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2.2
Methods
2.2.1
Creatinine Analysis
Creatinine determination was based on the Jaffe method and carried out as previously
described (Belaidi et al. 2011 ) . Brie fl y, 50 ml of diluted urine samples were mixed
with 150 ml alkaline picrate solution (1.2% picric acid in 0.75 M sodium hydroxide)
and the formation of an orange-red complex between creatinine and alkaline picrate
was quantified by measuring the absorbance at 490 nm.
2.2.2
HPLC
HPLC analyses were carried out on an Agilent 1200 SL system (Agilent Technologies
GmbH, Boeblingen, Germany). The chromatographic conditions were identical to
the previously reported SSC quantification method (Belaidi et al. 2011 ) . Automated
pre-column derivatization with O-phthaldialdehyde (OPA) was used and the ana-
lyzed compounds were separated on a reversed-phase C18 column: XBridge
(150 × 4.6 mm, 3.5 mm, Waters GmbH, Eschborn, Germany). For detection the UV
absorbance at 338 nm was recorded and compound identification was achieved by
comparing the retention time with that obtained for a standard. Peak area was used
for calibration. SSC, hypotaurine, and taurine amounts were determined by stan-
dard addition and normalized to creatinine concentration.
2.3
Results
2.3.1
HPLC Determination of Hypotaurine and Taurine
in Urine Samples
HPLC analysis of amino acids with OPA derivatization is one of the most sensitive
methods for amino acid quantification with detection limits in the femtomole range.
We previously developed a method for SSC determination in urine samples using
pre-column derivatization with OPA, which resulted in fast and accurate measure-
ment (Belaidi et al. 2011 ). In this study we extend the method to the measurement
of hypotaurine and taurine in addition to SSC. Under the chromatographic condi-
tions described above, separation was completed within 15 min using isocratic elu-
tion. Hypotaurine and taurine yielded sharp peaks eluting at 13.8 and 14.3 min,
respectively, whereas SSC eluted at 8 min (Fig. 2.1a ). Urine analysis in a sample
derived from a healthy individual revealed the presence of very low amounts of SSC
(4 mmol/mol creatinine), whereas hypotaurine and taurine levels were 25 and
30 mmol/mol creatinine, respectively (Fig. 2.1a ). In contrast, analysis of a urine
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