23– 6. 4. Kenchaiah S, Narula J, Vasan RS. Risk factors for heart failure. Med Clin North Am 2004;88:1145–72. 5. Vasan RS, Beiser A, D’Agostino RB, Levy D, Selhub J, Jacques PF, et al. Plasma homocysteine and risk for congestive heart failure in adults without prior myocardial infarction. JAMA 2003;289:1251–7. 6. Ventura P, Panini R, Verlato C, Scarpetta G, Salvioli G. Hyperhomocysteinemia and related factors in 600 hospitalized elderly subjects. Metabolism 2001;50:1466 –71. 7. Eagle KA, Guyton RA, Davidoff R, Ewy GA, Fonger J, Gardner TJ, et al. ACC/AHA guidelines for coronary artery bypass graft surgery: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines (Committee to Revise the 1991 Guidelines for Coronary Artery Bypass Graft Surgery). American College of Cardiology/American Heart Association. J Am Coll Cardiol 1999;34:1262–347. 8. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1987;27:43–52. 9. Stanger O, Herrmann W, Pietrzik K, Fowler B, Geisel J, Dierkes J, et al. DACH-LIGA homocysteine (German, Austrian and Swiss Homocysteine Society): consensus paper on the rational clinical use of homocysteine, folic acid and B-vitamins in cardiovascular and thrombotic diseases: guidelines and recommendations. Clin Chem Lab 2003;41:1392– 403. 10. Remme WJ, Swedberg K, Task Force for the Diagnosis and Treatment of Chronic Heart Failure, European Society of Cardiology. Guidelines for the diagnosis and treatment of chronic heart failure. Eur Heart J 2001;22: 1527– 60. 11. Willenheimer R, Erhardt LR. Value of 6-min-walk test for assessment of severity and prognosis of heart failure. Lancet 2000;355:515– 6. 12. Itoh H, Taniguchi K, Koike A, Doi M. Evaluation of severity of heart failure using ventilatory gas analysis. Circulation 1990;81:II31–7. 13. Working Group on Cardiac Rehabilitation & Exercise Physiology and Working Group on Heart Failure of the European Society of Cardiology. Recommendations for exercise testing in chronic heart failure patients. Eur Heart J 2001;22:37– 45. 14. Rostagno C, Olivo G, Comeglio M, Boddi V, Banchelli M, Galanti G, et al. Prognostic value of 6-minute walk corridor test in patients with mild to moderate heart failure: comparison with other methods of functional evaluation. Eur J Heart Fail 2003;5:247–52. 15. Wieczorek SJ, Hager D, Barry MB, Kearney L, Ferrier A, Wu AH. Correlation of B-type natriuretic peptide level to 6-min walk test performance in patients with left ventricular systolic dysfunction. Clin Chim Acta 2003;328:87–90. 16. Opasich C, Pinna GD, Mazza A, Febo O, Riccardi R, Riccardi PG, et al. Six-minute walking performance in patients with moderate-to-severe heart failure; is it a useful indicator in clinical practice? Eur Heart J 2001;22:488 – 96. 17. Cahalin LP, Mathier MA, Semigran MJ, Dec GW, DiSalvo TG. The six-minute walk test predicts peak oxygen uptake and survival in patients with advanced heart failure. Chest 1996;110:325–32. 18. Lucas C, Stevenson LW, Johnson W, Hartley H, Hamilton MA, Walden J. The 6-min walk and peak oxygen consumption in advanced heart failure: aerobic capacity and survival. Am Heart J 1999;138:618 –24. 19. Hobbs FD, Davis RC, Roalfe AK, Hare R, Davies MK. Reliability of N-terminal proBNP assay in diagnosis of left ventricular systolic dysfunction within representative and high risk populations. Heart 2004;90:866 –70. 20. Nielsen LS, Svanegaard J, Klitgaard NA, Egeblad H. N-Terminal pro-brain natriuretic peptide for discriminating between cardiac and non-cardiac dyspnoea. Eur J Heart Fail 2004;6:63–70. 21. McDonagh TA, Holmer S, Raymond I, Luchner A, Hildebrant P, Dargie HJ. NT-proBNP and the diagnosis of heart failure: a pooled analysis of three European epidemiological studies. Eur J Heart Fail 2004;6:269 –73. 22. Mueller C, Scholer A, Laule-Kilian K, Martina B, Schindler C, Buser P, et al. Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea. N Engl J Med 2004;350:647–54. 23. Maisel AS. The diagnosis of acute congestive heart failure: role of BNP measurements. Heart Fail Rev 2003;8:327–34. 24. Clerico A, Emdin M. Diagnostic accuracy and prognostic relevance of the measurement of cardiac natriuretic peptides: a review. Clin Chem 2004;50: 33–50. 25. Luchner A, Hengstenberg C, Lowel H, Trawinski J, Baumann M, Riegger GA, et al. N-Terminal pro-brain natriuretic peptide after myocardial infarction: a marker of cardio-renal function. Hypertension 2002;39:99 –104. 26. Joseph J, Joseph L, Shekhawat NS, Devi S, Wang J, Melchert RB, et al. Hyperhomocysteinemia leads to pathological ventricular hypertrophy in normotensive rats. Am J Physiol Heart Circ Physiol 2003;285:679 – 86. 27. Joseph J, Washington A, Joseph L, Kennedy RH. Hyperhomocysteinaemiainduced atrial remodelling in hypertensive rats. Clin Exp Pharmacol Physiol 2004;31:331–7.
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28. Joseph J, Washington A, Joseph L, Koehler L, Fink LM, Hauer-Jensen M, et al. Hyperhomocysteinemia leads to adverse cardiac remodeling in hypertensive rats. Am J Physiol Heart Circ Physiol 2002;283:2567–74. 29. Kennedy RH, Owings R, Shekhawat N, Joseph J. Acute negative inotropic effects of homocysteine are mediated via the endothelium. Am J Physiol Heart Circ Physiol 2004;287:812–7. 30. Chen P, Poddar R, Tipa EV, Dibello PM, Moravec CD, Robinson K, et al. Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease. Adv Enzyme Regul 1999;39:93–109. 31. Weiss N, Heydrick SJ, Postea O, Keller C, Keaney JF Jr, Loscalzo J. Influence of hyperhomocysteinemia on the cellular redox state—impact on homocysteine-induced endothelial dysfunction. Clin Chem Lab Med 2003;41:1455– 61. 32. Loscalzo J. The oxidant stress of hyperhomocyst(e)inemia. J Clin Invest 1996;98:5–7. DOI: 10.1373/clinchem.2005.049841
Evaluation of Oxidative Stress in Serum of Critically Ill Patients by a Commercial Assay and Gas Chromatography–Mass Spectrometry, Giuliana Cighetti,1* Rita Paroni,2 Silvia Marzorati,3 Erica Borotto,3 Riccardo Giudici,3 Gabriele Magnanini,2 and Gaetano Iapichino3 (1 Department of Medical Chemistry, Biochemistry and Biotechnology; 2 Department of Medicine, Surgery and Dental Science; and 3 Institute of Anaesthesia and Intensive Care Medicine, University of Milan, Milan, Italy; * address correspondence to this author at: Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Via Saldini 50, 20133 Milan, Italy; fax 39250316040, e-mail
[email protected]) The formation of reactive oxygen species (ROS), as a result of an imbalance of the oxidant/antioxidant system, and their reactivity toward various molecular targets lead to oxidative damage contributing to different human pathologies (1, 2 ). Nonneutralized ROS trigger the lipid peroxidation process of cell membranes, thus generating hydroperoxides (intermediate compounds) and malondialdehyde (MDA), the most abundant carbonyl-terminal molecule in the circulation. The direct in vivo detection of ROS is difficult because of their very short lifetimes; therefore, changes in hydroperoxide and/or MDA concentrations are often used as an indicator of oxidative stress in clinical laboratory settings (3 ). MDA exists in 2 forms in tissues and blood: free and bound to ⫺SH and/or ⫺NH2 groups of proteins, nucleic acids, and lipoproteins (4 ). Free MDA (F-MDA), the chemically active form, serves as an indicator of recent damage (4, 5 ), and the bound fraction excreted by urine is indicative of an older injury (6 ). The analytical assay generally used to quantify MDA, based on detection of the product from the reaction between MDA and thiobarbituric acid (TBARS), measures only total MDA (T-MDA; free ⫹ bound) (4, 7 ). This method, although criticized for its low specificity, is often used in clinical laboratories despite its complexity. Thus, the commercial availability of a specific, simple, rapid, and low-cost assay to measure oxidative stress by assay-
