Multiple screening of urolithic organic acids with copper nanoparticle-plated electrode: Potential assessment of urolithic risks Chung-Wei Yang a , Jyh-Myng Zen b , Yu-Lin Kao c , Cheng-Teng Hsu b , Tung-Ching Chung a , Chao-Chin Chang d , Chi-Chung Chou a, * a Veterinary Medical Teaching Hospital and Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan b Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan c Department of Urology, Chung-Shan Medical University Hospital, Taichung 402, Taiwan d Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan article info Article history: Received 29 May 2009 Available online 21 August 2009 Keywords: Urolithiasis Creatinine Organic acids Electrochemical Nanoparticle-plated electrode abstract There is yet to be a reliable prediction of urolithiasis. To facilitate early diagnosis, a simple and rapid high performance liquid chromatography method with electrochemical detection using disposable copper- nanoparticle-plated electrodes (Cu n -SPE) was developed for multiple detection of creatinine and 4 uro- lithic organic acids. A total of 206 normal and urolithic human and canine urines and urolith samples were collected for direct analysis of creatinine, cystine, uric acid, oxalic acid, and citric acid without sam- ple cleanup and derivatization processes. Urinary organic acids were separated in 11 min and were devoid of ascorbic acid interference. The detection limits (S/N > 3) were at the nanomolar level with lin- ear dynamic ranges spanning 2–3 orders of magnitude. Recoveries in urine ranged from 99.5% for creat- inine to 86.5% for citric acid. The analytical variations (RSD) were less than 6.2% in phosphate buffer and 7.7% in urine. Important differences in organic acid levels/profiles between animal species and among normal and urolithic urines/urolith were unveiled and corresponded well (70–90%) with the urolithic risk in a retrospective assessment. The simplicity and reproducibility of this method using disposable Cu n -SPE has made routine urine analysis possible and can be of great clinical and diagnostic potential in the screening of urolithiasis and abnormal states related to excess secretion of organic acids and amino acids in humans and animals. Ó 2009 Elsevier Inc. All rights reserved. Urolithiasis refers to the causes and effects of the presence of uroliths (stones) or excessive amounts of crystal anywhere in the urinary tract [1,2]. This condition affects 1–20% of the general pop- ulation, with an increasing lifetime incidence of 2–5% in Asia, 8– 15% in Europe and North America, and 20% in Middle East [3]. The prevalence of human urolithiasis in Taiwan is 9.6%, accounting for 15–24% of human patients in urological departments [4]. In vet- erinary medicine, the prevalence of canine and feline urolithiasis is 1.2–2% [1,5]. Currently, there are few effective medical options to treat urolithiasis other than urine pH modification and surgical intervention to remove the urolith, but the reoccurrence of urolith- iasis is as high as 80% in humans and 20–50% in canines [1,2,5]. In order to develop rational and effective approaches to prevent and/ or treat urolithiasis and its reoccurrence, early recognition of the types of uroliths that are in formation or have high potential of for- mation is strongly desirable such that appropriate practice could be initiated to create a favorable microenvironment for dissolution of uroliths [6–8]. Techniques commonly used for the diagnosis of urolith involve the use of X-ray and ultrasonography, but they do not differentiate among different urolith types [9]. Urine sediment examination could provide some useful information but it is con- sidered nonquantitative and the sensitivity is usually unsatisfac- tory. Thus, a fast, inexpensive, noninvasive, and routinely applicable method to identify major urinary metabolites compris- ing the urolith is very valuable. The major organic acids in human urolith are uric acid (UA) 1 and oxalic acids (OA) [3,4,6], while in canine, OA and struvite (ammonium magnesium phosphate) are the predominate types [1,5], accounting for more than 80% of the prevalence in both spe- cies. Cystine (Cys) stone is a distant second to OA and UA [3,5]. Other important urinary metabolites involved in the formation and/or dis- sociation of urolith include stone promoters (glycolate, glycerate) and inhibitors (citric acid, CA) [8,10–12]. Since a high concentration 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.08.020 * Corresponding author. Address: College of Veterinary Medicine, National Chung-Hsing University, 250-1 Kuo-Kuang Road, Taichung, Taiwan. Fax: +886 4 22862073. E-mail address: ccchou@nchu.edu.tw (C.-C. Chou). 1 Abbreviations used: CA, citric acid; Crt, creatinine; Cys, cystine; OA, oxalic acid; PBS, phosphate-buffered solution; UA, uric acid. Analytical Biochemistry 395 (2009) 224–230 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio