Determination of LDH in urine: Automation and study of interferences

ANALYTICAL.

BICCHEMISTRY

Determination

(1965)

l&290-303

of LDH in Urine: Automation Study of Interferences1 G. P. HICKS

From the Department

AND

and

S. J. UPDIKE

of Medicane, University Hospitals, Madison, Wisconsin

University

of Wisconsin,

Received June 3, 1964

The measurement of lactic dehydrogenase (LDH) in urine has been proposed as a screening procedure for the detection of diseases of the urinary system (l-3). The promise of urine LDH as a screening test has been discussed (14). Automation can greatly facilitate the evaluation of this new test and make its use as a routine screening test feasible. The successful determination of urine LDH depends on the recognition and, if necessary, removal of interferences before measurement of the enzyme. Interferences have been removed by dialysis of the specimen (3, 5). This paper presents an automated system for the determination of urine LDH and a rapid gel filtration technique for the removal of urine interferences in place of more time-consuming dialysis procedures. PRINCIPLES

The reactions for the LDH

AND

EQUIPMENT

Chemistry determination

are:

LDH

lactate + NAD

ti

pyruvate + NADHz

(1)

PMS

dye-ox f NADHl (blue)

F= dye-red + NAD (colorless)

(2)

The oxidation of lactate by nicotinamide adenine dinucleotide (NAD) is catalyzed by LDH to give pyruvate and reduced NAD (NADH,) as shown in reaction 1. In reaction 2, NADH, reduces a blue dye (dye-ox) in the presence of a catalyst, phenazine methosulfate (PMS). When the lactate, NAD, dye-ox, and PMS are at nonrate-limiting concentrations, the rate of dye reduction is proportional to the LDH activity. The rate ‘This investigation was supported in whole from the National Institutes of Health. 290

by Public

Health

Service Grants

DETERMINATION

OF

291

LDH

is measured as a decrease in the absorbance at 600 rnp. Details of the LDH reaction system and selection of optimum reagent conditions have been considered elsewhere (6). Most conventional methods of LDH determination are based on the reverse of reaction 1, which is more complete than the forward reaction. Several advantages accrue to the use of the forward reaction, Most important, lactate and NAD are much less expensive and more stable as reagents than pyruvate and NADH,. It has also been shown that the range of linearity and reproducibility are superior (7). Equipment

Equipment developed specifically for the automation of enzymecatalyzed procedures (8) which has been applied to the determination of serum LDH (6) can be used for the automation of urine LDH. Figure 1 illustrates a new flowing system principle recently developed

\

PHOTOMETER,

FIQ. 1. Principle

of flowing

system.

for the equipment (9, 10). For the determination of LDH, an LDH reagent containing all components necessary for the enzyme reaction except the enzyme, and an LDH sample containing the enzyme, are metered together at constant rates and mixed to initiate the enzymic reaction. The reaction stream is split into two delay lines of equal flow rates, one line being thermostated at 40°C and the other remaining at room temperature. After a fixed time delay, the two reaction streams flow simultaneously through photometer cells. At steady state, the absorbance difference between the cells is directly proportional to the

292

HICKS

AND

UPDIKE

rate of reaction at 40°C. The absorbance difference is continuously measured and recorded by a differential filter photometer having a full scale sensitivity of 0.01 absorbance unit (6). After calibration with a known enzyme standard, unknown sample values can be read directly from the recorder chart with no calculation or working curves. The new “parallel” system (so-called because the reaction stream is split into two parallel delay lines) has several advantages over the original “series” flowing system which measured the absorbance between two fixed points on a single reaction stream (6, 8). With the parallel system, longer time delays can be used with no sacrifice of the number of analyses per hour. The increased time delays, where needed, can greatly increase the sensitivity of the determination. The flowing system for LDH is illustrated in more detail in Fig. 2.

FULSER

FIQ. 2. Details of flowing system.

The LDH reagent and parallel streams are metered at 1.0 ml/min by a Harvard 600-1200 peristaltic pump (Harvard Apparatus Company, Dover, Mass.) using 0.065-in. Tygon tubing at 12 rpm. The sample flow rate is the difference between the sum of the flow rates of parallel streams and reagent stream, 1.0 ml/min. Each delay line is 10 in. of 0.085in. stainless-steel tubing embedded in a lead block. The high temperature block is thermostated at 40 -t- 0.05’C by a mercury regulator and cartridge heater embedded in the block. The low temperature block is protected from large temperature fluctuations by circulating a small stream of tap water through a loo-in. steel delay line contained in the block. The, parallel streams are pulsed 5 times per second after the photometer cells, promoting rapid mixing and decreasing laminar flow

DETERMINATION

OF

LDH

293

effects throughout the flowing system. An automatic sample turntable introduces diluted serum or urine samples at the rate of 20 per hour. The sample changer pinches off the sample stream and transfers the sample aspirator tip to the next sample in less than 1 set, preventing air from entering the system and causing no significant effect on the flowing system (11). REAGENTS Dye Solution: 34 mg of 2,6-dichlorophenolindophenol dissolved in 100 ml water and filtered. Dye solution is stored and used at room temperature for one week, after which any remainder is replaced with fresh solution. Phosphate Buffer: 840 ml of 0.1 M Na,HP04 and 160 ml of 0.1 M KH,PO, are mixed to give 1 liter of phosphate buffer, pH 7.4. LDH Reagent: 200 mg of NAD, 5 mg of PMS, 4 ml of sodium lactate, 60% syrup, and 20 ml of Dye solution (all from Sigma Chemical Company, St. Louis, MO.) are added to 200 ml of 0.1 M phosphate buffer, pH 7.4, and mixed. The reagent is aspirated for several minutes before use to prevent the formation of air bubbles in the high-temperature delay line. The reagent, which is light sensitive, is stable at room temperature for a day if kept in a brown bottle. For convenience, the NAD and PMS are preweighed into vials and stored in the dark at room temperature for up to one month with no noticeable deterioration. Serum Standard: A commercially available serum enzyme control (Enzatrol, Scientific Products, Evanston, Ill.) is reconstituted with 5 ml of distilled water according to directions on the label. PROCEDURES Preparation oj Serum Samples: Reconstituted serum standard is divided into 0.5-ml aliquots and frozen in 25.0-ml calibrated test tubes. The frozen aliquots are not kept for more than two weeks. For use, a frozen aliquot is thawed, diluted with phosphate buffer to the 25-ml mark, mixed, and poured directly into a sample cup. One tube is enough for 5 determinations. To prepare a single sample, 0.1 ml of serum is diluted to 5.0 ml with phosphate buffer, mixed, and poured into a sample cup. Routine Preparation of Urine Samples: Prior to analysis, interferences which inhibit LDH and reduce the dye in the absence of lactate must be removed from the urine. A rapid separation of interferences can be achieved by Sephadex gel filtration with the column shown in Fig. 3. A 40 X 2 cm chromatography column (Cat. No. S-18820, E. H. Sargent, Chicago, Ill.) is packed with 40 ml (11 cm) of Sephadex G-50,

294

HICKS

AND

UPDIKE

r

II cm L

FIQ. 3. Gel filtration column.

block form, 100-270 mesh (Pharmacia Fine Chemicals, New Market, N. J.), which has been decanted in water and equilibrated with phosphate buffer. A 2 cm sponge disk, cut from a reinforced household sponge cloth, is placed on top of the column. The sponge has a twofold purpose: First, it allows the application of a sample to the column with no disturbance to the gel bed. Second, when the column of fluid falls to the sponge, the column flow is stopped by the capillary action of the sponge, preventing the column from going dry for up to 24 hr, thereby greatly simplifying the routine use of the column procedure. Columns are stored by stoppering each end of the column and leaving 1 or 2 cm of buffer over the sponge disk. Periodically, columns are repacked by removing the sponge disks, filling the columns with phosphate buffer, stoppering the ends, inverting to mix well and allowing to settle overnight. The Sephadex gels have been used for many samples with no noticeable deterioration, However, because the nature of the interferences is not well understood, the gel is renewed every month. To prepare a urine sample, 5.0 ml of centrifuged urine is placed on the column and the effluent collected until the flow stops. This first collection is discarded. A 25-ml calibrated test tube is placed under the column, 20 ml of phosphate buffer is placed on the column, and the effluent is collected until the flow stops. The collection, which quantitatively contains the LDH fraction, is diluted to 25 ml, mixed, and poured into a sample cup. The 25-ml tube is adequate for 5 determinations and is the equivalent of the LDH in 5 ml of urine diluted to 25 ml. A single sample corresponds

DETERMINATION

OF

LDH

295

to the LDH in 1.0 ml of urine diluted to 5.0 ml with phosphate buffer. To regenerate the column for use, 40 ml of buffer is placed on the sponge. The eluate is collected until flow stops and discarded. In a very few instances when a yellow color remains on the column after one wash, washings are repeated until all of the color is eluted. Typical flow rates through the columns are 1 to 2 ml/min, requiring about 30 min total time for sample preparation. Since the columns do not require continuous attention, one technician can manage 10 to 20 columns routinely. The properties of the column are discussed later. While all of the work described in this paper was done with the block form Sephadex, the new spherical bead form, G-50, fine grade, gives comparable separation, but with 3 to 5 times the flow rate. Dialysis of Urine Samples: Low molecular weight interferences may also be removed by dialysis (5). To compare the results of the new gel filtration technique, some urine samples were prepared by dialysis; 5 ml of a centrifuged urine specimen is pipetted into about 10 in. of an 8-mm diameter cellophane dialysis tube which is tied at both ends. Twenty of these samples are dialyzed 4 hr against 8 liters of 0.001 M Tris buffer, pH 8.5 (Sigma Chemical Co., St. Louis, MO.), with constant stirring. The buffer is changed at 1 and 2 hr. After dialysis, the sample is transferred to a 25-ml graduated cylinder, diluted up to 25 ml, mixed, and poured into a sample cup. Instrument Conditions: The setting of the optical balance adjustment and other conditions of the filter photometer system are essentially the same as previously described (6, 8). Aside from switches for pulsing, automatic sample change, etc., which are self-explanatory, only two controls on the instrument are ever adjusted during a run. A zero control sets the baseline on the chart recorder when only buffer is in the sample cup. A sensitivity control allows the sensitivity of the filter photometer to be adjusted to calibrate the recorder chart to read directly in the desired units of enzyme activity when a standard sample is introduced. The use of these controls is discussed later. RESULTS

Figure 4 is a recording designed to demonstrate the characteristic responses of the instrument. First, from right to left, a baseline was established at zero with buffer. Next, two identical standard serum samples were run. At steady state, the sensitivity control was adjusted to make the standard sample (900 LDH units/ml) read 0.9 on the chart. Thus, for serum samples, full scale on the chart represents 2000 LDH units/ml. For urine samples, which are diluted only one-tenth as much as serum (5 ml of urine per 25 ml compared to 0.5 ml of serum per

296

HICKS

AND

TJF’DIKE

Fm. 4. Chart recording showing instrument responses. The chart W&B made with a series of serum standards and urine samples as explained in the text. The value for each sample response is read as the height of the steady-state plateau above the buffer baseline.

25 ml), full scale represents 200 LDH units/ml. LDH units, according to a widely accepted calorimetric method, are used throughout this paper (12, 13). International units are obtained by multiplying the calorimetric units by 0.48. After calibration, four urine samples, two buffers, and another standard serum sample were run. The mixed order of the samples, as shown in Fig. 4, allows the comparison of various transitions between different types of samples. Sample values are read as the height of the steadystate plateau above the buffer baseline. It can be seen that the change from one’ steady state to a new steady state occurs in less time for Borne samples. For all samples, however, the allotted period of 3 min for each sample is adequate to reach steady state. The longer a sample is metered, the closer its approach to steady state will be, so the most valid reading on a sample which does not actually reach a plateau is the last portion of the sample response prior to the transition to the next sample. In actual use, urine samples up to 400 units/ml cause no serious error when followed by samples in the range of 40 units/ml. Samples which are in the range of 200-400 units/ml can be read directly by cutting the sensitivity in half with the sensitivity control, reading the chart value, and multiplying the chart reading by two. Samples over 400 units/ml are diluted and repeated. The four urine samples in Fig. 4 are known dilutions of a urine specimen containing 182 units of LDH per milliliter. A plot of the diluted values against the recorder chart readings is shown in Fig. 5. The points have a maximum deviation of +-4 LDH units from a theoretical calibration curve drawn through the origin and the standard serum sample. Values obtained by direct readout procedures, that is, values read directly from the chart with no calculation or calibration curve, can be expected to be within +4 LDH units/ml over the entire range. Gen-

DETERMINATION

OF

LDH

297

1.6 -

Ol(0-I LDH

UNITS

per ml of URINE

FIQ. 5. Dependence of chart reading on LDH activity. erally, errors due to direct readout can be minimized by calibrating with a standard in the region of greatest interest (6). It should be noted that the data of Figs. 4 and 5 include maximum errors due to unfavorable transitions between dissimilar samples as described above and are not encountered frequently in routine use. When ten 5-ml aliquots of a single urine specimen are prepared with Sephadex, pooled, and the LDH determined ten times on the pool, the relative standard deviation is &2$% or less. When ten aliquots of a single specimen are prepared with Sephadex and the LDH values determined on each individual sample, the relative standard deviation is rt5 to 7%. The difference in precision represents the errors in the column procedure and manipulation of the sample during preparation. LDH values were determined on several urine specimens after preparation by both gel filtration and dialysis. A plot of the Sephadex values against the dialyzed values for about 75 samples is shown in Fig. 6. Generally, there is good agreement between Sephadex and dialyzed values, as shown by the distribution of the points about a straight line representing perfect agreement. Closer examination of the data in Fig. 6 reveals that there are 24 points above the line and 47 points below the line, showing that dialysis tends to give higher values than gel filtration. For the four points circled, the dialysis values were considerabIy greater than the Sephadex values.

HICKS

AND

DIALYSIS FIQ.

6. Comparison

UPDIKE

VALUE

of Sephadex and dialysis values.

Data has been obtained which suggest that dialysis is much less efficient than gel filtration and that nonenzymic blank activity may not be completely removed by dialysis in some cases. The high efficiency of gel filtration is evidenced by the facts that: (1) LDH activity is collected in a clear colorless fraction while dialysis has little effect on the colored appearance of the urine ; (2) boiling samples prepared by gel filtration consistently destroys all LDH activity while dialyzed specimens occasionally show some residual activity, even though, as will be shown later, the nonenzymic activity is partially destroyed by heat. Because of the complex nature of urine, it is conceivable that nonenzymic blank activity may consist of several kinds of chemical reducing substances that are dialyzed with different efficiencies, which may explain the occasional high values obtained by dialysis. Token experiments suggest that a routine calorimetric method for LDH (12, 13) can be modified to determine LDH in Sephadex treated urine. Normally, 0.2 ml of diluted serum or 0.2 ml of dialyzed urine is incubated with 1.0 ml of a substrate mixture (12). To test the colorimetric procedure, 5.0 ml of urine was prepared according to the routine gel filtration procedure previously described; 1.0 ml of prepared urine

DETERMINATION

OF

299

LDH

(containing the LDH from 0.2 ml of urine) was added to the substrate mixture and incubated as usual (12). The method was calibrated by running diluted serum standards according to the usual directions, but 0.8 ml of phosphate buffer was added to the incubation mixture to make the total volume the same as for urine samples. While no detailed study of the calorimetric method was made, a comparison of the automated and modified calorimetric procedures for about 50 samples showed good agreement. INTERFERENCE

STUDY

Eflect of Dialysis on Urine LDH Activity: An experiment was performed to demonstrate the reduction of nonenzymic blank activity by dialysis. Eight 5.0-ml aliquots of a centrifuged urine specimen were dialyzed against 8 liters of 0.001 M Tris buffer with constant stirring as previously described. The buffer was changed at 1, 2, 4, and 6 hr. One aliquot was removed from the bath every hour. After all of the aliquots had been removed, the LDH activity was determined in each as described before. The data for two urine specimens is shown in Fig. 7.

HOURS

FIG. 7. Effect of dialysis on LDH

The apparent LDH activity up to about 4 hr, after which show that some nonenzymic dialysis and that the removal Fig. 7, no correlation between was found.

OF

DIALYSIS

activity

of two urine samples.

in each specimen decreased with dialysis the activity became constant. These data reducing activity is being removed by is complete in about 4 hr. As suggested by the nonenzymic blank and LDH activity

Separation of Interferences by Gel Filtration:

A urine specimen with

300

HICKS

AND

UPDIKE

a conveniently measurable nonenzymic blank activity was selected and augmented with purified rabbit muscle LDH (type 2, Sigma Chemical Co., St. Louis, MO.) to give a convenient LDH activity; 5.0 ml of the augmented urine specimen was added to the column in Fig. 3. When the urine had drained to the sponge and the column flow had stopped, about 30 ml of phosphate buffer was added to the column. The height of buffer was kept relatively constant while 50 l-ml fractions were collected. Each fraciton was diluted to 5.0 ml, poured into a sample cup, and assayed for LDH activity. The results are shown by the solid Fig. 8. too x-x-x

,

80

I

-X--X-X-

Serum

LDH

_Icc

Activity

of

X-%-X-X

‘X

'Gx ‘Lx/

Recovered Column

*'

s’

Fractions

60

YELLOW

COLOR

40

2j,pJ,,Jy 20

ml

23

COLLECTED

30

33

from

40

, 45

30

55

COLUMN

FIO. 8. Separation of LDH and blank activity by gel filtration: 5.0 ml of urine was placed on a Sephadex column and eluted with buffer; fifty l-ml fractions were collected and the activity was measured in each fraction; the blank peak was obtained even when the lactate and NAD were omitted from the LDH reagent while the LDH peak was not.

The separation of the LDH and blank activity is clearly shown. The fractions collected routinely for the LDH determination are indicated by the arrow above the LDH peak. The yellow color of urine is not usually eluted exactly with the blank activity. This experiment has been performed many times using urine augmented with serum and purified LDH, and nonaugmented urine, always with the same results. To demonstrate the removal of inhibitors, the

DETERMINATION

OF

301

LDH

experiment described above was repeated, except 14 X 2 ml fractions were collected in the region of the blank activity. Two O.&ml aliquots were taken from each fraction. One aliquot was diluted to 5.0 ml with buffer and assayed for LDH; 0.1 ml of serum (86 units on chart) was added to the other, and the mixture was diluted to 5.0 ml and assayed for LDH. The recovery of the serum LDH from each fraction was calculated. These results are shown by the curve above the blank activity in Fig. 8. The recovery of serum LDH from the fractions dropped to a low of about 74 units (86%) in the region of the blank activity. Recovery studies using rabbit muscle LDH gave similar results. The inhibition varies widely among different urine specimens and there appears to be no correlation between the amounts of blank activity and inhibition for different specimens. Effect of Heat on Nonenzymic Blank: Because the fraction containing nonenzymic blank activity can be conveniently collected from the column, a few studies were made to determine the effect of heat on the blank. The blank activity used for the study was obtained from a pool of five lo-ml fractions, each taken in the region of peak blank activity from a Sephadex separation on each of five urine specimens.

60+ tee

LDH

-X-X-X-

BLANK

‘X \

\ ‘%-

-x

\ ‘X

Room

TEMPERATURE,

“C

FIG. 9. Effect of heat on LDH and blank activity. The activities a series of LDH and blank solutions after heating were measured. was heated for 10 min at the temperature indicated.

remaining in Each sample

302

HICKS

AND

UPDIKE

The urine LDH used in the study was collected as a single fraction under the LDH peak (from 10 to 20 ml in Fig. 8) from a single urine specimen. The fraction was diluted about twofold to about 170 LDH units/ml. Aliquots (2 ml) of the urine LDH and blank specimens were incubated for 10 min at 5” intervals from 30 to 95’C. After heating, the specimens were cooled, diluted to 5.0 ml with buffer, and assayed for LDH. A plot of the percentage of activity remaining after heating against the temperature is shown in Fig. 9. The LDH activity was completely destroyed in 10 min at 65°C. The blank activity was partially destroyed at all temperatures after 4O”C, losing about 55% of its activity at 95°C. Because a significant portion of the nonenzymic blank activity in urine is destroyed by heat, it cannot be estimated in raw urine by simply boiling the specimen to destroy enzyme activity, a practice which is common for nonenzymic blanks. DISCUSSION This work has shown, in agreement with others (5), that raw urine may possess at least two kinds of interferences, a nonenzymic blank and inhibitor(s). The nature of these interferences in urine LDH determinations is not entirely clear from the literature. The methods for urine LDH which measure the absorbance at 340 rnp of NADH, produced by the forward reaction (reaction 1) have encountered high nonenzymic blank activity (5), while methods using the reverse of reaction (1) have not revealed the blank (1, 2). The work in this paper shows that the blank is a small molecular weight chemical reducing activity. This is further demonstrated by the fact that the blue dye solution is bleached when it is added directly to raw urine or blank fraction from the Sephadex column in the absence of lactate, NAD, or any other reagents. Since methods based on the reverse reaction use as a substrate NADH,, which is in the reduced form, the lack of the reducing blank activity is expected (2). The role of the inhibition is not clear. The methods based on the forward reaction show definite inhibition. The reverse reaction methods show good recovery of rabbit muscle LDH and linearity over a restricted range of sample size, which does not preclude the possibility of inhibition for larger sample sizes (2). The magnitude of errors introduced by inhibition is much less than by the nonenzymic blank. Urine samples were found in which the blank gave over ten times as much activity as the LDH. The most inhibition found in any urine was about 20%. The demonstration that a significant portion of the nonenzymic blank is destroyed by heat shows the problems that one can encounter when

DETERMINATION

OF

LDH

303

heating enzyme samples to demonstrate blanks. Gel filtration appears to be a very useful tool to isolate low molecular weight enzyme interferences for characterization. The rapid gel filtration procedure makes the automation of the LDH determination feasible and reliable. With this technique it should be possible to screen many patients for the presence of elevated urine LDH. Such a screening program is now under way in this laboratory. ACKNOWLEDGMENT

The authors thank Miss Gloria Nalevac and the members of the senior class, School of Medical Technology, University of Wisconsin, for technical assistance. REFERENCES 1. BRENNER, B. M., AND GILBEBT, V. E., Am. J. Med. Sci. 245, 31 (1963). 2. RIGGINS, R. S., AND KISER, W. S., J. Ural. 90, 594 (1963). 3. WACKER, W. E. C., AND DORFMAN, L. E., J. Am. Med. Assoc. 181, 972 (1962). 4. WACKER, W. E. C., DORFMAN, L. E., AND AMAWR, E., J. Am. Med. Assoc. 188, 671 ( 1964). 5. DORFMAN, L. E., AMADOR, E., AND WACKER, W. E. C., .I. Am. Med. Assoc. 184, 1 (1963). 6. BLAEDEL, W. J., AND HICKS, G. P., Anal. Biochem. 4, 476 (1962). 7. AMADOR, E., DORFMAN, L. E., AND WACKER, W. E. C., Clin. Chem. 9, 391 (1963). 8. BLAEDEL, W. J., AND HICKS, G. P., Anal. Chem. 34, 388 (1962). 9. BLAEDEL, W. J., AND HICKS, G. P., Advan. Anal. Chem. Znstr. 3, 105 (1964). 10. HICKS, G. P., Ph. D. Thesis, University of Wisconsin, 1963. 11. Gilson Medical Electronics, Middleton, Wisconsin. 12. BERGER, L., AND BROIDA, D., Tech. Bull. No. 600, Sigma Chemical Co., St. Louis, MO. 13. CABAUD, P. G., AND WROBLEWSKI, F., Am. J. Clin. Path. 30, 234 (1958).