Investigation On BPO Content In Flour And Effects Of BPO On Mice Liver And Kidney Tissue

ObjectivesBenzoyl peroxide (BPO) is a strong oxidizing agent and widely used as flour bleaching agent because of its properties of bleaching, shortening the aging period, increasing the production of wheat flour, and sanitizing by producing benzoic acid. At present BPO is widely used in flour, and the phenomenon of beyond the standard is serious. Their potential risk of liver or kidney damage in edible process has been one of the central safety issues. In the present study, we developed a simple, rapid and reliable high perfonnance liquid chromatographic (HPLC) method to determinate the total amount of BPO in commercial flour from Jinan, China. Then, we evaluated the adverse effects of BPO on mice liver and kidney tissue by gavage at a different dose of BPO.Methods1 Method research of determination of BPO in flourThe total BPO content was determined by HPLC with UV detector.Chromatographic condition:HPLC analyses were performed on a L-2000 liquid chromatograph system equipped with one pump and an UV detector, and a auto-sampler. The extract was separated using an Hypersil ODS2 (4.6mm×250mm, 5μm)LC Column, at room temperature. The detection wave length was set at 218nm. BPO was well solved using Potassium dihydrogen phosphate (0.02M)/acetonitrile (90:10,v/v). The flow rate was 1.0 ml/min, and injection volume was 20μl.2 Determination of BPO in commercial flour from Jinan, ChinaFrom December in 2008 to August in 2009 fifty-one samples of flour were purchased from local markets (Jinan, China). The total amount of BPO was determined by HPLC method. According to GB2760-2007, samples of flour were judged qualified that BPO content ranged from 0 to 60 mg/kg.3 The adverse effects of BPO on mice liver tissue3.1 The effects of BPO on mice liver antioxidant statusThe liver was grinded to make tissue homogenate. Malondialdehyde (MDA) levels, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities were all determined to investigate the effects of BPO on mice liver antioxidant status.3.2 The effects of BPO on mice liver ATPasesThe activities of Na+K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase in liver homogenate were determined in each group to investigate the effects of BPO on mice liver ATPases.3.3 The effects of BPO on contents of calcium, zinc, and magnesium essential elements in mice liverThe liver tissue was digested by microwave digestion. Calcium, magnesium, and zinc elements levels were analyzed by flame atomic absorption spectrophotometry to investigate the effects of BPO on essential elements in mice liver.3.4 The effects of BPO on mice liver function3.4.1 The effects of BPO on protein metabolism in mice liverwe evaluated the effects of BPO on protein metabolism of mice liver by examining the concentrations of total protein (TP), albumin (ALB), and globulin (GLO) in mice serum.3.4.2 The effects of BPO on total bile acid (TBA) metabolism in mice liverwe evaluated the effects of BPO on TBA metabolism of mice liver by examining the concentration of TBA in mice serum.3.4.3 The effects of BPO on serum enzymes in mice liverwe evaluated the effects of BPO on serum enzymes of mice liver by examining alanine aminotransferase (ALT), aspartate aminotransferase (AST),and alkaline phosphatase (ALP) in mice serum.3.5 Histopathological observation of the liver cellsAfter fixed, liver tissue was sliced, and then stained by hematoxylin-eosin (HE) to make pathological section. Then sections were observed under microscope.4 The adverse effects of BPO on mice kidney tissue4.1 The effects of BPO on mice kidney antioxidant statusThe kidney was grinded to make tissue homogenate. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities were all determined to investigate the effects of BPO on mice kidney antioxidant status.4.2 The effects of BPO on mice kidney ATPasesThe activities of Na+K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase in kidney homogenate were determined in each group to investigate the effects of BPO on mice kidney ATPases.4.3 The effects of BPO on contents of calcium, zinc, and magnesium essential elements in mice kidneyThe kidney tissue was digested by microwave digestion. Calcium, magnesium, and zinc elements levels were analyzed by flame atomic absorption spectrophotometry to investigate the effects of BPO on essential elements in mice kidney.4.4 The effects of BPO on mice kidney functionwe evaluated the effects of BPO on mice kidney function by examining creatinine (CRE) and urea levels in mice serum.4.5 Histopathological observation of the kidney cellsAfter fixed, kidney tissue was sliced, and then stained by hematoxylin-eosin (HE) to make pathological section. Then sections were observed under microscope.Results1 Method research of determination of BPO in flourSolution of BPO reference substance was scanned by UV-2450.218nm was selected to be the detection wavelength. The retention time of BPO was 13.8 min in the experimental conditions. It has a good correlation in the linear range from 1 to 10μg/ml (r=0.99991). The detection limit was 0.2μg/ml(S/N=3). The recovery of BPO was 98.1-101.7% at low, middle and high concentrations. The RSDs of intra-day were less than 2.98%, and those of inter-day were less than 3.14%. 2 Determination of BPO in commercial flour from Jinan, ChinaWe have analyzed 53 samples of flour from local markets in Jinan.35 samples of them were qualified (value:0-59.2mg/kg), and the percentage was 66.0%.18 samples of them were over the standard (value:68.6-284.6mg/kg), and the percentage was 34.0%. The overstandard rate produced in Shandong province and others was 33.3% and 36.4% respectively, and no significant difference was found between them (P>0.05). The overstandard rate about special and common flour was 36.8% and 32.4% respectively, and no significant difference was found between them (P>0.05).3 The adverse effects of BPO on mice liver tissue3.1 The effects of BPO on mice liver antioxidant statusCompared with the control group, a significant decrease in SOD activity was observed in middle and high dose group (P<0.05, P<0.01). With the BPO dose increased, the MDA content in liver showed an upward tendency, and a significant increase was observed in high dose group (P<0.05). The activity of GSH-Px was not significantly altered after exposure to BPO (P>0.05).3.2 The effects of BPO on mice liver ATPasesWith the BPO dose increased, Mg2+-ATPase activity in liver tissue showed an downward tendency. BPO significantly decreased the liver Mg2+-ATPase and Ca2+-ATPase activities in high dose group (P<0.05). BPO, at all of the doses assayed, produced non-significant effects on Na+K+-ATPase activity.3.3 The effects of BPO on contents of calcium, zinc, and magnesium essential elements in mice liverCompared with the control group, calcium content in BPO dose groups decreased, but there was no significant alternation (P>0.05). Compared with the control group, zinc and magnesium contents had no significant alternation after exposure to BPO (P>0.05).3.4 The effects of BPO on mice liver function3.4.1 The effects of BPO on protein metabolism in mice liverTP and ALB levels in mice serum decreased after BPO administration, and they decreased significantly in high dose (P<0.01). GLO level in mice serum did not reveal alterations after BPO administration (P>0.05).3.4.2 The effects of BPO on TBA metabolism in mice liverTBA level in mice serum increased after BPO administration, and it increased significantly in high dose group (P<0.01).3.4.3 The effects of BPO on serum enzymes in mice liverWith the BPO dose increased, ALT level increased first, and then decreased. Compared with the control group, ALT level in high dose group significantly increased (P<0.05). AST and ALP levels in mice serum did not reveal alterations after BPO administration (P>0.05).3.5 Histopathological observation of the liver cellsIn liver tissue, various degree of the inflammatory cells were observed, and cytolysis and necrosis appeared in the BPO groups.4 The adverse effects of BPO on mice kidney tissue4.1 The effects of BPO on mice kidney antioxidant statusCompared with the control group, a significant decrease in SOD activity and increase in MDA content were observed in high dose group in mice kidney (P<0.05). No statistical signification was found in other dose groups (P>0.05).4.2 The effects of BPO on mice kidney ATPasesCompared with the control group, Na+K+-ATPase activity increased significantly in mice kidney in BPO groups (P<0.05), whereas Mg2+-ATPase activity decreased (P<0.05). Compared with the control group, Ca2+-ATPase activity decreased in mice kidney after exposure to BPO, and statistical signification was found in high dose group (P<0.05).4.3 The effects of BPO on contents of calcium, zinc, and magnesium essential elements in mice kidneyBPO, at all of the doses assayed, produced non-significant effects on contents of calcium, zinc, and magnesium essential elements in mice kidney(P>0.05).4.4 The effects of BPO on mice kidney functionCompared with the control group, urea levels increased after BPO administration, and statistical signification was found in high dose group in mice serum (P<0.05). BPO, at all of the doses assayed, produced non-significant effects on content of CRE in mice serum (P>0.05).4.5 Histopathological observation of the kidney cellsIn all BPO groups, morphological change was various degree of coagulative necrosis in renal tubular.Conclusions1 The HPLC method for the detection of BPO in flour developed in this study was quick, accurate, sensitive and repeatable well, and sample processing was easy. BPO in flour can be separated and determined.2 BPO was added to most samples of commercial flour from Jinan city, and there was the phenomenon of overstandard (The rate was 34.0%). There were not differences between Shandong province and others, between special and common flour.3 When BPO reached some degree, it had adverse effects on liver antioxidant status and ATPases, damaged the metabolism of protein and TBA, and induced certain degree of pathological damages to mice liver.4 When BPO reached some degree, it had adverse effects on kidney antioxidant status and ATPases, damaged the kidney function, and induced certain degree of pathological damages to mice kidney.