Experimental and Toxicologic Pathology 64 (2012) 509–512 Contents lists available at ScienceDirect Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp Ochratoxin A levels in human serum and foods from nephropathy patients in Tunisia: Where are you now? K. Hmaissia Khlifa a,∗ , R. Ghali b , C. Mazigh a , Z. Aouni a , S. Machgoul a , A. Hedhili b a b Department of Biochemistry, Military Hospital of Tunis, Montﬂeury, Tunis 1008, Tunisia Centre of Urgent Medical Aid of Tunis, Laboratory of Biology and Toxicology, Research Unit, Toxicology and Environment, 99UR07-04 Montﬂeury, Tunis 1008, Tunisia a r t i c l e i n f o Article history: Received 16 April 2010 Accepted 3 November 2010 Keywords: Ochratoxin A Human nephropathy HPLC Foods analyze Tunisia a b s t r a c t Ochratoxin A is a natural mycotoxin with nephrotoxic properties that can contaminate food products. It has been detected in high amount in human serum collected from nephropathy patients, especially those categorized as having a chronic interstitial nephropathy of unknown etiology. In the present study, ochratoxin A levels were measured in commonly consumed food items and in serum samples from nephropathy and healthy subjects in Tunisia. To assess ochratoxin A, a high performance liquid chromatography method was optimized. The ochratoxin A assay showed very different scales of ochratoxin A serum and food contamination from 0.12 to 1.5 ng/mL and 0.11 to 6.1 ng/g respectively, and in healthy subjects and 0.11 to 33.8 ng/g for food and 0.12 to 3.8 ng/mL for serum in nephropathy patients suffering from chronic interstitial nephropathy of unknown etiology. The disease seems related to ochratoxin A serum levels and food contaminations, since the healthy group was signiﬁcantly different from the nephropathy group (P < 0.001) for both food and serum ochratoxin A contamination. Those results combined with data published already, emphasize the likely endemic aspect of ochratoxin A-related nephropathy occurring in Tunisia. © 2010 Elsevier GmbH. All rights reserved. 1. Introduction Ochratoxin A (OTA) is naturally occurring mycotoxin produced by mainly Aspergillus ochraceus and Penicillium verrucosum, and is receiving increasing attention (Zinedine et al., 2006; Köller et al., 2006). OTA is a well-known nephrotoxic agent and has been associated with fatal human kidney disease, referred to as Balkan Endemic Nephropathy (BEN) and with an increased incidence of tumors of the upper urinary tract (Zinedine et al., 2006; Grosso et al., 2003). The mycotoxin is carcinogenic, teratogenic, immunotoxic, genotoxic and possibly neurotoxic (Var and Kabak, 2007; Maarouﬁ et al., 1995a). Several studies have proved that human from other parts of the world are being exposed to OTA (Kuiper-Goodman, 1993; Bacha et al., 1993; Maarouﬁ et al., 1995b,c). In North African countries, the most suspected food susceptible to be contaminated by OTA is domestic cereals like sorghum and wheat, olives, spices and imported cereals. In Tunisia, the ﬁrst data have shown the presence of OTA in a large number of frequently consumed foods (Bacha et al., 1988; Maarouﬁ et al., 1995b,c; Achour et al., 1993). Later, a chronic interstitial nephropathy of unknown etiology (CINI) was characterized ∗ Corresponding author. Tel.: +216 71 241554; fax: +216 71 241554. E-mail address: azziz [email protected] (K. Hmaissia Khlifa). 0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.11.006 (Hassen et al., 2003) that shows striking similarities with BEN as described by Savin et al. (2001). Indeed, CINI is a slowly progressive, insidious, non inﬂammatory, and, it is becoming in the fourth or the ﬁfth decade of life a leading cause of renal failure and death. Studies performed so far have shown high rates of blood OTA contamination among nephropathy patients (Bacha et al., 1993; Maarouﬁ et al., 1995b,c; Hassen et al., 2003; Abid et al., 2003). The current study was performed to strengthen the suspected link between serum OTA, alimentary OTA contamination and the nephropathy. 2. Materials and methods 2.1. Study population All sera samples of patients or healthy subjects (with no familial case on nephropathy) considered in these studies were collected from Military Hospital of Tunisia. Series of fourthly four samples were taken from healthy subjects and series of 22 serum samples were collected from patients having chronic interstitial nephropathy of unknown etiology. Characteristics of patients such as age, sex and any other health troubles were registered when available (Table 1). Sera were separated after the blood sample had been centrifuged. The separated sera, in the amount of 6 mL, were immediately frozen at −80 ◦ C until analysis of ochratoxin A. 510 K. Hmaissia Khlifa et al. / Experimental and Toxicologic Pathology 64 (2012) 509–512 Table 1 Characteristics of nephropathic patients and healthy subjects. Healthy subjects Nephropathic subjects Number Mean age ± SD Sex (M/F) 44 24 54.6 ± 16.3 59.33 ± 20.09 22/22 10/14 2.2. Food sampling of methanol/acetic acid 2% solution. The eluate was evaporated to dryness under nitrogen stream at 60 ◦ C and reconstituted with 1 mL of acetonitrile. Fifty L were injected into the HPLC. OTA determination was carried out under isocratic conditions, with a mobile phase constituted of acetonitrile/water/acetic acid (50:48:2; v/v/v) at ﬂow rate of 1.2 mL/min. 2.4. OTA conﬁrmation Food sampling was performed from the free gift of nephropathy patients at the request of medical doctors of the nephrology department. The food given was that consumed every day or several times a week by Tunisian populations, by both nephropathy patients and healthy subjects. The patients were informed that the sampling was to discover a possible cause of their disease in their diet. Samples were stored in plastic bag at −5 ◦ C until grinding and analysis. The selected foodstuffs groups were: cereals and their derived products. 2.3. Analysis of ochratoxin A in human serum and food samples Sera acidiﬁed with acetic acid 1 M (pH = 4.5) were gently loaded (2 mL/min) under vacuum onto the SPE cartridge (C18, 50 mg, 1 mL) previously conditioned with 6 mL of methanol. The SPE column was then washed with 4 mL of distilled water. The bound OTA was slowly eluted from the column with 6 mL of acidiﬁed methanol (methanol/acetic acid, 95/5). The eluate was evaporated under a stream of nitrogen at 60 ◦ C and the residue was resuspended in 500 L of methanol. An aliquot (50 L) of this solution was performed with a Waters HPLC chromatographic system (Waters, Milford, MA) equipped with a Waters Spherisorb ODS2 (4.6–250 mm; 5 mm) kept at 30 ◦ C. The mobile phase was acetonitrile/water/acetic acid (50/49/1: v/v/v) at 1 mL/min. Identiﬁcation of OTA was accomplished by comparing retention times for standards and appropriate components identiﬁed in spiked and unspiked serum samples. OTA and its methyl ester (OTA-Me) were estimated using a ﬂuorescence detector (477 scanning ﬂuorescence detector. Waters, Milford, MA) at an excitation of 310 nm and an emission of 465 nm. Food sample was ﬁnely ground and homogenized. Ten grams of ground sample were mixed with 40 mL of acetonitrile/water (80/20: v/v) solution and 20 mL of hexane and then horizontally stirred for 20 min. The extract was ﬁltered through ﬁlter paper and all the ﬁltrates were collected and centrifuged. 8 mL of the lower layer was transferred to a 20 mL tube and evaporated to dryness at 60 ◦ C under a lower stream of nitrogen. After addition of 0.5 mL acetonitrile and 2 mL of phosphate buffer saline (PBS) the tube was mixed-vortex for 2 min before adding 18 mL of PBS. Diluted extract (equivalent to 2 g sample) was loaded onto the immunoafﬁnity clean-up column (IAC) at 2 mL/min ﬂow followed by 20 mL of PBS at 4 mL/min ﬂow-rate for washing. OTA was then eluted with 2 mL Positive samples were qualitatively conﬁrmed. For OTA concentrations less than 0.25 ng/mL, sample extracts were spiked by adding 20 mL of 20 ng/mL OTA standard solution and these were then reanalyzed. Chromatograms of spiked and unspiked samples were then compared. For OTA concentrations up to 0.25 ng/mL, conﬁrmation was done by OTA methyl ester formation. We mixed 300 L of SPE or IAC eluate with 300 L of BF3 solution, the mixture was heated at 70 ◦ C for 20 min, and 50 L was assayed for OTA-Me. Conﬁrmation was based on the disappearance of the OTA peak, and the appearance of a new peak corresponding to the OTA-Me. 2.5. Statistical analysis Values of ochratoxin A are presented as means ± standard error were calculated by the SPSS software (SPSS Institute Inc., 2000, Version 10.0). A multi sample ANOVA-test was used for determination of statistical signiﬁcance of means differences. P value < 0.05 was accepted as signiﬁcant. 3. Results The proposed HPLC method enabled the OTA quantiﬁcation in analysed food commodities with higher selectivity and sensibility. The reproducibility of the retention time was good (CV = 2.1%). The detection and quantiﬁcation limit (signal-to-noise ratio of 3) obtained with a direct standard solution injection was, respectively, less than 0.l and 0.2 ng/mL. Quantiﬁcation was performed using a linear calibration curve established in the range of 0.1–8 ng/mL with a correlation coefﬁcient (r) of 0.996. In the CINI ochratoxin A positive population, the biological parameters are signiﬁcantly disturbed (Table 2). OTA levels in food samples for healthy and nephropathy subjects are presented in Table 3. The concentrations of OTA in food samples collected from healthy subjects were in the range of 0.11–6.1 ng/g but a higher OTA levels (0.11–33.8 ng/g) were detected in food samples collected from nephropathy subjects. The assay of food samples from healthy and nephropathy subjects for OTA contamination showed that 20–64% were positive in the range of 0.11–33.8 ng/g (Table 3). Sorghum samples were highly contaminated (mean = 13.19 ± 10.21 ng/g) as were wheat and wheat derived (8.43 ± 8.87 ng/g). They were more contaminated than barley (mean = 1.64 ± 2.32 ng/g). This conﬁrmed that the Table 2 Biological parameters in healthy and in nephropathic NICI-OTA positive subjects. Biological parameters Uremia (mmol/l) Creatinemia (mol/l) Haemoglobin (g/dl) Protenuria (mg/24 h) Urinary sodium (mmol/24 h) Urinary urea (mmol/24 h) ␤2-microglobulin (ng/mL) Healthy subjects NICI-OTA subjects 5.15 ± 1.02 24. 94 ± 19.1 74.79 ± 16.89 380.2 ± 262.86 12.8 ± 3.2 8.9 ± 1.60 0.001 ± 0.01 0.4 ± 0.19 114 ± 20.6 103.2 ± 11 320 ± 19.5 412.8 ± 52.9 79. 30 ± 44.57 12719.16 ± 23721.14 Table 3 Levels of OA contamination of food samples collected from storages by the healthy families and the families of patients suffering from CINI. Healthy subjects Nephropathic subjects Food samples (number) Contamination frequency (%) 44 44 52.3 88.63 Min–max OTA level (ng/g) 0.11–6.1 0.11–33.8 Mean ± SD (ng/g) 0.77 ± 1.53 9.28 ± 8.76 K. Hmaissia Khlifa et al. / Experimental and Toxicologic Pathology 64 (2012) 509–512 Table 4 Correlation between OTA food contamination levels with serum contamination. Family Nephropathy (NICI) Level of OTA in food samples (ng/g) Level of OTA in serum (ng/mL) 1 + 3.1 2 + 3 + 4 + 5 + 6 7 + + 8 + 9 + 10 11 + + 12 + 13 + 14 15 + + 16 + 17 18 + + 19 + 20 + 21 + 22 + 23 + 24 + 17.72 14.66 16.52 14.65 6.61 6.43 5.91 2.0 6.59 0 15.83 22.99 29.96 3.63 0 2.62 1.71 6.49 0.40 0.23 6.49 5.40 13.77 10.72 13.64 10.72 0 10.01 6.1 23.27 33.85 28.74 12.75 1.24 0.55 0.54 0 0 0.86 1.24 0.58 0.27 0.89 0.72 2.81 1.4 1.21 0.69 0.12 3.26 0.16 1.27 2.9 0.12 1.15 1.19 0.78 0.49 1.39 3.19 3.8 0.29 0.38 0.12 0.12 0.12 0.18 main sources of OTA contaminated are cereals and cereal-derived. The results of food samples from healthy individuals revealed that none had more than 6.1 ng/g (Table 3). Our results showed that only 34% of these healthy subjects have OTA in blood, with very low values, 0.12–1.5 ng/mL (mean = 0.22 ± 1.39 ng/g). Two food samples were collected from each of the 24 nephropathy patients (Table 4), 88.63% were found OTA positive with concentration ranging from 0.11 to 33.8 ng/g. All samples were positives in 20 out 24 families. Subjects 21 had all food samples negative, which was well correlated with his low level of OTA blood levels (0.12 ng/mL). Among healthy donors, 34% show OTA concentration ranging from 0.12 to 1.5 ng/mL with a mean value of 0.22 ng/mL, whereas, among nephropathy patients 80% are OTA positivity in range of 0.12–3.8 ng/mL and a mean value of 1.25 ng/mL. This population was found to be statistically different from the healthy group. The patients group exhibited both a signiﬁcantly (P < 0.01) higher incidence of OTA-positivity than healthy subjects (80% vs 34%, P < 0.01) and a signiﬁcantly (P < 0.01) higher mean values of serum concentrations (1.25 ± 1.22 ng/mL vs 0.22 ± 0.39 ng/mL, P < 0.01). Table 4 summarizes the scales of OTA contamination in all food samples analyzed. 24 food samples collected from nephropathic patients contained more than 5 ng/g. 511 The percent of samples foods were suppressed the European limit for OTA in cereals (3 ng/g) in healthy and nephropathic subjects are respectively 11.3% and 56.83%. 4. Discussion and conclusion Studies from several countries in the world have attempted to investigate human exposure to ochratoxin A. Two approaches were undertaken: analysis of food and analysis of OTA in biological ﬂuids. This later approach was used to try to correlate human exposure to OTA to nephropathy (Maarouﬁ et al., 1995b; Eko-Ebongue et al., 1994; Radic et al., 1997; Wafa et al., 1998). The signiﬁcance of OTA levels in human plasma as a marker of OTA intake can however be questioned. In a previous survey in Tunisia, it was found that many cereals, cereals-derived, dried fruits, olives and red paper were contaminated by OTA and some other mycotoxins, such as aﬂatoxine, zearalenone, trichothecenes, citrinin and fumonisine (Hadidne et al., 1985; Bacha et al., 1988; Maarouﬁ et al., 1995b; Ghali et al., 2009). In the meantime, determination of OTA in human serum suggested a prevalence of about 62–100% in healthy population and a prevalence of 100% in subjects suffering from nephropathy, whatever the aetiology (Bacha et al., 1993; Hassen et al., 2004). The classiﬁcation among nephropathy patients revealed that the most contaminated ones were those suffering from chronic interstitial nephropathy of unknown causes (Abid et al., 2003; Hassen et al., 2004). These were treated in hospital, dialyzer and could be sampled regularly afterwards. To link the presence of OTA in serum with its occurrence in food as in the Balkans, a new sampling of the different dietary components of several nephropathy families and healthy families without nephropathy was performed under the healthy of physicians in accordance with their ﬁles. Sampling was performed at home, after informed consent had been obtained. The values from healthy food and serum samples were found to be similar to and even less than those found in Egypt, Algeria and European countries (Ózcelik et al., 2001; Zimmerli and Dick, 1995; Khalef et al., 1993; Wafa et al., 1998). It indicates that low levels of OTA in foodstuffs (less than 5.4 ppb) produce less than 3.43 ng/mL in serum, a value which could thus be considered to be safe for the moment, at least with regard to nephrotoxicity. It seems very important to follow these healthy subjects (for food and serum OTA contamination) to see if any of them will develop nephropathy in the future if the situation improves or does not became worst. Food collected from patients suffering from chronic interstitial nephropathy was found to be more than 88.63% contaminated in a range of 0.11–33.8 ng/g, whereas the control food had more lower than 6.1 ng/g. In a good correlation, 22% from the healthy donors show OTA concentration ranging from 0.12 to 1.5 ng/mL with a mean value of 0.22 ng/mL. In contrast among nephropathy patients 80% are OTA positivity in range of 0.12–3.8 ng/mL and a mean value of 1.25 ng/mL. Therefore the two groups were signiﬁcantly different (P < 0.05). Our data are different with a posterior Tunisian works (Maarouﬁ et al., 1995a), which showed the existence of the OTA in frequently consumed food commodities. In the same study the occurrence of OTA in food samples collected from healthy subjects show OTA concentration ranging from 0.1 to 16.6 ng/g and a higher OTA levels (from 0.3 to 46 830 ng/g) were detected in food samples collected from nephropathy patients (Maarouﬁ et al., 1995a). Studies in other Mediterranean countries, like Morocco (Zinedine et al., 2006), Egypt (El-Kady et al., 1995), France (Albert and Gauchi, 2002), Spain (Gonzalez et al., 2006), have shown a higher food OTA incidence. In the study OTA assay showed that the OTA contamination in foods and serum in healthy and nephropathic subjects were very different. The disease seems related to OTA serum levels and foods 512 K. Hmaissia Khlifa et al. / Experimental and Toxicologic Pathology 64 (2012) 509–512 contamination since the control group was signiﬁcantly different from the nephropathic group for both food and serum OTA contamination. 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