Scandinavian Journal of Urology and Nephrology, 2010; 44: 331–336

ORIGINAL ARTICLE

Effect of storage time at �20�C on markers used for assessment ofrenal damage in children: albumin, g-glutamyl transpeptidase,N-acetyl-b-D-glucosaminidase and a1-microglobulin

FELICIA TRACHTENBERG1 & LARS BARREGARD2

1New England Research Institutes, Watertown, Massachusetts, USA, and 2Hospital and University of Gothenburg,Gothenburg, Sweden

AbstractObjective. The objective of this study is to examine the influence of storage time at �20�C on the concentration of albumin,g-glutamyl transpeptidase (g-GT), N-acetyl-b-D-glucosaminidase (NAG), a1-microglobulin (A1M) and creatinine in alarge sample of healthy children. Material and methods. The New England Children’s Amalgam Trial followed 534 chil-dren, aged 6–10 at baseline, for 5 years, with annual urine collections. Urine samples were analysed for creatinine,albumin, g-GT, NAG and A1M concentrations. Repeated measures analysis of covariance was used to model the effect ofstorage time on these concentrations. Results. The g-GT concentration decreased significantly with storage time at�20�C.There was also a limited decrease in NAG. Albumin, A1M and creatinine concentrations did not appear to be affected bystorage time at �20�C. Conclusions. If it is necessary to interpret results from samples stored for a long time at �20�C, it isadvisable to account for storage time in statistical models.

Key Words: A1M, albumin, children, g-GT, NAG, renal markers, storage

Introduction

The excretion of various proteins in urine is widelyused for assessment of kidney function, in clinicalpractice as well as in epidemiological studies ofpopulations exposed to nephrotoxic compounds,for example mercury [1,2]. Albumin is the commonlyused marker of glomerular integrity, while low molec-ular weight proteins, or enzymes, such as beta-2-microglobulin, a1-microglobulin (A1M; also calledprotein HC), N-acetyl-b-D-glucosaminidase (NAG)or g-glutamyl transpeptidase (g-GT), are used forassessment of effects on renal tubular cells.In many cases, especially in epidemiological stud-

ies, it is necessary to store the urine samples for longperiods before analysis. Storage at�20�C is common,

although many recent studies have found detrimentaleffects at this temperature and recommend eitherstorage at 4�C with immediate analysis or storage at�70�C long term.Fifteen studies of albumin were found, yielding

varying results. Nine studies [3–10] found a decreaseafter storage at �20�C for as little as 1 week (althoughsometimes not until after a longer time). Further-more, two studies [11,12] found a decrease only forsamples with low/normal albumin excretion, but notfor those with higher (abnormal) excretion. Fourstudies [13–17] found no decrease after 2 months,6 months, 2 years and 2 years, respectively. However,these studies were all done on diabetics or patientswith renal disease (who would have abnormally highalbumin excretion). Therefore, these results do not

Correspondence: F. Trachtenberg, New England Research Institutes, 9 Galen Street, Watertown, MA 02472, USA. Tel: +1 617 923 7747 ext. 420.Fax: +1 617 926 8246. E-mail: ftrachtenberg@neriscience.com

(Received 30 April 2009; accepted 7 May 2010)

ISSN 0036-5599 print/ISSN 1651-2065 online � 2010 Informa HealthcareDOI: 10.3109/00365599.2010.492785

Scan

d J

Uro

l Nep

hrol

201

0.44

:331

-336

.D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

t de

Gir

ona

on 1

1/25

/14.

For

per

sona

l use

onl

y.

necessarily contradict the studies performed onhealthy subjects, which find an effect of storage.Two studies [18,19] found a decrease in g-GT

concentration with storage at �20�C. The formerfound a 60% drop in g-GT concentration after just1 day of storage. They found g-GT to be bestpreserved when urine samples were stored at�70�C after initial centrifugation.Two studies [8,9] of NAG showed a significant

decrease after 6 months, and one study [19] con-cluded that approximately 15% of NAG concentra-tion is lost in the process of freezing/thawing, but thereare no further decreases over time. Another study[20,21] of NAG found no effect of storage at�20�C for 1 month, but found a 50% decrease after1 year.Two studies of A1M found a significant effect of

storage at �20�C. Klasen et al. [15] found a decreasein concentration over 2 years of storage. Tencer et al.[16,17] found a non-significant drop of 14% after6 months, and a significant decrease of 22% after1 year.Finally, Schultz et al. [9] found that creatinine

concentration was unaffected after 6–8 months ofstorage at �20�C, and Manley et al. [8] found thatcreatinine concentration was relatively stable after6 months’ storage, but significantly decreased after2 years.Overall, most studies find that the concentrations of

these kidney function markers are underestimatedafter storage at �20�C. All such studies have beenperformed on adults, with the exception of one studyof albumin [12] and one study of albumin and NAG[9] on diabetic children. No such studies have beenconducted on healthy children.The aim of the current analysis was to examine the

influence of storage time at �20�C on theconcentration of albumin, g-GT, NAG and A1M,as well as creatinine, in a large sample of healthychildren.

Material and methods

These data were available as part of the New Eng-land Children’s Amalgam Trial (NECAT) [22,23].The NECAT study was designed to examine effectsof amalgam dental fillings in 534 children in Bostonand Maine, aged 6–10 at the beginning of the study,for 5 years. Outcome measures of this trial includedeffects on the kidney, as measured by creatinine-corrected albumin, g-GT, NAG and A1M. Eligibil-ity criteria for the trial included no evidence ofkidney disorders, and the sample was gender bal-anced and racially diverse (Table I). The study was

approved by the institutional review boards of allparticipating sites, and it conforms to the provisionsof the Declaration of Helsinki. Written parentalconsent and child assent was obtained for allparticipants.The original protocol called for collection of timed

overnight samples, with spot day-time samplescollected for those children who did not provide anovernight collection. However, as the children grewolder, compliance with the overnight collectionbecome more problematic and a decision was madeto switch to spot samples at the clinic for all children.Therefore, the proportion of samples collectedovernight versus in the day-time varied over thecourse of the study, from 92% overnight versus 8%day-time at baseline to 4% overnight versus 96% day-time at year 5.g-GT and creatinine concentrations were measured

yearly for all NECAT children (53% overnight and47% day-time samples overall); albumin, NAG andA1M concentrations were measured at years 3 and 5,with additional measurements for 57 children in asubstudy at year 4 (12% overnight and 88% day-time samples overall).Urine specimens were sent to Rochester General

Hospital and Strong Hospital clinical laboratories inRochester, NY, USA, for analysis of creatinine andg-GT, as well as to the Sahlgrenska UniversityHospital in Goteborg, Sweden, for analysis of cre-atinine, albumin, NAG and A1M. When the spec-imen was not sufficient for both, priority was givento the laboratories in Rochester. Creatinine concen-tration was measured in both places for use in thecreatinine adjustment of the concentrations of thekidney markers; however, the creatinine concentra-tion reported here is that from the laboratories inRochester. Creatinine was determined by the pho-tometric Jaffe method.

Table I. Demographic information on the 534 children.

Mean ± SDor n (%)

Age (years) at beginning of5-year study

7.9 ± 1.4

Gender

Male 247 (46.3)

Female 287 (53.8)

Race

Non-Hispanic white 323 (62.1)

Non-Hispanic black 98 (18.9)

Hispanic 38 (7.3)

Other 61 (11.9)

332 F. Trachtenberg & L. Barregard

Scan

d J

Uro

l Nep

hrol

201

0.44

:331

-336

.D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

t de

Gir

ona

on 1

1/25

/14.

For

per

sona

l use

onl

y.

Specimen storage

Urine samples were stored frozen at �20�C untilanalysis. The median storage time for the laboratoriesin Rochester, NY was 71 days, with only 7% ofsamples stored for longer than 6 months. The medianstorage time for the laboratory in Goteborg, Swedenwas 6 months for albumin and NAG and 9 months forA1M. Nine per cent of the samples were stored forlonger than 1 year before analysis of albumin andNAG, with 32% stored for longer than 1 year beforeanalysis of A1M.

Assay methods

g-GT was measured using the Dade Behring Dimen-sion clinical chemistry system: Flex reagent cartridge.NAG was determined with an automated photo-

metric method based on the formation of 3-cresolpurple at the reaction catalysed by NAG, usingreagents and a calibrator from Roche Diagnostics(detection limit 0.1 U/l).Urinary albumin and A1M concentrations were

determined by automated nephelometric immuno-chemical methods using reagents and calibratorfrom Beckman Coulter. Additional internal referencesamples were used in each analytical run. The detec-tion limits were 2.4 mg/l for albumin and 4 mg/l forA1M. However, since several previous studies onA1M have been performed using antibodies fromDako [24], and the concentrations in these two com-mercial antigens (calibrators) differ, the A1M con-centrations were corrected. Using the BeckmanCoulter antigens results in 40% higher levelscompared with those obtained using the Dakoreagents. To be able to compare the data withsome previous studies, A1M levels in mg/l weredivided by 1.4.Special measures were taken to prevent inhomoge-

neous albumin concentrations in the thawed urinesamples. The frozen samples were thawed in a waterbath (37�C), mixed and kept in the water bath foranother 30 min. After mixing again, 10 ml ofTween20 was added to 1 ml of urine. Sampleswere then kept cool overnight, gently mixed on thefollowing day, then placed in the 37�C water bath for3 � 60 min, and gently mixed between every 60 minperiod. After this procedure they were analysed within3 days.

Statistical analysis

Repeated measures analysis of covariancemodels, withcompound symmetric variance structures, were fitted

to determine the effect of storage time on the concen-trations (mg or U/l) of creatinine, albumin, g-GT,NAG and A1M. Log transformation was used whenexamination of the model residuals indicated that itwas needed. The model for albumin excluded sampleswith microalbuminuria (albumin > 30 mg/g creatinine,12% of samples), since these samples would have hadtoo large an effect on the results. All models controlledfor age, gender, race and lean body mass. Lean bodymass was calculated as Weight � (1 � % Body fat),with body fat measured by a body fat scale (modelTBF-551). The study design also controlled for uri-nary collection time (overnight versus day-time sample) and creatinine concentration (exceptfor creatinine models) [25].To verify overall findings, subanalyses were

performed excluding storage times greater than 1year. In addition, the effect of storage time on thedetectability of albumin, NAG and A1M concentra-tions was examined by logistic regression.

Results

Table I shows background demographic data on the534 children, and Table II shows descriptive data onthe kidney marker concentrations, their correspond-ing creatinine-corrected levels and storage times.Creatinine and g-GT were almost always detectable,while levels of A1M were mostly below the limit ofdetection (27% detectable). Albumin and NAG were88% and 93% detectable, respectively.Figure 1 shows plots of (detectable) creatinine,

albumin, g-GT, NAG and A1M concentrations ver-sus storage times. Table III reports the results ofrepeated measures analysis of covariance models ofthe detectable kidney function measures on storagetime. Creatinine concentration remained unchangedwith storage at�20�C. g-GT concentration decreasedsignificantly with storage time. NAG concentrationalso decreased significantly with storage time; how-ever, this decrease was no longer significant whensamples with storage times greater than 1 year wereexcluded (although the slope remains similar). Sam-ples stored for longer were significantly more likely tohave non-detectable NAG (p < 0.001). No significanteffect of storage time on albumin or A1M concentra-tions was found.Based on model results, in a day-time sample

from an average child (age 12), one can expect a40% decrease in g-GT concentration after 6 monthsof storage at �20�C, with a 64% decrease after 1 year.For NAG concentration, a 9% decrease after6 months and an 18% decrease after 1 year may beestimated.

Storage time at �20�C and renal markers 333

Scan

d J

Uro

l Nep

hrol

201

0.44

:331

-336

.D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

t de

Gir

ona

on 1

1/25

/14.

For

per

sona

l use

onl

y.

Table II. Concentrations and excretions for the kidney markers and their median storage time.a

N Concentration/excretion Storage time (days)

Creatinine

Concentration (g/l) 2909 1.2 (0.1 – 4.2) 71 (4–1007)

Albuminb

Concentration (mg/l) 813 7.2 (ND–1320.0) 177 (42–468)

Excretion (mg/g C) 718 7.4 (1.5–773.1)

g-GT

Concentration (U/l) 2912 26.0 (0.0–224.0) 71 (4–1007)

Excretion (U/g C) 2909 26.7 (0.0–246.7)

NAGb

Concentration (U/l) 813 1.2 (ND–9.2) 176 (22–468)

Excretion (U/g C) 754 1.4 (0.1–7.8)

A1Mb

Concentration (mg/l) 813 ND (ND–17.0) 262 (42–563)

Excretion (mg/g C) 223 4.7 (1.1–29.5)

Data are shown as median (range).g-GT = g-glutamyl transpeptidase; NAG = N-acetyl-b-D-glucosaminidase; A1M = a1-microglobulin; ND = not detectable.a Creatinine-corrected excretions exclude samples with non-detectable concentrations; b albumin, NAG and A1M were not measured in allyears.

8

6

4

2

0

100 200 300 400

16

14

12

10

8

6

4

100 200 300 400 500

Storage time (days)Storage time (days)

NA

G c

on

cen

trat

ion

(U

/L)

4

3

2

1

0

0

Cre

atin

ine

con

cen

trat

ion

(g

/L)

200 400 600 800 1000

A. B.

E.D.

C.

60

40

20

0

Alb

um

in c

on

cen

trat

ion

( m

g/L

)

100

Storage time (days)

200 300 400 400 600 800 1000

Storage time (days)

GG

T c

on

cen

trat

ion

(U

/L)

0

0

50

100

150

200

200

Storage time (days)

A1M

co

nce

ntr

atio

n (

mg

/L)

Figure 1. Concentration versus storage time: (A) creatinine; (B) albumin (excludes cases with microalbuminuria); (C) g-glutamyl trans-peptidase (GGT); (D) N-acetyl-b-D-glucosaminidase (NAG); and (E) a1-microglobulin (A1M). The regression line is log-transformed forGGT.

334 F. Trachtenberg & L. Barregard

Scan

d J

Uro

l Nep

hrol

201

0.44

:331

-336

.D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

t de

Gir

ona

on 1

1/25

/14.

For

per

sona

l use

onl

y.

Discussion

The results show that storage time at �20�C has alarge effect on g-GT concentration and, to a lesserextent, NAG concentration. Albumin and A1M donot appear to be affected by storage time at �20�C,apart from a possible decrease by the freezing/thawing procedure per se, irrespective of storagetime. In addition, no effect of storage on creatinineconcentration was found, which suggests that both theconcentrations and creatinine-adjusted levels of renalmarkers will be affected to a similar extent. Analysesof the creatinine-adjusted levels confirmed this (datanot shown). Little information was found in theliterature on the effect of long-term storage at�70�C on these biomarkers.Although other studies have found that albumin

and A1M decrease with storage at �20�C, no signif-icant decrease with time of storage was found. Onepossible reason for this is that the process of freezingand thawing the samples make them inhomogeneous,decreasing the recovery of these proteins in theanalysis, while there is no substantial further break-down with time. In the present study special measureswere taken to dissolve and recover all proteins afterthawing. Another possibility is that because the studydid not analyse the same samples over time, it maylack power to detect a significant decrease with time.Inconsistent results in the literature regarding albu-min may be due to assay-dependent differences inreactivity to fragments of albumin [26].Although it was not possible to analyse the same

urine samples at various storage times, the conclu-sions are relevant to large epidemiological studies thatstore urine samples for varying amounts of timebefore analysis. In addition, these results extend the

conclusions of previous research on adults to healthychildren.Based on the conclusions of this study and the

studies of others, the authors recommend storingurine samples at �70�C, if storage is necessary[20]. Future study of storage at �70�C is recom-mended to show the need for standardized freezingprocedures of no less than �70�C. Furthermore, if itis necessary to interpret results from samples stored at�20�C, it is advisable to account for storage time instatistical models.

Acknowledgements

We acknowledge Elsa Cernichiari for the laboratoryanalyses of creatinine and g-GT. The study was by acooperative agreement (U01 DE11886) between theNew England Research Institutes and the NationalInstitute of Dental and Craniofacial Research,National Institutes of Health, who also participatedin the design and conduct of the study. Trial regis-tration: clinicaltrials.gov; identifier NCT00065988.

Declaration of interest: The authors report noconflicts of interest. The authors alone are responsiblefor the content and writing of the paper.

References

[1] World Health Organization. Concise International ChemicalAssessment Document 50. Elemental mercury and inorganicmercury compounds: human health aspects. Geneva: WHO;2003.

[2] Cardenas A, Roels H, Bernard AM, Barbon R, Buchet JP,Lauwerys RR, et al. Markers of early renal changes inducedby industrial pollutants. I. Application to workers exposed tomercury vapour. Br J Ind Med 1993;50:17–27.

[3] Erman A, Rabinov M, Rosenfeld J. Albumin determinationin frozen urines – underestimated results. Clin Chim Acta1988;174:255–61.

[4] Elving LD, Bakkeren JA, Jansen MJ, de Kat Angelino CM,de Nobel E, vanMunster PJ. Screening for microalbuminuriain patients with diabetes mellitus: frozen storage of urinesamples decreases their albumin content. Clin Chem 1989;35:308–10.

[5] Giampietro O, Penno G, Clerico A, Cruschelli L, Cecere M.How and how long to store urine samples before albuminradioimmunoassay: a practical response. Clin Chem 1993;39:533–6.

[6] Hara F, Nakazato K, Shiba K, Shimoda J, Kojima T,Fukumura Y, et al. Studies of diabetic nephropathy. I. Effectsof storage time and temperature on microalbuminuria. BiolPharm Bull 1994;17:1241–5.

[7] MacNeil ML, Mueller PW, Caudill SP, Steinberg KK. Con-siderations when measuring urinary albumin: precision, sub-stances that may interfere, and conditions for sample storage.Clin Chem 1991;37:2120–3.

Table III. Repeated measures analysis of covariance models ofkidney markers on storage time, controlling for age, gender, race,lean bodymass, creatinine concentration (except for creatinine) andsample time (overnight vs day-time sample).

Storage time (days)

Coefficient (SE) p

Creatinine concentration (g/l) 0.00015 (0.00015) 0.30

Albumina concentration (mg/l) �0.00038 (0.0037) 0.92

g-GT concentration (U/l)b �0.0028 (0.00022) <0.001*NAG concentration (U/l) �0.0010 (0.00044) 0.02c *

A1M concentration (mg/l) �0.00043 (0.0011) 0.71

g-GT = g-glutamyl transpeptidase; NAG = N-acetyl-b-D-glucosa-minidase; A1M = a1-microglobulin.a Excludes cases with microalbuminuria (12%); b log-transformed;c in a reanalysis excluding cases stored for over 1 year, p = 0.12[coefficient (SE) = –0.00090 (0.00057)].*Statistically significant.

Storage time at �20�C and renal markers 335

Scan

d J

Uro

l Nep

hrol

201

0.44

:331

-336

.D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

t de

Gir

ona

on 1

1/25

/14.

For

per

sona

l use

onl

y.

[8] Manley SE, Burton ME, Fisher KE, Cull CA, Turner RC.Decreases in albumin/creatinine and N-acetylglucosamini-dase/creatinine ratios in urine samples stored at �20 degreesC. Clin Chem 1992;38:2294–9.

[9] Schultz CJ, Dalton RN, Turner C, Neil HA, Dunger DB.Freezing method affects the concentration and variability ofurine proteins and the interpretation of data on microalbu-minuria. The Oxford Regional Prospective Study Group.Diabet Med 2000;17:7–14.

[10] Brinkman JW, de Zeeuw D, Duker JJ, Gansevoort RT,Kema IP, Hillege HL, et al. Falsely low urinary albuminconcentrations after prolonged frozen storage of urinesamples. Clin Chem 2005;51:2181–3.

[11] Osberg I, Chase HP, Garg SK, DeAndrea A, Harris S,Hamilton R, et al. Effects of storage time and temperatureon measurement of small concentrations of albumin in urine.Clin Chem 1990;36:1428–30.

[12] Shield JP, Hunt LP, Morgan JE, Pennock CA. Are frozenurine samples acceptable for estimating albumin excretion inresearch? Diabet Med 1995;12:713–6.

[13] Lamb E, Dawnay A. Effects of sample preparation andstorage on urine albumin determination. Ann Clin Biochem1989;26:560–2.

[14] Collins AC, Sethi M, MacDonald FA, Brown D,Viberti GC. Storage temperature and differing methods ofsample preparation in the measurement of urinary albumin.Diabetologia 1993;36:993–7.

[15] Klasen IS, Reichert LJ, de Kat Angelino CM, Wetzels JF.Quantitative determination of low and high molecular weightproteins in human urine: influence of temperature and stor-age time. Clin Chem 1999;45:430–2.

[16] Tencer J, Thysell H, Andersson K, Grubb A. Long-termstability of albumin, protein HC, immunoglobulin G, kappa-and lambda-chain-immunoreactivity, orosomucoid andalpha 1-antitrypsin in urine stored at �20 degrees C. ScandJ Urol Nephrol 1997;31:67–71.

[17] Tencer J, Thysell H, Andersson K, Grubb A. Stability ofalbumin, protein HC, immunoglobulin G, kappa- and lamb-da-chain immunoreactivity, orosomucoid and alpha 1-anti-trypsin in urine stored at various conditions. Scand J Clin LabInvest 1994;54:199–206.

[18] Matteucci E, Gregori G, Pellegrini L, Navalesi R,Giampietro O. Effects of storage time and temperature onurinary enzymes. Clin Chem 1991;37(8):1436–41.

[19] Loeb WF, Das SR, Trout JR. The effect of erythritol on thestability of gamma-glutamyl transpeptidase and N-acetyl glu-cosaminidase in human urine. Toxicol Pathol 1997;25:264–7.

[20] Matteucci E, Gregori G, Pellegrini L, Navalesi R,Giampietro O. How can storage time and temperature affectenzymic activities in urines? Enzyme 1991;45:116–20.

[21] Matteucci E, Pellegrini L, Uncini-Manganelli C, Cecere M,Saviozzi M, Giampietro O. More on effects of storage timeand temperature on urinary enzymes: a 1-year study. Enzyme1992;46:249–51.

[22] Bellinger DC, Trachtenberg F, Barregard L, Tavares M,Cernichiari E, Daniel D, et al. Neuropsychological and renaleffects of dental amalgam in children: a randomized clinicaltrial. JAMA 2006;295:1775–83.

[23] The Children’s Amalgam Trial: design and methods.Control Clin Trials 2003;24:795–814.

[24] Jarup L, Hellstrom L, Alfven T, Carlsson MD, Grubb A,Persson B, et al. Low level exposure to cadmium and earlykidney damage: the OSCAR study. Occup Environ Med2000;57:668–72.

[25] Trachtenberg F, Barregard L, McKinlay S. The influence ofurinary flow rate in children on excretion of markers used forassessment of renal damage: albumin, gamma-glutamyltranspeptidase, N-acetyl-beta-D-glucosaminidase, andalpha1-microglobulin. Pediatr Nephrol 2008;23:445–56.

[26] Sviridov D, Drake SK, Hortin GL. Reactivity of urinaryalbumin (microalbumin) assays with fragmented or modifiedalbumin. Clin Chem 2008;54:61–8.

336 F. Trachtenberg & L. Barregard

Scan

d J

Uro

l Nep

hrol

201

0.44

:331

-336

.D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

t de

Gir

ona

on 1

1/25

/14.

For

per

sona

l use

onl

y.