Urine samples obtained from 19 preterm infants were split and urinary malodialdehyde was measured in each sample using either HPLC or spectrophotometric methodology, The infants had a birth weight of 1384 +/- 533 g, gestational age of 30.2 +/- 3.6 weeks. Urine was collected between 12 and 240 hours, frozen at -70° C, and examined at a later date. The pH adjusted samples were incubated with thiobarbituric acid and analyzed by spectrophotometry at 532 nm. The HPLC method involved incubation of urine, TBA, glacial acetic acid, extracting the mixture with chloroform, centrifuging, and injecting the aqueous layer into the HPLC, which was run using 60:40 0.04 M Acetate/Methanol carrier. The MDA-TBA adduct was eluted at 8 min using absorbance at 532 nm for detection. Both sets of measurements were normalized to urine creatinine concentrations. We used a pure TBA-MDA adduct standard to establish our standard calibration curves.
For the population, MDA-TBA levels were 33.5 +/- 21.4 ng/mg creatinine by HPLC and 192.1 +/- 329.2 ng/mg creatinine by spectrophotometry (r=0.361, p>0.05). There were no correlations between uMDA by either method and birth weight, gestational age, and postnatal age. The results confirm that the spectrophotometric analysis measures a mixture of MDA and non-MDA substances. HPLC method should be used as the gold standard for MDA-TBA measurement.
