Kidney International, vol. 8 (1975), p. 176—1 84

Urinary lactic dehydrogenase isoenzyme 5 in the differential diagnosis of kidney and bladder infections HUGO F. CARVAJAL, RICHARD B. PASSEY, MICHAEL BERGER, LUTHER B. TRAVIS AND WILLIAM B. LORENTZ Department of Pediatrics, Division of Pediatric Nephrology, and Department of Pathology, University of Texas Medical Branch and the Shriners Burns Institute, Galveston, Texas

zymes 4 et 5 prédominent. Du fait qu'un recouvrement (a 3 écarts types) en cc qui concerne l'isoenzyme 5 n'a été observe que chez un malade un diagnostic difl'érentiel correct est possible chez 94% des enfants atteints de pyélonéphrite ou 97% de Ia population totale des malades.

Urinary lactic dehydrogenase isoenzyme 5 in the differential diagnosis

of kidney and bladder infections. Urinary lactic

dehydrogenase (U-LDH) isoenzyme assays were performed on

children with clinically proven kidney (N = 16) and bladder infections (N = 22) as well as normal controls (N = 24). Documentation of bladder and kidney infection was accomplished by means of the

bladder washout test, culture of ureteric urine (in patients with

Precise localization of the site of infection is a

urinary diversion), kidney function studies including the maximal urine concentration test, clinical symptomatology and radiologic appearance of the urinary tract. Total U-LDH in normal children (10.8 I mU/mI) was lower than in patients with bladder (27.0 3.9 mU/mI) or kidney (226 67.3 mU/mi) infections (P 100,000/mI)

Negative Negative Negative Negative

1.020

1.014

1.014 1.032 1.027 1.020 1.017 1.022 1.017 1.014 1.028 1.017 1.017 1.011 1.022

0.001 0.007

N

0 0—2 0—2 0—2

N N N N N N N N N

0—2 0—2 4—6 0—2

6.5 5.0 6.5 6.0 6.0 6.5 5.0 6.0 5.0

N N N

0—2 0—2 0—I

6.5 5.67

N

6.0 6.0 6.0 5.0

6.0 5.0

N N

N N

1—3

0—2 0—3 0—4 1—3

1—3

0—2 0—3

0—I

N

0—1

N

0—2 0—2

HPF 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

5

4

2

I

Total

0 0 0

0 0 0

0 0 0

0 0

14

0

4

4

19

19

1.5

1.5

1.5

1.5

0

0

3.5

0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0 0

4.9

0 0

0 0 0 0 0

1.8

1.8

1.8

1.8

4 1.8

5

3

2

8 10.5 12

12

9 9 9.2 14 1.8

14

0

1.8

1.8

0.5

1.8 1.0

1.8 1.0

0

5.7 1.0

1.0 0 1.6 1.0

2.4 3.0

2.4 2.5

2.4 2.5

0 2.5 0.5 2.4

1.2

1.2

1.2

0.8

0.8 0.9 0.2

0.8

0.8

1.1

0.3

6.8 1.3

1.0

1.2

6.4

0.12

0.3

0.59

1.4

1.2

HPF, high power field; N, negative.

9

1.8 14 1.8

1.8

1.1

14 14 15

0

1.0 1.2

9 9

1.0

1.8

1.6 1.0

14

24

0

0

14

14 24

1.8

0

0.8 0.9 0.2

as specific gravity; Prot, protein; WBC, white blood cells;

3

9

4.0

9 4 4

2.5

14

0.5

0.5

4

2.4 0

12

1.2

9 6 4 10.8

1.0 5.0

178

Carvajal et al

made every ten minutes until five additional specimens were collected (samples 3 to 7). All

Results

specimens collected during the procedure were subjected to quantitative bacterial culture. Patients were classified as having "bladder" infections if all cultures after bladder sterilization and washout were negative. Patients were classified as having "kidney" infection if the following criteria were met: a) culture 2 disclosed no bacterial growth; b) bacterial colony count

Normal controls. Total urinary LDH activity in 24 healthy children (11 boys, 13 girls) ranged from 4 to 19 mU/mi of urine; the mean was 10.8 mU/mi, and the SEM, 1.0 mU/mi of urine. The usual isoenzyme pattern was of the "fast zone" type (isoenzymes 1 and 2 predominating). lsoenzymes 3, 4 and 5 were present in amounts ranging from 0 to 5.7 mU/mI (Table I). The urine specific gravity ranged from 1.011 to 1.035 (mean, 1.022), and the urine pH, from 5 to 6.5 (mean, 5.3). Determinations for protein, glucose and ketones were negative. With the exception of an occasional white blood cell, examination of the sediment yielded negative results. Urine cultures were also negative (Table 1).

was greater than lOO/ml of urine on four of the specimens; and there was at least a tenfold increase between specimen 3 and specimens 4 to 7 [21]. Urinary LDH assays. Urine samples were obtained from control and study patients during the morning hours. The children were first asked to empty their bladders and discard the urine. A second urine sample was then obtained (usually within 30 mm). LDH assays were performed within two to three hours of collection, During the interim, the urine was stored at 4°C. Total LDH activity of undiluted and undialyzed

urine was assayed according to the method of Wroblewski and LaDue [22] on a Gilford 3400 system (Gilford Instruments Co., Oberlin, OH). LDH activity was expressed as milli-international units per milliliter (mU/mI).

The isoenzyme bands were visualized after electrophoresis on thin film agarose. The assay was carried out as follows: the urine was applied to the well in the thin film agarose (620E) according to the following scheme; if total LDH activity was less than 100 mU/mi, then 4 p1 was applied in 1 p1 aliquots; if the urine LDH activity was greater than 100 mU/mI but less than 500 mU/mI, then a total of 2 p1 was applied in 1 p1 aliquots, and if the total urine LDH activity was over 500 mU/ml, then 1 pl or a suitable

dilution was applied to correspond to this range. Electrophoresis was carried out for 25 mm, and immediately after, 1 ml of the mixed visualization reagent (1.2 mg/mI of p-nitroblue tetrazolium, 70

mg/mi of phenazine methosulfate, 2 mg/mI of nicotinamide adenine dinucleotide, 0.2M disodium lactate in 0.08M tris-hydroxymethylaminomethane buffer pH 8.5) was applied to the isoenzyme field and this was incubated in a stain tray at 37°C for 45 mm for color development. The enzymatic reaction was

stopped by a 1-mm wash in 10% acetic acid in methanol and the plate was then dried at 75°C for 30

mm. Quantitation of the LDH isoenzymes was achieved by scanning the pattern with the densitometer using a 605 flM filter. The percentage of each fraction was calculated and multiplied by the total activity to get the unit value for each fraction. The fraction that migrated the fastest to the anode was termed isoenzyme 1.

Kidney infections. Sixteen patients (nine girls, seven

boys) met the diagnostic criteria for active parenchymal infections (Table 2). Nine of these children (patients 25 through 33) had undergone

either cutaneous ureterostomy or ileal conduit diver-

sion and urine for culture was collected directly through the stoma utilizing a double-catheter techni-

que. The other seven patients had intact urinary tracts and positive bladder washout results. The latter had lower concentrations of urinary enzyme activity than the former, but no statistical differences between the two subgroups could be demonstrated (Table 2).

Total U-LDH activity in the 16 patients with kidney infections ranged from 117 to 1100 mU/mi (mean, 226 67 mU/mI). This level was significantly higher than in patients with bladder infections or normal controls (P < 0.005). Urinary LDH isoenzyme 5

ranged from 5.9 to 640 mU/mi (mean, 120 + 39 mU/mi) and was significantly higher than in patients

with bladder infections or normal controls (P < 0.005). Only one child (patient 37),—with pyelonephritis diagnosed on the basis of a positive blad-

der washout finding, fever and inability to maximally concentrate the urine — had urinary LDH isoenzyme 5 concentrations in the same range as patients with bladder infections (Fig. 1). On the other

hand, there were seven patients with kidney infec-

tions whose total urinary LDH concentrations overlapped with those in the bladder infection group (Fig. 2). After 16 hr of water deprivation, only three of the patients with kidney infections (patients 31, 36 and

39) were able to concentrate the urine above 700 mOsm/liter. The results of urinalysis, urinary LDH isoenzyme assays, urine culture and urinary concentrating ability in patients with kidney infections are summarized in Table 2.

Bladder infections. On the basis of the bladder

yr mo

yr

yr

1.1

SEM SD

F 9F 7M

F Proteus&Staph

E. co/i

Proteus Enterobacter Proteus

352 950

Pseudomonas E. coli& Proteus Providencia E. co/i E. co/i Pseudomonas

800 636 618 38 153

694

594 626 433 688 840 648

450

Proteus

550 433 600 600

.

1.017 1.009 1.012 1.014 1.006 1.0 19 1.005 1.010 1.010 1.013 1.010 0.001 0.003

1.011

1.008 1.010 1.009 1.010 1.009

SG

pH

0.52

0.20

5.99

6.0 6.0 6.5 7.0 6.0 7.5 6.0 5.0

7.0 7.0 7.5 7.0 7.5 6.0 7.0

7.0

16

N

N

N

Trace N N

N

N N 1+ N 1+ N Trace N

2+

Prot

30—40

2—3 0—3 15—20 3—5 7—12

5—10

TNTC TNTC

0

0CC

TNTC

0 10—20

3—5

0 0 0

TNTC TNTC

5—8

0—5 0—4

3—5

0 0 0

0CC

HPF

RBC

120 39 155

17

83.8 38.7 5.9 38.3 19.2

269.0

153.0

52.0

168.0

21.9

31.4

640.0 230.7

70.3

72.8

5

infections

TNTC TNTC TNTC

15—20

HPF

WBC

children with kidney

Urinalysis

and laboratory data in

Urinary concentrating abilityc

Proteus

Enterobacter

M F F M M M F M F F F

Pseudomonas E. co/i

M

M

M

Sex

.

Urine culture (colony count >100,000/mI)

sex

63

16

49

19.1 9.6 8

4.4

0 158.0 14.7 6.9

0

65.0

127.0 11.6 25.9

214.0

31.8

86.9

4

Not

44

II

4

33

4.5

14.0 2.9 11.8

6.1

15.2 165.0 82.9 3.8 59.4 17.0 0 0 76.0

67.6

3

5 22

17

2

7.6

2.7

2.9

31.0 3.6 5.9

0 0

0 53.1 7.0

44.6

30.8 8.2 72.0

2

2.4 14.0 0.7 0.9 3.3 7 7 2 7

4.0

0 0

25.9 11.0

5.9 6.4

9.0

15.9 3.7

I

Urinary LDH isoenzymes, mU/mi

aPatients 25 through 33 had undergone urinary diversion procedures; patients 34 through 40 had intact urinary tracts. bSG specific gravity; Prot, protein; WBC, white blood cells; HPF, high power field; N, negative; TNTC, too numerousto count; occ, occasional. tested simultaneously.

4.7

8.8

l6yr

7yr 8 yr

6yr

5 yr 9 yr

4yr Syr

15

4 yr

15

lSyr 8yr 7yr

15 18

Age

40 Mean

32 33 34 35 36 37 38 39

31

25 26 27 28 29 30

No.

Subject

Table 2. Age,

73 38 38 226 67 271

81 17

Ill

275 130 1100 494 67 187 268 52 153 537

Total

-1

-

Carvajal et a!

180

.640 U_LDIl isoenzyme 5 acthdty

•230

above 700 mOsm/liter (range, 505 to 660 mOsm/ liter), In three children (patients 45, 53 and 59),

concentrating ability was not tested (Table 3). The urinalysis usually disclosed pyuria but no clear-

cut correlation could be established between the number of white blood cells per high power field and total urinary LDH activity or any of its isoenzymes (Table 3). Discussion

Abnormally high levels of urinary LDH activity have been reported in patients with bladder carcinoma [23], prostatic malignancies [23], toxic and ischemic nephropathy [24, 25], renal neoplasia [23], nephrosis [26], acute and chronic pyelonephritis [27] and several other nephritides [23, 28]. Differentiation among these conditions on the basis of urinary LDH concentrations has not been possible and little signifiTotal U—LDI I activity

Normal

Bladder

controls

infections

• 1100

infections

Fig. 1. LDH isoenzyme 5 activity (mU/mi) in 24 normal children, 22 children with bladder infections and 16 children with kidney infections. The horizontal line depicts the mean concentrations

for each group and the height of the bar, 3 so above the mean.

washout test, 22 girls were diagnosed as having blad-

der infections (Table 3). Total U-LDH activity 3.9 ranged from 6 to 76 mU/mi (mean, 27 mU/mi). These values were significantly higher than

total U-LDH in normal controls (P < 0.005) but considerably lower than the corresponding values in

patients with kidney infections (P < 0.005). LDH isoenzyme 5 concentrations ranged from 0 to 11.4 0.8 mU/mi) and were also mU/mi (mean, 3.1 higher than in normal controls (P < 0.005) but lower than in patients with kidney infections (P < 0.005). Thirteen of the 22 children in the bladder infection group concentrated the urine normally (range, 851 to 1135 mOsm/liter) while 6 children (patients 41, 43,

46, 48, 58 and 61) failed to concentrate the urine

Normal

controls

Bladder infections

Kidney infections

Fig. 2. Total urinary LDH activity (in U/mi) in 24 normal children, 22 children with bladder infections and /6 children with kidney infections. The horizontal line depicts the mean level for each group and the height of the bar, 3 so above the mean.

E. coil

E.coil Proteus

F

F F F F F F F 22F

10

8

61

aj bNot

so

7 8.3 0.8 3.9

Enterobacter E. coil Enterobacter

E. coil E. coil

44

19

635 987 836

505 — 880

891 899 1016

1135

—

545 935 670 1053 1006 1010 912

—

538 853 572 851

Urinary concentrating abilityb

0.006

1.019 1.006 1.014 1.019 0.001

—

1.018 1.022 1.014 1.020 1.014 1.018 1.025 1.009 1.026 1.027 1.023 .022 1.013 1.027 1.023 1.025 1.025 1.012

SG

0.14 0.66

5.0 5.0 6.5 6.5 6.0 5.0 5.0 7 6 6 5.6 5.86

5.0

6.0 6.0 6.0 6.0

6.0 6.0 6.0 7.5 6.0 5.0

pH

TNTC 12—16

N

8—24 2—5

0

15—20 10—18

1—5

0CC 10—20 0CC 3—5 0CC 0CC 8—10 tO—IS

TNTC

5—10 10—20 10—20

WBC HPF

N N

N N N N N N N N

Trace N N N N N N N N Trace

Prot

Urinalysis

0 0 0

0

0 0

0—6 2—4 0—2

3.6

0.8

3.1

2.8

0 3.8

0 0

0

0 0

0

3.0

2.8

0 0 0

6.5

0

0 3.8 3.5

1.2

0 0 0

2.8 2.3 0.7 3.3

9

13

0 11.4

1.2

0

0 3—4 12—20

1.2

0

0

3.8 0

3.8

1.2 1.2

1.8 1.8

0 2.7 2.7

3.8

3.1

5.6 3.6

5.6

0

0

0 9.5

3

0

0

4

0

5.7

8.2 5.4 3.6 5.5 10.8

5

I

4.6

1.0

10.0 8 8 2.8 5.0

0 0

0

2.8 14.5 3.5 16.6

16 18

52

19 19

46

6

1.2

3.8 16.3

0 3.8 8.1 1.2 3 11.2 11.0

0

2.8

0

28 56.1 14.4 5.6 8.2 1.4 1.4 4.8

0 1.4 1.4

0 16.4 9.6 5.6

2

Urinary LDH isoenzymes, mU/mi

0 0

10—15

0

0

0 0 0

0CC

0cc 0

HPF

RBC

specific gravity; Prot, protein; WBC, white blood cells; HPF, high power field; TNTC, too numerous to count; occ, occasional. tested simultaneously.

SCM

62 Mean

12

3

9 7 7

OM

E.coil E.coil

F F

E. coil

E. coil

Proteus & Enterobacter E. coil E. coil E. coh

E. coil

E. coil

7

16

52 53 54 55 56 57 58 59 60

51

Proteus E. coil E. coil

E. coil

IS 8 4 4 7

16

46

6

3

45

47 48 49 50

F F

Sex

F F F F F F F F F

6

12

4

42

43 44

Il

yr

Age

41

Subject No.

.

Urine culture (colony Count >100,000/mI)

Table 3. Age, sex and laboratory data in 22 children with bladder infections

27 3.9 18.4

14

62 24 48

19 19

28 6 9 40 57

19

23

13 13

24 28 28 9

76

28

Total

'

.

Carvajal et a!

182

findings were then compared with those of 24 healthy

children. Urinary tract infection regardless of site Kidney infection N = 16

f Mean SEM

125

* p