Advanced oxidation protein products in plasma: stability during storage and correlation with other clinical characteristics.

Proteins are susceptible to free radical damage. We measured advanced oxidation protein products (AOPP) in the plasma of 56 hospitalised patients. Concentrations of AOPP were expressed as chloramine-T equivalents by measuring absorbance in acidic conditions at 340 nm in the presence of potassium iodide. We also determined erythrocyte sedimentation rate (ESR), circulating urea, creatinine, glucose, uric acid, electrolytes, lipids, total proteins and fractions and fibrinogen. Twenty-four samples were processed both immediately and after 7, 15, 30, 90, 180 and 438 days of storage at both at -20 degrees C and -80 degrees C (aliquots were frozen and thawed only once) to evaluate AOPP stability. The remaining 32 samples were also processed for thiobarbituric-acid-reactive substances (TBARS). Mean AOPP concentration in all 56 patients was 48.3+/-37.2 microM. Mean basal concentration of AOPP in the 24 plasma samples (55.0+/-47.1 microM) showed no significant change at each intermediate determination, yet significantly increased after 438 days of storage both at -80 degrees C (96.6+/-83.2, p<0.01) and, markedly, at -20 degrees C (171.3+/-94.6, p<0.001). TBARS concentration was 1.59+/-0.65 micromol/l. Multiple regression analysis evidenced that AOPP concentration was positively correlated (multiple r=0.62, p<0.001) with serum urea and triglycerides, but negatively correlated with patient age (indeed, serum albumin and total proteins decreased with increasing age, r=0.3, p<0.05). TBARS concentration was associated with ESR and serum glucose (multiple r=0.73, p<0.001), yet positively with AOPP (r=0.39, simple p<0.05). We conclude that AOPP remain stable during sample storage both at -20 degrees C and -80 degrees C for 6 months. Renal failure and hypertriglyceridemia probably enhance the in vivo process of AOPP formation. Oxidative damage as measured by TBARS may be increased because of exposure to hyperglycemia causing nonenzymatic glycation of plasma proteins.

With regard to glycohemoglobin measurement: are we sure that high-performance liquid chromatography currently works in the clinical routine?

The clinical usefulness of glycated hemoglobin (HbA1C) depends crucially on the accuracy and precision of its assay. When we compared an immunological bench-top analyzer (DCA 2000, Bayer Diagnostici, Milan) to the high-performance liquid chromatography (HPLC) reference method used in a routine hospital laboratory (Diamat and Fast Diamat, Bio-Rad Lab., Milan) by assaying multiple control sera, we found so many sources of systematic analytical errors in the routine use of HPLC as to compromise between-assay precision. DCA 2000 showed intra- and interassay coefficients of variation (CV) of 1.1% and 2.3% with the normal standard serum, 1.0% and 4.2% with the pathological one; Diamat yielded CVs of 1.3% and 7.0%, 1.3% and 5.7%, respectively. Although the measurement of 161 blood samples showed that Diamat usually overestimated HbA1C (paired t-test, P<0.001), a great variability of Diamat performance became evident when the relationship Diamat vs DCA was evaluated day by day over 17 days of observation (analysis of variance, ANOVA, P<0.001). Intra- and interassay CVs of Fast Diamat initially (new instrument still on approval) were 0.6% and 2.5% (normal standard serum), 0.3% and 1.9% (high standard serum), yet after 6 months of routine laboratory use, they became 3.1% and 3.2%, 1% and 12.3%, respectively. Main sources of error were: inaccurate autodilution, unsuitable parameter settings, disregard of the maintenance schedule. We conclude that the tendency to overlook major critical aspects in the routine use of HPLC is detrimental to the quality of HbA1C determination and implies the loss of HbA1C value in clinical practice. Both carefully supervising laboratory quality and checking the likelihood of the analytical result with the clinical setting appear even more important.