INTRODUCTION

There is increasing evidence that stress atwork can have detrimental effects on perfor-mance, well-being, and health (Cooper, 1998;Ganster & Perrewé, 2001; Kahn & Byosiere,1992; Marmot & Wilkinson, 1999; Sonnentag& Frese, 2003). However, the vast majority ofstudies in occupational stress research have usedself-reports for measures of both independent(stressors) and dependent variables (e.g., strain;see Kahn & Byosiere, 1992; Sonnentag & Frese,2003; Zapf, Dormann, & Frese, 1996). Thusstressor-strain relationships may be overestimat-ed because of correlated measurement error(common method variance; see Semmer, Zapf,& Greif, 1996).

Many authors therefore recommend measur-

ing independent and dependent variables withdifferent methods (multimethod approach; seeKahn & Byosiere, 1992). Physiological mea-sures are good candidates for such an approach.One cannot regard them as “the” more objec-tive measures, given that they also suffer fromtypical errors such as artifact susceptibility andmeasurement error attributable to devices, de-tection range of analytical procedures, handlingof instruments, occasional influences, and soforth (see Beehr, 1995; Fried & Ferris, 1987).Because these errors are not correlated with er-rors of self-report, relationships may be under-estimated, as Semmer et al. (1996) have shownfor job observation methods (which are anothercandidate for alternatives to self-report). Never-theless, if handled carefully, physiological mea-sures do offer the potential to avoid common

Two Urinary Catecholamine Measurement Indices forApplied Stress Research: Effects of Time and Temperatureuntil Freezing

Achim Elfering, Simone Grebner, and Norbert K. Semmer, University of Berne, Berne,Switzerland, and Christa Byland and Hans Gerber, University Hospital, Berne, Switzerland

Research on stress at work often involves the analysis of urinary adrenaline and nor-adrenaline. It is usually assumed that samples have to be cooled quickly and storedat refrigerator temperature before freezing. This is often difficult to achieve in fieldstudies. This experimental study therefore tests the robustness of results when sam-ples are not cooled immediately. Samples of 9 men and women, collected at 3 pointsin time, were immediately frozen, kept for a variable delay in a warm room, orstored in a refrigerator before freezing. Two indices were calculated: (a) the ratioof hormones to liquid volume, period of excretion, and body weight; and (b) theratio of hormones to urinary creatinine. The reliability of high performance liquidchromatography analysis was satisfactory, as was the comparability of the 2 indices.Unfavorable storage up to 24 hr did not cause bacteria-driven decreases of cate-cholamines, regardless of storage temperature or sampling time. Results suggest highstability for at least 24 hr without cooling, provided the samples are immediatelyacidified. Cooling may therefore be handled less restrictively than has been assumed.The application of this research is to facilitate research in settings where samples arecollected at different places, such as participants’ homes or different workplaces.

Address correspondence to Achim Elfering, Psychology Department, University of Berne, Muesmattstr. 45, 3000 Berne 9,Switzerland; achim.elfering@psy.unibe.ch. HUMAN FACTORS, Vol. 45, No. 4, Winter 2003, pp. 563–574. Copyright ©2003, Human Factors and Ergonomics Society. All rights reserved.

SPECIAL SECTION

564 Winter 2003 – Human Factors

method variance, to yield better estimates ofrelationships, and to increase the understandingof the processes involved.

However, as soon as one leaves the laborato-ry and moves into the field, a number of pitfallsexist that might lead to serious errors, thus ham-pering the interpretability of field study results.One of these potential sources of errors concernsthe necessity for storage and transportation(Lundberg, Melin, Fredrikson, Tuomisto, &Frankenhaeuser, 1990). Clinical chemistry showsthat it is not possible to assign fixed correctionfactors for such interferences to urine analysis.Therefore it is recommended that samples beacidified immediately and frozen as soon as pos-sible (Shoup, Kissinger, & Goldstein, 1984).What seems to be feasible, however, is to keepthe samples at refrigerator temperature for awhile before freezing them. Thus Boomsma,Alberts, van Eijk, Man in ’t Veld, and Schale-kamp (1993) showed that catecholamines (CAs)are stable at 4°C in unpreserved urine for 1month, a finding that was recently replicatedfor a 10-hr storage period by Miki and Sudo(1998). Miki and Sudo also stored samples atroom temperatures; here they found (a) a strongdecrease in CA values in unpreserved samplesand (b) a tendency toward increasing, ratherthan decreasing, values in acidified samples.These increases were, however, within a rangeof 10% for a delay of 1 day.

From the study by Miki and Sudo (1998), itseems clear that (a) immediate freezing is es-sential for unpreserved samples and (b) delaysof more than a day at room temperature arerisky for both acidified and unpreserved speci-mens. More refined studies are needed, however,to clarify whether results are robust with regardto delays until freezing of up to 24 hr. This is acrucial time period for field studies: Freezingwithin 24 hr usually can be guaranteed underfield conditions. Delays of several hours, how-ever, are often difficult to avoid in field work inwhich urine samples are picked up at the partic-ipants’ homes or workplaces in different areas(Elfering, Grebner, Semmer, & Gerber, 2002;Grebner, 2001).

Because this time lag of up to 24 hr beforefreezing is so crucial, we tested the role of delayas a potential source of error and compared itwith the measurement error that arises from

the accuracy of the laboratory analysis itself. Inthis way, the consequences of different violationsof the typical recommendations can be tested,and their effects on the trustworthiness of fieldresults with regard to catecholamines can beestimated.

Measurement Error

High performance liquid chromatography(HPLC) with electrochemical detection hasbecome the standard laboratory procedure ofanalysis. The inter- and intraassay deviation isusually reported to be lower than 10% whenstandardized probes are analyzed. Therefore,we expect errors for parallel samples that areimmediately acidified (pH 3) and frozen (–20°C)to be normally distributed and to be below 10%,independent of gender and voiding time. Intra-class correlation coefficients should be greaterthan r = .80.

Storage until Freezing

Urine samples are usually acidified to a pHlevel of about 3 as quickly as possible and frozenuntil laboratory analysis. If they cannot be fro-zen immediately, the acidified samples shouldbe stored at 2° to 8°C until freezing. This proce-dure is recommended in order to inhibit growthof bacteria and hydrolysis, which could cause asubstantial decrease in the concentration of hor-mones (Colombo, 1994). However, as men-tioned, Miki and Sudo (1998) found that storageat room temperature was associated with slightincreases in CA levels after 1 day, increasingeven more over 1 week, when samples werestrongly acidified (either 0.5 or 1.0 pH). Thismight be attributable to evaporation.

In the present study, we compared 24-hr stor-age at 5°C (refrigerator) with storage at roomtemperature (25°C, electric light). Bacterialactivity, hydrolysis, and evaporation should bemore evident at the higher storage tempera-ture. If temperature is an important source ofbias within this period, deviations caused byhigh-temperature conditions should be higherthan those caused by the genuine measurementvariance. However, compared with a room-temperature sample, a refrigerator-stored sampleshould correspond more closely to immediatelyfrozen samples, given that refrigerator storagehas been shown to preserve CA for up to 1 year

URINARY CATECHOLAMINE INDICES 565

in acidified specimens (Boomsma et al., 1993).Storage at room temperature was varied in four8-hr steps (0, 8, 16, and 24 hr) until freezingat –20°C. Bacterial activity, hydrolysis, andevaporation should cause some systematic linearor curvilinear changes in time. We thereforetested for trends.

Indices of Hormone Concentration

Standardization of urinary CA levels mayrefer to (a) body weight (or, alternatively, bodyvolume, expressed in square meters of body sur-face) or (b) concentration of urinary creatinine.The first index is useful when urine volumesand voiding intercepts are controlled (e.g., whenall urine in 24 hr is collected) because other-wise there would be a great impact of individualwater consumption and metabolism on hormoneconcentration. Creatinine is a waste product inthe blood created by normal breakdown ofmuscles during activity. Therefore it dependson muscle mass, which is correlated with age,height, and body weight. The kidneys take cre-atinine out of the blood and into the urine. Thecreatinine ratio as an index is appropriate whenit is not possible to control urine volumes andvoiding intercepts during the observation peri-od (spot samples). It has the disadvantage thatcreatinine excretion depends on many otherfactors that should be controlled for. For exam-ple, creatinine is also a waste product from meatprotein in the diet (Kohse & Wisser, 1987).

In field studies, urine specimens are oftenstored in screw-capped tubes. These may causeevaporation because often they are not absolute-ly leakproof (Sullivan, May, & Maberly, 2000).As the ratio of ingredients does not changewhen evaporation occurs, the creatinine indexshould be more robust with regard to evapora-tion effects.

Basically, we expect a high correlation of theindices (r > .80), similar to the findings report-ed by Frankenhaeuser et al. (1989). However,storage conditions might restrict comparabilityof indices by affecting them differentially (seeJames, Crews, & Pearson, 1989).

Before studying the robustness of urinarymeasurement, we need to address the questionof whether measuring catecholamines in salivarather than urine would be a valid alternative.Collecting saliva samples has obvious practical

advantages, both for researchers and for par-ticipants. Indeed, McClelland, Ross, and Patel(1985) showed that salivary noradrenaline reactsto a stressful situation to a significant degree.However, in contrast to steroid hormones (e.g.,cortisol), for which salivary and serum levelsare highly correlated, salivary catecholaminecontent is only slightly correlated with plasmacontent (Lac, 2001). More research is neededto define standards for salivary catecholaminesbefore they can be used independently fromserum and urine standards. At the present time,“the steroid hormones are actually the onlycompounds for which the salivary assay is avalid and largely recognized alternative consid-ering the number of published works using thismethod” (Lac, 2001, p. 663). Thus, at least forthe foreseeable future, there is continued needfor urinary extraction of catecholamines in ap-plied stress research.

METHOD

Participants

A sample of 9 healthy volunteers participated(n = 5 women, mean age = 25.2 years, SD = 2.8years; n = 4 men, mean age = 28.5 years, SD =5.1 years). The urine samples were collected ona working day that began at 9:00 a.m. and wasexpanded, for reasons of measurement, until9:00 p.m. Altogether, 162 aliquots were ana-lyzed. There was no loss attributable to missingvalues, and no data had to be excluded.

Procedure

Participants were asked to empty their blad-der at 12:00 a.m., at 5:00 p.m., and at 9:00 p.m.The urine samples (100 ml) were acidified im-mediately by hydrochloric acid (HCl) to a pHlevel of 3. Subsequently, each sample was divid-ed in six aliquots of 15 ml each. Two aliquots(Measures A and B) were immediately frozenat –20°C. They were used to calculate the mea-surement error of the HPLC analysis. Threealiquots were stored at room temperature (25°C)until freezing for 8, 16, and 24 hr, respectively.They were used to test the influence of delayon hormone concentrations. The last aliquotwas stored in a refrigerator at 5°C for 24 hrbefore it was frozen so that the influence of

temperature until freezing on CA could be test-ed. This procedure was repeated two times (at5:00 p.m. and at 9:00 p.m.) to test for differenturine properties and to simulate a prototypicaldesign of field studies measuring occupationalstress.

Laboratory Analysis

The aliquots were analyzed in the ChemicalLaboratory of the University Hospital in Berne.Analytical improvements and the recent intro-duction of a reliable and suitable method for thesimultaneous determination of urinary adrena-line, noradrenaline, and dopamine by HPLCwith electrochemical detection allow quantifica-tion of concentrations in nmol/L in the routinelaboratory procedure (Rosano, Swift, & Hayes,1991). The aliquots were thawed and centrifugedfor 10 min at 2000 revolutions/min to removesolid particles. The isocratic (i.e., without changeof the elution buffer during the elution) HPLCsystem used consisted of a pump (Waters 510,Waters Division of Millipore) equipped with anHPLC column (Chromsystems Instruments &Chemicals GmbH, Munich, Germany), a degas-ser (OmniLab), and an autoinjector (234 Gilson,Gilson Medical Electronics). Electrochemicaldetection was performed on an EC2000 (ThermoSeparation Products). Data control, acquisition,and evaluation – including integration, quali-fication, and reporting – were performed withHP-ChemStation software (Hewlett-Packard,Inc.).

The tests were performed with a Chrom-systems kit (Chromsystems Instruments andChemicals GmbH, Munich) according to themanufacturer’s instructions. The kit consistedof sample clean-up columns, dilution buffer,elution buffer internal standard (3,4-dihydrox-ybenzylamine 1.5 ng/µl), calibration standard(noradrenaline 25µg/L, adrenaline 5µg/L, dopa-mine 100 µg/L, internal standard 50 µg/L),mobile phase, and an equilibrated and testedcation-exchange HPLC column for CA. Theurine samples were first pretreated by the sam-ple clean-up columns: 3 ml urine (pH < 5)with 6 ml dilution buffer and 100 µl internalstandard were well mixed and then brought upto a pH between 3 and 7 by adding NaOH. Sub-sequently, the prepared samples were placedon the sample clean-up column. The column

was washed with HPLC water after the wholesample had been absorbed. Finally, the CAswere eluted with 6 ml elution buffer. The elu-ate was stabilized with 50 µl HCl 37%. Thecalibration solution was then injected severaltimes until retention time, peak height, andbaseline were stable. Then 20 µl of the stabi-lized eluate was injected in the HPLC system.The detector potential was set to 500 mV andthe flow rate to 1 ml/min. The detection limitis 10 nmol/L, linearity exists between 10 and100 nmol/L, and the coefficient of variance ofthe method is below 10% with a recovery ratebetter than 80%.

Creatinine was determined by the Jaffé reac-tion method. A sample and its sample controlwere measured simultaneously at wavelengthsof 505 and 415 nm, respectively (twin methodon Hitachi 911, Roche Diagnostics, Rotkreuz,Switzerland).

Calculation of Indices

Adrenaline and noradrenaline values wereexpressed as (a) pmol/L/min/kg (picomoles[10–12 mol] per liter urine per minute since lastvoiding per kilogram body weight) and (b) asthe ratio of hormone and creatinine level inurine (nmol/L CA per mmol/L creatinine).

Statistical Procedure

Correlational procedures for the error analysisincluded unadjusted Pearson intraclass correla-tions (Asendorpf & Wallbott,1979). Distributionof errors was tested by Kolmogorov-Smirnovtests. Comparison of means was done by Stu-dent’s t test for paired samples. Tests of variancehomogeneity were carried out by a homogeneitytest for correlated measures (Ferguson & Ta-kane, 1989). The “time until freezing” trend hy-pothesis was tested by a nonparametrical trendtest for correlated measures (Ferguson, 1965).The statistical decision of whether the 24-hrrefrigerator-stored aliquot was a better predictorof “true” hormone level than the 24-hr room-stored aliquot was based on a test for two cor-related correlations (Olkin & Finn, 1990).

RESULTS

Measurement Error

Analyses referring to the comparison of thetwo immediately frozen aliquots (Measures A

566 Winter 2003 – Human Factors

URINARY CATECHOLAMINE INDICES 567

and B) are displayed in Part 1 of Table 1 (leftcolumns). Differences between these aliquotscan be considered as reflecting the measurementerror of the HPLC analysis. Mean deviations(N = 27) for both hormones and creatininewere below 10% (adrenaline: 8.2%, SD =6.5%; noradrenaline: 6.2%, SD = 5.8%; crea-tinine: 3.0%, SD = 3.7%). Moreover, Pearsonintraclass coefficients are higher than r(9) = .80for the two indices of both hormones at all void-ing times. As expected, differences betweenreliability measures in both indices were notstatistically significant, and the distributions didnot deviate from the assumption of normality(Table 1, Part 1).

Storage Temperature

Storing the acidified urine samples for 24 hrat 5°C (refrigerator) versus 25°C (room) didnot generate substantial differences in levels ofadrenaline or noradrenaline (Table 1, Part 2).Mean deviations (N = 27) for both hormonesand creatinine were below 10% (adrenaline:6.9%, SD = 6.4%; noradrenaline: 4.3%, SD =3.9%; creatinine: 1.4%, SD = 1.2%). Generally,means and standard errors of means displayedin Figures 1 and 2 do not differ from values ofimmediately frozen aliquots. As shown in Part 2of Table 1, differences between temperature con-ditions in both indices were normally distributedand did not differ significantly. Moreover, thelowest Pearson intraclass coefficient was .85,and all others were higher than .90.

As shown in Part 3 of Table 1, the differencesbetween the two not-frozen samples were notsignificantly larger than the differences betweenthe two frozen aliquots of the reliability condi-tion. Furthermore, a comparison of variancesin the reliability-versus-temperature conditionyielded no significant differences, with one ex-ception, and this was in the unexpected direc-tion: For noradrenaline, variances of both theweight index and the creatinine index tended tobe even smaller in the temperature condition ascompared with the reliability condition. Furtheranalyses (not shown in the table) indicated thatin both indices there were no significant differ-ences in predictive power of the two differentlystored aliquots when used as predictors of the“true” hormone level, defined as the mean levelof reliability measures. A test of differences be-

tween correlated correlations (Olkin & Finn,1990) did not reach statistical significance foreither index of CA at any voiding time.

Delay until Freezing

For adrenaline, individual hormone concen-trations rarely decreased with time of delayedfreezing. For the weight index, individual de-creasing values occurred in 3 of 27 cases, andfor the creatinine index they occurred in 1 of 27cases. There were no individual negative trendsfor noradrenaline for either index. Trend analy-sis for the sample as a whole (N = 9) showedno negative trends for either of the hormones,for times of urine sampling, or for either indexof hormone concentration (Table 2). However,two out of six linear trend components of theweight index were significantly positive, one foradrenaline and one for noradrenaline. Therewas no significant linear trend component forthe creatinine index. Figure 1 shows that posi-tive linear trends were stronger for women thanfor men, a pattern that was supported by gender-specific trend analysis. One linear trend com-ponent appeared in women for the creatinineindex.

Correspondence between the Two Indices

Overall, the two indices of hormone levelwere highly correlated. For immediately frozenaliquots, coefficients were higher than r(9) = .80,with only one exception: 9:00 p.m. noradrena-line, r(9) = .70 (Table 1, far right column). Cor-relation coefficients based on values that areaggregated across voiding times are shown inTable 3. For adrenaline, all correlations of thetwo indices within a given condition, whichare displayed in the diagonal, were at least .80.Across conditions, the two indices also corre-late highly. Note that the lowest values, whichwere between .73 and .76, all involved the sam-ple that was kept in the refrigerator for 24 hr(24R); by contrast, the indices for the samplestored at room temperature correlated at least.80 with the indices based on the two frozensamples. For noradrenaline, values are similarfor the frozen samples but somewhat lower forthe indices based on the refrigerator and room-temperature samples.

TAB

LE 1

: M

easu

rem

ent

Err

or

and

Bia

s fr

om

Tem

per

atur

e d

urin

g 2

4-hr

Sto

rag

e in

Tw

o In

dic

es o

f U

rinar

y C

atec

hola

min

es

1. R

elia

bili

ty:

2. T

emp

erat

ure:

3. C

om

par

iso

n M

easu

res

A a

nd B

5°C

and

25°

Ca

of

1 an

d 2

4. In

dic

esb

5°C

≠25

°C,

∆5°

C–2

5°C,

A-B

≠5°

C–2

5°C

,s2 (1

)≠

s2 (2),

Tim

eIn

dex

r ICc

A ≠

B, t

(8)d

∆

A-B

, Ze

r ICc

t(8)

dZe

t(8)

dt(

7)f

r

Ad

rena

line

(N=

9)

12 a

.m.

Min

/kg

.96

–1.0

200.

50.9

1–0

.240

1.08

–0.5

00–0

.060

.98

Cre

atin

ine

.92

–0.3

900.

59.8

5–0

.230

1.02

–0.1

500.

585

p.m

.M

in/k

g.9

7–2

.07*

.0.

60.9

3–0

.150

1.11

–1.4

10–0

.180

.81

Cre

atin

ine

.92

–1.4

100.

48.9

2–0

.690

1.02

–0.6

600.

399

p.m

.M

in/k

g.9

9–0

.820

0.82

.98

0.06

0.52

–0.3

90–1

.520

.85

Cre

atin

ine

.99

0.80

0.51

.96

–0.0

200.

420.

43–1

.380

No

rad

rena

line

(N=

9)

12 a

.m.

Min

/kg

.97

–1.2

900.

53.9

9–1

.340

0.78

0.23

**2.

43**

.94

Cre

atin

ine

.96

–0.5

700.

76.9

9–1

.190

1.01

–0.0

40**

2.41

**5

p.m

.M

in/k

g.9

7–1

.430

0.75

.98

0.45

0.82

–1.7

700.

68.8

4C

reat

inin

e.9

6–0

.720

0.51

.98

–0.4

000.

80–0

.340

0.79

9 p

.m.

Min

/kg

.90

–0.3

200.

76.9

31.

090.

81–0

.860

0.49

.70

Cre

atin

ine

.96

0.33

0.66

.96

0.94

0.74

–0.5

40–0

.320

a Thi

s an

alys

is c

once

rns

only

tho

se s

amp

les

that

wer

e st

ored

for

24 h

r, ei

ther

at

room

tem

per

atur

e (2

5°C

) or

in t

he r

efrig

erat

or (5

°C).

bC

orre

latio

n b

etw

een

the

wei

ght

ind

ex a

nd t

he c

reat

inin

e in

dex

for

the

first

mea

sure

tha

t w

as im

med

iate

ly f

roze

n (M

easu

re A

). c P

ears

on

intr

acla

ss c

orr

elat

ion

(MS

bet

wee

n p

artic

ipan

ts–

MS

with

in p

artic

ipan

ts) /

[(M

Sb

etw

een

par

ticip

ants

+ M

Sw

ithin

par

ticip

ants) ×

(n–

1)].

dTh

e t

test

fo

r co

rrel

ated

mea

sure

s.e Z

valu

e o

f th

e K

olm

og

oro

v-Sm

irno

v te

st o

f no

rmal

dis

trib

utio

n in

diff

eren

ce v

alue

s. f T

est

of

diff

eren

ces

in v

aria

nce

for

corr

elat

ed m

easu

res

(Fer

gus

on

& T

akan

e, 1

989)

.*

p<

.10.

**

p<

.01.

568

URINARY CATECHOLAMINE INDICES 569

DISCUSSION

Occupational scientists should study thestress process in both the laboratory and the nat-ural setting, as ergonomists measure the effectsof physical load in the laboratory and at theworkplace (Backs & Boucsein, 2000; Meijman,1993; Meijman & Kompier, 1998). Physiologicalresponses at the workplace can serve as indica-tors of the stress process and may be used as an

outcome variable in the evaluation of workplaceinterventions (Semmer, 2003a, 2003b). To beemployed for such purposes, measurement inthe field has to be reliable. The aim of thismethodological study was to test the effects ofdeviations from the recommended handlingprocedures for the analysis of urinary catecho-lamines. Conditions were construed to reflectpossible influences of field conditions (e.g., inapplied stress research), in which organizational

Figure 1. Urinary adrenaline and noradrenaline excretion as related to urine volume, time since last voiding,and body weight (pmol/L/min/kg). Trends show mean values and standard errors of the mean for women andmen for three voiding times. *p < .05, linear trend in nonparametric trend analysis, two-tailed.

570 Winter 2003 – Human Factors

constraints and the often-encountered need forimprovisation sometimes make it difficult toimmediately store acidified urine samples atrefrigerator temperatures until freezing. If stor-ing them at higher temperatures proves to havesubstantial effects, such samples must be disre-garded for analysis. If results are comparable,however, the strictness of recommendationsmight be relaxed.

Reliability of the HPLC analysis was accept-able. For two common indices of hormone level,

the effects of storage temperature and delayedfreeze were generally in the range of the reliabil-ity of HPLC analysis and therefore almost negli-gible. For the weight- and the creatinine-relatedindices of catecholamines, we observed no sys-tematic decrease that corresponded to the delayin freezing. This presumably reflects the effect ofacidification, which prohibited bacteria growth.Contrary to what would be expected on thebasis of bacterial activity, we observed a patternof small increases in hormone concentrations.

Figure 2. Urinary adrenaline and noradrenaline excretion as related to excretion of urinary creatinine(nmol/L/mmol creatinine). Trends show mean values and standard errors of the mean for women and menfor three voiding times. *p < .05, linear trend in nonparametric trend analysis, two-tailed.

URINARY CATECHOLAMINE INDICES 571

The increase in our data is very small, but it oc-curs within periods shorter than 24 hr. Thispattern is in line with the tendency toward in-creasing values reported by Miki and Sudo(1998). As in our study, their values were onlyslightly higher after 1 day. After 1 week, how-ever, the values had increased substantially.

Miki and Sudo (1998) did not comment ontheir results of increasing values. However, it isknown that storage in screw-capped plastictubes does not rule out evaporation effects. Forinstance, a World Health Organization manualfor urine sampling in the field recommendsestimating evaporation effects by storing andhandling a prepared standard quantity in thesame manner as the specimens (Sullivan et al.,2000). Mild evaporation might have occurred

also in the present samples that were storedfor up to 1 day at 25°C. This interpretation issupported by the results of a trend analysis(Table 2), which showed linear increase as thedominant trend component, which would beexpected if evaporation is occurring. Becausethe ratio of hormones and creatinine shouldremain constant even under conditions of evap-oration, the alternative creatinine index shouldnot be affected by evaporation. In line with thisreasoning, significant effects were observedonly for the weight index. For the creatinineindex, trends were smaller and failed to reachstatistical significance (Table 2).

In spite of the mild evaporation effect, nosubstantial variance was added over and abovemeasurement error when samples were stored

TABLE 2: Trend Components (z Values) from Nonparametric Trend Analysis of Adrenaline and Noradren-aline Level Indices for Delays of 0, 8, 16, or 24 hr until Freezing

Trend Component (z)

Adrenaline Noradrenaline

Hormone-Level Index,Voiding Time (n = 9) Linear Quadratic Cubic Linear Quadratic Cubic

pmol/min/kg12:00 a.m. 1.17 1.33 –0.230 2.28 0.26 0.0005:00 p.m. 1.10 –0.970 –0.120 1.72 –0.130 –0.34009:00 p.m. 2.07 0.00 0.52 1.54 –1.080 0.12

nmol/mmol creatinine12:00 a.m. 0.34 0.90 0.11 1.25 0.13 1.0205:00 p.m. 1.47 –0.650 –0.110 1.47 0.65 –0.34009:00 p.m. 0.79 1.94 –1.020 1.37 0.13 –1.140

Note. Positive values indicate increasing trends and negative values indicate decreasing trends. Critical z value for the trend hypothesisis z = ±1.96, p < .05, two-tailed.

TABLE 3: Correspondence between Two Indices of Urinary Adrenaline and Noradrenaline for ImmediatelyFrozen and Differently Tempered Aliquots

nmol/mmol Creatinine

Adrenaline Noradrenaline

A B 24R 24 A B 24R 24

pmol/min/kg A .87 .84 .76 .84 .84 .79 .69 .66B .83 .88 .73 .80 .79 .85 .73 .7024R .74 .74 .80 .75 .84 .81 .73 .7024 .82 .82 .73 .81 .90 .88 .75 .73

Note. A, B: Two aliquots that were frozen immediately. 24R = 24 hr stored in a refrigerator (5°C) before freezing; 24 = 24 hr stored atroom temperature (25°C) before freezing. Data were aggregated across voiding times (N = 27).

572 Winter 2003 – Human Factors

for 24 hr, and this applies not only to storagein a refrigerator (where this result was to be ex-pected) but also to storage at 25°C. As a con-sequence, the refrigerator-stored sample failedto be a better predictor of the “true” hormonelevel than the room-temperature one. Note thatmild evaporation need not restrict the validity ofthe room-temperature sample in correlationalanalyses, given that it causes only a linear trans-formation of the level of hormones and doesnot affect individual differences, which arecrucial in regression analysis. Nevertheless, oneshould prevent evaporation by controlling clo-sure of samples and humidity in the room.

The most frequent indicator of urinary hor-mone level in applied stress research is standard-ization by voiding intervals and body weight.Correspondence between this indicator and theratio of hormone level to creatinine was satis-factory, which is in line with results reported byFrankenhaeuser et al. (1989). Correspondenceof indices may slightly decrease when compar-ing samples that were immediately frozen withothers that were not. Altogether, however, ourconclusions with regard to the creatinine indexare rather favorable, given its lower liability toevaporation effects. This is in line with the posi-tion advocated by Lundberg (2000) but in con-trast to Baum, Lundberg, Grunberg, Singer, andGatchel (1985), who did not recommend creati-nine as a valid reference value. However, it isrecommended that several factors be controlledwhen using creatinine in defining excretion ratesin urine. Time of day is a trivial factor, but creati-nine levels can be influenced by strenuous exer-cise (elevating it by 30%–50% above baseline),dietary intake of meat or polyunsaturated fat(10%–30%), time of menstrual cycle (10%–15%), and pregnancy (5%–20%). Also, age, in-fection, trauma, and renal insufficiency shouldbe controlled for (see Vesper et al., 2002, for areview, and Hansen, Garde, Christensen, Eller, &Netterstrom, 2001, for useful reference values).

It is noteworthy that for 1 participant in oursample, creatinine was very high in the eveningand differed strongly across voiding times. Pro-tein intake presumably caused this variation, asthis participant had muscular hypertrophy anda high protein metabolism (Kohse & Wisser,1987; Parra, Ramira del Angel, Cervantes, &Sanchez, 1980).

Creatinine is generally stable in urine storedat 4°C for at least 5 to 7 days (Vesper et al.,2002), and the present data also show high sta-bility for 24-hr storage at room temperature.

A special problem would be created for ap-plied stress research if creatinine levels werestrongly influenced by stress, because then boththe catecholamines and the reference value (i.e.,creatinine) would change. There are not manystudies on this issue. Those that do exist reportthat effects of stress on creatinine excretion arerather small (Light & Turner, 1992; Scrimshaw,Habicht, Piche, Cholakos, & Arroyave, 1966).We could not analyze this issue in the data set ofthis study. However, this analysis was performedin a sample of young people entering the work-force (see Grebner, 2001), and no intraindivid-ual differences in creatinine were found whencomparing measurements taken at the sametime of day on a working day versus a rest day.Because stress levels can be assumed to be dif-ferent on these days, these results indicate anegligible influence of stress on creatinine.

This study has some limitations as well asstrengths. A weak point concerns the issue wejust discussed – that is, we did not directly con-trol for stress levels and thus had to rely on otherdata to support our argument that a strong in-fluence of stress on creatinine is unlikely. A sec-ond point is that participants were young andhealthy students, who are not representative ofmost working populations.

As for our study’s strong points, first, we in-cluded participants of both sexes and collectedurine at different times of day. Note that the twostudies that are comparable to ours (Boomsmaet al., 1993, and Miki & Sudo, 1998) both ana-lyzed urine samples that were collected onlyonce. In addition, Boomsma et al. used pooledurine, and Miki and Sudo had only 2 partici-pants. Furthermore, in the Boomsma et al. study,urine was enriched in epinephrine. One canexpect the sensitivity of the measurement to bebetter in higher concentrations. However, froma point of view concerned with field researchwith participants who are mostly in a normal,nonclinical range, we were more interested inthe effects of handling and storage in a morephysiological range of hormone concentration,which is potentially associated with less sensi-tivity of the analysis system. By analyzing urine

URINARY CATECHOLAMINE INDICES 573

from 9 individuals of both sexes and collectingspecimens at different times, we controlled forinterindividual and intraindividual variationsin excretion of catecholamines. For instance,the association of stress-related excretion ofcatecholamines and working conditions oftendiffers between sexes, especially in achieve-ment situations. Psychological factors seem tobe more important than biological ones for thesedifferences (Baum et al., 1985), and our studyindirectly supports this position by ruling outthe possibility that these differences reflect selec-tive biases in reliability.

A second strength is that our study was per-formed under field conditions. This implies thatparticipants had to collect the urine, note thevolumes, acidify the samples, record intermedi-ate voidings, and follow the instructions. There-fore, the study also tests the appropriateness ofthe whole sampling technique under realisticfield conditions. Finally, we compared two differ-ent indices of catecholamine measurement.

CONCLUSIONS

Our main finding is a substantial robustnessof urinary catecholamines with regard to tem-perature conditions over 24 hr. This robustnesssuggests that researchers can be more flexiblewith regard to the delay between the collectionand storage of specimens. This is particularly im-portant with respect to temporal or economicrestrictions in complex, work-related field stud-ies. In such studies samples often are collectedat different locations, such as workplaces in dif-ferent companies or, if data from a day off arecollected to compare with those collected atwork, at the participants’ homes (see Korunka,Huemer, Litschauer, Karetta, & Kafka-Lützow,1996). Under such circumstances, substantialnumbers of people are needed to collect samplesfrom participants, unless their workplaces aswell as their homes are close together. Employ-ing so many people to work in parallel dramati-cally increases the costs involved and thereforeis often not feasible.

Furthermore, if a few hours’ delay until cool-ing did matter, the research team would have tobe equipped with transportable cooling facili-ties, thus increasing costs even further. If a fewhours’ delay does not matter, however, a small-

er team can be employed to work sequentiallyrather than in parallel, and this is a much morerealistic option in field research. Thus resultssuch as ours can greatly increase the feasibilityof collecting urinary catecholamine data undernormal field conditions.

Note, however, that the robustness can be as-sumed only for samples that have been properlyacidified. Researchers should therefore takeevery precaution to make sure that errors inacidification can be avoided. For instance, thismay be facilitated by using colored hydrochloricacid and transparent urine containers, so thatparticipants and researchers have visual feed-back on whether acidification was executed asprescribed. Avoiding this type of error may bemore important than keeping within short timelimits before cooling and freezing the samples.

Results such as ours need to be replicated.If they are, however, they will have importantimplications for field researchers. Put simply,the prescription would be as follows: “You donot have to be unduly restrictive with regard tothe time that elapses before cooling urinarycatecholamine samples; up to 24 hr, you willstill have interpretable results. You have to bevery careful, however, to make sure that thesamples are properly acidified very quickly.”Because immediate acidification is easier toachieve under field conditions than is immediatecooling, this is good news for those who want tostudy occupational stress in the field.

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Achim Elfering received a Ph.D. in psychology fromthe University of Frankfurt, Germany, in 1997. He isa lecturer in work and organizational psychology atthe University of Berne, Switzerland.

Simone Grebner received a Ph.D. in psychologyfrom the University of Berne, Switzerland, in 2001.She is a research and teaching assistant in work andorganizational psychology at the Department ofPsychology, University of Berne, Switzerland.

Norbert K. Semmer received a Ph.D. in psychologyfrom the Technical University of Berlin, Germany, in1983. He is a professor of the psychology of work andorganizations at the University of Berne, Switzerland.

Christa Byland received her M.D. in 1986 from theUniversity of Berne. She is head of quality control atBlood Transfusion Service SRC Berne Ltd., a blooddonation center in Berne, Switzerland.

Hans Gerber received his M.D. in 1977 from theUniversity of Berne, where he holds an associateprofessorship in medicine. He is also director of theBrunnhof Institute of Pathology and Medical Analy-tics in Berne, Switzerland.

Date received: January 15, 2002Date accepted: June 27, 2003