1-s2.0-S0940299310001843-main

Experimental and Toxicologic Pathology 64 (2012) 509– 512
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology
jo u rn al h omepage: www.elsevier.de/etp
Ochratoxin A levels in human serum and foods from nephropathy patients in
Tunisia:
Where are you now?
K. Hmaissia Khlifaa,∗,
R. Ghalib, C. Mazigha, Z. Aounia, S. Machgoula, A. Hedhilib
aDepartment
of Biochemistry, Military Hospital of Tunis, Montfleury, Tunis 1008, Tunisia
bCentre
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, ochra-
toxin
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 asso-
ciated
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 inter-
stitial
nephropathy of unknown etiology (CINI) was characterized
∗Corresponding
author. Tel.: [phone]; fax: [phone].
E-mail
address: azziz [email protected] (K. Hmaissia Khlifa).
(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 contami-
nation
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 nephropa-
thy
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 imme-
diately
frozen at −80 ◦C until analysis of ochratoxin A.
0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2010.11.006

510 K. Hmaissia Khlifa et al. / Experimental and Toxicologic Pathology 64 (2012) 509– 512
Table
1
Characteristics
of nephropathic patients and healthy subjects.
Number Mean age ± SD Sex (M/F)
Healthy subjects
44 54.6 ± 16.3 22/22
Nephropathic
subjects 24 59.33 ±
20.09 10/14
2.2. Food sampling
Food
sampling was performed from the free gift of nephropa-
thy
patients at the request of medical doctors of the nephrology
department.
The food given was that consumed every day or sev-
eral
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 col-
umn
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 evapo-
rated
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 sys-
tem
(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. Iden-
tification
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
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 determi-
nation
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
Positive
samples were qualitatively confirmed. For OTA con-
centrations
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, Ver-
sion
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 sensibil-
ity.
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 sam-
ples
collected from nephropathy subjects.
The
assay of food samples from healthy and nephropathy sub-
jects
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 contami-
nated
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 5.15 ± 1.02 74.79 ± 16.89 12.8 ± 3.2 0.001 ± 0.01 114 ± 20.6 320 ± 19.5 79. 30 ± 44.57
NICI-OTA
subjects 24. 94 ± 19.1 380.2 ± 262.86 8.9 ± 1.60 0.4 ± 0.19 103.2 ± 11 412.8 ± 52.9 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.
Food samples (number) Contamination frequency (%) Min–max OTA level (ng/g) Mean ± SD (ng/g)
Healthy subjects 44 52.3 0.11–6.1 0.77 ± 1.53
Nephropathic
subjects
44 88.63 0.11–33.8 9.28 ± 8.76

K. Hmaissia Khlifa et al. / Experimental and Toxicologic Pathology 64 (2012) 509– 512 511
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 + 17.72 3.1
14.66
2
+ 16.52 2.81
14.65
3
+ 6.61 1.4
6.43
4 + 5.91
1.21
2.0
5
+ 6.59 0.69
0
6
+ 15.83 0.12
7
+ 22.99 3.26
29.96
8
+ 3.63 0.16
0
9
+ 2.62 1.27
1.71
10
+ 6.49 2.9
11
+ 0.40 0.12
0.23
12
+ 6.49 1.15
5.40
13
+ 13.77 1.19
10.72
14
+ 13.64 0.78
15
+ 10.72 0.49
0
16
+ 10.01 1.39
6.1
17
+ 23.27 3.19
18 +
33.85 3.8
28.74
19
+ 12.75 0.29
1.24
20
+ 0.55 0.38
0.54
21
+ 0 0.12
0
22
+ 0.86 0.12
1.24
23
+ 0.58 0.12
0.27
24 + 0.89
0.18
0.72
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 nephropa-
thy
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 sam-
ples
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.
The
percent of samples foods were suppressed the European
limit
for OTA in cereals (3 ng/g) in healthy and nephropathic sub-
jects
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 contam-
inated
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 sam-
pled
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 differ-
ent
(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 sam-
ples
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 contam-
ination.
So, OTA levels found merits special concern and must be
taken
in account to deduce the OTA daily intake, to restore a spe-
cific
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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