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) B2, 8-iso-prostaglandin (PG)F2α, 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 °C, and measured for a second time for 11-dehydro-TxB2 (n = 677) and/or 8-iso-PGF2α (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-TxB2 and creatinine were stable across all cycles (11-dehydro-TxB2: 100.4 ± 21%; creatinine: 101 ± 7% of baseline at cycle ten; n = 17), while 8-iso-PGF2α 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 °C over 10 years. Multiple freeze–thaw cycles increase urinary 8-iso-PGF2α in urine samples without antioxidants. These data are relevant for studies using urine samples stored over long-term and/or undergoing multiple freezing–thawing.

By means of reactive oxygen species, AA is non-enzymatically oxidized into F 2 isoprostanes that are excreted by the kidney (Fig. 1) 11 .In particular, the 8-iso-prostaglandin (PG)F 2α is the most abundant in human urine 12 , reflecting in vivo lipid peroxidation 2,13 .Consistently, urinary 8-iso-PGF 2α is increased in conditions at high cardiovascular risk as well as high oxidative stress such as cigarette smoking, diabetes mellitus hypercholesterolemia, and obesity 14 .In addition, urinary 8-iso-PGF 2α has been reported as an independent biomarker of future cardiovascular events and mortality 15,16 .In the large longitudinal Framingham cohort, urinary 8-iso-PGF 2α and 11-dehydro-TxB 2 were significantly correlated 9 .
Thus, urinary 11-dehydro-TxB 2 and 8-iso-PGF 2α metabolites have been measured in large longitudinal studies 8,9,17 , possibly years after collection, however their long-term stability has never been assessed while is rather relevant.Both metabolites contain cycloalkanes, double bonds, as well as oxygen and hydroxyl groups (Fig. 1) 18 , which may affect their long-term stability and antigenic properties in immunometric measurements.
Thus, the aims of this study were: (i) to investigate the effect of long-term storage on the concentration of 11-dehydro-TXB 2 , 8-iso-PGF 2α and creatinine (as a control molecule), in urine and chromatographic extracts of urine, all stored at −40 °C, and (ii) to assess the effect of repeated freeze-thaw cycles in urine samples and in chromatographic extracts.
For 11-dehydro-TxB 2 , 677 suitable urine samples, extracted as described above, were assayed by a standard enzyme-linked immunosorbent assay (ELISA) as previously published 25 , using a specific rabbit polyclonal antiserum 26 with a detection range from 3.9 to 500 pg/mL and a sensitivity calculated as 80% B/B 0 (i.e. the relative maximum binding in a sample to maximum binding capacity) of 10 pg/mL.The inter-assay variability, calculated as the coefficient of variation of repeated measurement of a commercial standard (11-dehydro-TxB 2 ELISA Standard, Cayman Chemical, Ann Arbor, MI, USA), was 9% (n = 1344 determinations) over the entire study duration.The accuracy of the ELISA was assessed using a commercial standard of 1.5 ng/mL (Cayman Chemicals) that measured 1.57 ± 0.14 ng/mL (n = 20 measurements).The cross-reactivity of the anti-11-dehydro TxB 2 antiserum against other prostanoids that can be measured in urine, namely PGE 2 2,3-dinor T X B 2 , TXB 2 , 6-keto PGF 1α , and the isoprostane 8-iso-PGF 2α was < 0.05%, and against PGD 2 was 0.3%.
The stability of 11-dehydro TxB 2 and 8-iso-PGF 2α as also assessed in chromatographic extracts stored in PBS at −40 °C between 1 week and 10 years.Extracts were assayed again by ELISA for 11-dehydro TxB 2 (n = 748 samples) and/or for 8-iso-PGF 2α (n = 212 samples) as described.
Both ELISA methods used in the current study had been previously validated against gas chromatography/ mass-spectrometry (GC/MS) and showed a strong correlation between the analysis of identical urine samples for 11-dehydro TxB 2 and 8-iso-PGF 2α 25,27 .

Creatinine measurements
For creatinine, 610 urine samples from 53 healthy individuals 19,20 , 189 patients with diabetes mellitus [Petrucci  et al. submitted], 110 patients with hematologic 21,22 , and 258 with solid cancers 10 , were assayed for a second time for creatinine using a commercial kit (Creatinine Colorimetric Detection Kit; Enzo Life Sciences, Farmingdale, NY, USA).The inter-assay coefficient of variation using a commercial standard (Creatinine Standard, Cayman Chemical) was 7% over the study duration (n = 523 determinations).

Freeze-thaw experiments
Forty urine samples from 37 volunteers were collected and aliquoted into 1.5 mL samples without antioxidant.One aliquot was immediately processed (baseline sample) and the remaining aliquots that were frozen at −80 °C for 20 min and thawed in water bath at 25 °C for 10 min multiple times.In 24 samples the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO) (Merck KGaA, Darmstadt, Germany, 10 mM final concentration), was added immediately after collection and samples underwent multiple freeze thaw cycles as already described.
To evaluate the oxidative stress we measured hydrogen peroxide (H 2 O 2 ) levels, an established index of reactive oxygen species generation 28 ,with a commercial kit (Peroxide Assay Kit, Merck KGaA) that measures H 2 O 2 using a chromogenic Fe 3+ − xylenol orange reaction based on the Fenton reaction.The colorimetric intensity is proportional to the H 2 O 2 concentration in the sample.
The effect of freeze-thaw cycles was also investigated in the chromatographic extracted samples in PBS, by pooling extracts and making aliquots for multiple freeze-thaw cycles, as described.

Statistical analysis
Since the second measurement was dependent on both the availability of a first measurement performed for a specific protocol as well as on an appropriate remaining sample volume, we did not formulate a specific hypothesis for sample size over time.Data from the repeated measurements and the freeze-thaw experiments were plotted as % of the first or baseline measurement, respectively, as indicated.Data were expressed median and interquartile ranges.Comparisons were performed by ANOVA for repeated measurements or paired t-test, as appropriate.Correlations were analysed by Pearson or Spearman's rank test according to data distribution.The significance was set at P < 0.05.Analyses were performed using GraphPad Prism 7.04.
All samples were received anonymized in the central laboratory at the Catholic University School of Medicine in Rome (to GP and BR), since were labelled only using alphanumeric codes, according to the original protocols.Each study protocol was approved by the referent Ethics Committee, namely: South Central-Oxford C, UK, ref 14/SC/0171 10  All protocols were performed in accordance with the Helsinki declaration 29 and written informed consent for study participation and measurements of urinary metabolites was obtained from all study participants.

Chromatographic extracted samples from urine
As a control for the medium for the considered metabolites, we also performed immunoassays in chromatographic extracts of urine samples which were eluted and in PBS.In these extracts (n = 748), only the 11-dehydro-TXB 2 was stable over time (Fig. 4a) with similar concentrations (543[210-1172] and 552.5[199-1181] mg/mL, first and second determination, respectively), with highly correlated values (rho = 0.99, P < 0.0001, Fig. 4b), while 8-iso-PGF 2α in chromatographic frozen extracts (n = 212) showed a significant trend toward a decrease starting approximately from month 4 of storage (Fig. 4c), even though the values of the 2 measurements were still significantly correlated (rho = 0.85, P < 0.0001, n = 212, Fig. 4d).
The urinary pH values with and without antioxidant were not affected by the 10 freeze-thaw cycles (data not shown).

Discussion
This study investigated for the first time the stability of the 2 major urinary enzymatic and non-enzymatic metabolites of AA, i.e., the 11-dehydro-TxB 2 and the 8-iso-PGF 2α , respectively, as well as of creatinine as a control molecule, in a large number of urine samples stored at -40º C for several years and in chromatographic urinary extracts, as control for the storage medium for these analytes (urine versus PBS in purified extracts).We also assessed the effect of multiple freeze-thaw cycles on the same metabolites in both urine and extract samples.To the best of our knowledge, this study has the largest sample size and the longest storage interval assessing stability.
In over 700 urine samples, we observed a substantial stability of 11-dehydro-TxB 2 , 8-iso-PGF 2α , and creatinine levels between few weeks and 10 years, with a variability of the repeated values that remained within the coefficient of variation of the methods.We included different type of subjects from different studies, to evaluate whether in protein-and glucose-enriched urine, as in the case of diabetes mellitus, there were differences in the stability of the studied metabolites and creatinine.The stability of 11-dehydro-TxB 2 and 8-iso-PGF 2α in urine samples stored at −40 °C over 10 years, was observed in all studied groups, i.e. healthy, diabetes mellitus and cancer subjects (Table 1).Previous studies on the 8-iso-PGF 2α reported stability in urine stored at −20 °C for up to 6 months 30 , at −70 °C for a maximum of 2 years (Table 2) 31 , and a recent study showed that both 11-dehydro-TxB 2 and 8-iso-PGF 2α are stable in urine samples stored at both −20 and −70 °C over 3 years (Table 2) 32 .Thus, our data are consistent with and enlarge evidence from previous, smaller studies.Notably, our urine samples were stored at −40 °C for 10 years, which is a condition more feasible and cheaper as compared to lower storage temperatures, e.g.−80 °C, especially in large biobanks.
To compare the stability of these molecules in different suspension media, in addition to the physiological urine milieu, we re-measured the chromatographic extracts from urine, which contain the purified lipid fraction in PBS.The 11-dehydro-TxB 2 levels were stable also in PBS milieu, while the 8-iso-PGF 2α levels progressively decreased starting from approximately 4 months of storage.This progressive decrease in 8-iso-PGF 2α may be related to its chemical structure that includes a cyclopentane 33 which is possibly less stable in PBS at pH 7.4 than the oxane ring of 11-dehydro-TxB 2 18,33 .Eleven-dehydro-TxB 2 has extra chemical resonance forms due to the double-bonded oxygen which may further stabilize the molecule 33 , while the number and position of the hydroxyl groups in the 8-iso-PGF 2α molecule may increase its reactivity and instability 34 .Since the 8-iso-PGF 2α was stable in urine but not in chromatographic extracts, we can hypothesize that the molecule is less stable at higher pH, as for PBS versus urine and/or in solutions containing salts like ethylene-diamine-tetra-acetic acid.Further investigations will be needed to clarify this difference in stability.Concerning urinary creatinine, previous studies had investigated its stability in different storage conditions and time-intervals, as summarized in Table 2. Creatinine levels were stable in urine samples kept at 37 °C for 30 days, while at > 55 °C creatinine levels decreased (Table 2) 35 , likely due to temperature-driven degradation 36 .We had previously studied urine samples kept for 7 days at room temperature and observed stable concentrations over this short time interval as well (Table 2) 24 .Creatinine was also stable in samples stored at −22 °C over 15 years (Table 2) 37 , which is consistent with our findings.Notably, we studied urine samples from different conditions (healthy, diabetes mellitus and cancer) and creatinine values were consistently stable in all groups (Table 1).
Since the same urine samples stored in biobanks are usually used multiple times, undergoing multiple freeze-thaw cycles, we investigated the effect freeze-thaw cycles on both urine and extracted samples.Freeze-thaw cycles had been reported to variously affect some urinary metabolomic profiles 38 , with acylcarnitine and hexose 39 , acetate, benzoate and succinate significantly increasing while formate and urea decreasing after 8 freeze/thaw cycles 40 .Urinary albumin appeared stable in urine samples up to 5 freeze-thaw cycles and decreased from cycle 6 (Table 2) 41,42 .Urinary creatinine was reported stable over 8 cycles 40 which is consistent with our data.8-iso-PGF 2α and 11-dehydro-TxB 2 have been previously studied in urine and PBS medium after a maximum of 3 freeze-thaw cycles (Table 2) 31,41,43,44 .Our data confirm and expand the stability of 11-dehydro-TxB 2 and creatinine in urine up to 10 consecutive freeze-thaw cycles, while a significant progressive increase in 8-iso-PGF 2α and hydrogen peroxide concentrations were observed starting from 5 to 6 cycle in samples without antioxidant.Approximately 40% of 8-iso-PGF 2α has been shown to be excreted in human urine as glucuronide conjugate and increasing pH has been reported to increase glucuronidase hydrolysis and the concentration of un-conjugated F 2 isoprostane 45 .However, our experiments showed no changes in pH in urine samples by freezing and thawing, so it is unlikely that the increase in 8-iso-PGF 2α results from release of unconjugated compound.Increased concentration of H 2 O 2 triggered by multiple freezing-thawing may lead to a non-enzymatic oxidation of AA from cell membrane residues or other contaminants in urine.Interestingly, PLA 2 has been found in urine of mice [46][47][48] , healthy subjects and patients 46,48,49 and reported to be activated by freeze-thaw cycles by urinary invertase 39 .Moreover, freezing-thawing of cells and biological fluids has been reported to increase different reactive oxygen species, mostly H 2 O 2 and superoxide anion [50][51][52] .Consistent with this hypothesis, in the chromatographic extracts eluted in clean PBS with no contaminants, freeze-thaw cycles did not affect the 8-iso-PGF 2α .Whichever the origin of free AA in urine, our data indicate that it may undergo oxidation into 8-iso-PGF 2α as indicated by the increased concentration of H 2 O 2 that reflects the oxidation level in the sample 28 .Consistently, 8-iso-PGF 2α was stable when the antioxidant 4-hydroxy-TEMPO was added, which also blocked H 2 O 2 increase.
Our study has some limitations: we did not investigate the stability of those biomarkers in urine samples and chromatographic extracts under different storage temperatures.However, since these metabolites were stable at −40 °C, it can be assumed that −80 °C storage would give similar results, while the stability at −20 °C may be shorter.Previous studies have reported the stability of 8-iso-PGF 2α at −70 °C for up to 2 years 31 .Furthermore, we

Figure 2 .
Figure 2. Eleven-dehydro-TxB 2 , 8-iso-PGF 2α , and creatinine in urine samples stored for up to 10 years.Panel (a) shows 11-dehydro-TxB 2 (pg/mL) values in urine samples stored from 1 week up to 10 years.Values are represented as % of the first extraction.Panel (b) represents the absolute values of 11-dehydro-TxB 2 (pg/mL) on the first versus the second extraction.Dotted line is the correlation.Panel (c) shows 8-iso-PGF 2α (pg/mL) values in urine samples stored from 2 weeks up to 10 years.Values are % of the first extraction.Panel (d) represents the absolute values of 8-iso-PGF 2α (pg/mL) on the first versus the second extraction.Dotted line is the correlation.Panel (e) shows urinary creatinine values in urine samples stored from 1 week up to 10 years.Values are % of the first creatinine measurement.Panel (f) represents the absolute values of creatinine (mg/mL) on the first versus the second measurement.Dotted line is the correlation.PG: prostaglandin; Tx: thromboxane.

Figure 3 .
Figure 3. Eleven-dehydro-TxB 2 and 8-iso-PGF 2α expressed as pg/mg creatinine in urine samples stored for up to 10 years.Panel (a) shows 11-dehydro-TxB 2 (pg/mg creatinine) values in urine samples stored from 1 week up to 10 years.Values are % of the first extraction.Panel (b) represents the absolute values of 11-dehydro-TxB 2 (pg/ mg creatinine) on the first versus the second extraction.Dotted line is the correlation.Panel (c) shows urinary 8-iso-PGF 2α (pg/mg creatinine) values in urine samples stored from 2 week up to 10 years.Values are % of the first extraction.Panel (d) represents the absolute values of 8-iso-PGF 2α (pg/mg creatinine) on the first versus the second extraction.Dotted line is the correlation.PG: prostaglandin; Tx: thromboxane.

Figure 4 .
Figure 4. Eleven-dehydro-TxB 2 and 8-iso-PGF 2α levels in chromatographic extracts stored for up to 10 years.Panel (a) shows 11-dehydro-TxB 2 (pg/mL) values in chromatographic extracts stored in PBS from 1 week up to 10 years.Values are % of the first measurement.Panel (b) represents 11-dehydro-TxB 2 (pg/mL) absolute values on the first versus the second measurement.Dotted line is the correlation.Panel (c) shows 8-iso-PGF 2α (pg/mL) repeated values in chromatographic extracts stored in PBS from 2 week up to 10 years.Values are % of the first measurement.Panel (d) represents 8-iso-PGF 2α (pg/mL) absolute values on the first versus the second measurement.Dotted line is the correlation.PG: prostaglandin; Tx: thromboxane.

Table 2 .
Effectprojects associated with large clinical datasets analysing samples stored in biobanks for several years and undergoing multiple cycles of freezing and thawing.