Occurrence of AFM
1
in urine samples of a Brazilian population and association
with food consumption
Alessandra de Cássia Romero
a, Tânia Raquel Baroni Ferreira
a, Carlos Tadeu dos Santos Dias
b,
Maria Antonia Calori-Domingues
a, Eduardo Micotti da Gloria
a,*a
Department of Agroindustry, Food and Nutrition, ESALQ, Universidade de São Paulo – USP, Av. Pádua Dias, 11, CEP 13418-900, Piracicaba, São Paulo, Brazil
bDepartment of Exact Sciences, ESALQ, Universidade de São Paulo – USP, Av. Pádua Dias, 11, CEP 13418-900, Piracicaba, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 6 April 2009
Received in revised form 10 August 2009 Accepted 17 August 2009 Available online xxxx Keywords: Aflatoxin M1 Biomarker Urine Food consumption
a b s t r a c t
This survey evaluated the presence of AFM1in human urine samples from a specific Brazilian population, as well as corn, peanut, and milk consumption measured by two types of food inquiry. Urine samples from donors who live in the city of Piracicaba, State of São Paulo, Brazil were analyzed to detect the presence of aflatoxin M1(AFM1), an aflatoxin B1metabolite, which may be used as aflatoxin B1exposure biomarker. The AFM1analysis was performed using immunoaffinity clean-up and detection by high-per-formance-liquid chromatography with fluorescence detector. A total of 69 samples were analyzed and 45 of them (65%) presented contaminations P1.8 pg ml 1, which was the limit of quantification (LOQ). Sev-enty eight percent (n = 54) of the samples presented detectable concentrations of AFM1(>0.6 pg ml 1). The AFM1concentration among samples above LOQ ranged from 1.8 to 39.9 pg ml 1. There were differ-ences in food consumption profile among donors, although no association was found between food con-sumption and AFM1concentration in urine. The high frequency of positive samples suggests exposure of the populations studied to aflatoxins.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Human exposure to aflatoxins is a concern worldwide because aflatoxins are potent cancer-promoting agents, especially liver can-cer (International Agency for Research Cancer (IARC) 1993). One way of evaluating aflatoxin exposure through diet is to estimate the aflatoxin concentration in foods and the level at which these foods are consumed. However, several other factors may impair this estimation such as heterogeneity in relation to food contami-nation, difficulties in measuring food consumption, seasonal varia-tions associated with food contamination and effect of food preparation on contamination level, among others (Hall & Wild, 1994; Polychronaki, 2007).
An alternative for a more precise evaluation of total aflatoxin exposure is to measure the presence of aflatoxin metabolites in body fluids such as milk, urine and blood (Hall & Wild, 1994). The detection of several aflatoxin metabolites in body fluids has already been reported in the literature (Essigmann et al., 1977; Gorelick, 1990; Mykkanen et al., 2005; Sabbioni, Skipper, Buchi, & Tannenbaum, 1987). However, these metabolites can only be used as evidence of aflatoxin exposure after their validation as
biomarkers. For aflatoxins, the presence of adducts, aflatoxin N7 guanine in urine, aflatoxin lysine in blood and aflatoxin M1in urine is considered a valid alternative for aflatoxin exposure verification (Groopman & Kensler, 1999).
Aflatoxin M1(AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) originated from animal and human metabolism after the ingestion of aflatoxin B1, which may be present in body fluids such as urine, milk, and blood (Groopman, 1994). Only from 1.2% to 2.2% of the aflatoxin present in the diet is excreted as AFM1in human urine (Zhu et al., 1987).
The use of AFM1present in urine as biomarker of human expo-sure to aflatoxins has already been reported in several world pop-ulations. In Taiwan, the analysis of urinary excretion performed by
Hatch et al. (1993) with 250 people resulted in concentrations ranging from 0.003 to 0.108 ng ml 1. Data obtained byCheng, Root,
Pan, Chen, and Campbell (1997)with people from China and Tai-wan revealed 64–66% of positivity, with concentrations ranging from 3.2 to 108 ng for 12 h and from 2.7 to 17 ng for 12 h among people who live in China and Taiwan, respectively. In Sierra Leone, Africa, concentrations ranged from 0.1 to 374 ng ml 1, according to a study byJonsyn-Ellis (2000). In Ghana, concentrations reported byJolly et al. (2006)reached maximum levels close to 12 ng mg 1 of creatinine. In the Czech Republic,Malir et al.(2006) reported the occurrence of AFM1in 57.6% of urine samples analyzed, with con-centrations ranging from 0.019 to 19.219 ng g 1of creatinine.
0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.08.004
* Corresponding author. Tel./fax: +55 19 34225678. E-mail address:emgloria@esalq.usp.br(E.M. da Gloria).
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consump-In Brazil, there are several reports on the occurrence of aflatoxin in foods aimed at human and animal consumption. However, corn and peanut, as well as corn and peanut-based products are the main foods related to aflatoxin contamination (Caldas, Silva, & Oliveira, 2002; Gloria, Fonseca, & Souza, 1997; Gloria, Romero, Carvalho, Calori-Domingues, & Gonçalves, 2006; Kawashima & Va-lente-Soares, 2006; Rodriguez-Amaya & Sabino, 2002; Sabino et al., 2000). Milk and dairy products are also frequently reported as con-taminated in Brazil with low aflatoxin levels (Gonçalez et al., 2005;
Oliveira & Ferraz, 2007; Oliveira, Germano, Bird, & Pinto, 1997); Oliveira, Rosmaninho, & Rosim, 2006; Shundo & Sabino, 2006.
However, there are few investigations on the presence of afla-toxin exposure biomarkers in the Brazilian population. Scussel, Haas, Gong, Turner, and Wild (2004)analyzed blood samples from 50 blood donors in a hospital in the city of São Paulo, Brazil, and observed the presence of AFB1-lysine in 62% of the samples ana-lyzed, with concentrations ranging from <3 to 57.3 pg mg 1.Navas
(2003)reported the analysis for AFM1of samples of human breast milk collected in a hospital in the city of São Paulo as well. Only one sample presented a contamination level of 0.024 ng ml 1. The detection/quantification limit for the methodology proposed by that author was 0.01 ng ml 1.
This study aimed to conduct a survey on the occurrence of afla-toxin M1in the urine of individuals that live in Piracicaba, São Pau-lo, Brazil. Donor feeding habits were evaluated by means of two types of food inquiries and consumption data were tested for cor-relation with contamination levels observed in urine samples.
This study has been approved by Comitê de Ética em Pesquisa (Research Ethics Committee), Faculdade de Odontologia de Piraci-caba - Universidade Estadual de Campinas (Protocol No. 095/2005).
2. Material and methods 2.1. Donor selection
Sixty-nine individuals were randomly selected among residents from different regions of the city of Piracicaba, São Paulo, Brazil as urine donors.
2.2. Food Inquiries
The individuals selected as donors answered two types of food inquiries, namely a feeding frequency inquiry (FFI) on the monthly consumption by individuals, and a 24-h recollection (IR24), in which the consumption of all foods ingested on the day before ur-ine collection were recorded. Among the FFI component items and items reported by individuals in the IR24, foods were selected and grouped as corn-, peanut-, and milk-based foods, whose contami-nation by aflatoxins has been more frequently reported in the sci-entific literature.
2.3. Sampling
Each donor collected an early-morning urine sample using a 300 ml polyethylene flask. The sample volume ranged from 50 to 300 ml among donors. After collection, samples were identified and immediately sent to the laboratory, and were stored at
18 °C until analysis.
2.4. Aflatoxin M1analysis in urine
The extraction and purification of urine samples for AFM1 deter-mination were performed as recommended byKussak, Andersson, and Andersson (1995), with some modifications. A 30 ml volume from the urine sample was filtered through a glass microfibre filter
paper (Whatman Schleider & Schuell, Maidstone, England, product number 934-AH). Later, 20 ml of filtered extract was transferred to a 50 ml capacity vial and 20 ml of sodium acetate buffer (pH 5.0) was added. The pH of the mixture was measured and corrected to 5.0 using an appropriate volume of a 0.1 M glacial acetic acid solution whenever necessary. The mixture was directly passed through an immunoaffinity column (NeocolumnÒ, Neogem Europe, UK) at a flow rate of approximately 1.0–1.5 ml min 1. After adding the mixture the column was washed with 40 ml of ultrapure water (Milli QÒ, Millipore, Bedford, MA, USA). The column was dried by applying positive pressure with a syringe and bound AFM1 was eluted with 2.0 ml of HPLC-methanol which was recovered in a 4 ml vial previously treated with acid. The eluate was evaporated under nitrogen gas and reconstituted with 500
l
l of the mobile phase before liquid chromatograph analysis.Detection and quantification of sample extracts were performed by high-performance-liquid chromatography (HPLC) with a liquid chromatography system equipped with a LC-10AT Shimadzu pump (Kyoto, Japan), a Shimadzu RF-10AXL fluorescence detector (exci-tation 365 nm and emission 460 nm), an injection volume of 100
l
L, and a reverse phase column (250 4.6 mm, particle size of 3l
m) and precolumn (Synergi FusionÒ, Phenomenex Inc., Tor-rance, CA, USA) kept at room temperature. The mobile phase con-sisted of an isocratic mixture of water and acetonitrile at a volume ratio of 75:25 and a flow rate of 1.0 ml min 1. A calibration curve was prepared using standard AFM1solutions in mobile phase at concentrations of 0.005, 0.01, 0.02, and 0.03 ng ml 1. The standard obtained (Sigma, St. Louis, MO, USA, product code 6428, 10l
g) as purified crystalline AFM1was dissolved in HPLC-grade acetonitrile and its concentration was determined by spectrophotometry according toTrucksess (2005, chap. 49).2.5. Aflatoxin M1identity confirmation
The AFM1 identity was confirmed by the formation of AFM1 hemiacetal derivative (AFM2a). The sample extract was evaporated under nitrogen gas and re-dissolved with 200
l
l of HPLC-n-hexane and 200l
l of trifluoroacetic acid (TFA). The mixture was vortexed during 1 min and left to rest for 10 min. The mixture was evapo-rated under nitrogen gas and re-suspended in 500l
l of mobile phase before injection into the liquid chromatography system. The disappearance or reduction of the original AFM1 peak and the appearance of a new peak at an earlier retention time con-firmed the presence of AFM1in the sample.2.6. Method evaluation
The limit of detection (LOD) was considered as the lowest AFM1 concentration that could be reliably detected (>3:1 signal to noise ratio) in blank urine sample chromatograms. The quantification limit (LOQ) was considered as three times the LOD.
The method was studied for its recovery rate using human urine sample as blank and for this a non detect AFM1urine sample was used. Seven replicates were made. AFM1 standard solution was added to samples immediately before analyze to give the concen-tration of 4.0 pg ml 1of AFM
1. The recovery rate was considered as the concentration of AFM1 quantified over the concentration added. The repeatability measured by relative standard deviation was checked over replicates of urine samples used in the recovery study.
2.7. Statistical analysis
The results obtained were analyzed via the LSD Means Compar-ison Test (p < 0.05) between the individuals total and the 90 per-centile of AFM1concentrations in urine. The correlation between Please cite this article in press as: Romero, A. d. C., et al. Occurrence of AFM in urine samples of a Brazilian population and association with food
consump-consumption of food groups and AFM1concentrations in urine was tested via Spearman’s correlation Test. For statistical analysis pur-poses, AFM1contamination results lower than the limit of detec-tion of the methodology employed were considered numerically equal to zero, while values between the limits of quantification (1.8 pg ml 1) and the detection (0.6 pg ml 1) were considered as the mean between those extremes, that is, 1.2 pg ml 1. Statistical analyses were performed using Statistical Analysis System soft-ware (SAS, 2004).
3. Results and discussion 3.1. Method evaluation
The AFM1 retention time considering the liquid chromato-graphic conditions used was 14.0 min. The linearity of the calibra-tion curve was 0.9907. The limit of deteccalibra-tion (LOD) considering a final sample extract of 500
l
l and an injection volume of 100l
l was 0.6 pg ml 1 and the limit of quantification (LOQ) was 1.8 pg ml 1. The recovery rates ranged from 73% to 97% with a mean value of 83% and relative standard deviations for seven spiked samples (4 pg ml 1) of 20.7%. The AFM1identity confirma-tion method by TFA described showed that the original AFM1peak in the positive sample disappeared and a new peak appeared at a shorter retention time of 5.1 min.
3.2. Occurrence of aflatoxin M1
The AFM1levels detected in urine samples are presented in
Ta-ble 1. From 69 samples analyzed, 54 (78%) presented detectable AFM1concentrations. Forty-five samples (65%) had concentrations ranging from 1.8 to 39.9 pg ml 1.
3.3. Food intake
The FFI was used to estimate food intake habits among the indi-viduals sampled and confirmed that the foods generally reported as contaminated with aflatoxins in Brazil were also part of the reg-ular diet of donors.
Milk and milk-based products were the foods consumed at the highest frequencies and quantities, and were consumed by all indi-viduals (69), followed by corn and corn-based products (67) and peanut and peanut-based products (39).
IR24 was used to obtain the ingestion of foods on the day that preceded collection of the urine sample. Consumption of milk and milk-based products were the most frequent among the 69 individuals, and was reported by 61 individuals. Corn and peanut consumption had lower frequencies, and were reported by 12 and 14 individuals, respectively (Table 2).
The consumption of corn and corn-based product food group significantly differed from peanut and peanut-based products, as well as from milk and milk-based product in the FFI I and II IR24
II inquiries. In IR24 I, corn and corn-based products and peanut and peanut-based products did not differ from each other, but were both different on average from milk-based products.
The comparison between means for all individuals and only those that reported consumption (Table 2) indicates that for FFI the mean of all individuals could be used as an instrument repre-sentative of the group. For IR24, the corn and peanut groups had significantly higher means when the analysis was made only be-tween individuals that consumed those foods, demonstrating that for the evaluation of exposure to aflatoxins via those foods, it would also be important to consider consumption means only be-tween individuals that reported some level of consumption.
3.4. Association between consumption of risk foods and urine AFM1 The correlation analysis for food intake and detected AFM1 lev-els, obtained by Spearman’s correlation Test, showed statistically non-significant r values (Table 3), indicating that no correlation trend exists between those factors. Consequently, in spite of the differentiated consumption of donors in relation to food groups, no association was found between the specific consumption of any of those food groups and AFM1concentrations in the urine of individuals, as well as between the total consumption of those food groups and AFM1.
A correlation analysis between consumption AFM1levels was also performed; comparisons were made for the group of donors with the highest AFM1 levels observed and all other donors. To accomplish that, the 90 percentile for AFM1concentration in urine showed that 10% of the highest AFM1concentrations in urine ran-ged from 9.8 to 39.9 pg ml 1while the other 90% showed values lower than 9.7 pg ml 1. The means comparison between both groups of individuals considering the 90 percentile of contamina-tion did not detect significant differences in food intake between individuals, although the mean AFM1contamination values were significantly different between groups.
Table 1
Distribution of AFM1contamination among urine samples from donors, State of São
Paulo, Brazil. AFM1(pg ml1) n % <0.6 15 21.7 0.6–1.7 9 13.1 1.8–9.7 37 53.6 9.8–39.9 8 11.6
Mean*(standard deviation): 5.96 (±6.39)
Median*: 3.96
Coefficient of variation*
: 107%
*Values for the interval between 1.8 and 40.0 pg ml1.
Table 2
Comparison between consumption means reported in the FFI and IR24 inquiries for food groups considered as risk groups for contamination by aflatoxins.
Food groups Mean intake in inquiries (g/day)*
FFI IR24
I II I II
Corn and corn-based products 23.2a,**,A,** 23.9a,A
13.3a,A
76.7a,B
Peanut and peanut-based products
5.1b,A 9.1b,A 7.1a,A 35.0b,B
Milk and milk-based products 286.1b,B
286.1b,B
307.7b,A
348.0b,A *In both inquiries, I corresponds to the mean of all individuals and II corresponds
to the mean of individuals that reported some level of intake only.
** The same lower case letters in the same column indicate that the means are not
different from each other. In each inquiry, equal upper case letters on the same row indicate that the means are not different from each other (LSD Test, p < 0.05).
Table 3
Spearman’s correlation Test between AFM1and consumption reported in the FFI and
IR24 inquiries for food groups.
Food groups Spearman’s correlation Test
FFI IR24
r p r p
Corn and corn-based products 0.0174 0.8869 0.1809 0.1368 Peanut and peanut-based products 0.1774 0.1448 0.1076 0.3788 Milk and milk-based products 0.0876 0.4741 0.1352 0.2681
consump-The lack of association between AFM1levels present in urine and consumption of certain types of food investigated in this study shows that those foods do not make an important or expressive contribution toward exposure to aflatoxins through the diet. Per-haps in association with other foods they may contribute partially and fortuitously toward total exposure. However, the possibility that the source of exposure to aflatoxins here detected comes from a particular type of food or food group not addressed in the food inquiries formulated in this study cannot be ruled out (seeTable 4). Studies conducted in regions of Asia and Africa were successful in obtaining correlations between food ingestion and excretion of biomarkers for aflatoxins or with aflatoxicosis signals ( Azziz-Baumgartneret al., 2005; Gan et al., 1988; Lewis et al., 2005; Lye et al., 1995; Zhu et al., 1987). However, it is observed that those re-gions are greatly susceptible to the occurrence of aflatoxins in foods, providing greater food exposure. In regions of China, it is estimated that the daily ingestion of AFB1from corn consumption is 184.1
l
g; however, AFB1ingestion from total diet is even higher, estimated between 6.5 and 2027 ng kg 1d 1. In regions of Africa, the ingestion of aflatoxins by peanut consumption ranges from 9.9 to 99.2 ng kg 1d 1, but exposure via total consumption may reach from 3.5 to 183.7 ng kg 1d 1(Williams et al., 2004). There are few studies in Brazil dealing with human exposure estimation resulting from the presence of aflatoxins in foods.Oliveira, Ger-mano, Bird, and Pinto (1997)estimated exposure to AFM1 in 4-month-old children and obtained values of 3.7 ng kg 1d 1 for those individuals after analyzing milk samples for infant consump-tion in day-care centers in São Paulo, Brazil. Similarly, in a study on the occurrence of aflatoxins in foods,Amaral, Nascimento, Sekiy-ama, Janeiro, and Machinski (2006) estimated exposure via afla-toxin concentrations obtained in corn flour and consumption data available in the scientific literature, and obtained an exposure value of 0.14 ng kg 1d 1 provided by the consumption of that corn-based food.The AFM1 values in urine observed in this study were lower than those previously reported for other countries however, the number of positive samples observed was within the same range (Cheng et al., 1997;Hatch et al., 1993; Jolly et al. 2006; Jonsyn-El-lis, 2000; Malir et al., 2006). It can be seen that although AFM1in urine is a validated biomarker to evaluate exposure to aflatoxins, in this study the collection of a punctual urine sample that was not representative of a 12 or a 24 h period, prevented the estimation of ingestion of aflatoxins and, consequently, a correla-tion between total AFM1excretion per time period with food con-sumption in the previous day. The possibility of AFM1glucoronide and sulphate (Eaton, Ramsdell,& Neal, 1994) conjugated excretion must be considered with an additional factor which can affect the correlation (Mykkanen et al., 2005) despite free AFM1 be a vali-dated biomarker. Nevertheless, the high rate of positive samples observed constitutes important information because it shows that those populations are exposed to aflatoxin.
Since the presence of AFM1in urine is considered a short-term AFB1exposure biomarker (Groopman, 1994) and aflatoxin contam-ination level is heterogeneous and could be seasonal (Council for Agriculture Science & Technology (CAST), 2003) perhaps a 24 h ur-ine sample and a different sampling frequency could more
ade-quately show AFM1 contamination in the urine of those populations.
4. Conclusions
This study is the first to report AFM1in urine samples from Bra-zilian populations. In the BraBra-zilian populations mentioned here, concentration levels were not as high as those reported for other countries but their frequencies, resulting from the low AFM1 detec-tion limit achieved in this study, were as high as or even higher than those, showing that most donors were exposed to aflatoxin. The lack of association between consumption of the foods investi-gated in this study, considered as normally contaminated with aflatoxins in Brazil, and AFM1levels in urine suggest that exposure of this population via their diet did not have those foods individu-ally as the main sources of exposure. Further AFB1biomarker stud-ies are required to better evaluate the true levels of aflatoxin exposure over a long period, with urine collections that would quantitatively represent AFM1excretion among individuals.
Since the city of Piracicaba is considered a region with high so-cial and economic indicators (Instituto Brasileiro de Geografia e Estatística – IBGE (Internet)., 2007), the observed high frequencies of aflatoxin exposure suggests that maybe other less developed re-gions of the country present disturbing levels of aflatoxin exposure, corroborating the need for new studies to evaluate individual exposure to aflatoxins in Brazil.
Acknowledgment
The authors would like to thank Fundação de Amparo à Pesqu-isa do Estado de São Paulo (FAPESP), Brazil, Grant No. 2005/53308-6, for financial support.
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