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Article history: A number of animal and in vitro studies have demonstrated that cholesterol oxidation products (COPs)

leads to formation of 7a- and 7b-hydroperoxycholesterol or

006),sterolacidsre, it

Similarly, other studies have reported the presence of COPs in

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Contents lists availabl

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Journal of Food Composition and Analysis 21 (2008) 4894951987).7a-OOH and 7b-OOH (Smith, 1996). These hydroperoxidesdecompose rapidly to 7a- and 7b-hydroxycholesterol or 7a-OHand 7b-OH, which are commonly found in foods. Both isomeric

meat and meat products. In general, a close association existsbetween unsaturated fatty acids and cholesterol oxidation; theseevents result in the formation of fatty acid radicals in meat, whichare also likely to lead to cholesterol oxidation (Park and Addis,

Pie et al. (1991) found that the amount of COPs can reach 12%of the total cholesterol during daily cooking in beef, veal and pork.

Corresponding author. Tel.: +522299341469x116; fax: +522299345701x201.

E-mail address: [email protected] (H.S. Garcia).0889-15

doi:10.1identied in nature (Dutta, 2004; Smith, 1996). Cholesteroloxidation can be initiated by abstraction of hydrogen, predomi-nantly at C-7, followed by addition of an oxygen molecule, which

can be suggested that the oxidized lipids, which are formed fromhighly unsaturated fatty acids in marine sh products, may affectthe oxidation of associated cholesterol (Oshima et al., 1993).cytotoxic, mutagenic and carcinogenic (Schroepfer, 2000; OBrienet al., 2000; Ryan et al., 2005). Furthermore, COPs have beenidentied as the primary factor that triggers the atheroscleroticlesion (Garca-Cruset et al., 2002). More than 70 COPs have been

(Espe et al., 2001) and salted-dried shrimp (Sampaio et al., 2contain high levels of COPs. It has been suggested that cholein seafoods is oxidized together with polyunsaturated fattyor PUFA from triacylglycerols during storage in air; therefo1. Introduction

Cholesterol is widely distributed in foods mainly from animalorigin, and it can undergo oxidation in the presence of light,oxygen and during storage, giving rise to the formation ofcholesterol oxidation products or COPs (Paniangvait et al., 1995).While cholesterol is a compound of profound biological impor-tance, some of its oxidized forms or COPs have proven to be

7a-OH and 7b-OH can also undergo dehydration during heating toform 7-ketocholesterol or 7-K (Smith, 1996; Tai et al., 1999).

Processes such as cooking, dehydration and deep-frying, areregarded as some of the main causes of cholesterol oxidation inmost foodstuffs of animal origin (Savage et al., 2002). Some typesof commercially available processed seafood products, such assmall sun-dried sh (Chen and Yen, 1994), boiled and driedanchovies (Oshima et al., 1996), cold-smoked Atlantic salmon2 February 2008

Accepted 3 March 2008

Keywords:

Cholesterol oxidation

Mexican food products

Cholesterol oxides

Dried beef

Machaca

Salted-dried shrimp

Deep-fried pork rinds

Chicharron75/$ - see front matter & 2008 Elsevier Inc. A

016/j.jfca.2008.03.008several COPs have carcinogenic, cytotoxic and mutagenic effects. Although some works have reported

the presence of COPs in a number of dried and deep-fried foods, other high-risk food products have not

yet been evaluated. The aim of this study was to analyze the content of COPs of three widely consumed,

typical Mexican food products: deep-fried pork rinds (chicharron), dried beef (machaca) and sun-dried

shrimp, which are subjected to severe thermal treatments and have relatively high cholesterol content.

Lipids were extracted according to Folchs method, then subjected to cold saponication, puried by

aminopropyl solid-phase extraction, silanized and analyzed by gas chromatography. The most abundant

COPs found in all samples were 7a-hydroxycholesterol, 7b-hydroxycholesterol and 7-ketocholesterol.The total amount of COPs varied from 45 to 57mg/kg in chicharron and machaca, whereas the sun-dried

shrimp showed higher COPs content (131254mg/kg). The extent of cholesterol oxidation of chicharron,

machaca and sun-dried shrimp were 2.3%, 5.1% and 14.5%, respectively.

& 2008 Elsevier Inc. All rights reserved.Received 20 May 2007

Received in revised formare the primary factor that triggers the atherosclerotic lesion. Furthermore, it has been recognized thatOriginal Article

Cholesterol oxidation in traditional Mexfood products

Ida Soto-Rodrguez a,b, Perla J. Campillo-VelazquezMara T. Rodrguez-Estrada c, Giovanni Lercker c, Ha UNIDA, Instituto Tecnologico de Veracruz, Mexicob Facultad de Bioanalisis, Universidad Veracruzana, Mexicoc Dipartimento Scienze degli Alimenti, Universita` di Bologna, Italy

a r t i c l e i n f o a b s t r a c t

journal homepage: ww

Journal of Food Comll rights reserved.an dried and deep-fried

Jorge Ortega-Martnez a,o S. Garcia a,

e at ScienceDirect

lsevier.com/locate/jfca

sition and Analysis

formation in raw pork was lower than in cooked pork portions.

Sigma-Aldrich were: lup-20(29)-ene-3b,28-diol (betulin) used as

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ompinternal standard for COPs and cholesterol quantication,5a-cholestane used as standard for SPE recovery of COPs,cholest-5-en-3b-ol (cholesterol), cholest-5-en-3b-ol-7-one (7-ke-tocholesterol), 5a,6a-epoxy-cholestan-3b-ol (5,6a-epoxycholester-ol), 5b,6b-epoxy-cholestan-3b-ol (5,6b-epoxycholesterol),cholestan-3b,5a,6b-triol (cholestanetriol), cholest-5-en-3b,7b-diol(7b-hydroxycholesterol), cholest-5-en-3b,20a-diol (20a-hydroxy-cholesterol) and cholest-5-en-3b,25-diol (25-hydroxycholesterol).The standard cholest-5-en-3b,7a-diol (7a-hydroxycholesterol)was supplied by Steraloids (Newport, CT, USA).

Solid-phase extraction (SPE) cartridges (500mg amino propylstationary phase/3mL) were purchased from Alltech (MountainView, CA). The silylation agent was a mixture of dried pyridine,hexamethyldisilazane and trimethylchlorosilane (all Sigma pro-ducts) in a ratio of 5:2:1 by volume. Methanolic HCl (3M) waspurchased from Supelco (Mexico City).

2.3. MethodsThree samples of machaca (M1, M2 and M3) were purchasedfrom local markets in the Mexican cities of Chihuahua andHermosillo. Chicharron were obtained from two small producerfacilities and one larger plant at Veracruz (Mexico). According tothe manufacturer, the latter sample of chicharron (marked as CH3)was deep-fried in vegetable oil, whereas the other two sampleswere deep-fried in lard (CH1 and CH2). Three samples of sun-dried shrimp (SH1, SH2 and SH3) were purchased from localmarkets in the cities of Tepic and Veracruz (Mexico). All sampleswere packed in plastic bags under vacuum and stored at 18 1Cuntil analysis.

2.2. Reagents, solvents and standards

Analytical grade chemicals were purchased from Sigma-Aldrich (Mexico City, Mexico). COPs standards supplied byChen and Yen (1994) reported the presence of several COPs invarious small sh, which were dehydrated under direct sunlightwithout special packaging during storage or retail.

Considering the above reports, it is necessary to assess thedegree of cholesterol oxidation in animal-derived food productsthat are subjected to severe thermal treatments. The aim of thiswork was to evaluate the extent of cholesterol oxidation andquantify the content of individual COPs in three widely consumed,traditional Mexican food products of animal origin: dried-beef(machaca), sun-dried-shrimp and deep-fried-pork rinds (chichar-ron). These products were also chosen because their preparationprocedures make them prone to oxidation. Mexico is an importantmeat and shrimp producer and consumer; in fact, during 2005,pork, beef and shrimp productions were of 1 billion tons, 1.6billion tons, and 134,755 tons, respectively (COFEMER, 2005;INEGI, 2004).

2. Materials and methods

2.1. SamplesKowale et al. (1996) reported that COPs in mutton increasedduring cooking. Monahan et al. (1992) found that the rate of COPs

I. Soto-Rodrguez et al. / Journal of Food C4902.3.1. Moisture

Moisture was measured according to the method 950.46 formoisture in meat as described by the AOAC (1990).2.3.2. Determination of water activity

Water activity (aw) was measured with an Aqualab automaticanalyzer Series 3TE (Pullman, WA) at 22 1C.

2.3.3. Lipid extraction

Lipid extraction was performed according to a modiedversion (Boselli et al., 2001) of the method of (Folch et al., 1957).Samples (5 g) were minced, weighed and homogenized with200mL of a chloroform:methanol solution (1:1, v/v) for 3min in ascrew-cap glass bottle. The bottle was placed in a water bath at60 1C for 20min before adding 100mL more of chloroform. After a3-min homogenization, the entire content was ltered throughWhatman No. 1 paper lter. The ltrate was mixed thoroughlywith 100mL of a 1M KCl solution and left overnight at 4 1C forproducing phase separation. The lower phase was collected anddried in a vacuum evaporator. The fat content was determinedgravimetrically. Two replicates of the lipid extraction procedurewere performed per sample.

2.3.4. Cold saponication

Cold saponication was performed according to Sander et al.(1989). A 200mg lipid portion of the Folch extract was added with12.5mg of betulin for both COPs and cholesterol quantication. Tenmilliliters of 1N KOH solution in methanol were added andmanually shaken until the mixture became free of dispersed fatparticles. The cold saponication was performed at roomtemperature for 20h, after which 10mL of distilled water wereadded. The saponied mixture was then transferred to aseparation funnel. For the extraction of the unsaponiable matter,10mL of diethyl ether were added, shaken and the diethyl etherfraction was then separated; the extraction with diethyl ether wasrepeated twice. The three portions of diethyl ether were mixedand washed with 5mL of 0.5N KOH and 5mL of saturated NaCl.Finally, the extracts were ltered using Whatman No. 1 paperlter through a bed of anhydrous sodium sulfate, and laterconcentrated in a vacuum evaporator. The unsaponiable extractwas dissolved in 1mL of hexane:isopropanol (4:1, v/v), fromwhich 100 and 900 mL were used for the determination ofcholesterol and COPs, respectively.

2.3.5. Determination of cholesterol by gas chromatography (GC)

For the determination of cholesterol, 1/10 of the unsaponiablematter collected was subjected to silylation (Sweeley et al., 1963)by adding 0.1mL of the derivatizing mixture (pyridine:hexa-methyldisilazane:trimethylchlorosilane, 5:1:2, v/v/v) at 40 1C for15min, dried under nitrogen stream and dissolved in 100 mL of n-hexane. One microliter of the silylated solution was injected intoan HP6890 gas chromatograph (Hewlett-Packard Co., Wilmington,DE), equipped with a split-splitless injector and a ame ionizationdetector (FID). A HP-1 fused-silica capillary column (30m0.32mm i.d.0.25mm lm thickness) supplied by Hewlett-Packardcoated with 100% dimethyl-polysiloxane, was used. Oven tem-perature was programmed from 180 to 300 1C, at a rate of3 1C/min. Nitrogen was used as carrier gas at a ow rate of 3mL/min; the split ratio was 1:40. The injector and FID temperatureswere both set at 300 1C.

2.3.6. Determination of cholesterol oxidation products (COPs) by gas

chromatography (GC)

For determination of COPs, the remaining 9/10 of theunsaponiable matter were taken to dryness, resuspended in100 mL of hexane:ethyl acetate (95:5, v/v) and puried by SPE

osition and Analysis 21 (2008) 489495(Rose-Sallin et al., 1995). The unsaponiable extract was loadedinto the SPE cartridge, which had been previously equilibratedwith 3mL of hexane. The cartridge was eluted with the following

solvent sequence: 6mL of hexane:ethyl acetate (95:5, v/v); 10mLof hexane: ethyl acetate (90:10, v/v) and 10mL of acetone. COPseluted with the acetone fraction, which was collected andsilanized (Sweeley et al., 1963), dried under nitrogen stream and

determine statistical differences among samples (po0.05). Sta-tistical analysis of the data was performed by Minitab v. 14software (State College, PA).

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dy

M

I. Soto-Rodrguez et al. / Journal of Food Composition and Analysis 21 (2008) 489495 491dissolved again in 100 mL of n-hexane. One microliter of thesilylated COPs was injected into the GC under the same conditionsas reported for the determination of total cholesterol.

2.3.7. Identication and quantication of cholesterol and COPs

Peak identication of cholesterol and COPs were carried out bycomparing the peak retention times with those of the commercialstandards and by spiking the samples with a small amount of aCOPs standard mixture.

The SPE recovery of each COP was calculated by using 5a-cholestane as reference standard. Ten milligrams of 5a-cholestaneand 10mg of each COP were dissolved in 100 mL of hexane:ethylacetate (95:5, v/v) and puried by SPE as described above. TheCOPs recoveries were: 7a-hydroxycholesterol, 95%; 7-ketocholes-terol, 91%; 5,6a-epoxycholesterol, 91%; 5,6b-epoxycholesterol,90%; cholestanetriol, 90%; 7b-hydroxycholesterol, 90%; 20a-hy-droxycholesterol, 91% and 25-hydroxycholesterol, 90%.

Quantication of cholesterol and COPs from GC data wasperformed by using relative response factors (Guardiola et al.,2004).

RRF WXSAISWISAXSwhere RRF is the relative response factor; WXS is the COPs orcholesterol standard weight; WIS is the internal standard weight;AXS is the COPs or cholesterol standard area and AIS is the internalstandard area. Betulin was used as internal standard for bothcholesterol and COPs quantication.

2.3.8. Fatty acids analysis by gas chromatography

Fatty acids methyl esters were prepared from the productsaccording to Ortega et al. (2004): 50mg of each fat extract weremixed with 1mL of 0.2M methanolic HCl. This mixture washeated to 60 1C for 4h and then 200 mL of distilled water wereadded. The resulting mixture was extracted with 2mL ofn-hexane, dried with anhydrous sodium sulfate and centrifugedat 4000g for 2min. One microliter of the methylated preparationwas injected into a HP6890 gas chromatograph (Hewlett-Packard,Wilmington, DE), equipped with a Supelco SP-2560 capillarycolumn (100m0.25mm i.d.0.2mm lm thickness) that wascoated with 100% bis-cyanopropyl polysiloxane (Supelco Inc.,Bellafonte, PA). Injector and FID temperatures were set at 220 and230 1C, respectively. The temperature program was as follows:starting at 100 1C, heating to 180 1C at 20 1C/min, followed byheating from 180 to 220 1C at 15 1C/min. The nal temperature(220 1C) was held for 30min.

2.3.9. Statistical analysis of the data

The data are reported as mean values of four replicates (n 4)of each analytical determination, unless otherwise stated. Meanand standard deviation of the different analytical parametersdetermined in each sample, are shown in Tables 27. One-wayanalysis of variance (ANOVA) was performed. Tukeys honestsignicant multiple comparison test was carried out, in order to

Table 1Moisture and water activity of the three typical Mexican food products of this stu

COPs Chicharron (CH1) (mean7SD)Moisture (g/100g) 1.6670.08 1aw 0.37070.002 0achaca (CH2) (mean7SD) Sun-dried shrimp (CH3) (mean7SD)3. Results and discussion

The three typical Mexican food products had average moisturecontent and water activities (aw) as depicted in Table 1. Chicharron,is prepared by deep-frying of pork rinds in lard, and oftenconsumed with other portions of deep-fried pork meat, corntortillas and chili sauce. Chicharron from lots 1 and 2 corre-sponded to samples that were traditionally prepared, using lard atfrying temperatures between 140 and 175 1C; in contrast, lot 3 wasproduced in a larger-scale facility, which used vegetable oilinstead of lard, but there was no way to know the fryingtemperature used.

The amount of cholesterol determined in each of the three lotsof chicharron was signicantly different as shown in Table 2.Bragagnolo and Rodrguez-Amaya (2002) observed levels ofcholesterol of 49mg/100 g in pork loin and 33mg/100 g in backfat, on adult animals. Other parameter that could have inuencedthe concentrations of cholesterol is the loss of moisture duringcooking. Kowale et al. (1996) and Echarte et al. (2001) reportedthat when pork loins were deep-fried, a signicant loss of waterand cholesterol was measured. It is then suggested that differentfrying conditions could cause differences in cholesterol contentsin the samples of chicharron.

When meat is cooked, undesirable changes, such as loss ofnutrients, modications in the protein structure and especiallylipid oxidation, may occur (Rodrguez-Estrada et al., 1997).Cholesterol, which can be found in different concentrations infoods of animal origin, can actually be oxidized during theprocessing or storage of food, generating COPs. Six COPs wereidentied in the three lots of chicharron (Table 2), that have beenpreviously reported in other types of deep-fried meat (Paniangvaitet al., 1995). The differences in the concentrations of the COPs inthe samples of chicharron can be attributed to several factors, suchas animal breed, age, type of feed, fat/muscle ratio, slaughteringconditions, holding period, storage conditions and packaging(Larkeson et al., 2000). In addition, the frying conditions (timeand temperature) used for the preparation of chicharron, couldhave been a key factor for the development of different levels ofCOPs. Although Echarte et al. (2001) observed that the increase inthe frying temperature did not promote a rise in the oxidativeprocess of loin fried at 160, 170 and 180 1C, the authors concludedthat the frying process could have contributed to the oxidation ofcholesterol. On the other hand, although samples CH1 and CH2were obtained immediately after frying, it must be consideredthat this meat product is usually exposed to light for long periodsduring retailing, so the product can photo-oxidize and developeven higher levels of COPs when it reaches the consumer (Kim andNawar, 1993). In fact, it is well known that cholesterol oxidation inmeat is favored by exposure to light (Boselli et al., 2005).

Consistent with a previous report by Lercker and Rodrguez-Estrada (2000), 7-ketocholesterol was the most abundant COP inall three lots of chicharron. The other COPs in lots CH1 and CH2were present in approximately the same ratio (see Table 2).3.2370.09 25.0770.20.69070.001 0.74070.007

Since machaca samples were obtained as a nished product, itwas not possible to determine the content of cholesterol of theraw meat from which the machaca was prepared. However, the

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Table 4COPs content (mg/100g machaca), cholesterol (mg/100 g machaca) and extent of

cholesterol oxidation (%) of machaca samples

COPs M1 (mean7SD) M2 (mean7SD) M3 (mean7SD)

7a-Hydroxycholesterol 0.3470.04a 0.4870.08b 0.5670.04c

7b-Hydroxycholesterol 0.7570.01a 0.3570.05b 0.8970.03c

5,6b-Epoxycholesterol 0.4970.02a 0.7770.08b 0.3170.04c

5,6a-Epoxycholesterol 0.6770.05a 0.8470.08b 0.5270.03c


7-Ketocholesterol 2.270.10a 1.470.1b 2.670.1c

Cholestanetriol 0.1770.01a 0.1270.01b 0.2970.01c

Total COPs 4.8970.2 4.2670.5 5.5070.3Cholesterol 95.1976.70a 72.1671.84b 120.28 77.20c

Oxidized cholesterol (%) 5.13 5.90 4.57

Each value corresponds to the mean of four replicates7the standard deviation(SD) is reported. Means in the same row followed by different superscripts are

signicantly different according to analysis of variance and Tukeys multiple mean

comparison test (po0.001).

ompHowever, in lot CH3 this ratio was not kept with respect to 7b-hydroxycholesterol, 5,6a-epoxycholesterol and cholestanetriol.This probably could be attributed to the frying method employed,which used vegetable oil.

Since phytosterols are also prone to oxidation (Guardiola et al.,2004), it would be interesting to further evaluate the presence oftheir oxidation products (POPs) in chicharron prepared withvegetable oil, such as sample CH3. An elaborated analyticalmethod for the simultaneous chromatographic separation ofcomplex mixtures containing COPs and POPs in this type ofproduct, will become necessary.

The total COPs content detected in chicharron ranged from 4.5to 5.7mg/100 g (1.121.42104mol as 7-ketocholesterol) ofsample, which may represent an important toxicological risk,because the minimum amount of 7-ketocholesterol for a corre-sponding negative biological activity of the other COPs (aboutone-fth of that of 7-ketocholesterol) (Caboni et al., 1989) hasbeen estimated as in 107 molar basis (Sevanian, 1991). In fact,negative biological effects of COPs seem to manifest at lowerconcentrations (Sevanian, 1991, Schroepfer, 2000; Ryan et al.,2005) if compared with the daily intake that this food productwould actually represent among Mexican population.

Regarding the extent of cholesterol oxidation (see Table 2), nosignicant differences were found among the 3 lots of chicharron.Although several authors have found a direct relationshipbetween time and temperature of processing (Kim and Nawar,1993), the products analyzed in such work were heated at 140 and

Table 2Individual COPs content (mg/100g chicharron), cholesterol (mg/100g chicharron)

and extent of cholesterol oxidation (%) of chicharron samples

COPs CH1 (mean7SD) CH2 (mean7SD) CH3 (mean7SD)







Total COPs 5.7270.17a 5.2370.14b 4.4870.17c

Cholesterol 245.073.5a 228.072.2b 197.3073.0c





I. Soto-Rodrguez et al. / Journal of Food C492175 1C for samples CH1 and CH2, respectively, prepared undercontrolled laboratory conditions and such variables could not beused to explain the levels of cholesterol oxidation. According toreports by Echarte et al. (2001, 2003), this percentage of oxidationcould also be attributed to the methods of cooking, like deep-frying or microwave.

Seven major fatty acids were identied in chicharron samples(Table 3). Basically, the same content of saturated, monounsatu-rated and polyunsaturated fatty acids were found in CH1 and CH2samples. However, in sample CH3, the content of C18:1, C18:2, andC18:3 were the highest of all three. These results could beattributed to the absorption of vegetable oil used for fryingsample CH3, while samples CH1 and CH2 were immersed in lardas frying medium.

Another typical product from northern Mexico is machaca. Toproduce machaca, fresh beef is usually frozen and held at 8 to12 1C overnight; the meat is then thawed, sliced and sun oroven-dried for approximately 8h at 6895 1C. Once the dehydra-tion process has been completed, the meat pieces are salted,garlic-seasoned, nely minced, packed and stored at roomtemperature.Table 3Main and total fatty acids (FA) of chicharron samples, expressed as % relative area

Fatty acid CH1 (%) CH2 (%) CH3 (%)

Myristic acid (C14:0) 1.570.1a 1.470.0b n.d.Palmitic acid (C16:0) 25.770.8a 23.670.0b 9.670.1c

Palmitoleic acid (C16:1) 2.970.1a 2.870.0a 1.070.0b

Stearic acid (C18:0) 12.670.4a 10.370.0b 4.170.0c

Oleic acid (C18:1) 44.771.7a 45.670.1a 57.770.2b

Linoleic acid (C18:2) 11.170.3a 14.970.0b 20.770.1c

Linolenic acid (C18:3) 1.570.0a 1.470.1a 6.970.1bPSFA 39.8 35.3 13.7PMUFA 47.6 48.4 58.7PPUFA 12.6 16.3 27.6

MUFA/SFA 1.9 1.4 4.3

PUFA/SFA 0.3 0.5 2.0

Each value corresponds to the mean of four replicates7the standard deviation.Means in the same row followed by different letters are signicantly different

according to analysis of variance and Tukeys multiple mean comparison test

(po0.001).Abbreviations: n.d., not detected; SFA, saturated fatty acids; MUFA, monounsatu-

rated fatty acids; PUFA, polyunsaturated fatty acids.

osition and Analysis 21 (2008) 489495average content of cholesterol of the nished product was96.075.3mg/100 g (see Table 4), which is consistent with datareported for charqui, a type of dehydrated meat (Kesava et al.,1996; Kowale et al., 1996).

Seven major COPs were found in the three lots of machaca(Table 4) and these oxidation products had been previously foundin other types of dry meats (Torres et al., 1994; Kesava et al., 1996;Kowale et al., 1996). It can be noted in Table 4 that the levels of7-ketocholesterol were higher that those of the other COPs foundin the product because the starting oxidized products, 7-hydro-peroxycholesterol and 7-hydroxycholesterol, are transformed intothe more stable 7-ketocolesterol by dehydration or dehydrogena-tion, respectively (Smith, 1996). A similar effect was found inother studies in which meat has been salted and dried (Torres etal., 1994). Probably, temperature to which the meat is subjectedfor the preparation of machaca plays an important role in theformation of COPs. In previous studies, Larkeson et al. (2000)observed an increase in COPs formation when precooked meatwas subjected to re-heating; or when it was kept frozen for longperiods (Pie et al., 1991; Hwang and Maerker, 1993; Vicente andTorres, 2007).

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ompIn the three samples of machaca, the proportion of each COPwith respect to the total COPs content was signicantly different(Table 4). According to other authors, this variation could be dueto various factors, such as aw, as the decrease of this parameterincreases lipid oxidation in meat (Torres et al., 1994; Sun et al.,2002). Additionally, it has been described that lipid oxidation isinitiated by a large number of molecules (including some reactiveoxygen species) to form peroxy radicals; these radicals thenabstract a hydrogen from fatty acids to form acyl radicals andhydroperoxides, which in turn enhance cholesterol oxidation(Smith, 1996). Thus, the type of fatty acid is important, because itaffects the generation of hydroperoxides that can oxidize thecholesterol, giving rise to different COPs (Smith, 1996; Gandemer,2002). Moreover, the control of lipid oxidation and the balancebetween volatile compounds arising from lipid oxidation shouldbe controlled to produce high quality dry-cured meat productslike machaca (Gandemer, 2002).

The total COPs detected in machaca ranged from 4.2670.50 to5.5070.30mg/100 g of sample, which represent an appreciabletoxicological risk (Caboni et al., 1989; Sevanian, 1991; Schroepfer,2000; Ryan et al., 2005). Regarding the extent of cholesterol

Table 5Main and total fatty acids of machaca samples, (FA) (expressed as % relative area

Fatty acid M1 (%) M2 (%) M3 (%)

Myristic acid (C14:0) 3.270.0a 2.370.0b 2.770.0c

Palmitic acid (C16:0) 27.470.1a 28.570.1b 28.370.1b

Palmitoleic acid (C16:1) 4.470.2a 4.170.0b 4.570.0a

Stearic acid (C18:0) 17.270.2a 17.270.1a 15.570.2b

Oleic acid (C18:1) 43.970.2a 41.970.2b 41.770.1b

Linoleic acid (C18:2) 3.970.1a 6.070.2b 7.370.1c

Linolenic acid (C18:3) n.d. n.d. n.dPSFA 47.8 48.0 46.5PMUFA 48.3 46.0 46.2PPUFA 3.9 6.0 7.3

MUFA/SFA 1.01 0.96 0.99

PUFA/SFA 0.08 0.12 0.16



comparison test (po0.001).Abbreviations: n.d., not detected; SFA, saturated fatty acids; MUFA, monounsatu-

rated fatty acids; PUFA, polyunsaturated fatty acids of machaca samples.

I. Soto-Rodrguez et al. / Journal of Food Coxidation (5.2%), signicant differences were not detected amongthe three samples of machaca (see Table 4).

Seven major fatty acids were identied in machaca samples(Table 5) showing no signicant changes in the ratios of fattyacids. These results are consistent with a study on dry reindeermeat (Sampels et al., 2004), where smaller PUFA content afterdrying, as well as smaller PUFA/SFA and UFA/SFA ratios, weresuggested as indicators of an oxidation process.

Sun-dried seafood products are also common in Mexico,especially dried shrimps, from either coast. Processing consistsof fresh shrimps boiled in brine (5% NaCl) for 510min and thendried by placing them on concrete plates for 310 days underdirect sunlight; the dried shrimps are then packed and handled atroom temperature. The brine is used only once for each portion ofcooked shrimp. The range of cholesterol content in the 3 samplesof shrimps (Table 6) was from 110.075.4 to 149.279.2mg/100 g.These data was similar to the range reported by Sampaio et al.(2006) (148.33758.44191.26717.01mg/100 g). Variations in thecholesterol level of shrimps can be explained by several factors,such as age, water temperature, season, geographical location, andmoisture content of the product (Sampaio et al., 2006). On theother hand, the processing technology used for the production ofsun-dried shrimp can induce cholesterol oxidation, since salting,direct light-exposure and large surface contact with oxygen forrelative long time periods, are known as lipid pro-oxidant factors.Table 6 reports the amount of the main COPs detected in sun-dried shrimps. Eight major COPs were found in the three lots ofshrimps. Sampaio et al. (2006) found only 7-K, 25-OH, 7a-OH and7b-OH in 50 batches of shrimps. Neither cholestanetriol nor epoxyderivatives were detected in that study, because the COPs analysiswere performed by a HPLC-UV method that is unable to detectthese oxidation products.

Sampaio et al. (2006) found a total COPs content that rangedbetween 6.74 and 54.87mg/g in salted-dried shrimps from Brazil,which is smaller than those found in the present study (see Table6). The samples analyzed by Sampaio et al. (2006) contained anoticeable degree of cholesterol oxidation, but only 4 cholesteroloxides were monitored. In the current study, a much higher totalCOPs content (13.0625.40mg/100 g) was found in the same typeof material, but eight different COPs were detected and monitored.

The content of 7-ketocholesterol and 7-hydroxycholesterol(a and b) were greater than the other COPs found in dried shrimps.Probably, this could be related to the formation of large amountsof peroxy radicals (Sampaio et al., 2006), and singlet oxygen byphoto-oxidation, that could favor cholesterol oxidation (Boselliet al., 2005). Peroxy radicals and singlet oxygen attack preferablythe positions 5 and 7 of cholesterol and thus 7-hydroperoxy-

Table 6COPs content, cholesterol content (mg/100g sun-dried shrimp) and extent of

cholesterol oxidation (%) of sun-dried shrimp samples

COPs SH1 (mean7SD) SH2 (mean7SD) SH3 (mean7SD)








25-Hydroxycholesterol 0.1670.03a 0.0970.01b 0.2070.01c

Total COPs 15.9070.02a 13.0670.70b 25.4070.01c

Cholesterol 149.2373.20a 131.1576.40b 110.075.40c





osition and Analysis 21 (2008) 489495 493cholesterol (a and b), and 5a-hydroperoxycholesterol are initiallyformed (Osada et al., 1993; Oshima et al., 1993). These hydroper-oxides are unstable and easily converted into 7-hydroxy-cholesterol (a and b) and 7-ketocholesterol (Osada et al., 1993).A similar oxidation mechanism has been suggested for other foodproducts that contain considerable levels of unsaturated fattyacids, such as spray-dried egg yolk (Nourooz-Zadeh and Appelqvist,1987). In the case of dried shrimp, the formation of peroxyradicals from unsaturated fatty acids might have played animportant role as the average value of PUFA was 41.6% (Table 7).

Another factor that may have favored cholesterol oxidation ofthe sun-dried shrimp is the pro-oxidative effect of salt. Sampaioet al. (2006) suggested that the culinary processes that usedimmersion of the shrimp in brine may collaborate to lipidoxidation.

The total COPs content detected in dried shrimps represent animportant toxicological risk (Caboni et al., 1989; Sevanian, 1991;Schroepfer, 2000; Ryan et al., 2005). The oxidation of cholesterolwas from 9.95% to 23% with respect to intact cholesterol (seeTable 6), which is one of the highest reported and should raiseconcerns about the consumption of this product. The COPs/cholesterol ratio (about 0.1) is similar to values reported for saltedshrimp (Sampaio et al., 2006).

ARTICLE IN PRESS

omp4. Conclusions

The present study shows that some traditional Mexican foods

Table 7Main and total fatty acids of sun-dried shrimp samples,(FA) expressed as % relative

internal area

Fatty acid SH1 (%) SH2 (%) SH3 (%)

Myristic acid (C14:0) 1.970.0a 1.470.0b n.d.Palmitic acid (C16:0) 19.870.3a 24.470.3b 22.670.0c

Palmitoleic acid (C16:1 n7) 5.270.1a 3.970.0b 6.170.0c

Hexadecadienoic acid (C16:2 n4) 5.370.1a 1.670.6b 3.070.6c

Hexadecatetraenoic acid (C16:4 n1) 0.770.0a 0.370.0b n.d.Stearic acid (C18:0) 10.870.0a 9.770.1b 11.970.6c

Oleic acid (C18:1 n9) 7.870.1a 17.670.3b 10.970.6b

cis-Vaccenic acid (C18:1 n7) 6.670.1a 7.270.2b 7.670.1b

Linoleic acid (C18:2 n6) 1.770.2a 10.670.1b 2.870.1c

Linolenic acid (C18:3 n3) 2.370.1a 0.970.1b n.d.Octadecatetraenoic acid (C18:4 n3) 1.670.4a 0.770.1b n.d.Arachidonic acid (C20:4 n6) 9.270.1a 2.370.0b 8.470.1c

Eicosatetraenoic acid (C20:4 n3) 0.770.1a 0.470.1b n.d.Eicosapentaenoic acid (EPA, C20:5 n3) 15.070.2a 9.470.1b 16.670.5c

Docosapentaenoic acid (C22:5 n3) 2.070.0a 1.370.0b n.d.Docosahexaenoic acid (DHA, C22:6 n3) 9.770.1a 8.270.1b 10.270.1cPSFA 32.5 35.5 34.5PMUFA 19.6 28.7 24.6PPUFA 48.2 35.7 41.0

MUFA/SFA 0.60 0.80 0.71

PUFA/SFA 1.48 1.00 1.10



comparison test (po0.001).Abbreviations: n.d., not detected; SFA, saturated fatty acids; MUFA, monounsatu-

rated fatty acids; PUFA, polyunsaturated fatty acids of machaca samples.

I. Soto-Rodrguez et al. / Journal of Food C494(chicharron, machaca and sun-dried shrimps) have signicantamounts of COPs. Considering that these food products are widelyconsumed in Mexico, a large part of the population is thusexposed to COPs, and this fact could be associated to the incidenceof atherosclerosis and other ailments. Development of moresuitable processing and storage procedures is, therefore, necessaryin order to reduce the amount of COPs in these Mexican dried anddeep-fried food products.

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ARTICLE IN PRESS

I. Soto-Rodrguez et al. / Journal of Food Composition and Analysis 21 (2008) 489495 495

Cholesterol oxidation in traditional Mexican dried and deep-fried food productsIntroductionMaterials and methodsSamplesReagents, solvents and standardsMethodsMoistureDetermination of water activityLipid extractionCold saponificationDetermination of cholesterol by gas chromatography (GC)Determination of cholesterol oxidation products (COPs) by gas chromatography (GC)Identification and quantification of cholesterol and COPsFatty acids analysis by gas chromatographyStatistical analysis of the data

Results and discussionConclusionsReferences

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