Effect of very long-term storage and multiple freeze and thaw cycles on 11-dehydro-thromboxane-B2 and 8-iso-prostaglandin F2α, levels in human urine samples by validated enzyme immunoassays

Biological samples are often frozen and stored for years and/or thawed multiple times, thus assessing their stability on long-term storage and repeated freeze-thaw cycles is crucial. The study aims were to assess:-the long-term stability of two major enzymatic and non-enzymatic metabolites of arachidonic acid, i.e. urinary 11-dehydro-thromboxane-(Tx) B-2, 8-iso-prostaglandin (PG)F-2 alpha, and creatinine in frozen urine samples;-the effect of multiple freeze-thaw cycles. Seven-hundred and three urine samples measured in previously-published studies, stored at -40 degrees C, and measured for a second time for 11-dehydro-TxB(2) (n = 677) and/or 8-iso-PGF(2 alpha) (n = 114) and/or creatinine (n = 610) were stable over 10 years and the 2 measurements were highly correlated (all rho = 0.99, P < 0.0001). Urine samples underwent 10 sequential freeze-thaw cycles, with and without the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (10 mM); urinary 11-dehydro-TxB(2) and creatinine were stable across all cycles (11-dehydro-TxB(2): 100.4 +/- 21%; creatinine: 101 +/- 7% of baseline at cycle ten; n = 17), while 8-iso-PGF(2 alpha) significantly increased by cycle 6 (151 +/- 22% of baseline at cycle ten, n = 17, P < 0.05) together with hydrogen peroxide only in the absence of antioxidant. Arachidonic acid metabolites and creatinine appear stable in human urines stored at -40 degrees C over 10 years. Multiple freeze-thaw cycles increase urinary 8-iso-PGF(2 alpha) in urine samples without antioxidants. These data are relevant for studies using urine samples stored over long-term and/or undergoing multiple freezing-thawing.