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Storage at -80 degrees C decreases the concentration of HPLC-detected urinary albumin: possible mechanisms and implications.

BACKGROUND
Urinary albumin is now measured by high-performance liquid chromatography (HPLC) which also detects albumin missed by traditional immunochemical methods. A predictive effect of HPLC-detected albuminuria on mortality has just been reported in the AusDiab study, measuring albuminuria with HPLC after 7 years of -80 degrees C storage. However, there are already some data suggesting that HPLC-detected albuminuria is affected by -80 degrees C storage. We aimed to measure changes in HPLC-detected albuminuria after 2.5 years and find the factors which may be responsible for this alteration.


METHODS
Urinary albumin was measured by the US Food and Drug Administration approved HPLC Accumin kit. Total free sulfhydryl groups (TFSG) of urine samples were measured by Ellman's reagent.


RESULTS
We found a significant 24% average decrease in HPLC-detected albuminuria and a correlation between the magnitude of decrease and urinary pH. We found a correlation between changes of urinary albumin dimeric to monomeric ratio of stored urine and pH; however, only changes of monomeric form were found to be significant. A correlation was also found between the TFSG of fresh urine samples and pH. Less TFSG could be detected, and a correlation between TFSG and pH was absent in stored urine.


CONCLUSIONS
We conclude that measurement of albuminuria by HPLC in long-term -80 degrees stored urine gives unreliable results. Decrease of HPLC-detected albuminuria is pH-dependent and may be due to the reducing capacity of urine. Prospective studies need to decide whether the predictive properties of HPLC-detected albuminuria decrease during longterm storage.