Dec 7, 1990 - We measured three renal tubular brush-border enzymes. (lactate dehydrogenase, LDH, EC 1.1.1.27; gamma-glu- tamyltransferase, GGT, EC ...
assay. Cliii Chem 1984;30:395-8. 5. Seth J. The external quality assessment of hormone assays. J Cliii Biochem Nutr 19872:111-39. 6. Sufi SB, Jeffcoate SL, Hall PE, Goncharov N. Five years’ experience with WHO matched assay reagent programme in research in human reproduction. Op. cit. (ref 3):555-71. 7. Malan PG, Simpson JG, Stevens RAJ, Grove-White JF, Whitworth AS. Calibration of standards for immunoaesay, including an appendix and spectrophotometric standardization of solutions of thyroxine and triiodothyronine. In: Hunter WM, Come JET,ads., Immunoassay forclinical chemistry. 1983:48-65.
8. Healy MJR. Outliers
in clinical chemistry quality-control schemes. Cliii Chem 1979;25:675-7. 9. Pilo A, Zucchelli GC, Piro MA. External quality control forT3 and T4 assays in Italy outline ofthescheme and results ofthefirst semester experience. J Nucl Med Allied Sci 1981;25:1O7-14. 10. Vanderhnde RE, Rej R, Fasce CF Jr. Enzyme determinations: criticisms of some recent reports [Letter]. Cliii Chem 1973;19: 282-3. 11.Swift AD, Ratcliffe JG. UK EQAS forthyroid stimulating hormone-past, present and future. Commun Lab Med 1985;1: 94-6.
CLIN.CHEM.37/8, 1436-1441(1991)
Effects of Storage Time and Temperature Elena
Matteucci,
Giovanna
Gregori,
Lulsa
Pellegrini,
on Urinary Enzymes Renzo
Navalesi,
and Ottavio
Glampietro1
within 4-6 h (19,21) afier centrifugation. Others report We measured three renal tubular brush-border enzymes (lactate dehydrogenase, LDH, EC 1.1.1.27; gamma-gluthat urine may be stored deep-frozen without enzyme tamyltransferase, GGT, EC 2.3.2.2; and alkaline phosloss (7). phatase, AP, EC 3.1.3.1) in morning urine samples from Here we report the effects of different storage times 48 healthy subjects to check whether different storage and temperatures on some enzyme activities in urine times and temperatures could modify enzyme concentrasamples, centrifuged or not, collected from healthy subtions. Short-term (24 h) storage time at room temperature jects. or 4#{176}C does not affect urinary enzyme activity. A few days of freezing, at -20 or -70 #{176}C, dramatically iowers LDH Materials and Methods and AP values; GGT is partially preserved only at -70 #{176}C, Enzymes studied. We chose to study three brushif the sample has been previously centrifuged. Urinary border enzymes, because we are involved in programs enzymes investigated in this study are extremely iabiie at dealing with tubular diabetic nephropathy (7, 15,21). low temperatures. These enzymes are gRmmsi-glutalnyltransferase (GGT; EC 2.3.2.2), lactate dehydrogenase (LDH; EC 1.1.1.27), AddItIonal K.yphrsaes: lactate deMydrogenase glutamyltrans.. and alkaline phosphatase (AP; EC 3.1.3.1).2 ferase alkaline phosp’iatase sample handllng Equpment. We used in these manual assays a DU-8 ultraviolet-visible computing spectrophotometer (BeckMeasurement of urinary enzymes is currently used as man Instruments, Inc., Fullerton, CA) with a temperaa noninvasive test of renal integrity in clinical nephrolture-controlled cuvet holder; Pipetman 1000 from Gilogy (1-24). However, several methodological problems ford Medical Electronics (France) S.A., and micropiarise if an unfavorable environment for the enzymes is pettes from Socorex (Swiss made); silica cells from used, e.g., urine. Urine as a medium is extremely Helma Italia srl; and a Model J6B centrifuge (Beckvariable in volume, composition, pH, ionic strength, and man). the presence of interfering substances, blood, epithelial Reagents. GGT activity was measured at 610 nm with cells, and microorganisms. Hence, generally approved .
.
standardized procedures to measure enzymes in urine are lacking. A decisive problem concerns the stability of enzyme activity during the collection and storage of urine samples before assay. Most published papers fail to report any details about how to treat urine samples during collection or how to store them. Some investigators (1,3, 6, 7,11,14,19,21,23) suggest centrifugation before the assay, with the assay to be performed promptly (22) or
Cattedra di Malattie delMetabolismo, Istituto di Clinica Medica II,Universit#{224} degli Studi di Pisa, Italy. ‘Address correspondence to this author, at: Istituto di Clinica Medica II, Via Roma, 67, 56100 Pisa, Italy. Received December 7, 1990; accepted May 10,1991.
1436 CLINICAL CHEMISTRY, Vol. 37, No.8,
1991
gsrnm-glutamyl-3,5-dibromo-4-hydroxyanilide
(gam-
ma-glu-DBHA) substrate in a Sera-Pack kit (code 6679; Miles Italiana S.p.A., Ames Division, Cavenago Brianza, Italy). AP activity was measured at 405 nm with sodium p-nitrophenyl phosphate as substrate (Sera Pack kit; code 6677). LDH activity was measured at 340 nm via the conversion of pyruvate to lactate, which is proportional to the rate of NADH oxidation (Sera-Pack kit;code 6393). Control sera (Sera-Chek”; code 6656) were kindly provided by Miles Italiana. p-Nitrophenol 2Noxj’
abbreviations: LDH, lactate
GGT, gamma-glutamyltransferase;
dehydrogenase; AP, Rlknhine phoephatase; and
gamma..glu-DHBA, gam-glutamyl-3,5-dibromo-4-hydroxyanilide.
stock solution (10 mmol/L) was obtained from Sigma Diagnostics, St. Louis, MO 63178. Plan of the study. To obtain “freshurine,”we decided to collect the second urine sample inthe morning. Urine specimens from 48 healthysubjects (22women, 26 men; ages 24-39 years) were processed immediately after collection: some aliquots of all specimens were centrifuged (900 x g) for 10 mm and decanted to remove the sediment, some aliquots were treated as native urines. Sediment was examined, and aliquots of urine were taken for determination of creatinine (by the Jaff#{233} reaction with the Astra 8 analyzer from Beckman) and albumin
excretion
(by immunonephelometry)
(25).
We investigated three protocols for handling the urine samples: Protocol A, day before/after (21 subjects): Urine specimens, centrifuged or not, were subdivided into three aliquots. The first one was processed without delay for determinations of catalytic activities; the second was kept at room temperature for one day, and the third was stored at 4#{176}C for 24 h. These later two aliquots were analyzed for enzymes the next morning. Protocol B, freezing effect (21 subjects): Urine specimens, with and without centrifi.igation, were processed without delay. Meanwhile, aliquots of each were frozen at -20 and -70 #{176}C for about one month before enzyme assay.
Protocol C, time-course (6 subjects): We measured enzyme activities during sample storage at 20 (room temperature), 4, -20, and -70 #{176}C. We carried out this study to detect precisely the “break point” of enzymic activity change at the different temperatures investigated. We assayed centrifuged urine specimens for GGT, AP, and LDH activities immediately after collection and successively after 24 (for practical purposes, only those frozen at -20 and -70 #{176}C), 36 (only those at 20 and 4#{176}C), 48,72, and 96 h and after 7,14,22,and 28 days of storage (at all four temperatures). Enzyme activity monitoring for AP was stopped before the 28th day, because the recovered activity had dropped heavily (by >90% of the basal value) much earlier than this; this decrease had occurred after only one week for frozen GGT and LDH (see Table 5). Enzymic reaction. All absorbance readings were performed at 37#{176}C, at the suggested wavelength. Absorbance changes were monitored at 1-mm intervals for 6-10 mm. Then we calculated the mean value of the absorbance changes per minute (iSA/mm) on the linear portion of the curve. No modifications of reagent and sample volumes were necessary for GGT analysis, enzyme activity being sufficiently high in fresh urine. Low urinary AP and LDH activities made it necessary to adjust total/sample volume ratio to 1.2 mL/0.10 mL. Calculation. By definition, an enzymic unit (U) isthe activity of enzyme that converts 1 jmol of substrate in 1 mm under standard conditions. For the enzymes in this study, 1 U corresponds to the production of 1 mol of blue quinone monoimine dye (GOT) or of 4-nitrophe-
no! (AP) or to the consumption
of 1 mol
of NADH
(LDH).
Enzyme activity was calculated from this equation: activity, UIL = (iSA/min) x factor. In the manual procedure, the factor is total volume, niL x 103/(sample volume, mL x molar absorptivity). For statistical evaluation, we used paired Student’s t-test, simple regression, and correlation test (paired x, y) when appropriate. Calibration. Enzyme activity (U/L) is calculated by measuring the absolute absorbance of the conversion product appearing during the stoichiometric enzymesubstrate reaction and using itsspecific molar absorptivity at the wavelength of measurement. For GOT, because we lacked the blue quinone monoimine dye produced in the Ames reactions (26), we measured only Ames Sera-Chek control sera. For AP, we measured the absorbance of a p-mtrophenol working standard solution, 100 tano1/L. The micromolar absorptivity of p-nitrophenol was 18.2 cm2/mol at 405 nm. As for LDH, because the absolute absorbance of NADH is measured directly, no standard curve is required. Enzyme reference materials and quality control. Unlike with serum, no control material could be provided for urine samples, because of enzyme degradation in this medium. On the other hand, the solubility of lyophilized enzyme in urine was so low that recovery studies appeared not practicable. The only control we easily could use for GGT determination in urine was a dilution test: a urine sample with the highest GOT activity was progressively diluted with isotonic saline, and measured for enzyme activity. Similar tests on urine for AP and LDH were not feasible, owing to their low concentrations in urine. Hence, for those two enzymes, we assessed quality control by diluting the serum control to simulate the concentration ranges more frequently observed in urine. For precision and accuracy current checks, we used Ames Sera-Chek lyophilized control sera. The coefficient of variation (CV), intra- and interassay, was calculated from the differences between two to four separate determinations of enzyme activities for each sample. Table 1. Urinary Excretion
of Creatinine, AlbumIn, GGT, AP, and LDH in 48 Healthy Control Subjects Msan
Creatlnine,g/L Pjbumin,mg/L GGT
SD
Range
0.94
0.81 0.52
U/L
113.17
63.81
10.1-238.8
U/g creatinine U/mmolcreatinine
114.72
64.24
10.3-240.8
1.56
0.16-4.4 0.6-2.7
3.96
2.9-19.4
9.01
5.14
7.28 0.82
7.70 0.87
1.3-22.6 0.9-51.9
U/L
21.34
U/gcreatinine
15.76 1.78
16.48 12.97
AP U/L U/gcreatinine U/mmol creatlnine
8.56
0.1-5.9
LDH
U/mmol creatinine
1.47
1.9-65.1 1.0-65.6 1.1-7.4
CLINICAL CHEMISTRY, Vol.37, No.8, 1991
1437
Results Mean values for urinary creatinine, albumin, and enzyme excretion in the 48 healthy subjects in this study are reported in Table 1. We observed no significant differences according to sex or age. Although AP and LDH mean values from our study fit reasonably with those reported by others (1,6,20,21), the mean urinary output of GOT we measured exceeds many times that in the literature; however, comparison may not be simple because of differences in mode of urine collection, storage, assay, and expression of data. In this regard, we point out that, for serum, Foesati et al. (26) documented a 20% discrepancy between GOT results by the gRrnrnRglu-DBHA method and the gasnms-glutsrnyl-4-nifroanilide method (27). For urine, we also found a difference between the enzymic values determined by these two
300
y
R - 0.99
217,869x
100
0 0.4
0.0
0,8
1,2
Dilution
Fig. 2. Unearity of GGT dilution In urine Urinewith a high GGT concentration (206 U/L)was progressively diluted with isotonicsailne, and enzyme activity was measured y- -2,717+54,433x
R-0.99
TRANSPEPTIDASE
1,5
‘I
4,574 +
200
$0 GAMMA-GLUTAMYL
*
-
50
1,0
20
0,5
10 0,2
-
0,4
0.6
0,0 0
1
3
2
0.8
1,0
1,2
Dilution 4
5
6
TIme (minutes) 40
ALKALINE PHOSPHATASE 1,6 1,4
‘I
y.5,575+
175,711x
R-0.99
30
TrT!T
1,2
20
1,0
0,8 0,02
0
2
4
6
8
0,06
0,10
0,14
Dilution
10
Time (minutes) y - 2,146 + 178,892x R - 0,98
LACTATE DEHYDROGENASE 2,4 2,2
-.---.--,__._...,
I; 1,8
1,6
0
0 2
4
6
8
0,00
0,10
0,20
0,30
TIme (minutes) Fig. 1. Reaction ratesfor the enzymes studied (only some of the basal determinations are reported), displayed as change In absorption with time
Fig. 3. GGT, AP, and LDHdilutiontests in control serum The control serum (GGT 51 U/L, AP 148 U/L, LDH 319 U/L) was diluted with Isotonic saline until within the urlna,y enzyme concentration range and
Gamma-gkitam)l transpeptidase
enzymlcactivitywas measured
1438
=
GGT
CLINICAL CHEMISTRY. Vol.37, No.8,
1991
Dilution
Table 2. Day before/after: Protocol A
Table 3. “FreezIng” Effect (Protocol B) on Enzyme ActivIty
Mean (SD), U/L Sampl.a
AP
GGT
Basal (fresh wines) C 105.6(63.67) NC 110.0(65.66) After 24 h at 20#{176}C C 96.9(64.72)
NC
102.7(69.78)
S.mpl.
8.0(3.57) 10.2 (5.06)
Basal (fresh urine)
16.8(10.05)
C
116.0(68.97)
LDH
AP 8.8 (3.38)
24.9
(19.65)
After a month at -20 “C 9.9 (6.70) 11.4(8.17)
15.0(10.50) 17.7(11.16)
C NC
16.2(37.85)b 8.4(16.39)b
3.7 (2.58)b
2.4
(l.73)b
5.2
2.2
(2.1#{216})b
2.4 2.3
(2.16)b
(4.25)b
After a month at -70 “C
C 102.8(65.27) 11.3(5.05) 103.4(70.14) 11.9(4.59) NC a c, centrifuged;NC, not centrifuged. =
GGT
15.5 (9.46)
After 24 h at 4 #{176}C
n
Mean (SD), U/I.
LDH
13.8 (8.77) 15.2 (9.04)
21.
C
83.5 (60.80)c 5.0(3.lO)b 60.0 (47.48)b 4.4 (3.47)L #{149} C, centrifuged;NC, not centrifuged. b.c Significantlydifferent from basal values by paired t-test:
NC
(2.74)b
b p
