Article pubs.acs.org/JAFC
Increased Accumulation of Protein-Bound Nε‑(Carboxymethyl)lysine in Tissues of Healthy Rats after Chronic Oral Nε‑(Carboxymethyl)lysine Mei Li,† Maomao Zeng,† Zhiyong He,† Zongping Zheng,† Fang Qin,† Guanjun Tao,† Shuang Zhang,† and Jie Chen*,†,‡ †
State Key Laboratory of Food Science and Technology, and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, 1800 Lihu Road, Wuxi Jiangsu 214122, People’s Republic of China ABSTRACT: In recent years, chronic diseases related to advanced glycation end products (AGEs) have attracted more attention. Because diet is an important exogenous source of AGEs, this study aimed to investigate the effects of chronic oral administration of pure Nε-(carboxymethyl)lysine (CML) (a major AGE) at 60 mg kg−1 per day on healthy Sprague−Dawley rats. After administration for 12 weeks, the levels of protein-bound CML were increased to 202 ± 17, 167 ± 47, 217 ± 44, 107 ± 4, 144 ± 23, and 33 ± 7 μg/g dry matter in the kidneys, heart, liver, lungs, spleen, and pancreas, respectively, in comparison with control values of 98 ± 1, 90 ± 15, 140 ± 42, 76 ± 18, 115 ± 15, and 30 ± 4 μg/g dry matter. The difference was significant (p < 0.05) for the kidneys, heart, liver, and lungs, whereas no significant increase was seen in the spleen and pancreas. Furthermore, serum blood urea nitrogen (BUN), creatinine (CREA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) values increased significantly (p < 0.05), as evidence of impaired kidney and liver function. Additionally, the rats’ fasting blood glucose (FBG) levels remained within the normal range, indicating that chronic intake of CML does not promote a rise in blood glucose. These results clearly indicate that a CML-rich diet might be a potential health risk in humans, particularly with respect to kidney and liver function. KEYWORDS: advanced glycation end products, Nε-(carboxymethyl)lysine, serum markers, fasting blood glucose, tissues
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tendon.29,30 These findings are helpful for understanding the CML accumulation in healthy human bodies. However, most samples used in these studies were of an AGE-rich diet;26,29,31,32 thus, it has been difficult to clearly elucidate which compound or compounds are responsible for the increase in CML in body tissues and a biological effect of CML on healthy organs. Therefore, this work was performed to systematically investigate the distribution and accumulation of protein-bound CML in healthy tissues after chronic administration of pure CML. The intent was to clarify the biological effect of exogenous CML on the healthy tissues and thus predict and prevent AGEs-related chronic diseases in healthy individuals. The purpose of the present study was 2-fold: to thoroughly investigate the changes in protein-bound CML levels in healthy tissues of Sprague−Dawley rats under chronic oral administration of pure CML, and to investigate the effects of exogenous CML on the function of healthy tissues and the blood glucose levels of these rats.
INTRODUCTION Advanced glycation end products (AGEs) comprise a heterogeneous group of end-stage compounds produced through nonenzymatic reactions between reducing sugars and amino compounds.1 They are believed to be associated with a variety of chronic diseases such as Alzheimer’s disease,2,3 atherosclerosis,4 diabetes,5 heart failure,6 and tumor.7 In addition to being formed in living organisms,8 AGEs are generated in food processing and storage.9 It has been reported that daily diet plays an important role in the accumulation of AGEs in the body.10−13 Nε-(Carboxymethyl)lysine (CML), as the first stable AGE found in nature,1,14 is frequently used as a major AGE marker in food analysis15−19 and clinical studies.7,20−22 In the healthy body, CML binds to tissue proteins23 by glycoxidation or lipid peroxidation1 to form protein-bound CML, which may induce several vascular and inflammatory complications.24−26 Previous studies have mainly investigated the protein-bound CML levels in the tissues of patients with chronic diseases and found that the protein-bound CML content of some specific tissues was significantly elevated in comparison to that in healthy subjects. The vulnerable tissues were the kidneys of patients with diabetic nephropathy,20,27 the vessels of patients with atherosclerosis,4,28 and the hearts of patients with diabetes.6 These studies focused mainly on the protein-bound CML levels in pathological tissues. In recent years, researchers have begun to focus on the effects of dietary AGEs on healthy tissues and found that ingestion of bread crusts and Maillard reaction products increase the CML contents in heart, bone, and © 2015 American Chemical Society
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MATERIALS AND METHODS
Chemicals. Sprague−Dawley rats (12-week-old males, specific pathogen-free and weighing 357 ± 37 g) were purchased from Shanghai Slaccas Laboratory Animal Co. Ltd. (Shanghai, China). CML standard and Nε-(carboxy[2H4]methyl)lysine (CML-d4) standard were Received: Revised: Accepted: Published: 1658
October 19, 2014 December 27, 2014 January 22, 2015 January 22, 2015 DOI: 10.1021/jf505063t J. Agric. Food Chem. 2015, 63, 1658−1663
Article
Journal of Agricultural and Food Chemistry
For the SPE, an Oasis MCX cartridge (3 mL, 60 mg; Waters, Milford, MA) was preconditioned with 3 mL of methanol and 3 mL of ultrapure water, followed by 3 mL of 0.1 M HCl at a flow rate of 1 mL/min. The reconstituted sample was loaded onto the preconditioned cartridge at a flow rate of
