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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. So, OTA levels found merits special concern and must be
taken in account to deduce the OTA daily intake, to restore a specific guideline, and to detect the foods responsible of the presence
of OTA in serum, in order to reduce OTA inputs and toxic effects,
especially in patients with nephropathy.
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