2.3.2. Measurement of PPO activity
The PPO activity, that is natural present in the crude extract
of the I. batatas, was determined. The o-quinones were obtained
when different volumes of PPO (0.04–0.2 ml) were mixed with
2.8 ml of 0.05M catechol and buffer phosphate to 5.0 ml. The
classical spectrophotometric methodwas used for the absorption
measurements of the pigments at 410 nm.
One unit of PPO activity is defined as the amount of enzyme
that causes an increase of 0.001 absorbance units per minute,
under the conditions above described [16].
2.3.3. Total protein determination
The protein concentration was determined by triplicate. The
Biuret method [17], using bovine serum albumin as standard,
was applied.
2.3.4. Sampling of urines
Urine samples (100 ml) were obtained during a work shift
from 10 laboratory-workers (7 women and 3 men). The samples
were immediately refrigerated until further treatment.
2.3.5. Hydrolysis of urine samples
About 6ml of urine were introduced into a Hach® tube and
0.6 ml of 12M HCl was added. The hydrolysis was carried out
heating between 100 and 110 ◦C during 90 min. The hydrolyzed
urine was cooled at room temperature and then, 4MNaOH was
added up to pH 7. Finally, the hydrolyzed sample was diluted
with buffer solution to 10 ml and total phenols were determined
by the proposed method.
On the other hand, the same samples were spiked with different
concentrations of catechol standard solution before doing
the hydrolysis. These samples were used for the recovery study.
2.3.6. Creatinine determination
The concentration of creatinine in urine was determined to
give all the analysis results asmgof phenol/g creatinine [18]. The
method is based on the Jaff´e reaction. The complex absorbance
was measured at 510 nm.
2.3.7. FIA system and procedure
A two-channel reverse FIA manifold with spectrophotometric
detection was developed (Fig. 1). An injected volume