24-hour Urine Catecholamines (PCS1, PCS2, PMBC)

Levels of catecholamines (epinephrine [E], norepinephrine [NE], and dopamine [DA]) in urine were determined using ion-exchange high pressure liquid chromatography (HPLC).  HPLC is a very sensitive assay that can detect as little as 0.05 picomoles of each catecholamine.  For this assay, individual urine samples are pre-cleaned by passing them though a column containing ionically charged beads.  Catecholamine molecules are retained on the column and all other materials or contaminants are washed through the column.  The bound catecholamines are then eluted and collected from the column with an acidic buffer.  A portion of the pre-cleaned sample is injected into the HPLC system which consists of an ion-exchange column, a high pressure pump, and an electrochemical cell detector.  The ion-exchange column has a charge that is opposite to the charge of the catecholamine molecules (when suspended in the acidic solution).  As the sample is passed (under high pressure) through the ion-exchange column, the catecholamines are slowed down due to their charge.  Because E, NE, and DA differ in magnitude of charge, the charge of the ion-exchange column will slow differentially the passage of each catecholamine.  Thus, catecholamines can be separated into E, NE, and DA based on the relative time necessary for the molecules to pass through the column.  Once through the column, the catecholamines pass through an electrochemical detector that oxidizes and then reduces the molecules using electrical energy.  The changes in potential between the oxidized and reduced states are measured by the detector.  The levels of the individual molecules directly correlate with the amount of potential such that the higher the potential, the greater the level of catecholamines.  Comparing the potentials to a standard cure that has been constructed using known concentrations of the different catecholamines yields final values. 

Sample Collection

In PCS1, PCS2, and PMBC, subjects provided 24-hour urine samples during their first 24 hours in Quarantine.  Specific collection procedures, however, differed slightly between PMBC and the two earlier studies.  In PCS1 and PCS2, urines were collected throughout a single, continuous 24-hour period.  In PMBC, urines also were collected throughout a continuous 24-hour period, but separately for the first 15 hours and the last 9 hours, respectively (see specific procedures below).  In PMBC and PCS2, an additional 24-hour collection was conducted during the pre-Quarantine baseline.    

Quarantine Day 0 Sample Collection Procedures: PCS1, PCS2, PMBC

PCS1 & PCS2

Specimens were collected in dark urine collection bottles containing 2 g of sodium metabisulfate (a preservative).  Subjects were instructed to begin collecting urine during their first morning void on Quarantine Day 0 and to continue the collection until the same time on the following morning (Day 1).  Subjects were further instructed to record the date and time of their first and last collections on the label affixed to the exterior of the collection bottle.  Specimens were returned to study staff at the end of the collection period.

PMBC

On Day 0 of quarantine, subjects were again provided with 1–liter specimen bottles containing sodium metabisulfate, and were instructed to collect all urine voids during the target period, which was divided into two stages. Stage 1 began at 5:00 p.m. and ended at 8:00 a.m. the following morning. Stage 2 began at 8:00 a.m. and ended at 5:00 p.m. on the same day.  Subjects were instructed to save overnight samples and the first morning void in one container, and voids collected during Stage 2 in a second container.

Pre-Quarantine (Baseline) Sample Collection Procedures: PCS2 & PMBC

PCS2

Baseline 24-hour urine samples were collected during a typical day at home.  Subjects were provided with dark urine collection bottles containing sodium metabisulfate (preservative), and instructed to begin collection in the morning after their first void, and to continue to collect all urine produced during the subsequent 24 hours. 

PMBC

Approximately two days prior to their baseline collection, subjects attended a training session during which they were instructed on how and when to collect their urine samples (see Urine and Saliva Collection Instructions).  Samples were collected in 1-liter plastic specimen collection bottles containing sodium metabisulfite.  Subjects were instructed to begin collection in the morning after their first void, to collect all urine produced during the subsequent 24-hour period, and to record any lapses in this collection.  They also were instructed to keep the sample refrigerated when possible and not add water or any other substance to increase the volume of the sample.  A report describing foods, activities and other behaviors that could affect catecholamine levels also was to be completed (see Urine Collection Record).

Following all collections, the total volume of each sample was measured and recorded.  After thorough stirring, 50ml of urine was extracted from the sample and placed in a 100-ml plastic container with 1 ml of 6N hydrochloric acid (or amount necessary to achieve a pH of 5 or less).  Samples were then stored at -70°C until later analysis.

Sample Preparation

PCS1 & PCS2

After being thawed and centrifuged, samples were prepared for analysis by high performance liquid chromatography (HPLC) using a standard protocol (see Frequently Asked Questions).

PMBC

Samples were thawed and centrifuged prior to analysis by solid phase extraction (SPE) and HPLC.  In preparation for SPE, 3–ml aliquots of urine in duplicate, 3 ml of a standard solution (Bio-Rad) and 3 ml of a normal quantitative urine control (Bio-Rad) each were mixed with 50 μl of internal standard (DHBA, Bio-Rad), 5 ml of dilution reagent (30 mM ammonium acetate, 2.7 mM EDTA; pH 7.5) and 100 μl of 0.5M NaOH. The pH of each mixture was adjusted to 6.5 ± 0.5 by adding 0.5M NaOH. The solutions were absorbed onto a SPE column filled with Bio-Rex 70 cation exchange resin (50–100 mesh) and allowed to drain. The columns were washed with dilution reagent and distilled water and eluted with 6 ml of 3.6 mM ammonium pentaborate. The eluate was collected and injected into the high-performance liquid chromatograph.

Assay

PCS1 & PCS2

Catecholamine assays were performed at the Clinical Analytical Laboratory of the University of Pittsburgh School of Medicine using high performance liquid chromatography (HPLC) with electrochemical detection. The interassay coefficients of variation were 1.49% and 3.49% for NE and E, respectively.

PMBC

HPLC assays for urinary catecholamines were performed by the Immunologic Monitoring and Cellular Products Laboratory (IMCPL) at the University of Pittsburgh Cancer Institute using a Beckman Ultrasphere C18 ODS 5 μm column (4.6 × 15 cm). The system included a pump (ESA Model 583), an autosampler (ESA Model 542), and an electrochemical detector (ESA Model 5200A). The mobile phase consisted of 50 mM NaH₂PO₄, 33.6 μM EDTA, 0.43 mM SOS, and a pH of 3.5. The MeOH ratio was 95:5, and the flow was set at 1.2 ml/min. Peak heights for E, NE, and the internal standard were measured with ESA 501 software.  Reliability of the assay was measured by rerunning samples and periodically running known standards or test samples.  All coefficients calculated by correlating duplicates, later replicates, and known values exceeded .97.

Reference

Baum, A., & Grunberg, N. (1997). Measurement of stress hormones. In: Cohen, S., Kessler, R. C., & Underwood, G. L., editors. Measuring stress.  New York: Oxford University Press; p. 75–92.