Urinary ketone is associated with the heart failure severity

Clinical Biochemistry 45 (2012) 1697–1699

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Urinary ketone is associated with the heart failure severity Ji Hyung Chung a, Jin-Sup Kim b, Oh Yoen Kim c, Seok-Min Kang a, Geum-Sook Hwang b,⁎, Min-Jeong Shin d,⁎⁎ a

Cardiovascular Product Evaluation Center, Cardiovascular Research Institute, Yonsei University Health System, Seoul, Republic of Korea Korea Basic Science Institute, Seoul and Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea Department of Food Science and Nutrition, Dong-A University, Busan, Republic of Korea d Department of Food and Nutrition, Korea University, Seoul, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 2 April 2012 Received in revised form 6 August 2012 Accepted 11 August 2012 Available online 24 August 2012 Keywords: Heart failure Urine Ketones Ejection fraction Echocardiography

a b s t r a c t Objectives: To test the hypothesis that urinary metabolites characterizing heart failure (HF) are associated with the magnitude of echocardiographic measurements and ultimately the severity of HF. Design/methods: Patients with systolic HF (n = 46) and control subjects (n = 32) participated in this study. Patients with type 2 diabetes mellitus were excluded. Echocardiographic measurements were performed, and selected urinary metabolites were quantified. Results: Urinary levels of acetate (pb 0.05), acetone (pb 0.01), cytosine (pb 0.001), methylmalonate (pb 0.001), and phenylacetylglycine (pb 0.01) were significantly higher, while 1-methylnicotinamide (pb 0.05) were significantly lower in HF patients than in controls. There were significant differences in E/E′ (pb 0.05), urinary levels of acetate (pb 0.005), acetoacetate (pb 0.05), acetone (pb 0.05) and ketones (pb 0.01) according to the New York Heart Association (NYHA) classification in HF patients. Multiple linear regression analysis revealed that urinary ketones were found to be independent factors for both left ventricular ejection fraction and E/E′ after adjusting for confounders. Conclusion: Our results showed that urinary levels of ketone bodies are associated with the magnitude of echocardiographic parameters. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction As one of major worldwide health burdens, the prevalence of heart failure (HF) is on the rise [1]. In an effort to reduce the morbidity and mortality of HF, there have been attempts to identify novel biomarkers for HF to provide a reliable, noninvasive method to confirm and to estimate the severity of HF [2], including N-terminal-pro-B-type natriuretic peptide, C-reactive protein, and red blood cell distribution width [3]. We previously investigated metabolic pathways, characteristics of human HF using 1NMR-based urinary metabolomic analysis in conjunction with multivariate statistics [4]. The results show that urinary 6 metabolites [1-methylnicotinamide (1-MNA amide), acetate, acetone, cytosine, methylmalonate and phenylacetylglycine (PAG)] were significantly different between the control and HF patient groups. To further substantiate the previous findings [4], we aimed

to examine whether urinary 6 metabolites along with acetoacetate are associated with echocardiographic parameters and to determine whether selected metabolites can predict the severity of HF. Materials and methods Subjects The present study was an extension of a previous study that characterized the urinary metabolites specific to HF patients [4]. This study consisted of HF patients (n = 46, 68.6 yrs, 26 males) and age, gender-matched healthy controls (n = 32, 66.6 yrs, 15 males). Venous blood and urine samples were collected for further analysis after a fasting period. Echocardiographic measurement

Abbreviations: E/E′, early mitral inflow velocity to early diastolic mitral annular velocity ratio; HF, heart failure; LVEF, left ventricular ejection fraction; NMR, nuclear magnetic resonance; NYHA, New York Heart Association; MNM amide, methylnicotinamide; MMA, methylmalonate; PAG, phenylacetylglycine. ⁎ Corresponding author. Fax: +82 2 920 0779. ⁎⁎ Corresponding author at: Department of Food and Nutrition, College of Health Science, Korea University, Seoul, Republic of Korea. Fax: +82 2 940 2850. E-mail addresses: [email protected] (G.-S. Hwang), [email protected] (M.-J. Shin).

Standard two-dimensional echocardiography was performed in all patients. Left ventricular ejection fraction (LVEF) was calculated by the modified Quinones method. Peak velocity of early diastolic filling (E) was obtained from the mitral inflow velocities by pulsed wave Doppler at the apical four-chamber view. Peak early diastolic velocity of mitral annulus (E′) was obtained from the tissue Doppler imaging of the septal mitral annulus.

0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.08.013

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J.H. Chung et al. / Clinical Biochemistry 45 (2012) 1697–1699

Quantification of urinary metabolites All NMR spectra were phased and baseline corrected, and the selected metabolites were identified through the use of Chenomx NMR suite version 7.1 (Chenomx Inc., Edmonton, AB, Canada). The ambiguous peaks due to overlaps were confirmed using 2D NMR experiments and spiking experiments. Metabolite concentrations were determined using the 600-MHz library from Chenomx NMR Suite 7.1, which compares the integral of a known reference signal (DSS-d6) with signals derived from a library of compounds containing chemical shifts and peak multiplicities. The library is based on a database of individual metabolite spectra acquired using the NOESYPRESAT sequence and contained 302 metabolites [5]. Each urinary metabolite concentration was normalized to creatinine levels (μM/mM creatinine) in each urine sample.

renal failure were 56.5%, 47.0% and 13.0%, respectively (Supplementary Table 1). Comparisons of urinary metabolites Consistent with the previous findings [4], creatinine-corrected levels of acetate, acetone, ketones as sum of acetoacetate and acetone, cytosine, methylmalonate, and PAG in urine were significantly higher, while levels of 1-MNM amide were significantly lower in HF patients, compared to those in healthy controls (Supplementary Table 2). On the other hand, creatinine-corrected levels of acetoacetate in urine were similar between the groups (8.9 ± 0.9 vs. 12.2 ± 1.6 μM/mM creatinine, N.S.). Comparisons of urinary metabolites according to severity of HF

Statistical analysis Statistical analysis was performed using Window SPSS (ver12.0, Statistical Package for the Social Science, SPSS Ins., Chicago, IL). The Kolmogorov–Smirnov test was used to test the normality of distribution, and skewed variables were logarithmically transformed for statistical analysis. Differences in continuous variables between the groups were tested by independent t-test (student t-test), and one-way analysis of variance (ANOVA) followed by Bonferroni corrections for post-hoc test. Non-continuous variables were tested by the chi-square test. Pearson correlation coefficient and stepwise multiple regression analysis were used to evaluate the associations between variables. Results are expressed as mean± S.E or %. A two-tailed value of P b 0.05 was considered statistically significant.

Study subjects were classified by the New York Heart Association (NYHA I: n = 29, 68.3 ± 1.6 yrs, males 20; NYHA II: n = 8, 68.7 ± 1.2 yrs, males 3; NYHA III: n = 9, 71.2 ± 2.4 yrs, males 3). Age, BMI, use of medications and co-morbidity were similar among the groups. The ratio of transmitral Doppler early filling velocity to tissue Doppler early diastolic mitral annular velocity (E/E′) in NYHA III patients were significantly higher than those in NYHA I patients (NYHA I: 12.8 ± 0.9, NYHA II: 18.7 ± 3.5, NYHA III: 18.9 ± 3.8, p b 0.05). Urinary levels of acetate, acetoacetate, acetone and ketones as sum of acetoacetate and acetone in NYHA III patients were significantly higher than those in NYHA I patients (Fig. 1). Associations between echocardiographic parameters and urinary metabolites

Results The mean LVEF was 35.1 ± 1.7% in all HF patients. The proportions of HF patients with coronary artery disease, hypertension, and chronic

LVEF was negatively correlated with urinary levels of acetone (r = − 0.425, p b 0.005), acetoacetate (r = − 0.307, p b 0.05) and ketones (r = − 0.383, p b 0.05). On the other hand, E/E′ was positively

Fig. 1. Comparisons of quantified metabolites in HF patients according to the NYHA classification. Data are given as means ± SE. Tested by analysis of variance (ANOVA) with Bonferroni correction for post-hoc test. Sharing the same alphabet indicates no significant difference among the groups. Urinary metabolite concentrations were normalized to creatinine (μM/mM creatinine) levels, and ketones were defined as sum of acetoacetate and acetone.

J.H. Chung et al. / Clinical Biochemistry 45 (2012) 1697–1699 Table 1 Stepwise multiple regression analysis to identify factors influencing LVEF and E/E′. Dependent variable

Independent variable

Adjusted β-coefficients

p-value

R2

p-value

LVEF

BMI Ketones Ketones Uric acid

0.381 0.361 0.357 0.399

b0.0 b0.05 b0.05 b0.01

0.303

b0.001

0.450

b0.001

E/E′

Independent variables are age, BMI, gender, uric acid, coexisting conditions and medication. BMI: body mass index; E/E′: early mitral inflow velocity to early diastolic mitral annular velocity ratio; LVEF: left ventricular ejection fraction.

associated with urinary levels of acetate (r=0.458, pb 0.01), acetoacetate (r=0.388, pb 0.05), acetone (r=0.383, pb 0.05), ketones (r=0.456, pb 0.005) and serum levels of uric acid (r=0.412, pb 0.05). Stepwise multiple regression analysis revealed that urinary ketones were independent contributing factors for both LVEF (adjusted β-coefficient=0.361, pb 0.05) and E/E′ (adjusted β-coefficient=0.357, pb 0.05) after adjusting for age, BMI, gender, associated biochemical measures and urinary metabolites, medications, and coexisting condition (Table 1). Discussion The main objective of this study was to examine whether metabolic changes are associated with the echocardiographic parameters and to determine whether selected metabolites can predict the severity of HF. Among the selected urinary metabolites, significantly higher urinary levels of acetate and ketones (acetoacetate + acetone) were present with increasing HF severity as measured by the NYHA classification. A few clinical studies [6,7] reported that increases in ketone bodies in the blood and breath were observed in HF patients. Our study is distinct from the earlier studies in that the results added novel knowledge on the association between urinary ketones and cardiac function evaluated by echocardiography. The mechanism explaining the elevation of ketone bodies in HF patients has not been clear, however, increased free fatty acid (FFA) in the failing heart has been speculated to be involved in this process. Usually, the plasma concentrations of ketone bodies are very low, however, the levels of ketone body can increase significantly under starvation, uncontrolled diabetes and HF [7,8]. Furthermore, high levels of circulating stress hormones elevated in HF are able to stimulate lipolysis, leading to increase circulating FFA [9] which is known to be both the principal substrate for ketogenesis and the major stimulant for this process to occur [10]. Taken together, enhanced FFA due to either increased stress hormones or diabetic in HF may trigger the stimulation of ketogenesis. On the other hand, alterations in the substrate metabolism observed in HF contribute to contractile dysfunction and the progression of left ventricular (LV) remodeling that are characteristic of HF [11]. That is, perturbations in myocardial energy metabolism such as mitochondrial dysfunction and a reduction in the fatty acid oxidation rate accompanied by enhanced ketogenesis in HF may contribute to myocardial damage and exacerbate the progression of HF [12]. Recently, it was reported that exhaled breath acetone levels were significantly different as a function of severity of HF determined by NYHA classification [13], suggesting as a promising non-invasive diagnostic method of HF. Our study shows the association of urinary ketones with the HF severity

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determined by echocardiographic parameters such as LVEF and E/E′ as well as NYHA classification, substantiating the importance of ketones in the development and progression of HF. Considering elevated E/E′ was an independent predictor in systolic heart failure patients [14], our result showing urinary ketones as major determinants for both LVEF and E/E′ indicates that the degree of urinary ketone may reflect HF severity. The main limitation of this study includes a small sample size. However, we showed for the first time that urinary levels of ketone bodies are associated with the magnitude of echocardiographic parameters and suggested ketone levels as a surrogate marker of HF severity. While the relevance of urinary ketones in clinical practice for determining the severity of HF still remains unclear, our results raise the possibility that urinary ketones might reflect the progression of HF. Conflict of interest None. Acknowledgment This research was supported by the Korea Basic Science Institute (T32409) and the Creative Allied Project (CAP) grant funded by the Korea Research Council of Fundamental Science and Technology (KRCF). Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.clinbiochem.2012.08.013. References [1] Bui AL, Horwich TB, Fonarow GC. Epidemiology and risk profile of heart failure. Nat Rev Cardiol 2011;8(1):30-41. [2] Braunwald E. Biomarkers in heart failure. N Engl J Med 2008;358(20):2148-59. [3] van Kimmenade RR, Januzzi Jr JL. Emerging biomarkers in heart failure. Clin Chem 2012;58(1):1127-38. [4] Kang SM, Park JC, Shin MJ, Lee H, Oh J, Ryu do H, et al. 1H nuclear magnetic resonance based metabolic urinary profiling of patients with ischemic heart failure. Clin Biochem 2011;44(4):293-9. [5] Weljie AM, Newton J, Mercier P, Carlson E, Slupsky CM. Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Anal Chem 2006;78(13):4430-42. [6] Kupari M, Lommi J, Ventila M, Ventilä M, Karjalainen U. Breath acetone in congestive heart failure. Am J Cardiol 1995;76(14):1076-8. [7] Lommi J, Kupari M, Koskinen P, Näveri H, Leinonen H, Pulkki K, et al. Blood ketone bodies in congestive heart failure. J Am Coll Cardiol 1996;28(3):665-72. [8] Lopes-Cardozo M, Mulder I, van Vugt F, Hermans PG, van den Bergh SG, Klazinga W, et al. Aspects of ketogenesis: control and mechanism of ketone-body formation in isolated rat-liver mitochondria. Mol Cell Biochem 1975;9(3):155-73. [9] Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, et al. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med 1984;311(13):819-23. [10] Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev 1999;15(6):412-26. [11] Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 2005;85(3):1093-129. [12] Lommi J, Kupari M, Yki-Jarvinen H. Free fatty acid kinetics and oxidation in congestive heart failure. Am J Cardiol 1998;81(1):45-50. [13] Marcondes-Braga FG, Gutz IG, Batista GL, Saldiva PH, Ayub-Ferreira SM, Issa VS, et al. Exhaled acetone as a new biomarker of heart failure severity. Chest 2012;142(2): 457–4466. [14] Sharp AS, Tapp RJ, Thom SA, et al, Francis DP, Hughes AD, Stanton AV, Tissue Doppler E/E′ ratio is a powerful predictor of primary cardiac events in a hypertensive population: an ASCOT substudy. Eur Heart J 2010;31(6):747-52.