Journal of Chromatography B, 879 (2011) 3214–3219 Contents lists available at ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb Determination of time-dependent accumulation of d-lactate in the streptozotocin-induced diabetic rat kidney by column-switching HPLC with fluorescence detection  Mei-Hsiang Lin a , Hsiang-Yin Chen a , Tzu-Hsin Liao a , Tzu-Chuan Huang a , Chien-Ming Chen b , Jen-Ai Lee a,∗ a School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing St., Taipei 11031, Taiwan b Department of Electro-Optical Engineering, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Rd., Taipei 106, Taiwan a r t i c l e i n f o Article history: Received 30 November 2010 Accepted 8 February 2011 Available online 13 February 2011 Keywords: d-Lactate l-Lactate Diabetic rat Nephropathy Streptozotocin Methylglyoxal a b s t r a c t For better understanding the complete metabolism and the physiological role of d-lactate, the concen- trations of d-lactate in the serum, liver and kidney of normal and diabetic rats were determined by our established column-switching HPLC method with pre-column fluorescence derivatization. Eight- week-old male Sprague–Dawley rats were administered with streptozotocin (STZ) (80 mg/kg) or citrate buffer intraperitoneally. The tissues were then removed and homogenized after 4, 8, 12 and 16 weeks of drug administration, respectively. The homogenates were centrifuged at 1200 × g for 10 min, then the supernatants were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on an ODS column followed by a Chiralpak AD-RH chiral column for enantiosep- aration. The results showed that the d-lactate content elevated in all the 3 examined tissues under diabetic stages. In addition, d-lactate concentrations in rat kidney were accumulated significantly and time-dependently in diabetic groups after receiving STZ for 4, 8, 12 and 16 weeks (2.99, 13.11, 18.19, 23.23 vs. 0.79 mol/mg protein as control group). Moreover, the kidney of induced 12-week diabetic rat renal showed some histological changes of progressive diabetic nephropathy. The results suggest that d-lactate may be used as a marker of diabetic nephropathy. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The d-lactate, an endogenous product from excessive use of reg- ular energy resources, has been tested to use as a marker of several diseases. It has reported to increase in bacterial infection [1,2], acute intestinal ischemia [3–6], and appendicitis [7,8]. The most recent application is on its relationship with the severity of diabetics. Sig- nificant increases of serum, plasma and urinary d-lactate under diabetic stage were found in our previous publications [9–11]. In diabetic stage, with the catalysis by glyoxalase I and II being the major producing pathway [12,13], the plasma level of d-lactate was found to elevate proportionally with the increasing production Abbreviations: DM, diabetes mellitus; TFA, trifluoroacetic acid; NBD- PZ, 4-nitro-7-piperazino-2,1,3-benzoxadiazole; I.S., internal standard; o-PD, o-phenylenediamine; 2-MQ, 2-methylquinoxaline; 5-MQ, 5-methylquinoxaline; AGEs, advanced glycation end products.  This paper is part of the special issue “Analysis and Biological Relevance of d- Amino Acids and Related Compounds”, Kenji Hamase (Guest Editor). ∗ Corresponding author. Tel.: +886 2 2736 1661x6125; fax: +886 2 2736 1661x6120. E-mail address: jenai@tmu.edu.tw (J.-A. Lee). of its precursor, methylglyoxal (MG), in plasma [14–17]. However, the concentrations of d-lactate and MG in other biological sam- ples at normal and diabetic stage are warranted to determine for establishing the model of application in diabetes. Measuring the concentrations of d-lactate in biological sam- ples encounters many barriers. Lactic acid (2-hydroxypropionic acid), the chemical formula of C 3 H 6 O 3 , has a chiral center to form two enantiomers, l-(+)-lactic acid and d-(-)-lactic acid. With a pK a value of 3.86, lactic acid mostly dissolves to form lactate in the physiological fluids. The automated metabolite analyzers can only determine the content of l-lactate by using l-lactate dehy- drogenase. And the traditional enzymatic method by d-lactate dehydrogenase (d-LDH), although widely used to measure d- lactate in biological samples, cross reacts with many endogenous substances, such as pyruvate, S-lactonyl glutathione, l-lactate, fructose 1,6-bisphosphate, and 3-phosphoglyceric acid, reducing its accuracy and precision significantly [18,19]. To overcome the above drawbacks, many high-performance liquid chromatography (HPLC) methods have been developed and modified by our pre- vious efforts [9,20]. We previously determined the d-lactate in urine of normal and diabetic rats by HPLC with an octadecylsil- ica (ODS) column connected to an amylose-based chiral column. 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.02.015