Food Chemistry 133 (2012) 760–766

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Food Chemistry

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Inhibition of heterocyclic amine formation by water-soluble vitaminsin Maillard reaction model systems and beef patties

Daniel Wong, Ka-Wing Cheng, Mingfu Wang ⇑School of Biological Sciences, The University of Hong Kong, Hong Kong, PR China

a r t i c l e i n f o

Article history:Received 5 September 2011Received in revised form 10 November 2011Accepted 26 January 2012Available online 4 February 2012

Keywords:Heterocyclic aminesVitaminsChemical model systemsBeef patties

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.foodchem.2012.01.089

Abbreviations: HA, heterocyclic amine; MeIQdazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethMRPs, Maillard reaction products; PhIP, 2-amino-1-mb]pyridine; SPE, solid phase extraction.⇑ Corresponding author. Tel.: +852 2299 0338; fax:

E-mail address: mfwang@hku.hk (M. Wang).

a b s t r a c t

The inhibitory activities of 11 water-soluble vitamins against heterocyclic amine formation were exam-ined in a PhIP and a MeIQx producing chemical model. Six investigated vitamins (pyridoxiamine, pyri-doxine, nicotinic acid, biotin, thiamine and L-ascorbic acid) out of the 11, exhibited significantinhibition (>40%) in both models. Pyridoxamine was the most potent inhibitor, and its inhibitory effectincreased with increasing concentration in the model, although not in a linear manner. The activity ofpyridoxamine, niacin and ascorbic acid was investigated using fried beef. Moderate inhibition (�20%)of the formation of PhIP, 4,8-DiMeIQx and MeIQx was found for niacin and ascorbic acid; whereas pyri-doxamine reduced the levels of all three HAs by �40%. GC–MS analysis showed that pyridoxamine sig-nificantly reduced the level of PhIP intermediate, phenylacetaldehyde. LC–ESI–MS/MS analysis revealedthat pyridoxamine directly reacts with phenylacetaldehyde to form an adduct, whose structure was char-acterised by MS and NMR spectroscopy.

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1. Introduction view of their potent mutagenic activity and the fact that they can

Our diet plays an important role for human’s uptake of exoge-nous harmful substances. Heterocyclic amines (HAs) are a largegroup of structurally close genotoxic compounds formed fromreactions between creatinine, sugar and amino acids at high tem-peratures (Abdulkarim & Smith, 1998). Thus, they are found pre-dominantly in meat and fish based products, especially whenthese food products are subjected to heat treatment (Sugimura,1997). There are mainly two categories of HAs, polar and non-polarHAs. The latter category has been shown to be responsible for mostof the HA-associated mutagenic activity detected in food(Sugimura, Wakabayashi, Nakagama, & Nagao, 2004).

HAs have been shown to be potently mutagenic in bacteria muta-genicity testing (Gooderham et al., 2001). Some of them were alsofound to induce tumorigenesis in various organs including the colon,prostate, and mammary glands in animal models (Baranczewski,Gustafsson, & Moller, 2004) and in humans (Layton et al., 1995). Epi-demiological studies also tend to support a positive correlation be-tween the consumption of well-done red meat and the risk ofcertain cancers (Cheng, Chen, & Wang, 2006; Sugimura, 1997). In

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, 2-amino-3,4-dimethylimi-ylimidazo[4,5-f]quinoxaline;ethyl-6-phenylimidazo[4,5-

+852 2288 0340.

be formed even during ordinary household cooking (Warzechaet al., 2004), a lot of attention has been focused on HAs aiming to de-velop strategies which can effectively lower HA content in foods, andthus help to reduce human exposure to HAs (Cheng, Chen & Wang).Reducing the formation of HAs has been one of the most widelyrecognised approaches to attenuate HA-associated health risk.

Vitamins are essential nutrients for humans. They have bothbeen used as dietary supplements and as additives to enhancethe nutritional values of different food products. Recent studiesshowed that vitamin B6 could inhibit the formation of Maillardreaction products (Khalifah, Baynes, & Hudson, 1999). It also inhib-ited the formation of advanced glycation end-products both in vitroand in vivo (Booth, Khalifah, Todd, & Hudson, 1997; Reddy & Beyaz,2006; Voziyan & Hudson, 2005). These studies have providedstrong evidence that certain vitamins may be able to interact withthe Maillard reaction to thus impact the types and concentrationsof reaction products formed. In the present study, the effects of 11water-soluble vitamins on the formation of heterocyclic amineswere examined in 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxa-line (MeIQx)- and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyr-idine (PhIP)-forming model systems, respectively. The testvitamins included thiamin (vitamin B1), riboflavin (vitamin B2),nicotinic acid (vitamin B3), pantothenic acid (vitamin B5), pyridox-ine (vitamin B6), pyridoxal (vitamin B6), pyridoxamine (vitaminB6), biotin (vitamin B7), folic acid (vitamin B9), vitamin B12, andL-ascorbic acid (vitamin C). Activities of some of the promisinginhibitors were further evaluated in fried beef.

D. Wong et al. / Food Chemistry 133 (2012) 760–766 761

2. Materials and methods

2.1. Reagents and chemicals

Triethylamine, HCl, thiamin hydrochloride (vitamin B1, VB1),riboflavin (vitamin B2, VB2), nicotinic acid (vitamin B3, VB3), d-pantothenic acid hemicalcium salt (vitamin B5, VB5), pyridoxinemonohydrochloride (vitamin B6, PN), pyridoxal hydrochloride(vitamin B6, PL), pyridoxamine dihydrochloride (vitamin B6, PM),biotin (vitamin B7, VB7), folic acid (vitamin B9, VB9), cobalamine(VB12), L-ascorbic acid (vitamin C, VC) were purchased from Sig-ma–Aldrich Company (St. Louis, MO, USA). HA standards, 2-ami-no-3,4,8-trimethy-limidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-ami-no-1-methyl-6-henylimidazo [4,5-b]pyridine (PhIP) were fromToronto Research Chemicals (Toronto, Canada).

Phenylalanine, glucose, creatinine, diethylene glycol, NH4OH,NaOH, silicone oil, ammonium acetate, triethylamine, HCl, werealso obtained from Sigma–Aldrich company (St. Louis, MO). Pro-pyl-sulphonic acid (PRS) Bond-Elut cartridges (500 mg), C-18 car-tridges (100 mg), Bond-Elut reservoir, and packing materials(diatomaceous earth) were obtained from Varian (Harbor City,CA, USA). All solvents used were of analytical grade and obtainedfrom BDH Laboratory Supplies (Poole, UK). The Reacti-Therm IIIheating module (model 18840) and screw cap Tuf-Bond Teflon fit-ted glass reaction vials (10 ml capacity) were purchased fromPierce (Rockford, IL, USA). Ground beef samples were purchasedfrom the local supermarket.

For instrumentations, HPLC analysis was performed on aShimadzu HPLC system with a LC-20AT separation module, anautosampler (SIL-20A) and a diode array detector (modelSPD-M20A). GC/MS analysis was performed on an Agilent gas chro-matograph (6890N) equipped with an autosampler (G2614A) andcoupled to an Agilent mass spectrometer (5973N, EI mode). LC-MS was performed on a QTRAP2000 mass spectrometer (AppliedBiosystems, Foster City, CA) with an Agilent separation model(Agilent 1100; Agilent Technologies, Santa Clara, CA).

2.2. Effects of vitamins on HA formation in chemical model systems

Effects of water-soluble vitamins on PhIP and MeIQx formationwere investigated in a PhIP- and a MeIQx-producing model system,respectively, according to the literature with slight modifications(Cheng, Chen, & Wang, 2007; Gross & Gruter, 1992; Murkovic, We-ber, Geiszler, Frohlich, & Pfannhauser, 1999). In the PhIP model,0.4 mmol phenylalanine, 0.4 mmol creatinine, and 0.2 mmol glu-cose with or without the addition of 0.2 mmol of a test vitaminwere mixed in 3 ml of diethylene glycol containing 14% of distiledwater in screw cap-sealed reaction vials. The temperature metre ofthe heating block was set to 130 �C and preheated for 2 h beforeinserting the vials into the cavities. The heating time of the vialswas 2 h after the pre-heating treatment had completed and thetemperature was checked every 15 min. Fluctuation of tempera-ture was within 3 �C. The vials were immediately cooled in anice–water mixture at the 2 h time point. The content of each vialwas diluted with 47 ml of 2 M NaOH solution. Five millilitres of di-luted aliquot was transferred into a 100-ml beaker. Solid phaseextraction (SPE) was applied for the extraction of HAs from chem-ical models. The aliquot was mixed thoroughly with diatomaceousearth before packing into a Bond-Elut reservoir fitted with a bot-tom frit. Elution was then performed with 48 ml of dichlorometh-ane directly into an attached PRS Bond-Elut cartridge. The PRScartridges were dried under maximum vacuum for 5 min and weresequentially washed with 6 ml of 0.1 M HCl, 15 ml of 40% methanol

in 0.1 M HCl, and finally 2 ml of distiled water. HAs absorbed onthe PRS cartridges were then eluted into Bond-Elut C-18 cartridgeswith 20 ml of ammonium acetate solution (0.5 M, pH 8). Prior touse, the PRS and C-18 cartridges were conditioned according tothe procedures by Gross and Gruter (1992). The C-18 cartridgeswere washed with 2 ml of distiled water and dried under positivepressure. The final elution was carried out with 1.2 ml of MeOH–NH4OH (9:1, v/v) into HPLC sample vials. The eluate was dried un-der nitrogen gas and the residue was dissolved in 100 ll of meth-anol. The samples were filtered before being subjected to HPLCanalysis. The above procedure was also applied to study thedose-dependent effect (0.2, 0.1 and 0.05 mmol) of pyridoxamineon PhIP formation in model system.

The MeIQx model system study was performed following thesame procedure as that applied for the PhIP system, except thatphenylalanine was replaced by glycine in the former system.

2.3. Effects of vitamins on HA formation in beef patties

An accurately weighed amount (30 + 0.2 g) of ground beef wasformed into a beaker and powers of test vitamins (0.2 mmol) werethoroughly mixed with the beef. After mixing, the ground beef wasformed into a disk shape using a glass Petri dish (6.2 � 1.2 cm). Thebeef patties were then fried on a Teflon-coated frying pan with asurface temperature of 200 �C for 6 min (3 min for each side). Eachof the control and vitamin-pretreated groups had three samplesand the experiment was repeated three times. The three beef pat-ties for each group were combined and homogenised in 150 ml of1.0 M NaOH for 2 min to form a dense paste. Six portions (eachequivalent to 5 g of beef) were weighed into separate beakers.Next, 20 ml of 1.0 M NaOH was added to each beaker and stirredto form a suspension. The suspension was mixed thoroughly withdiatomaceous earth and packed into a Bond-Elut reservoir. Subse-quent steps were the same as those for the analysis of samplesfrom chemical model systems (Cheng et al., 2007).

2.4. HPLC analysis of HAs

HPLC analysis was performed on a Shimadzu HPLC system. Sep-aration of HAs was carried out on an YMC-Pack Pro C-18 column(5 lm, 150 � 4.6 mm). The elution programme was adopted fromthe literature (Cheng et al., 2007) with slight modification. The mo-bile phase was composed of water (0.01 M triethylamine, pH wasadjusted to 3.2 using phosphoric acid) (solvent A) and ACN (solventB) at a flow rate of 1 ml/min. A gradient elution programme wasused: 0–10 min, 95% A: 5% B; 10–20 min, 75% A: 25% B; 20–30 min, 45% A: 55% B; 30–35 min, 20% A: 80% B. The total runningtime was 35 min and the post-running time was 15 min for equil-ibration of the column. HAs were monitored at two wavelengths,265 and 312 nm. Before commencing an analytical run, the columnwas conditioned with the initial mobile phase composition for30 min and the LC-DAD system tested for system stability usingblank and standard HA solutions. Peak identification was accom-plished by comparing the retention times and UV spectral charac-teristics of the HPLC peaks with those obtained from standardsolutions of HA mixture analysed under the same conditions.Quantitative determination was performed using an external cali-bration curve. Correlation coefficients (r2) for HA standard curveswere: 0.9942 for PhIP and 0.9891 for MeIQx, respectively. Recover-ies for HPLC analysis of the three HAs were as follows: PhIP 59.1%,MeIQx 54.3% and 4,8-diMeIQx 57.1%. These recoveries datamatched the range of previous published work by Balogh, Gomaa,and Booren (2000).

Table 1Chemical model reactions.

Reactants Reactant concentration (mM)

A B C D E F G H I

Phenylalanine 20 20 20 20 20 20Glucose 10 10 10 10 10Creatinine 20 20Pyridoxiamine 5 5 5 5 5 5Phenylacetaldehyde 20

Model reactions A–H were carried out in phosphate buffer (0.1 M, pH 7.0), whileModel I was carried out in di(ethylene) glycol containing 14% water. All systemswere heated at 130 �C for 30 min.

762 D. Wong et al. / Food Chemistry 133 (2012) 760–766

2.5. Effect of pyridoxamine on the generation of Maillard intermediatesin phenylalanine-containing chemical model systems

The methodology was adopted from previous studies (Chenget al., 2008; Gross, Gruter & Heyland, 1992). To investigate the ef-fect of PM on the degradation of phenylalanine, phenylalanine andPM in a molar ratio of 4:1 were dissolved in 10 ml of phosphatebuffer (0.1 M, pH 7.0) in screw cap Tuf-Bond Teflon fitted glassreaction vials (40 ml capacity). The reaction mixture was heatedat 130 �C for 30 min. The reaction mixtures were subsequentlycooled under room conditions for 1 h and then prepared for GC–MS analysis.

For analysis of volatile compounds, 10 ml of selected reactionmixtures was spiked with the internal standard, n-dodecane(0.02 ll of n-dodecane/8 ml reaction mixture) and extracted with10 ml of diethyl ether under vortex for 1 min. The extraction pro-cess was repeated three times, each with 10 ml of diethyl ether.The diethyl ether extract was combined and dried with anhydroussodium sulphate. The sample was then filtered under vacuum.Nitrogen gas was used to evaporate diethyl ether and the residuewas dissolved in methanol for GC–MS analysis.

To further study the role of PM in the PhIP-producing Maillardmodel system, a range of chemical model reactions composed ofdifferent combinations of PhIP precursors with or without thepresence of PM were carried out (Table 1). The reactants were dis-solved in phosphate buffer (0.1 M, pH 7.0, 10 ml) or diethylene gly-col containing 14% water in screw cap Tuf-Bond Teflon fitted glassreaction vials (40 ml capacity) and heated at 130 �C for 30 min.After cooling to room temperature, the reaction mixtures were fil-tered and subjected to LC–MS analysis.

2.5.1. GC–MS analysisThe diethyl ether extracts were analysed on an Agilent gas chro-

matograph (6890N) equipped with an autosampler (G2614A) andcoupled to an Agilent mass spectrometer (5973N, EI mode). Sepa-ration was performed on a DB-Wax capillary column(30 m � 0.25 mm i.d., 0.25 lm film thickness). Analyses were car-ried out using the following parameters: injection, 2 ll of samplein splitless mode; inlet temperature, 200 �C; column flow, 1 ml/min (He); temperature programme, 50 �C for 4 min, ramp at 5–230 �C and hold for 5 min; and MS temperature, 230 �C. Identifica-tion of phenylacetaldehyde was by comparison with mass spec-trum generated by EI–MS from an authentic standard solution.Peak area was adjusted relative to the peak area of the internalstandard (n-dodecane).

2.5.2. LC–MS/MS identification of formed adductsFiltrates obtained from chemical model reactions were analysed

on an LC–MS instrument equipped with an electrospray ionisation(ESI) source interfaced to a mass spectrometer (Qtrap 2000 AppliedBiosystems). Liquid chromatography was run on an Agilent HPLCsystem with a degasser, a quaternary pump, a thermostatted auto-sampler, and a diode array detector. Separation of Maillard reac-tion products was carried out with a Varian Inertsil ODS C-18column (3 lm, 15 � 4.6 mm). The mobile phase composed of0.1% formic acid aqueous solution (solvent A) and analytical gradeacetonitrile (B) of the following gradients: 0 min, 5% B:95% A;35 min, 80% B:20% A; 37 min, 5% B:95% A; and 50 min, 5% B:95%A. Effluent from the UV detector was split 4:1 with one part(200 ll/min) directed to the MS for spectrometric analysis andthe remaining to waste. The MS conditions were as follows: nega-tive ion mode; spray voltage, 3.5 kV; scan range, 120–800 Da; andcapillary temperature, 300 �C. Enhanced product ion scan (EPI) wasused for the analysis.

2.6. Isolation, purification, and structural elucidation ofphenylacetaldehyde–pyridoxamine adducts

The methodology followed the procedures described by Chenget al. (2008), with slight modifications. Pyridoxamine and phenyl-acetaldehyde in a molar ratio of 1:20 were dissoloved in di(ethyl-ene) glycol, respectively. The total reaction time was 6 h. At the 6 htime point, the reaction was quenched by inserting the vials into anice–water mixture. Liquid–liquid extraction was performed forremoving di(ethylene) glycol and for concentrating the samples.The solvent system was composed of sample–H2O–ethyl acetate–hexane in a ratio of 1:1:2:1 (v/v). The solvent mixture was vor-texed for 2 min and then centrifuged at 10,000 for 5 min. Theextraction steps were repeated three times. The clear supernatantwas transferred to a flask and concentrated on a rotary evaporatorunder vacuum. The extract was thus dissolved in distiled waterand loaded onto an Amberlite XAD-16 column (40 � 4 cm i.d.).The concentrated extracted was suspended in 70% methanol andloaded onto a Sephadex LH-20 column (40 � 4 cm i.d.). Elutionwas performed with 70% methanol and the eluate was collectedusing an automatic fraction collector. Profiles of the fractions werechecked by LC-DAD analysis and similar fractions were combined.The aqueous solvent was then removed on a rotary evaporator.This open-column chromatographic process eventually led to thepurification of the target adduct, whose structure was character-ised by NMR spectroscopy (Bruker, AVANCE 600).

2.7. Spectral data of PM-phenylacetaldehyde adduct

1H NMR (300 MHz): d 8.20 (s, H-2), 7.15 (dd, H-16, H-12), 6.80(dd, H-15, H-13), 6.75 (tt, H-14), 5.06 (s, 2H, H-17), 4.88 (t, H-9),4.72 (s, 2H, H-7), 3.30 (d, 2H, H-10), 2.60 (s, 3H, H-18). 13C NMR(75 MHz): d 158.49 (C-9), 155.01 (C-2), 143.04 (C-5), 141.47 (C-6), 139.22 (C-4, C-3), 130.46 (C-11), 130.05 (C-16, C-12), 120.54(C-15, C-13), 116.26 (C-14), 59.90 (C-17), 59.37 (C-7), 49.36 (C-10), 14.75 (C-18).

2.8. Statistical analysis

Statistical analyses were performed using the SPSS statistics17.0 package (SPSS, Chicago, IL, USA). Paired samples t-test was ap-plied to determine whether a particular treatment of the samplewould result in a significantly different content of HAs comparedwith the control set. p < 0.05 was selected as the level of decisionfor significant differences.

3. Results and discussion

3.1. Effects of water-soluble vitamins on the formation of HAs inchemical model systems

According to Skog, Johnansson, and Jagerstad (1998) and Hidalgo,Gallardo, and Zamora (2005), HAs can be formed when creatine,

D. Wong et al. / Food Chemistry 133 (2012) 760–766 763

amino acid and monosaccharide are allowed to react at elevatedtemperatures (usually above 100 �C). In this study, the effects ofa group of water-soluble vitamins on HA formation were comparedusing chemical model reactions known to be capable of generatingtwo of the most important mutagenic HAs (Cheng et al., 2006),PhIP and MeIQx. As shown in Fig. 1, seven out of the 11 test vita-mins, including VB1, VB3, VB7, PM, PN, PL and VC, significantly re-duced the formation of PhIP and MeIQx. Noticeably, at a molarratio of 1:2 (Vitamin: amino acid), PM and VB7 both caused >70%reduction in the levels of PhIP and MeIQx in the respective modelsystem relative to the control.

Ever since the early 1980s when researchers started to look intothe mechanism whereby certain chemical agents inhibited the for-mation of mutagenic HAs, antioxidative activity had been proposedto be the most important pathway to modulate HA formation (Oz &Kaya, 2011; Kikugawa, Hiramoto, & Kato, 2000; Tai, Lee, & Chen,

Fig. 1. Inhibitory effects of water-soluble vitamins on heterocyclic amine formationin PhIP-producing (A) and MeIQx-producing (B) chemical model systems. Datavalues are means and vertical error bars are standard deviations of threeindependent experiments. Paired samples t-test was applied to determine whethera particular treatment of the sample would result in a significantly different contentof HAs compared with the control. p < 0.05 was selected as the level of decision forsignificant differences. Bars with an asterisk indicate significant difference from thecontrol, and the control set was set to have 0% inhibition.

2001). In contrast to this applauding mechanism of action, recentstudies (including those conducted by our group) have reportedthe failure to establish linear correlation between antioxidantactivity and HA formation-inhibitory activity of different groupsof chemical agents (Cheng et al., 2007, 2008). Of note, some ofthe well-known antioxidants such as tocopherol were found to en-hance the formation of HAs (Lan, Kao, & Chen, 2004). In the presentstudy, many of the water soluble vitamins, especially the B vita-mins, which are known to have weak antioxidant capacities, dem-onstrated potent inhibition against the formation of HAs. In ourprevious study, which employed a large group of dietary phenoliccompounds representing a wide range of antioxidant capacities, nolinear correlation was found between the antioxidant capacitiesand HA-formation inhibitory activities of the phenolic compounds(Cheng et al., 2007). Other researches provided evidences for effec-tive HAs inhibitors with strong antioxidant capacities (Kato, Hara-shima, Moriya, Kikugawa, & Hiramoto, 1996; Vitaglione & Fogliaon,2004; Johansson & Jagerstad, 1993, 1996). For example, antioxi-dant like vitamin E had been studied for its inhibitory effect againstHAs formation and up to 72% inhibition of PhIP was achieved (Bal-ogh et al., 2000). The only antioxidant examined in our study wasvitamin C. VC achieved around 70% inhibition in the PhIP modeland 35% inhibition in the MeIQx model. The results obtained weresimilar to the previous research conducted by Balogh et al. (2000).

In contrast, our mechanistic studies established that polyphe-nolic antioxidants such as naringenin inhibited PhIP formation bydirectly trapping phenylacetaldehyde, a key intermediate on thepathway to the formation of PhIP (Cheng et al., 2008) Taking intoconsideration that the weak antioxidant capacities of PM and PNwere unlikely to account for their potent inhibitory effects on theformation of PhIP, it was hypothesised that these vitamins mayact through a different pathway to modulate the formation ofHAs. In this regard, their terminal primary amino or hydroxylgroup may play important roles, such as interacting with reactiveMaillard intermediates to stop them from propagating in pathwayswhich would otherwise lead to HA formation. This research ques-tion will be further discussed in later sections on the elucidation ofthe mechanism of inhibition of the promising HA-formationinhibitors.

3.2. Effect of VC, VB3 and PM on the formation of mutagenic HAs inbeef patties

In the area of food-borne genotoxicants associated with heattreatment, the most commonly employed approach to identifyinhibitors is by screening a group of potential candidates usingchemical model reactions, followed by corroboration of the activityof the promising inhibitors in real food systems. The same ap-proach was also applied in the present study. Thus, the activitiesof VC, VB3 and PM were further investigated using fried beef pat-ties. Although VC was shown in a previous study to only mildly re-duce the formation of HAs in fired fish (Tai et al., 2001), it wasincluded in this experiment because of two reasons: (1) it exhib-ited strong inhibition of HA formation in the chemical model sys-tems; (2) it may demonstrate a different modulating effect infried beef which belongs to a very different type of food systemfrom fish. Three HAs, including PhIP, MeIQx and 4,8-diMeIQx, weredetected and quantified by HPLC-DAD analysis with correspondingyields of 7.44 ± 1.12, 2.46 ± 0.32 and 7.39 ± 0.84 ng/g, respectively.As presented in Table 2, VC and VB3 only showed moderate inhib-itory effects (�20%) on the formation of the three HAs, whereas PMcaused >40% reduction. Based on the means from three indepen-dent experiments, the relative inhibitory potency follows the sameorder (pyridoxamine > niacin > ascorbic acid) for all the three polarHAs identified.

Table 2Effect of vitamins (0.2 mmol) on the formation of HAs in beef patties fried at 200 �C for 3 min on each side.

Treatment HAs (ng/g beef patties) and inhibition (%)

PhIP 4,8-diMeIQx MeIQx Total (HAs)

7.44 ± 1.12a 2.46 ± 0.32a 7.39 ± 0.84a 17.3Ascorbic acid 6.02 ± 0.13b (19) 2.12 ± 0.04b (14) 6.13 ± 0.25b (17) 14.3 (17)Niacin 6.03 ± 0.27b (19) 2.09 ± 0.09b (15) 5.99 ± 0.20b (19) 14.1 (18)Pyridoxamine 4.24 ± 0.45c (43) 1.53 ± 0.16c (38) 4.29 ± 0.49c (42) 10.0 (42)

a Each value is expressed as mean ± SD (n = 3).b The figures in the brackets represent the inhibition % value compared to the control.c Means with different letters in the same column are significantly different (p < 0.05).

Fig. 2. MS/MS spectrum of the m/z 271 [(M+H)]+ adduct ion identified in chemicalmodel I and the structure of the purified adduct.

Fig. 3. Postulated pathways for the inhibitory activity of pyridoxamine against PhIPformation.

764 D. Wong et al. / Food Chemistry 133 (2012) 760–766

3.3. Dose-dependent effect of pyridoxamine on PhIP formation

Results from both chemical model and real food analyses iden-tified PM to be the most promising HA-formation inhibitor amongthe group of vitamins tested. Further study was performed in thechemical models and beef patties to examine the dose–responsebehaviour of PM. At a concentration as low as one-eighth of theprecursor amino acid of PhIP, PM was able to reduce the level ofPhIP by 46%. Interestingly, doubling the dose of PM did not signif-icantly enhance its inhibitory effect on PhIP formation. However,when the concentration of PM was further increased to 80% of thatof PhIP precursors, the level of PhIP in the model system was

reduced by more than 80% compared with the control. In the beefpatty study, 0.05, 0.1 or 0.2 mmol of PM was added to each beefpatty, and the levels of HAs formed after frying were comparedto those in beef patties without pretreatment with PM. For allthe three polar HAs identified, the inhibition rate increased withthe level of PM added to the beef patties. In other words, positive(although not linearly proportional) correlations exist betweenthe degrees of inhibition and the amounts of PM present in the beefpatties. For decades, both synthetic and natural agents have beenthe subjects of ample research works which aim to identify potentinhibitors of HA formation. The ultimate goal is to attenuate theload of genotoxic HAs in food products by adding these inhibitorsat different steps of the food preparation process. In particular, nat-ural agents represent more promising candidates in view of theirclose association with our daily cuisine and their acceptance bythe public. Flavonoids, especially those derived from certain fruitextracts (Cheng et al., 2007), have been reported to be among themost promising inhibitors of mutagenic HA formation. The presentstudy has demonstrated that PM may exhibit similar inhibitory po-tency to some of the most promising natural inhibitors identifiedthus far when applied over a similar concentration range.

3.4. Effect of pyridoxamine on the formation of PhIP Maillardintermediates

To investigate the effect of PM on the formation of PhIP Maillardintermediates, GC–MS analysis was performed for samples fromPhIP-producing model systems with or without the addition ofvitamins. In agreement with a study by Cheng et al. (2008), phenyl-acetaldehyde was identified as the chief volatile compoundformed. No other volatiles related to the formation of PhIP, suchas phenyethylamine (Zochling, Murkovic, & Pfannhauser, 2002),were found. Quantitative analysis showed that PM at one-fourththe concentration of phenylalanine was capable of reducing the le-vel of phenylacetaldehyde in the model by nearly 60% relative tothe control. The key intermediate, phenylacetaldehyde, is espe-cially important since its yield of PhIP was found to be ten timeshigher than that of phenylethylamine according to Zochling et al.(2002). Therefore, it is probable that PM inhibits PhIP formationby directly trapping or by interrupting the formation of key inter-mediary compounds derived from phenylalanine.

3.5. Identification of adducts formed from pyridoxamine andphenylalanine degradation products

LC–ESI–MS analyses were conducted for samples from a widerange of model system reactions in order to identify adductsformed between PM and phenylalanine degradation products.Examination of the LC–UV and LC–MS chromatograms revealedthe generation of an adduct with molecular weight correspondingto that formed between PM and phenylacetaldehyde in modelscontaining phenylalanine plus PM with/without glucose or creati-nine (models E, F, G and I). The predicted molecular weight of the

D. Wong et al. / Food Chemistry 133 (2012) 760–766 765

adduct is 270 (m/z [M+H]+ 271), which is larger than that of anyprecursor in the model reaction, but not equal to that of the Maillardreaction products, oxidation products or phenylacetaldehydeoligomers. Such an adduct was not detected in systems containingphenylalanine or PM alone (model A, B), phenylalanine and glucose(model C), phenylalanine, glucose and creatine (model D), or PMand glucose (model H). The adduct was present only in models E,F, G and I. Models E, F and G all contain PM and phenylalanine,while model I contained PM and phenylacetaldehyde. In particular,this adduct was present in significant quantity in model system I,probably due to the greater abundance of phenylacetaldehyde inthis model relative to the others.

3.6. Characterisation of the structure of the pyridoxamine-phenylacetaldehyde adduct

To better understand the chemistry of the proposed reaction ofadduct formation, the structure of adduct was further character-ised by LC–MS/MS and NMR spectroscopy. MS/MS was performedwith the parent m/z ratio set at 271 in Enhanced product ion mode(EPI). Collision-induced dissociation (CID) of this ion generated m/z153 fragment ion, which probably arose from the lost of C8H8Nfrom this adduct (Fig. 2). This fragmentation pattern, together withliterature data (Cheng et al., 2008; Arribas-Lorenzo, Pintado-Sierra,& Morales, 2011), suggest that the m/z 271 adduct likely resultedfrom electrophilic substitution of phenylacetaldehyde on the pri-mary amine functional group of PM, followed by elimination of awater molecule to form a stable adduct. A mechanism of actionis thus tentatively proposed (Fig. 3) to be responsible for PM’sinhibitory activity against the formation of PhIP. At high tempera-ture, phenyalanine may degrade to form Strecker degradationproducts. In the presence of PM, the reactive carbonyl, phenylacet-aldehyde, may be scavenged to form adduct, thus preventing itsentry into pathway(s) which lead to PhIP formation.

The adduct was subsequently purified from the PM-phenylacet-aldehyde model system. Its structure was elucidated by 1D NMRspectroscopy. In the 1H NMR spectrum, 5 proton signals (d 7.15(dd, H-16, H-12), 6.80 (dd, H-15, H-13), 6.75 (tt, H-14)) were assign-able to the benzene ring of the structural moiety of the original phe-nylacetaldehyde. The alkyl chain of the –CH2–CH@N–CH2– moietydisplayed signals at d 4.88 (t, H-9), 4.72 (s, 2H, H-7) and 3.30 (d,2H, H-10). The pyridine ring of the PM moiety contributed to oneproton signal at d 8.20 (s, H-2). The methyl (Ar–CH3) and thehydroxymethyl group (Ar–CH2OH) attached to the pyridine ring ac-count for the signal at d 2.60 (s, 3H, H-18) and 5.06 (s, 2H, H-17),respectively. Its 13C NMR spectrum exhibited 16 carbon signals,which matched well with the proposed structure. Six carbon signalswere identified at d 130.46 (C-11), 130.05 (C-16, C-12), 120.54 (C-15,C-13) and 116.26 (C-14), and were assignable to the benzene ring ofphenylacetaldehyde. The alkyl chain of the –CH2–CH@N–CH2– moi-ety displayed signals at d 158.49 (C-9), 59.37 (C-7) and 49.36 (C-10).The 5 carbons of the pyridine ring contributed to signals at d 155.01(C-2), 143.04 (C-5), 141.47 (C-6) and 139.22 (C-4, C-3). The methyland the hydroxymethyl group attached to the pyridine ring gave riseto signals at 14.75 (C-18) and 59.90 (C-17), respectively. Based onthe MS and NMR data, and with reference to previous studies onthe characterisation of PM-derived Maillard reaction products(Arribas-Lorenzo et al., 2011), the structure of the adduct is thusidentifiedas5-(hydroxymethyl)-2-methyl-4-({[(1E)-2-phenylethylidene]amino}methyl)- pyridin-3-ol.

4. Conclusion

In this study, the activities of a group of water-soluble vitaminsagainst the formation of mutagenic HAs were compared in

chemical models to identify potent inhibitors of HA formation. Sev-eral of them, including VB1, VB3, VB7, VC and PN, were found toeffectively reduce the content of both PhIP and MeIQx in the reac-tion systems. The strong inhibitory effects observed for these vita-mins suggest that antioxidant activity is not likely a keymechanism of action. The activities of some of the potent inhibitorswere further confirmed in fried beef patties. Eventually, PM wasidentified to be the most promising inhibitor. Further mechanisticinvestigation showed that this vitamin inhibits the formation ofPhIP, most probably via direct trapping of phenylacetaldehyde, akey intermediary compound on the pathway of PhIP formation.The proposed action mechanism was further supported by purifi-cation and structural elucidation of the product formed from thereaction between PM and phenylacetaldehyde.

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