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Endocrinology and Metabolism |
1 Departments of Chemical Endocrinology, 2 Endocrinology, and 3 General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
aAddress correspondence to this author at: 479 Dept. of Chemical Endocrinology, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands, Fax 31-24-3541484; e-mail a.ross{at}ace.umcn.nl.
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Abstract |
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Methods: Spot urine samples were collected from 8 healthy volunteers. Aliquots were immediately frozen at –20 °C, or acidified to pH 4 and then frozen either directly or after 24 h at room temperature. The remaining urine was left at room temperature for 24 h and then split into one portion that was acidified and one portion that was not. Aliquots were either frozen or allowed to stand at room temperature for an additional 24, 48, 72, 96, and 168 h before freezing. We also tested the efficacy of adding Na2EDTA and Na2S2O5, as an alternative to acidification for preservation of the catecholamines.
Results: No clinically relevant degradation (<5%) was observed for the fractionated metanephrines under any of the storage conditions. In contrast, in 
50% of the untreated samples catecholamines were partially degraded during the first 24 h at room temperature. Immediate acidification, however, prevented degradation, whereas acidification after 24 h prevented further decay. Addition of Na2EDTA and Na2S2O5 fully prevented degradation of catecholamines during the first 24 h in 4 of 5 cases. In the remaining case, degradation did not exceed 10%.
Conclusion: Preservation of samples for measurements of urinary fractionated metanephrines is not necessary if samples are assayed or frozen within 1 week, which is an important advantage if transport of samples is necessary. In contrast, urinary catecholamines require preservation measures during collection.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a. immediately frozen at –20 °C.
b. immediately acidified to pH 4 and frozen
c. immediately acidified to pH 4 and frozen after 24 h
d. left for 24 h at room temperature and then frozen, or frozen after a subsequent period of 24, 48, 72, 96, or 168 h of storage at room temperature without preservation
e. left for 24 h at room temperature, acidified to pH 4 and then frozen, or frozen after a subsequent period of 24, 48, 72, 96, or 168 h of storage at room temperature in an acidified condition
With this protocol we were able to test the effects on the stability of both metanephrines and catecholamines in samples that were left standing at room temperature for up to 8 days (d vs a or b) and acidifying during (c vs a or b) or after the first 24 h at room temperature (e vs a or b) and (e vs d).
The results of these experiments led to an additional study in which spot urine samples were collected from another 5 healthy volunteers (2 males and 3 females) for catecholamine assay. Each urine sample was divided into 2 equal parts, and to one part, as an alternative preservation method, Na2EDTA and Na2S2O5 (25 mg of each) were added immediately after collection.
chemicals and reagents
All reagents were of analytical grade and MilliQ reagent water was used. NE, EPI, internal standard isoproterenol, MN, and NMN were obtained from Sigma. Na2EDTA and Na2S2O5 were obtained from Merck. For measurements of the catecholamines and metanephrines, we used the same reagents and materials as previously described (16)(17), but the reagent set from Bio-Rad Laboratories (17) has been replaced by a newer version.
instrumentation
An Alliance Separations Module Model 2695 (Waters Associates) was used for HPLC measurements of the catecholamines and metanephrines in urine. Chromatographic data were processed with Empower Pro Workgroup, including Oracle9i® database Software version 1154. Separation and detection were performed as previously described (16)(17).
sample preparation
Urine samples for measurement of the catecholamines were processed as previously described (16), with the following exceptions: 25 µL of urine were added to 1 mL 0.01 mol/L HCl before liquid-liquid extraction, which was performed only once. We made the final extraction in 75 µL of 0.08 mol/L acetic acid. To convert the catecholamines into their diphenyl-quinoxalin derivatives, we used twice the amounts of the solutions. After derivatization, 25 µL of the solution was injected into the chromatographic system. Processing of the urine samples for measurement of metanephrines was as described (17), without modification.
sensitivity, precision and accuracy
Analytical characteristics of assay performance have been described (16)(17). Most relevant to the present experiments were the within-run CVs for measurements of the catecholamines and metanephrines, which were calculated from 24 duplicate measurements of the 1st sample of each volunteer. These were 1.8% for NE, 1.9% for EPI, 2.9% for NMN, and 2.4% for MN.
calculations
We used Empower Pro Workgroup, including Oracle9i® database Software package version 1154 from Waters Associates for HPLC-FD and HPLC-ECD peak analysis. With each series of samples, a standard mixture was analyzed. Peak-height ratios for each analyte relative to the corresponding internal standard isoproterenol (catecholamines) or 4-O-methyldopamine (metanephrines) were determined in both the standard mixture and the urine samples. These ratios were used to calculate the concentrations in the urine samples.
statistics
We calculated 1-sided 95% confidence intervals from the within-run CVs for percentage of baseline concentrations. Degradation was assumed to have taken place from the point in time that only values below the confidence limits were observed. Degradation was considered relevant when it exceeded 10%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). When these urine samples were acidified to pH 4 immediately after voiding, no degradation of the catecholamines was found (Table 1
). Moreover, acidification prevented decay in one sample that suffered from degradation (F2; NE –18% and EPI –24%) when stored frozen without acidification (not shown). In contrast, no degradation of fractionated metanephrines was found in any of the 8 unpreserved urine samples, irrespective of acidification, including the 4 in which catecholamine decay occurred (Table 2
).
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After the first 24 h at room temperature (equivalent to a usual collection period), further degradation of the catecholamines was observed during the next 7 days in the same 4 unpreserved urine samples (Fig. 1
). The decreases ranged from –25% to –78% for NE and –39% to –89% for EPI. Moreover, in 2 of the other unpreserved urine samples a moderate decrease was found after 96 h (NE –6% and EPI –11%) and after 192 h (NE –5% and EPI –11%). There were only 2 unpreserved urine samples in which no degradation was found after storage at room temperature during the study period. In contrast, acidification to pH 4 after the first 24 h at room temperature prevented further decay (Fig. 1
).
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No degradation of fractionated metanephrines was found in any of the unpreserved urine samples for the next 7-day period at room temperature (Fig. 1
, Table 2
).
When the stability of the catecholamines was examined with and without the addition of Na2EDTA and Na2S2O5 immediately after voiding, there was a slight decrease of NE (–5.4%) and EPI (–6.2%) in 1 of 5 unpreserved urine samples, and during the first 24 h at room temperature a slight decrease of only EPI (–8.9%) in a preserved urine sample. After 4 days, a protective effect attributable to the addition of Na2EDTA and Na2S2O5 became evident. In 1 unpreserved sample, both NE and EPI had virtually disappeared, whereas the decay was limited, although not sufficiently, to –14% for NE and –19% for EPI when the additions had been made. After 8 days, NE and EPI had practically disappeared from an additional unpreserved urine sample, whereas the maximal change in the preserved urine samples was –25.8% for NE and –43.5% for EPI.
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Discussion |
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In our experiment 8 volunteers participated, and we purposely did not pool the obtained urine samples. We found degradation of the catecholamines in unpreserved urine samples after the first 24 h at room temperature in 3 of 8 cases for NE and in 4 of 8 cases for EPI. In contrast, no degradation was found in 4 of the other urine samples after the first 72 h of storage at room temperature, and only a small decrease was found in 1 unpreserved urine sample after 96 h storage at room temperature (NE –6% and EPI –11%) and in another urine sample after 192 h storage at room temperature (NE –5% and EPI –11%). In 2 urine samples no degradation was found even after 192 h of storage at room temperature. Apparently, catecholamine decay may vary widely between individual samples, even for samples from healthy persons, ranging from no deterioration to complete degradation of the analytes. Therefore, inclusion of too few individuals, or the use of pooled material, may yield misleading results, an observation that may explain the seeming contradiction between our results and those of Miki and Sudo (7) and Boomsma (12). For stability studies in general, we advise against reliance on pooled urine, serum, or plasma. The cause of this variability (initial pH, microbial growth, proportion of unconjugated analyte) may be the subject of further study.
As confirmed by earlier observations (5)(6)(7)(8)(9)(10)(11)(12), acidifying to pH 4 is an effective means to prevent catecholamine decay, even when it is not done immediately after voiding (Fig. 1
). For those urine samples in which degradation starts during the first 24 h and without acidification continues at room temperature for the next 7 days, acidification after the first 24 h abolishes further degradation. Chan et al. (13) reported that unpreserved pooled urine samples stored at –80 °C were stable, with <10% variation for at least 22 days. In one of our cases, we observed degradation during storage at –20 °C in the unpreserved but not in the acidified sample. To achieve acidification in practice, HCl may be added in advance to the polyethylene collection container, but this leads to hydrolysis and poses a hazard to the patient when the acid is still concentrated (7)(10)(13) during the first phase of collection. The catecholamine concentrations found under such conditions result from the opposing effects of deconjugation and decay. Moreover, insufficient antioxidative capacity may lead to catecholamine instability. Therefore, addition of Na2EDTA and Na2S2O5 instead of HCl during collection, followed by acidification to pH 4.0 with HCl after receiving the urine sample is mandatory. Boric acid (6) or formate buffer(10) also can be used for this purpose.
The special requirements to stabilize catecholamines during collection and storage at room temperature do not appear to be necessary for fractionated metanephrines in urine (Table 2
), at least for a 1-week period. Nevertheless, acidification to pH 4 immediately after receipt of the 24-h urine sample can be considered as a general safety measure. We did not find data in the literature allowing for a direct comparison with the present observations. Our findings are highly relevant to the biochemical diagnosis of pheochromocytoma according to recommendations emerging from the First International Symposium on Pheochromocytoma (2005, Bethesda, MD). First, concentrated acid need not be added to the devices used for urine collection; thus the risk for the patient of injury by the acid is eliminated. Second, if samples are assayed or frozen for assay within 1 week, urinary metanephrines are sufficiently stable at room temperature to permit shipment by regular mail. Third, the absence of additives may permit measurement of other analytes such as urinary sodium and potassium, which are essential to the interpretation of aldosterone excretion when assessed in the same sample, as well as albumin and total protein.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  R. T Peaston and S. Ball Biochemical detection of phaeochromocytoma: why are we continuing to ignore the evidence? Ann Clin Biochem, January 1, 2008; 45(1): 6 - 10. [Abstract] [Full Text] [PDF]
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