Bisphenol A ≥ 99% purity (Merck, Sigma-Aldrich, Darmstadt, Germany) was dissolved
in absolute ethanol and corn oil for the dietary (po) and subcutaneous (sc) routes
of administration, respectively. The volume administered to the sheep was adjusted
to the body weight recorded on the day of the administration. For the dietary administration,
approximately 1 mL of BPA solution in absolute ethanol was applied onto the pellet
ration to obtain the single dose of 100 µg/kg body weight, and applied with the morning
feed of pellets (400 g). For the sc administration, the injection of BPA solution
was performed in the shoulder area (2.9 mL) at the same dose. Both solutions were
stored at the ambient temperature in sealed amber glass bottles for the entire duration
of use. All materials used for the solution preparation, sample processing and assays
were either made of glass or of BPA-free plastics.
All animal procedures were carried out in accordance with ethical standards and approved
by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector
and Plant Protection with permission no. U34401-3/2015/8. The study was performed
on one stable, healthy lactating Istrian Pramenka sheep with a single suckling lamb
in a sheepfold at the Centre for Sustainable Recultivation at Vremščica belonging
to the Veterinary Faculty of the University of Ljubljana, Slovenia. The ewe was six
years old and weighed 59 kg, while the suckling lamb was four weeks old and weighed
12 kg. The ewe and lamb were kept under natural temperature and photoperiodic conditions,
with free access to water, hay and salt. In addition, the sheep was fed twice a day
with 400 g plant based pellets (SchafKorn Lac, Unser Lagerhaus Warenhandels Ges.,
Austria). Eventual contamination of the experimental environment was checked by preliminary
testing of drinking water and pellets by HPLC analysis, which revealed the slight
presence of BPA of 0.02 µg/L and 5 µg/kg in these two matrices, respectively. The
sheep and its lamb were, at both periods of the study, penned individually the day
before the first administration until three days after the last administration. The
lamb was kept with its mother, except on sampling days, when they were separated for
a few hours before sampling time to collect enough milk for analysis. The animals
were clinically healthy, as indicated by medical (temperature, breathing and rumination
frequency, pulse rate), haematological, biochemical and faecal examinations. Fourteen
days after the second experimental period, the sheep and its lamb were released in
their original herd.
A sheep was chosen for this study due to its physiological similarities with cows,
but easier manipulation. However, cows are mainly used in milk production in Europe
(accounting for 96.9% of the total milk produced) .
The experiment was divided into two periods, the first being the dietary administration
period and the second being the subcutaneous administration period. The same ewe was
used for both exposure routes, thus a 13 days wash-out period was permitted to ensure
that BPA was removed from the body of the ewe before the start of the second period.
Regarding the administration of BPA, in the first period the ewe received BPA in its
diet (100 µg/day/kg of body weight) for five consecutive days (dietary route of administration).
The ewe ingested all pellets within 2-9 minutes. During the second period, the same
ewe was injected in the shoulder area with 100 µg/kg of b. w. of BPA subcutaneously
per day for five consecutive days (subcutaneous route of administration).
On the first day of the dietary period of the experiment, the ewe`s blood samples
were taken at time 0 (before the first administration) and 0.083, 0.16, 0.33, 0.5,
1, 2, 4, 6, 8, 10 and 24 hours after the first administration. The blood samples were
then taken every day for the next seven days (trough concentrations). The sampling
time started when the ewe ingested the whole portion of pellets. Similar sampling
intervals were used in the second subcutaneous period of the experiment, with the
exception of the first blood sampling (0.083 h after the sc administration), which
was not taken. Blood samples from the suckling lamb were collected on the first day
10 hours after BPA administration to the ewe, and on every following day before the
next administration to the ewe. Jugular vein blood samples were collected in heparinised
glass vacuum tubes, cooled to 4 °C and transported to the laboratory where blood plasma
was separated by centrifugation at 2640×g for 15 min. The plasma was transferred and
stored in polypropylene (PP) tubes. Plasma samples were kept frozen at -20 °C until
A diagram illustrating the design of the study, including the two experimental periods,
BPA administration and blood sampling schedule in the ewe, is provided below (Figure
[Please insert Figure 2.]
Milk sampling was done in both periods. On the first day of the experiment milk was
collected six and 10 hours after the first BPA administration and every next day just
before the following administration. Before the first administration in each period
the ewe was milked and then the lamb was separated from the ewe during next six hours
to allow estimation of the amount of BPA excreted in milk. The sampling period continued
from the 5th until the 8th day of both periods, when there was no BPA administration. Milk was collected in
polypropylene (PP) containers and stored at -20 °C until analysis.
Blind samples of blood plasma from the sheep and suckling lamb and milk from the sheep
were taken just before the start of both periods, to provide a baseline for the analysis.
BPA and total sample analysis
A stock solution of BPA of 200 µg/mL was prepared in acetonitrile, while the intermediate
and working standard solutions ranging from 2,000 to 1.0 ng/mL were further prepared
in a mixture of acetonitrile and water at a ratio of 35 : 65 (v/v). Working standard
solutions ranging from 50,000 to 50 ng/mL for fortification of the total BPA samples
were prepared in water with a small portion (≤ 20%, v/v) of ethanol or acetonitrile.
All solutions were prepared using high purity deionised water obtained using a PureLab
Option and PureLab Classic water purification system (Elga, Woodridge, Illinois, USA).
The acetonitrile and methanol used were of HPLC gradient grade purity and purchased
from J.T. Baker (Center Valley, PA, USA). Only high quality glass or PP labware were
used for the sample analysis.
Samples of the sheep blood plasma and milk were tested for the presence of both free
(unconjugated) and total BPA (free and conjugated), of which the latter was determined
indirectly by conversion of the BPA-GLUC to free BPA. Sample aliquots of 1.5 and 5
mL were taken for the analysis of free BPA in the blood plasma and milk, respectively,
while aliquots of 1.0 and 2.5 mL were taken to determine the total BPA and were diluted
by 1.1 M Na-acetate buffer solution with pH values of 5.3 and 5.1 and volumes of 1.0
and 2.5 mL for the blood plasma and milk, respectively. Forty and 70 µL of ß-glucuronidase
from Helix pomatia, type HP-2, ≥100,000 units/mL including also ≤7,500 sulfatase units/mL (Merck, Sigma-Aldrich,
Darmstadt, Germany) were added to each sample of the blood plasma and milk, respectively.
Samples were then incubated in a shaking water bath at 37 °C for 4 h.
The blood plasma and milk samples were further extracted by 6 and 10 mL of acetonitrile,
respectively and ultrasonicated before being evaporated to dryness at 40–42 °C under
a stream of N2 using an N-evap 111 evaporator (Organomation Associates, Berlin, MA, USA). A further
clean-up procedure included solid phase extraction (SPE) by the use of molecularly
imprinted polymer (MIP) columns AFFINIMIP® SPE Bisphenols, 6 mL, 100 mg (AFFINISEP, Petit-Couronne, France), while the additional
use of a Chromabond HR-X phase, with 6 mL columns, 200 mg, and 85 µm particle size
(Macherey-Nagel, Düren, Germany) was previously utilised for all deconjugated sample
extracts, as described by Deceuninck et al. . Final SPE extracts were re-dissolved
in acetonitrile/H2O (35/65, v/v) as follows: both free BPA blood plasma and milk samples in 0.5 mL,
and total BPA blood plasma and milk samples in 1.0 and 0.5 mL, respectively. Fifty
µL of the final extract were taken for the high-performance liquid chromatography
HPLC measurements were performed using a Varian ProStar HPLC system (Varian Analytical
Instruments, Walnut Creek, CA, USA), comprised of a tertiary pump (240 model), automatic
injector (410 model), fluorescence detector (363 model), degasser and Galaxie 184.108.40.206
analytical software. Chromatographic separation was performed at room temperature
by the gradient binary pumping of water and acetonitrile at a flow rate of 1 mL/min
through a Hypersil Gold C18 analytical column, 150 x 4.6 mm, with a particle size
of 3 µm, which was protected with Hypersil GOLD 3µ Drop in the guards (Thermo Scientific,
Waltham, MA, USA). The mobile phase gradient was as follows: 0–2 min, 35% (v/v) of
acetonitrile, gradient to 12 min, 35–50% (v/v) of acetonitrile, held to 20 min, gradient
to 20.5 min, 50–35% (v/v) of acetonitrile, held to 21 min. The excitation and emission
wavelengths of the fluorescence spectrophotometry analysis were set at 230 and 315
nm, respectively . The results were evaluated in accordance with the external
standard method using a standard calibration curve as a function of chromatographic
peak areas and standard concentrations. Each sample series consisted of a matrix sample,
obtained before the first periodic BPA administration (a baseline sample), five to
seven animal study samples in duplicate and two baseline matrix samples fortified
with BPA to control the recovery rate. The measured sample concentrations were corrected
for the possible baseline matrix response and for the mean recovery of the respective
series and then used as final results.
Validation of the analytical methodology used was performed to demonstrate its fitness
for the stated purpose. Linearity was determined by the least-squares method to calculate
regression and correlation parameters for six to seven standard concentration points
per calibration curve (range 1.0–100 ng/mL), and for both matrices as a correlation
between measured and added concentrations (ranges 0.25–10 μg/L and 1.0–50 μg/L for
free and total BPA in blood plasma, respectively, 0.5–15 μg/L for both free and total
BPA in milk). Mean recovery was evaluated by analysis of four to six fortified blank
materials at two concentration levels at separate time points (blood plasma: free
BPA 2 and 10 μg/L, total BPA 25 and 50 μg/L; milk: free BPA 2 and 5 μg/L, total BPA
5 and 10 μg/L). The within-laboratory reproducibility of the method was evaluated
as the CV of the determined and recovery values. The LOD value was estimated as the
BPA concentration in the retention time window where the analyte was to be expected,
which corresponded to 3 × noise and was corrected for the blank matrix response.
Each entity (free, conjugated, and total) plasma concentration time course until the
second BPA administration was first analysed using a noncompartmental approach to
obtain the estimates of the area under the concentration–time curve extrapolated to
infinity (AUC), maximum concentration in plasma and time when it occurs (cmax and tmax, respectively). AUC was calculated using the linear trapezoidal method and extrapolated
to infinity by addition of the term Clast/λz, where Clast is the last quantified concentration measurement and λz is the terminal slope of the concentration profile in the semi-log plot calculated
by linear regression. tmax and cmax were reported as observed. AUCvalues were used to estimate clearance (CL) as CL = Dose/AUCsc and relative bioavailability after dietary administration (Fr) as Fr = AUCpo/AUCsc. The indexes po and sc refer to the route of administration (dietary and subcutaneous,
respectively) and Dose is the single BPA dose (100 µg/kg of b. w.). Note that CL
can be estimated only after intravenous administration. Our estimate of CL is therefore
apparent clearance, i.e. assuming complete bioavailability after subcutaneous administration.
Subsequently, all TK data after both routes of administration were simultaneously
fitted to a one- and two-compartment model with first-order absorption and elimination.
The estimated parameters were clearance (CL), volume of the central and peripheral
compartment (Vc and Vp, respectively), distribution clearance (Q), absorption rate constants after subcutaneous
and dietary administration (ka sc and ka po, respectively) and relative bioavailability (Fr). Parameter fitting was performed using ADAPT II software  with the maximum likelihood
method and a proportional variance model, Vi = ( × Yi)2, where Vi is the variance of the i-th data point and Yi is the value predicted by the model. The AIC value was used to select the model.
Permeation of free, conjugated and total BPA into milk was modelled as a first order
process dAm/dt = km × Cp(t), where dAm/dt is the transfer rate in µg/h, Cp(t) is the BPA plasma concentration at time t, and km is the transfer rate constant. km was estimated by simultaneous fitting of the amounts excreted into milk up to six
hours after the first subcutaneous and dietary administration, with TK parameters
for the plasma data fixed to previously estimated values. The amounts excreted in
milk up to 6 h were approximated by multiplication of the concentration in milk at
6 h by 0.25 L, i.e. assuming an average milk yield of 1 L/day. We tested the hypothesis
that only free BPA is transferred into milk and subsequently conjugated in the mammary
gland, i.e. fixing the TK parameters to the values estimated for the free BPA versus
the hypothesis that conjugated BPA is also transferred, i.e. fixing the TK parameters
to the values obtained for the conjugated and total BPA.