In vitro digestion/Caco-2cell model to estimate cadmium and lead bioaccessibility/bioavailability in two vegetables:The influence of cooking and
additives
Jin Fu,Yanshan Cui ⇑
College of Resources and Environment,University of Chinese Academy of Sciences,Beijing 100049,
China
a r t i c l e i n f o Article history:
Received 29January 2013Accepted 11June 2013
Available online 18June 2013Keywords:
Bioavailability Lead
Cadmium Vegetable
In vitro methods
a b s t r a c t
The estimation of heavy metal bioaccessibility and bioavailability in vegetables is helpful for human health risk assessment.Using an in vitro digestion/Caco-2cell model,the bioaccessibility and bioavail-ability of cadmium (Cd)and lead (Pb)in raw/cooked pakchoi (Brassica rapa L.,Chinensis Group )and Mal-abar spinach (Basella rubra L.)were studied.The effect of the addition of iron,calcium and acetic
acid to the samples was also determined.The results indicated that Cd bioaccessibility was higher in the gastric phase and Pb bioaccessibility was higher in the small intestinal phase.Cadmium and Pb bioavailability were 11.2%and 9.4%in the raw vegetables,respectively,and found to be higher significantly than the cooked vegetables with 6.1%for Cd and 3.2%for Pb.The results showed that it will be overestimating the risk of Pb and Cd based on the data of raw vegetables ingestion.Using bioavailability values,average Cd and Pb daily intake by adult were 23%and 28%respectively,of the base bioaccessibility values.Our study will be better understanding the possible health risks of some vegetables base on the bioaccessi-bility or bioavailability.
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1.Introduction
Heavy metal contaminated soils are found all over the world and a major concern is the presence of heavy metals in agricultural soils (Intawongse and Dean,2006;Khan et al.,2008).When grown in polluted soil,plants take up water,nutrients and heavy metals that then may enter the food chain (Cui et al.,2004;Williams et al.,2009).Approximately 50%of lead (Pb)taken up by human is through food,and half of that originates from plants (Nasreddine and Parent-Massin,2002).Human exposure t
o Pb and cadmium (Cd)can have chronic effects on several organs (WHO,2010a,b ).Additional evidence supports the hypothesis that people exposed to Cd have an increased possibility of having cancer (Amiard et al.,2008;Vinceti et al.,2007).
It has been reported that increased vegetable consumption is associated with a decreased cancer risk (Ferruzzi and Blakeslee,2007);therefore,increased consumption is recommended.The concentration of heavy metals in vegetables differs between spe-cies.Vegetables such as spinach,lettuce,tomato,radish and zuc-chini can accumulate certain Cd or Pb in their tissues (Cobb et al.,2000;Mattina et al.,2003).In general,heavy metal uptake in vegetables is directly proportional to the metal concentration in the contaminated soil.However,the total amount of heavy metals in the vegetables does not always reflect the amount that is available to the consumer.Therefore,there is an urgent need to determine the bioaccessibility and bioavailability of heavy met-als in vegetables to assess the human health risk.Bioaccessibility (BAC)describes the fraction of a contaminant that is ingested with food and released from its matrix into the digestive juice chime;it has the potential to be absorbed by the intestines during digestion.Bioavailability (BAV)is the proportion of a contaminant that is in-gested with food and absorbed by the intestine;it has the potential to reach the systemic circulation and exert toxic effects (He et al.,2010).
In order to estimate the bioaccessibility and bioavailability,sev-eral in vitro digestion methods have been proposed.These methods are an alternative to in vivo methods due to their lower cost,time and energy saving properties and their independence from physio-logical factors (Sahuquillo et al.,2003;Tang et al.,2006).Depend-ing on the physiological conditions used in the test and its ability to predict bioavailability,the physiologically based extraction test (PBET)have been used to estimate the bioaccessibility of heavy metals in soil and sea food (Amiard et al.,2008;Cabañero et al.,2007;Sloth and Julshamn,2008),the bioaccessibility of nutrient elements and carotenes in plants,and some factors that may affect bioaccessibility and bioavailability (Hemalatha et al.,2007;Kulkar-ni et al.,2007;Ornelas-Paz et al.,2008;Ryan et al.,2008).The Caco-2cell has been used in the development of in vitro research and mimics the process of intestinal cell retention and transport.
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Corresponding author.Tel.:+861088256467.
E-mail address:cuiyanshan@ucas.ac (Y.Cui).
It also offers a more reliable approximation of the in vivo condi-tions to estimate bioavailability at the intestinal level(Ekmekcio-glu,2002).However,some limits of Caco-2for assessing the bioavailability were also reported(Trapecar and Cencic,2012).Re-cently,the in vitro digestion method combined with the Caco-2cell model was established and some validation studies were per-formed(Dhuique-Mayer et al.,2007;Yun et al.,2004).And the method has been widely used to estimate the bioavailability of minerals and biologically active compounds in foods(Dhuique-Mayer et al.,2007;Tako and Glahn,2010;Tako et al.,2011).
Only a few studies have investigated the bioaccessibility and bioavailability of heavy metals in vegetables.Intawongse and Dean (2006,2008)found differences in the bioaccessibility of heavy met-als in lettuce,spinach,carrots and radishes.There is still a lack of information on the bioaccessibility and bioavailability of Pb and Cd in vegetables based on in vitro digestion and the Caco-2cell model.
Pakchoi and spinach are very common and widely consumed vegetables in China,which has a quick growth rate(harvest in about2–3months from sowing).Recently,Yan et al.(2009)mea-sured Cd levels in pakchoi grown in a metal contaminated area and then found that the Cd in pakchoi have a high contribution to the total daily intake(TDI)of Cd for human.In this study,the in vitro digestion method and the Caco-2cell model were used to study the bioaccessibility and bioavailability of Cd and Pb in
pak-choi and Malabar spinach.The aims of this study were to(1)deter-mine the bioaccessibility and bioavailability of Cd and Pb in the raw and cooked forms of these two vegetables;(2)determine the effect of additives(iron,calcium and acetic acid,which are coming from other food or as seasonings in Chinese cooking)on the bioac-cessibility and bioavailability of the heavy metals in the vegeta-bles;and(3)assess the health risk of the two heavy metals in the vegetables.
2.Materials and methods
2.1.Reagents
Enzymes and bile salts were purchased from Sigma Chemical(St.Louis,MO, USA),including pepsin(Porcine;catalogue No.P-7000),pancreatin(Porcine;cata-logue No.P-1750),and bile extract(porcine;catalogue No.B-8631).The following reagents were ultrapure or analytical grade:sodium chloride,citric acid,malic acid, lactic acid,glacial acetic acid,hydrochloric acid,sodium bicarbonate,iron trichlo-ride,calcium chloride,acetic acid,and alcohol.Deionized water(18.2M X cm) was made using a super-pure water machine(Pine-tree,Desheng Science and Tech-nology Park,Beijing,China)and was used to prepare the reagents and standards. Before being used,all glassware was treated with10%(v/v)HNO3for at least24h and then rinsed3times with deionized water.
For the batch extraction method,a commonly used gastric solution was used,as described by Ruby et al.(1996).We added2.00g of citric acid,2.00g of malic acid, 1.68mL of lactic acid and2mL of glacial acetic acid into a4L solution of0.15M NaCl.The pH was adjusted to1.5with12M HCl.Ten grams of porcine pepsin was then added to the gastric solution.Bile and pancreatic juice were prepared in the sample tubes with2.5g salt bile extract and0.75g pancreatin dissolved in 25mL of0.1mol LÀ1NaHCO3(Glahn et al.,1998).
Inorganic iron was obtained by dissolving4.5g iron trichloride(FeCl3)in25mL of water.0.5675M calcium chloride monohydrate(CaCl2ÁH2O)solution was used as an external source of calcium.
2.2.Samples
Pakchoi(Brassica rapa L.Chinensis Group)and Malabar spinach(Basella rubra L.) were planted in a heavy metal contaminated soil for60days.The soil with a total Pb concentration of5576mg kgÀ1and Cd concentration of38.4mg kgÀ1was collected from barren land contaminated with Pb/Zn mine tailings in Neimenggu,China.Prior to use,the vegetables were cleaned and homogenized after the roots were removed. Deionized water was used to clean vegetables and stainless tools were used to cut and homogenize the vegetable tissues.Filter paper was used to absorb the water of the washing vegetables and to keep them dry.
Fresh vegetable samples(0.25g)of the vegetables were predigested overnight with2mL of nitric acid in50mL centrifuge tubes(Corning Incorporated,Riverfront Plaza,Corning,New York,USA)at room temperature.This solution was heated in a microwave oven(CEM,Mars5,USA).Each treatment has three replicates.It took 5min to increase the temperature to55°C and it was maintained for10min.The sample was then heated for another5min to75°C and maintained for10min.Fi-nally,the temperature was increased to95°C over5min and maintained for 30min.The digestion solution was then diluted to afinal volume of25mL.Lead and Cd analyses were performed on an inductively coupled plasma mass spectrom-etry(ICP-MS,Agilent Technologies USA,7500a).Instrument operating conditions for ICP-MS are as follows:carrier gas1.05L minÀ1,RF power1300W,atomizer chamber2°C,sampling depth7mm,sample uptake rate0.4mL minÀ1,number of replicate3.A series of calibration standards was prepared from10l g LÀ1to 500l g LÀ1for Pb and Cd by diluting1000mg LÀ1stock Pb or Cd solutions(National Institute of Metrology,China).This standard solution was checked for every10 samples and the results agreed within±10%of the original value.Iron and calcium content were analysed on an inductively coupled plasma optical emission spec-trometer(ICP-OES,PerkinElmer Co.,USA).To ensure that the results obtained for the analyses were accurate,a certified reference material of GBW08505(tea leaves) was determined in the same way.Recovery rates for reference plant samples were between93%and110%.The concentrations of Cd,Pb,Ca,Fe were2.58mg kgÀ1FW
(fresh weight),9.42mg kgÀ1FW,3.18g kgÀ1FW,44.10mg kgÀ1FW respectively for pakchoi,and2.37mg kgÀ1FW,6.34mg kgÀ1FW,2.99g kgÀ1FW,21.90mg kgÀ1FW respectively for Malabar spinach.
2.3.Additives and cooking
Each vegetable was assigned to two balanced groups,cooking and raw.1.5 grams of homogenized vegetable was dipped in4.5mL of ultrapure water.Based on the Chinese dietary reference intakes(CNS,2000),the standard daily calcium and iron intake for Chinese adult is800mg and15mg,0.5mL of external sources of calcium,iron or acetic acid was added to afinal concentration of0.62g LÀ1(4% of the dietary reference intakes),0.38g LÀ1(14%of the dietary reference intakes), and1.67%(30%of the concentration of vinegar),respectively.The addition of acetic acid was simulated using vinegar.The volume of the control(no additives solution added,ultrapure water)was adjusted with ultrapure water in order to be equal to the volume of the treatments with additions(Mateo et al.,2007).Thefirst group of samples was cooked for30min(90°C water bath).The second group was left raw and used to determine metal bioaccessibility.Each treatment was repeated3times.
2.4.In vitro digestion protocol
The bioaccessibility of Pb and Cd in the vegetable samples was determined by the PBET(physiologically based extraction test)method,as previously described by Ruby et al.(1996)The IVG(in vitro gastrointestinal)method proposed by Rodri-guez et al.(1999)also made reference to the PBET.
Briefly,the prepared raw or cooked vegetable samples were subjected to simu-lated gastric digestion by incubating them in25mL gastric solutions(adjusted to a pH of1.5with HCl)for1h in a100rpm shaking incubator at37°C.Aliquots(5mL) were removed by syringe andfilter(0.45l m).And the samples stored at4°C,then the samples were analysed for total Pb and Cd concentrations by ICP-MS.To mimic digestion in the small intestine,1mL of small intestinal juice(0.5mL solution of salt bile extract(100g LÀ1)+0.5mL pancreatin solution(30g LÀ1))was added to the gastric solution after raising the pH to7with saturated NaHCO3.The samples were then placed in the100rpm shaking incubator for4h at37°C.Aliquots of10mL small intestinal solution were extracted and stored at4°C.These samples were then analysed for total Pb and Cd concentrations by ICP-MS and prepared for the measurement of Pb and Cd absorption by Caco-2cells.
In this experiment,each treatment has three replicates,and bioaccessibility was defined as the solubility of Pb or Cd in the simulated gastric or small intestinal solu-tion divided by the total Pb or Cd
in the vegetable.The bioaccessibility(BAC,%)of Pb and Cd in the gastric and small intestinal phases was calculated as follows:
BAC¼ðD IV V IVÞ=ðT S M SÞÂ100ð1Þ
where D IV is the bioaccessible concentration of Pb or Cd in the gastric phase(or the small intestinal phase)(mg LÀ1);V IV is the solution volume in the reaction vessel(L), T S is the total Pb or Cd concentration in vegetable(mgÁkgÀ1);M S is the mass of veg-etable in the reaction vessel(kg).
2.5.Caco-2cell culture and exposure
The Caco-2cell line was obtained from the Institute of Basic Sciences Chinese Academy of Medical Sciences School of Basic Medicine Peking Union Medical Col-lege.The cells(passage number25–30)were grown in25cm2flasks in the presence of Dulbecco’s modified Eagle’s medium(DMEM,Hyclone,USA)supplemented with 20%(v/v)fetal bovine serum(FBS,Hyclone),1%(v/v)non-essential amino-acid (NEAA)and1%antibiotics(complete medium).The cells were incubated at37°C, 5%CO2in a humidified atmosphere and the medium was changed every48h.Mon-olayers were subcultured with a7days passage frequency by treatment with0.25% trypsin–EDTA when they reached a confluence of about80%.To establish the Caco-2 intestinal model,the cells were seeded at
a density of5.0Â104cells cmÀ2on a transwell plate(Millpore24-well Millicell Hangcell Culture Insert,0.4l m pore size
216J.Fu,Y.Cui/Food and Chemical Toxicology59(2013)215–221
membrane,PET;Corning Incorporated,NY,USA).The media was changed every 2days for21days to obtain confluent,differentiated cell monolayers(Salvini et al.,2002;Dhuique-Mayer et al.,2007).Before the Caco-2uptake experiment, the growth medium was removed from each culture well and the cell layer was washed twice with37°C DMEM at pH7.The cellular integrity was checked by mea-suring trans-epithelial electrical resistance with a volt-ohm meter equipped with a chopstick electrode(Millicell ERS-2,Millipore,USA).In order to investigate Pb and Cd BAV in the two vegetables,the intestinal juice was heated for5min at90°C to denature the digestive enzymes thenfiltrated with a0.2l m Supor membrane(PN 4612,Pall Corporation,MI,USA).The sample was then diluted1:3with DMEM.The apical side of the cell monolayers received0.5mL of the dilution,and the basolat-eral side received0.5mL of FBS-free medium.
After4h of incubation(Versantvoort et al.,2005),the media from the apical chamber was harvested.Cell monolayers were washed twice with1mL phosphate buffered saline(PBS)and0.5mL c
oncentrated HNO3was added to the washing solution for24h.The concentrations of the heavy metals in the washing solution were determined by ICP-MS.Lead and Cd retention(cell monolayer)and transport (basal medium)percentages were calculated by the initial amount metals in the solutions minus the amount of metals obtaining from washing solution.Un-spiked cells were used throughout each assay.
In this experiment,each treatment has three replicates,and the bioavailability of the heavy metals was defined as the total amount of Pb or Cd absorbed by the Caco-2cells(retention and transport)from the small intestinal phase divided by the total amount of vegetable Pb or Cd.The bioavailability(BAV,%)of Pb and Cd in the treatment was calculated as follows:
BAV¼ð1ÀðU IV V IVÞ=ðT S M SÞÞÂ100ð2Þ
where U IV is the total concentration of harvested Pb or Cd in the washing solution after Caco-2cell incubation(mg LÀ1);V IV is the washing solution volume(L),T S is the total Pb or Cd concentration in vegetable(mg kgÀ1);M S is the mass of vegetable in the reaction vessel(kg).
2.6.Health risk assessment
The Joint Food and Agriculture Organization of the United Nations(FAO)/World Health Organization(WHO)Expert Committee on Food Additives(WHO,2010b) established a provisional tolerable monthly intake(PTMI)for cadmium at 25l g kgÀ1body weight(BW),which is0.83l g kgÀ1body weight provisional toler-able daily intake(PTDI).Provisional tolerable weekly intake(PTWI)of 25l g Pb kgÀ1bw was established(WHO,2010a),which is3.57l g Pb kgÀ1bw PTDI. The minimum daily consumption of fruit and vegetable is expected to be400g per person(FAO/WHO,2004).The health risk assessment(HRA,l g kgÀ1BW dÀ1)of Pb and Cd in the intestinal phase(HRA1)were calculated as follows:
HRA1¼BACÂW v=W bð3ÞHRA after Caco-2uptake was calculated as follows:
HRA2¼BAVÂW v=W bð4Þ
BAC is the bioaccessibility of Pb or Cd in the small intestinal phase.BAV is bio-availability of Pb or Cd estimated by Caco-2cells.W v(kg daysÀ1)is the total con-sumption of vegetables per adult per day and expected to be400g.W b(kg pÀ1)is the average Chinese adult body weight,63.9kg.The average Chinese children body weight is32.7kg,and consumption of vegetables per day for children is257g(Song et al.,2009).
2.7.Statistical analyses
All statistical analyses were performed using SPSS software(Ver16.0;SPSS,Chi-cago,IL,USA)and Excel2007.All data were reported as the mean or mean with standard errors(SE)from three replicates of each experiment.The means were con-sidered to be significant difference if p values were<0.05.
3.Results
3.1.Bioaccessibility of Cd and Pb in vegetables
In the gastric phase,the average bioaccessibility of Cd for pak-choi was68.2%,higher than the50.0%found in Malabar spinach (Fig.1).The average Cd bioaccessibilities were71.2%and65.2% for cooked and raw pakchoi,respectively,and54.1%and46.0%for
cooked and raw Malabar spinach,respectively.In the small intesti-nal phase,the average Cd bioaccessibility was36.8%and39.1%for the pakchoi and Malabar spinach,respectively,lower than those in the gastric phase(Fig.2).In the gastric phase,the additives did not have a significant effect on the bioaccessibility of Cd in the pak-choi(Table1).Cooking increased Cd bioaccessibility significantly f
or the two vegetables,and there were also significant change of Cd bioaccessibility between Malabar spinach and pakchoi.In the small intestinal phase,the additives significantly increased Cd bioaccessibility,and the interactions among vegetable species, cooking and additives were also highly significant(Table1).The highest increase was occurred with acetic acid addition,and the Effects of cooking and additives on the bioaccessibility of Cd in the gastric Multiple comparison tests were for cooked pakchoi,raw pakchoi,cooked spinach and raw Malabar spinach,respectively,and different letters
significant differences at p<0.05as calculated by the least significant difference(LSD)test.
Effects of cooking and additives on the bioaccessibility of Cd in the small phase.Multiple comparison tests were for cooked pakchoi,raw pakchoi,
Malabar spinach and raw Malabar spinach,respectively,and different indicate significant differences at p<0.05as calculated by the least significant difference(LSD)test.
Table1
Significance of differences among vegetable species,cooking,additives in the gastric and small intestinal phase for Figs.1–4.
Gastric phase Small intestinal phase
Cd Pb Cd Pb Vegetable species(V)*p<0.001p<0.001p=0.005p<0.001 Cooking(C)**p<0.001NS p<0.001p<0.001 Additives(A)***NS p<0.001p<0.001p<0.001 VÂC NS p=0.002p<0.001p<0.001 VÂA p=0.05p<0.001p<0.001p<0.001 CÂA p=0.026p=0.025p=0.002p=0.008 VÂAÂC p=0.001p=0.007p=0.001p<0.001 NS,not significantly at the level0.05.
*Analysis of variance of the species for the two vegetable.
**Analysis of variance of the cooking for the cooked and raw vegetables.
***Analysis of variance of the additives for FeCl
3
,CaCl2,acetic acid and CK(without additives addition).
J.Fu,Y.Cui/Food and Chemical Toxicology59(2013)215–221217
values were all1.6times that of the control in the cooked and raw pakchoi(Fig.2).Multiple comparison
tests showed that there were no significant change among the CK(control,without additives addition),calcium and iron addition for cooked pakchoi and raw pakchoi,respectively.For the Malabar spinach,decreases in Cd bio-accessibility were observed following the addition of acetic acid and calcium in the cooked samples and following all three additives in the raw samples.The addition of calcium in the raw Malabar spinach samples showed the lowest of Cd bioaccessibility,57%of the control.
In the gastric phase,the average Pb bioaccessibility was10.8% and15.6%for the pakchoi and Malabar spinach,respectively (Fig.3).The additives and vegetable species had significant effect on bioaccessibility of Pb(Table1).In the small intestinal phase, the average Pb bioaccessibility was20.2%for pakchoi and23.9% for Malabar spinach.These values were higher than those found in the gastric phase(Fig.4).The cooking significantly decreased the Pb bioaccesssibility for the two vegetables,and the significant difference of Pb bioaccesssibility also found between the two veg-etables(Table1).Furthermore,the significant interactions among vegetable species,cooking and additives were observed(Table1). In the small intestinal phase,multiple comparison tests showed that additives significantly increased Pb bioaccessibility for cooked pakchoi and raw pakchoi,respectively.While for the Malabar spinach,the calcium addition significantly decreased the P
b bioac-cessibility in the cooked vegetable,and calcium and iron addition significantly decreased the Pb bioaccessibility in the raw vegetable.
3.2.Bioavailability of Cd and Pb in vegetables
The average bioavailabilities of Cd and Pb in the raw vegetables were11.2%and9.4%,respectively,significant higher than that in the cooked vegetables(6.1%and3.2%,respectively)(Table2).The average Cd bioavailability in the two raw vegetables was2times that of the cooked vegetables.The Pb bioavailability in the raw Malabar spinach was4times that of the cooked vegetable.For pak-choi,the value was2times.Cd bioavailability was higher than the Pb bioavailability in both vegetables.The highest bioavailability difference was observed in the cooked Malabar spinach,the Cd bio-availability was2.5times that of Pb bioavailability.The additives had no significant decrease for the Cd and Pb bioavailability in all cooked or raw vegetables,and the significant change were also not observed between the two vegetables(Table2).
3.3.Health risk assessment of the heavy metal in vegetables
Based on the bioaccessibility data,the adult and children daily intake values were all higher than that
of the provisional tolerable daily intake(PTDI)of the FAO/WHO(0.83and3.57l g kgÀ1body weight for Cd and Pb,respectively)(Table3).The averages of the adult and children daily intake of Cd and Pb from the vegetables were6.9and3.1times,and of8.7and3.9times of the PTDI values respectively.The daily intake of Cd and Pb in the raw vegetables was higher than that of the cooked vegetables.The highest differ-ence in daily intake was observed for Pb in pakchoi.The value was 16.18l g kgÀ1BW daysÀ1for the raw samples,1.9times the cooked samples.Relative to the raw samples,the averages of daily intake of Cd and Pb in both vegetables decreased21%and36%,respec-tively,in the cooked samples.The addition of acetic acid increased the daily intake of Cd and Pb from eating pakchoi.The addition of calcium decreased the daily intake of Cd and Pb from eating Mala-bar spinach.
Based on the bioavailability data,the average of daily intake of Cd was160%the PTDI value.The average of daily intake of Pb was 87%the PTDI value(Table3).The average of daily intake of Cd and Pb was23%and28%,respectively,of the base bioaccessibility val-ues.The daily intake of Cd and Pb from the raw vegetables was higher than the cooked vegetables.The average daily intake of Cd and Pb from both cooked vegetables was54%and36%,respec-tively,of the raw values.The lowest daily intake of Cd(the addition of acetic acid to raw pakchoi)and Pb(the addition of acetic acid to cooked pakchoi)were51%and31%,respectively,of the control.
In2009and subsequently confirmed in2011,the Panel on Con-taminants in the Food Chain issued an opinion in which they rec-ommended that the PTWI should be reduced to a tolerable weekly intake(TWI)of  2.5l g kgÀ1body weight(EFSA,2012). Based on the bioavailability data,the average of daily intake of Cd was370%the TWI value.
4.Discussion
Our results showed that Cd bioaccessibility was higher in the gastric phase than the small intestinal phase.These observations agreed well with the study of Intawongse and Dean(2006);they found that Cd bioaccessibility of radish root in gastric phase (54.9%)was higher than that in small intestinal phase(24.8%). Most Cd accumulates in the vacuoles of plant cells,except that ab-sorbed by cell wall,so Cd is easily released from plant tissues(Hall, 2002;Zenk,1996).In the gastric phase,most Cd was dissolved and
Effects of cooking and additives on the bioaccessibility of Pb in the gastric
Multiple comparison tests were for cooked pakchoi,raw pakchoi,cooked
spinach and raw Malabar spinach,respectively,and different letters
significant differences at p<0.05as calculated by the least significant
difference(LSD)test.
Effects of cooking and additives on the bioaccessibility of Pb in the small
phase.Multiple comparison tests were for cooked pakchoi,raw pakchoi,
Malabar spinach and raw Malabar spinach,respectively,and different
indicate significant differences at p<0.05as calculated by the least
significant difference(LSD)test.
218J.Fu,Y.Cui/Food and Chemical Toxicology59(2013)215–221
Table 2
Bioavailability (%)of Cd and Pb based on the Caco-2cells uptake.Treatment
Pakchoi Malabar spinach Cd Pb Cd Pb Cooked
Raw Cooked Raw Cooked Raw Cooked Raw
CK    6.6±0.1a 15.9±2.3a    6.5±0.6a 12.6±4.6a 7.5±1.2a 13.5±2.2a    2.8±0.6a 13.0±2.2a FeCl 3  5.9±0.8ab 10.8±3.1a    2.7±0.6c 8.3±2.6a 7.6±0.2a 11.9±1.4a    2.5±0.4a    5.3±0.8b CaCl 2
4.7±0.3b 9.3±2.0a    4.2±0.4b    6.2±0.6a 7.1±1.8a 12.3±0.9a    2.9±0.6a 10.4±1.3a Acetic acid    4.8±0.2b
8.1±5.2a
2.0±0.9c
6.0±2.8a
4.0±0.3b
8.0±2.8a
2.2±0.3a
12.9±0.7a
reference group
Significance
Cd Pb Vegetable species (V)*NS
NS
Cooking (C)**p <0.001p <0.001Additives (A)***NS NS V ÂC NS NS V ÂA NS NS C ÂA NS NS V ÂA ÂC
NS
NS
Multiple comparison tests were only for each column,and different letters indicate significant differences at p <0.05as calculated by the least significant difference (LSD)test.Data are means the mean ±SE (n =3).NS,not significantly at the level 0.05.*
Analysis of variance of the species for the two vegetable.**
Analysis of variance of the cooking for the cooked and raw vegetables.***
Analysis of variance of the additives for FeCl 3,CaCl 2,acetic acid and CK (without additives addition).
Table 3
Comparison of estimated adult and children dietary intake of Cd and Pb through vegetable consumption (l g kg À1BW days À1).
Adult Children Pakchoi Malabar spinach Pakchoi Malabar spinach Cd Pb
Cd Pb
Cd Pb Cd Pb
Cooked
Raw Cooked Raw Cooked Raw Cooked Raw Cooked Raw Cooked Raw Cooked Raw Cooked Raw Bioaccessibility CK    4.09b    6.07b 7.31b 15.77a    6.35a 7.48a 9.12a 13.58a    5.13b 7.62b 9.18b 19.78a 7.97a 9.39a 11.46a 17.04a FeCl 3  4.21b    6.69b 7.14b 15.64a    6.51a    5.32bc 9.38a 8.70b    5.29b 8.39b 8.96b 19.63a 8.18a    6.68bc 11.78a 10.93b CaCl 2
4.17b    6.55b 8.30ab 1
5.90a    3.85b    4.39c
6.33b    6.17c    5.24b 8.22b 10.42ab 19.96a    4.83b    5.51c
7.95b 7.75c Acetic acid    6.30a
9.08a
11.50a
17.43a
5.22a
6.26b
9.05a
14.00a
7.90a
11.40a
14.43a
21.88a
6.55a
7.86b
11.36a
17.57a
Significance Cd Pb
Cd Pb
Vegetable species (V)*
NS p <0.001NS p <0.001Cooking (C)**p <0.001p <0.001p <0.001p <0.001Additives (A)***p <0.001p <0.001p <0.001p ?0.001V ÂC p <0.001p <0.001p <0.001p <0.001V ÂA p <0.001p <0.01p <0.001p <0.01C ÂA NS
NS
NS
NS
V ÂA ÂC p =0.05
p =0.05
p =0.05
p =0.05
Bioavailability CK    1.06a    2.56a    3.82a 7.41a    1.12a    1.99a    1.10a    5.18a    1.34a    3.21a    3.95a 9.3a    1.40a    2.50a    1.38a    6.50a FeCl 30.95ab    1.74a    1.59c    4.92b    1.15a    1.77a 0.98a    2.10b    1.19ab    2.19a    1.99c    6.18b    1.44a    2.22a    1.23a    2.64b CaCl 2
0.76b    1.49a    2.47b    3.68b    1.06a    1.82a    1.17a    4.45a 0.95b    1.87a    3.11b    4.43b    1.33a    2.28a    1.46a    5.56a Acetic acid 0.78b    1.30a    1.17c    3.54b 0.59b    1.18a 0.85a    5.14a 0.98b    1.63a    1.47c    4.44b 0.74b    1.48a    1.07a    6.45a
Significance Cd Pb Cd Pb Vegetable species (V)*
NS NS NS NS Cooking (C)**p <0.001p <0.001p <0.001p <0.001Additives (A)***NS NS NS NS V ÂC NS NS NS NS V ÂA NS NS NS NS C ÂA NS NS NS NS V ÂA ÂC
NS
NS
NS
NS
Multiple comparison tests were only for each column,and different letters indicate significant differences at p <0.05as calculated by the least significant difference (LSD)test.Data are the means of three replicates.NS,not significantly at the level 0.05.*
Analysis of variance of the species for the two vegetable.**
Analysis of variance of the cooking for the cooked and raw vegetables.***
Analysis of variance of the additives for FeCl 3,CaCl 2,acetic acid and CK (without additives addition).
J.Fu,Y.Cui /Food and Chemical Toxicology 59(2013)215–221
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