Chromatographic profiles of urinary isoenzymes in healthy children

ChicaChimica Acta, 169(1987)209-216

209

Elsevier CCA 03969

Chromatographic profiles of urinary isoenzymes in healthy children Hiroyoshi Matsukura a, Yoshifumi Suzuki a, Rika Takai a, Toshio Okada a, Saburou Naiki b and Nobuo Sakuragawa b a Department of Pediatrics and b Department of Central Laboratory, Faculty of Medicine, Toyama Medical and Pharmaceutical

University, Sugitani, Toyama (Japan)

(Received 2 April 1987; revision received 17 June 1987; accepted after revision 8 July 1987) Key words: Alkaline phosphatase;

gamma-Glutamyltransferase;

Lactate dehydrogenase

Summary

Urinary excretion of alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase was studied in a carefully selected group of 155 healthy children, 83 females and 72 males. Enzyme activity was assayed in randomly collected urine samples after gel filtration of the urine specimens. On chromatograms, urinary enzymes of alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase were separated into 4, 2 and 5 isoenzymes, respectively. Mean values of alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase activity were 4.59, 21.6 and 10.0 U/g creatinine. There were no sex-related differences besides lactate dehydrogenase which showed a higher excretion in females than in males. The excretion of urinary enzymes clearly decreased with increasing age.

Introduction

In previous papers, we described a simple and rapid separation method suitable for clinical laboratories, for the separation of urinary enzymes using high-performance liquid chromatography [1,2]. Chromatographic analysis and enzyme detection were fully automated and provided excellent reproducibility as described earlier [2]. Although there were many papers dealing with the increased excretion of urinary enzymes in various kidney diseases [3-71, only a few papers reported normal values of urinary enzymes [6,8]. This work was undertaken to analyze the chromato-

Correspondence to: H. Matsukura, Department of Pediatrics, Toyama Medical and Pharmaceutical University 2630 Sugitani, Toyama 930-01, Japan. OOO9-8981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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graphic profiles of urinary isoenzymes of alkaline phosphatase (AP, EC 3.1.3.1) gamma-glutamyltransferase (GGT, EC 2.3.2.2) and lactate dehydrogenase (LD, EC 1.1.1.27) in healthy children. Materials and methods Subjects We studied 155 children who were referred for screening for urinalysis from October, 1986 to January, 1987. The subjects consisted of 83 females (mean 9.3, range 1 mth-14 yr) and 72 males (mean 9.7, range 2 mth-15 yr). We excluded from the study all individuals presenting symptoms or giving a history of renal diseases of the lower urinary tract, or who were using drugs known to affect enzyme excretion in urine, such as analgesics or antibiotics [3]. Furthermore, only experimental data from subjects who had normal results for urinalysis were selected for the estimation of normal values. Sample preparation For routine analyses, a randomly collected urine sample was used for enzyme assay within 6 h. Ten milliliters of urine were centrifuged at 1500 X g for 20 min and then 2.5 ml of the supernatant were filtered through Sephadex G-25 medium (Pharmacia, PD-10). Apparatus and enzyme assay The chromatographic apparatus and the post-column enzyme detection system have been described previously [1,2]. All calculations of net area for peaks were evaluated by a Model X-Plan 360 Area Curve Meter (Ushikata Co., Tokyo, Japan). Enzyme activity was related to urinary creatinine concentration in order to correct differences in urinary volume output. The resulting expression was denoted ‘ U/g creatinine’. One way or two way analysis of variance was used to access each factor’s effect. The least significant difference method was also used for multiple comparisons using Bonferroni [9] or Scheffe methods [lo]. The sex-related difference was calculated applying Mann-Whitney’s U test. Values were given as mean k 1 SEM. For purposes of analysis, the healthy subjects were divided into 3 groups in relation to age. Results The typical chromatograms, of urinary isoenzymes in a healthy child are shown in Fig. 1. On chromatograms, urinary enzymes of AP, GGT, and LD were separated into 4, 2 and 6 peaks, respectively. Each separated isoenzyme was numbered in the order of electrophoresis. Multiple LD3 peaks were also observed. Values of urinary isoenzymes in healthy subjects are calculated and summarized in Tables I-III and Fig. 2. We found no sex-related differences for total enzyme activity in urine besides

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Alkaline

H

0

Lactate

e

0

’

Phosphatase

DehydrTgenase

I

0 T

10 I

Fig. 1. Chromatographic

I

H

1

20 30 E (H I N)

profiles

of urinary

enzymes

in a healthy

child.

LD. Total excreted lactate dehydrogenase activity was higher in urine of females (10.5 f 0.87 U/g creatinine) than in males (9.34 f 0.92 U/g creatinine, p -e 0.025). In viewing isoenzymes, such differences were noted in GGTl, LD2 and LD4 isoenzymes. In this study total enzyme activity in urine showed the highest value in

TABLE

I

Urinary

enzyme activity

Age o- 4 5-10 11-15

n 34 43 75

of alkaline

phosphatase

in 155 healthy

children

APal

AP2

AP3

AP4

(W

(W)

(4%)

(W

Total AP (U/g creatinine)

33.5 f 2.29 31.1 f 1.88 29.3 f 1.31

28.4 f 2.00 31.8kO.90 36.0 f 0.91

19.3+1.70 25.3i1.66 22.2 + 1.04

18.8 i- 2.37 11.8+2.37 12.5 + 1.27

7.06 + 1.13 b.d 4.59 kO.60 b,c 3.47 f 0.26 c,d

a Alkaline phosphatase Values represent mean f SEM. b~cp < 0.05, d p < 0.01.

212

TABLE II Urinary enzyme activity of gamma-glutamyltransferase

in 155 healthy children

Age

n

GGT”1 (W)

GGT 2 (W)

Ratio GGT l/GGT

0- 4 5-10 11-15

36 43 76

9.10f1.97 15.7 +2.29 18.3 k1.62

90.9 + 1.97 84.3 + 2.34 81.7+ 1.63

0.12kO.03 b 0.23 i 0.04 0.27 f 0.03 b

a gamma-Glutamyltransferase Values represent mean i- SEM.

bp <

2

Total GGT (U/g creatinine) 31.4;t 2.06 ‘S 22.9 + 1.29 c.d 16.5 f 0.64 d,e

0.05, c.d p < 0.01, e p < 0.001.

the period of infancy and there was also an actual decrease in the excretion with increasing age (Tables I-III and Fig. 2). For the output of AP isoenzymes as well as total enzyme activity, we found no sex-related differences. On the ~hromatogram, API, AP2 and AP3 isoenzymes consisted of a broad peak and the remaining AP4 isoenzyme was usually faint (Fig. 1). The value of AP3 isoenzyme (23.8 & 1.60%) was lower than either the value of APl (30.7 f 0.98% p < 0.001) or the value of AP2 isoenzyme (32.9 It 0.75%;. p < 0.001) throughout all age groups. In groups under 10 years of age these were no differences for values between APl and AP2 isoenzymes, whereas in the 11-15 yr group the value of AP2 isoenzyme (36.0 _t 0.91%) was sig~fic~tly higher than the value of APl isoenzyme (29.3 + 1.31%, p < 0.001). Despite the absence of the sex-related difference for total enzyme activity of GGT, the output of GGTl isoenzyme in urine was higher in females (38.2 f 0.42 U/g creatinine) than in males (2.70 rf: 0.38 U/g creatinine, p -c 0.025). In the O-4 year group we could not detect GGTl activity in 16 of 36 subjects (44%). The value of GGTl/GGT2 ratio was higher in the ll- to 15-yr group (0.27 rt 0.03) than the value in the 0- to 4-yr group (0.12 jlo.03, p < 0.05). Values of enzyme activity in urine of LD2 and LD4 isoenzymes were higher in females (3.90 & 0.86 U/g creatinine for LD2; 0.43 & 0.07 U/g creatinine for LD4) than values in males (2.38 rJI0.27 U/g creatinine for LD2, p -c 0.01; 0.33 f 0.83 U/g creatinine for LD4, p -=c 0.025). On chromatograms, LDl and LD2 isoenzymes were the most prominent (Fig. 1). The value of LDl isoenzyme (58.3 + 0.85%) was

TABLE III Urinary enzyme activity of lactate dehydrogenase in 155 healthy children Age

n

LD=l (W

LD2 (W

LD 3+3’ @)

LD4 (9)

LD5 (W

Total LD (U/g creatinine)

0- 4 5-10 11-15

32 42 75

60.4 f 2.33 5X6+1.42 58.9kl.10

24.3kl.48 29.4f0.96 27.7f0.12

8.911t1.10 9.31 kO.90 7.88kO.33

3.72+1.05 3.09kO.47 3.33kO.41

2.67 f 0.49 2.60k0.42 2.19rt0.25

15.2 rfr2.07 b,c 10.13f 1.20 b 7.8OkO.38 =

a Lactate dehydro8~~e Values represent mean + SEM.

bp <

0.05, ’ p < 0.01.

213 u/g creatinine Alkaline

Phosphatase

.

ctate

Dehydrogenase

.

I

I

I

I

I

I

o-2

3-4

5-6

7-8

9-l 0

11-12

I 13-l 5 years

Fig. 2. Changes of the excretion of urinary enzymes according to age. Horizontal bars represent mean values. Each point represents enzyme activity U/g creatinine. 0, Females; 0, males.

always higher than the value of LD2 (27.5 + 0.61% p -c0.001).Mean values of both LD4 (3.32 + 0.33%) and LD5 (2.45 f 0.20%) isoenzymes were < 4% and constant during this study. Discussion

The excretion of urinary enzymes had a wide variability according to age, sex, urine volume, and the time of urine collection, and this was a major obstacle in discriminating between normal and pathological condition. Nevertheless, we preferred to use randomly collected urine samples rather than 8-h or 24-h urine samples [3] because it is really difficult to obtain 24-h urine samples from healthy children, and we were afraid of enzyme deactivation of urine samples stored during 24 h. A few investigations have dealt with urinary enzymes in healthy subjects [ll-131 and most of these studies were done in the adult population. However, to our knowledge, very few studies concerning urinary isoenzymes have been undertaken so far. Hodson et al detected only one component and occasionally two components

214

of AP in normal urine [14]. In this study the successful separation of AP isoenzymes in normal urine was noted, which must be certainly responsible to a high-sensitivity of this procedure. Rambabu et al described that urinary GGT was resolved into two fractions on DEAE-cellulose c~omato~aphy [15], which was in good agreement with ours. However, it was stili difficult to decide whether the variants of urinary GGT were no use in clinical medicine as they suggested [15]. Estimation of urinary LD was of clinical significance in developing a diagnosis of acute renal conditions. Either LD4 or LD5 isoenzyme has been well known to be closely related with the occurrence of the renal involvement [l&17]. Multiple peaks for LD3 isoenzyme were also observed previously [X%20]. In normal urine specimens, enzyme activity of either LD4 or LD5 isoenzyme was usually very faint and occasionally was undetectable, especially in the 0- to 4-yr group (Tables I-III). It was of interest that these isoenzymes showed constant values for our observed age groups. For chromatographic profiles, particularly in the 0- to 4-yr age group, urinary isoenzymes often exhibited very poor chromato~ap~c separations, in which a few isoenzymes such as GGTl or LD4 isoenzyme failed to be detected despite the high enzyme activity in urine. A significant increase in the excretion of many unknown activators or inhibitors which had a great influence on the determination of enzyme activity in urine should occur in the period of infancy. Because with increasing age, the chromatogram became satisfactory. Sex-related differences have been reported for only a few enzymes in urine [12,21,22]. Maruhn et al reported that for all enzymes we investigated, the female subjects had a significantly higher excretion in urine compared to males upon relating enzyme activity to urinary creatinine concentration [8]. However, in this study we were able to find a pronounced sex-related difference only for the output of LD, which would possibly correspond to a higher excretion of urinary LD2 isoenzyme in females than in males because of its prominence. In addition, little attention has been paid to an age-related difference. Salgo et al described how urinary GGT showed no substantial differences in their observed age groups except the neonatal period [6]. Bartels et al also reported similar results [23]. In contrast to the above statement, our results showed a definite decrease in the excretion of urinary enzyme with increasing age. Furthermore, in comparison with values by Maruhn et al in the adult population [S], our results for urinary enzyme activities for AP and LD were considerably or slightly higher than their proposed normal values, and our data for GGT were closely constant with theirs. The evidence thus obtained supported the suggestion that urinary enzymes of AP, GGT, and LD showed the highest activity in the period of infancy and thereafter a definite decrease with increasing age. The output of urinary GGT would probably decrease to limits normal in the adult population at an earlier time compared to other two enzymes. Conclusion

We describe chromatographic profiles of urinary isoenzymes in healthy children. We realize that a sample of 155 subjects may be too small to make definite statements, but believe that newer developments in the separation of isoenzyme

215

patterns make it possible tointroduce increased diagnostic specificity, which inour opinion willbeaccomplished inthenearfuture, Acknowledgements

The author wishes to thank Mr. S. Anahara, Mr. T. Kawasaki (Pharmacia), Mr. Y. Takao (Technicon) and Mr. J.H. Rowland. References 1 Matsukura 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

21 22 23

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