Standardisation of isoenzyme assays with special reference to lactate dehydrogenase isoenzyme electrophoresis

Clin. Biochem. 7, 29-40 (1974)

S T A N D A R D I S A T I O N OF I S O E N Z Y M E A S S A Y S W I T H S P E C I A L R E F E R E N C E TO LACTATE DEHYDROGENASE ISOENZYME ELECTROPHORESIS

SIDNEY B. ROSALKI

Department of Diagnostic Chemical Pathology, St. Mary's Hospital, London, England (Received August 3, 1973)

CLBIA, 7 (1): 29-40 (1974) Clin. Biovhem. Rosalki, Sidney B

Department of Diagnostic Chemical Pathology, St. Mary's Hospital, London, England. STANDARDISATION OF ISOENZYME ASSAYS WITH SPECIAL REFERENCE TO LACTATE DEHYDROGENASE ISOENZYME ELECTROPHORESIS The many factors that may be involved in the standardisation of isoenzyme assays are illustrated by reference to lactate dehydrogenase separation by electrophoresis and isoenzyme demonstration by tetrazolium staining. Some proposals are made for a standardised lactate dehydrogenase isoenzyme procedure by this technique.

CONSIDERABLE PROGRESS IS BEING MADE towards the standardization of e n z y m e a c t i v i t y m e a s u r e m e n t s . This h a s r e s u l t e d f r o m t h e i n v e s t i g a t i o n o f conditions o p t i m a l f o r e n z y m e a c t i v i t y d e t e r m i n a t i o n a n d t h e r e c o m m e n d a t i o n of r e f e r e n c e m e t h o d s based on t h e s e conditions 1. Total e n z y m e a c t i v i t y d e t e r m i n a t i o n s , h o w e v e r , f o r m only a p a r t of e n z y m e diagnosis. I s o e n z y m e d e t e r m i n a t i o n s a r e r e q u i r e d to e x t e n d t h e i r d i a g n o s t i c value a n d a r e b e i n g i n c r e a s i n g l y requested. Despite this, t h e r e h a s b e e n little e f f o r t d i r e c t e d t o w a r d s i s o e n z y m e a s s a y s t a n d a r d i s a t i o n .

Correspondence: Dr. Sidney B. Rosalki, Department of Diagnostic Chemical Pathology, St. Mary's Hospital, Praed Street, London W. 2, England.

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ROSALKI

Isoenzymes may be defined as "the multiple protein forms in which enzymes catalysing the same reaction may exist ''2, and may be measured by a variety of procedures. In this communication only isoenzyme separation by electrophoresis, with isoenzyme demonstration by staining, will be considered, since these are techniques of major clinical importance yet are poorly standardised. Electrophoretic separations using agar gel or cellulose acetate membrane will be mainly discussed, as these are widely employed in the diagnostic laboratory, and lactate dehydrogenase (LD: L-lactate: NAD oxidoreductase, EC 1.1.1.27) isoenzyme assay has been chosen for a discussion of the factors that may be involved in standardisation. This choice reflects the major clinical importance of LD isoenzymes, but the principles involved in LD isoenzyme standardisation have wide general application and where appropriate this will be illustrated by reference to other isoenzymes.

STANDARDISATION OF PATIENT SAMPLE

Where samples are submitted to isoenzyme assay, it is desirable t h a t patient food intake, exercise and drug status are known. Recent food intake may be unimportant with LD, but this is not so, for example, with alkaline phosphatase, in which the serum level of the intestinal isoenzyme increases after fat ingestion 3. Prolonged severe exercise may result in increased activity of m a n y serum enzymes originating from skeletal muscle~, including LD, with enhancement of activity of isoenzyme fractions of muscular origin. Circadian variation of LD activity and the LDs, isoenzyme have been observeda apparently unrelated to food ingestion, but possibly related to the level of ambulant activity. Drug interference with the technique of isoenzyme determination must always be a possibility. However, the rapid electrophoretic migration of low molecular weight drugs renders this unlikely, and no examples of drug interference at the technical level are available.

STANDARDISATION OF SAMPLING TECHNIQUE

Sample collection without stasis is important. Stasis has been shown to cause an increase in serum LD isoenzymes originating from muscle 6. A haemolysed sample will be unsuitable for examination and show excess anodic isoenzymes. Serum for LD isoenzyme analysis should be obtained from blood allowed to clot in glass, since LD activity may be spuriously high in serum harvested from blood clotted in plastic tubes. This may be due to enzyme leakage

ISOENZYME ASSAYS

31

from blood cells as a result of delayed clotting and clot retraction, and in the absence of visible haemolysis. The use of serum rather than plasma avoids any possibility of isoenzyme inhibition by anticoagulants. Heparin does not inhibit LD, but if heparinised blood is used it should be well centrifuged to remove platelets. Plasma rich in platelets may have lower total LD levels than serum, owing to reduced release of platelet LD activatorsL Thorough initial centrifugation during plasma preparation, however, fully activates plasma LD to the level found in serum. In addition, by removing all platelets it ensures that platelets are not disrupted during plasma storage, with liberation of platelet LD isoenzymes of intermediate mobility (LDe and LDs). For isoenzyme examination serum samples should be stored between 10 ° and 20 ° and examined within 48 hours. Samples must therefore be collected and stored under aseptic conditions. Slow-moving LD isoenzymes (LD4 and LDs) are labile in refrigerated or frozen samples and their activity may diminish within 48 hours of storage at 4 ° or -10 °s. The author has observed that the storage of serum samples frozen may result in even more radical isoenzyme pattern alteration, with diminution of slow-moving LD isoenzymes and enhancement of anodic fractions (LD1). STANDARDISATION OF ELECTROPHORETIC MEDIUM Agar

Where agar is used as electrophorectic medium, the brand of agar used should be standardised, since some agar brands may show inhibition of slow-moving LD iseenzymes with i m p a i r m e n t of their mobility 9. Such inhibition may, however, be reversed by the addition of protein (gamma globulin or albumin) to the gel lo. Most separations in agar gel are carried out in gels of approximately 1% strength. Higher gel concentrations may yield unsatisfactory separations and may inhibit the penetration of staining reagents 11. Cellulose acetate

Differing brands of cellulose acetate membrane do not appear to affect LD isoenzyme activity or mobility, although the quality of separation may vary. However, with alkaline phosphatase, different brands of cellulose acetate may substantially alter the mobility relationships of the different isoenzymes. Addition of protein to the buffer in which the cellulose acetate is soaked prior to electrophoresis appears essential for the preservation of labile cathodic LD isoenzymes 12.

32

ROSALKI STANDARDISATION OF SAMPLE APPLICATION

In any isoenzyme staining system, staining intensity can only relate to isoenzyme activity within a defined range of the latter; it is therefore necessary to determine the total enzyme activity of the sample prior to sample application. With LD, it is logical to do this at 37 ° and using a lactate to pyruvate method, since it is under these conditions that isoenzyme activity by staining is being examined. For this same reason, there may be some advantage in carrying out such total determinations by a tetrazolium method13, 14 When total activity has been determined, sample application must ensure that staining intensity will remain linearly related to activity. This can be effected by control of the volume of the sample applied a n d / o r by the dilution of high activity samples. Where sample dilution is required, this may best be carried out in protein solutions. Dilutions in heated sera are not suitable for LD isoenzymes because of the pronounced heat stability of anodic LD fractions. It is important to assume that total enzyme activity is concentrated in a single isoenzyme fraction when the volume or dilution of the sample to be applied is considered. Whilst in normal sera, and some pathological sera, LD activity may be spread over several fractions, in other pathological sera activity may be concentrated in only one or two fractions, and this must be allowed for if the staining capacity/isoenzyme activity relationship is not to be exceeded 15 Although there is no evidence that activators need be added to serum samples for LD isoenzyme electrophoresis, this is not the case for other enzymes. For example, satisfactory isoenzyme electrophoresis of serum creatine kinase requires that a thiol compound be added to the serum sample examined 16. STANDARDISATION OF ELECTROPHORESIS BUFFER

The buffer used for electrophoretic separation must give adequate iso~ enzyme separation without enzyme inhibition. The barbitone buffer commonly used for protein electrophoresis may not be ideal for LD isoenzyme work, and with agar gel it has been shown to inhibit slow-moving isoenzymes 11. The addition of citrate (a known activator of LDs) may reverse LD5 inhibition by barbitone. STANDARDISATION OF SEPARATION CONDITIONS

It is not possible to specific conditions of voltage, current or duration of separation since these must vary with the apparatus in use. It would

ISOENZYME ASSAYS

33

seem that the duration may best be standardised by continuing the electrophoretic run until a serum sample marked with bromophenol blue has migrated a suitable distance. Separation conditions and current levels liable to produce heating should be avoided during the separation to prevent the possibility of enzyme inactivation, particularly of heat-sensitive slow-moving isoenzymes. Separations should therefore be carried out with adequate cooling to 4 ° .

STANDARDISATION OF LD ISOENZYME STAINING

For LD isoenzyme staining, the electrophoretic support medium is incubated with a staining mixture which includes the enzyme substrate lactate, and the coenzyme NAD. Reduction of the latter reduces a tetrazolium salt to a coloured formazan, phenazine methosulphate acting as an intermediate electron carrier. Formazan is deposited at the sites of isoenzyme activity so that isoenzymes are located as coloured bands 17,1s. Despite the limited number of essential components of the LD i s o e n z y m e stain, considerable variation in stain composition is found in the literature. Thus, stain has been prepared in unbuffered solution, or in one of a variety of buffers. Whatever the pH of buffer included in the stain, the final pH of the staining mixture should be checked and specified 19. Because spontaneous tetrazolium reduction may occur above pH 8 and result in excessive background staining, a pH more alkaline and optimal for the LD enzyme reaction in the direction lactate to pyruvate (L-P) cannot be used. The extent of pH-dependent tetrazolium reduction varies with the buffer chosen. Tris, phosphate and barbitone have all been satisfactorily utilised as stain buffer, the last in order to avoid the introduction of a buffer additional to that used for isoenzyme separation. However, since barbitone may inhibit LDs, citrate addition may be required to reverse such inhibition if barbitone is used. As substrate, lactate, sodium lactate and lithium lactate have all been recommended in the literature, generally without specifying the degree of r e a g e n t purity or the stereoisomer employed. The enzyme is active only against L-lactate, but racemic mixtures of lactate may contain variable proportions of the D and L forms and variable amounts of lactide. In addition, whilst the D form of lactate is not normally regarded as inhibitory to the enzyme reaction, racemic sodium lactate may inhibit tetrazolium reduction 13. Lithium L-lactate provides a stable soluble lactate salt available in adequately pure form and therefore appears particularly suitable as substrate. Nitroblue tetrazolium (NBT), iodonitrotetrazolium (INT) and thiazolyl blue (MTT) are the tetrazolium salts that have been most frequently

34

ROSALKI

employed. These should be of high purity since, for example, contamination of NBT with the monotetrazolium salt can produce staining colour variation within the separation mediumS. Different batches of I N T v a r y in the ease with which they are reduced 13, so t h a t if INT is used an easily reducible batch should be chosen. High quality INT is readily and cheaply available and i~ frequently chosen as stain, though its redox potential is less than t h a t of NBT, and background staining m a y be higher. NBT, therefore, appears more suitable. Most batches of commercially available phenazine methosulphate (P:MS) are satisfactory for LD isoenzyme staining. The reagent is, however, profoundly light-sensitive. It should be stored in the dark and discarded if discoloured. The NAD chosen for stain preparation should be of high quality and free from LD inhibitors. Many samples of NAD are contaminated with ethanol 2o, and this may be undesirable because of possible interaction with alcohol dehydrogenase in separated isoenzyme samples. A variety of compounds have been recommended as additives to the above basic staining solution. Many workers include cyanide as a t r a p p i n g agent for formed pyruvate when staining agar gels 21, but such addition does not appear to be required with cellulose acetate 19. Cyanide may, however, unite with NAD so t h a t the latter is no longer able to participate in the tetrazolium reaction, and may produce isoenzyme-dependent LD inhibition. For this reason its substitution by pyrophosphate has been recommended 11. The addition of protein or other colloids to the staining solution may enhance the degree and rapidity of staining. Once again, it is desirable t h a t the protein solution should be ethanol-free. Where the substrate stain is made up in gel form, a final a g a r concentration of 1% is suitable for good gel formation and adequate stain diffusion. Higher agar concentration may result in excessive spontaneous tetrazolium reduction in the staining reagent 11. Where agar is included, it may be desirable to add EDTA in order to complex copper ions, which m a y be present in the agar, and which may be inhibitory to LD522. With lactate dehydrogenase and particularly with other isoenzyme separations it is important to run "blank" staining preparations from which substrate is omitted. For routine LD isoenzyme staining, however, 'this is generally ignored. When such staining is carried out with lactate omitted from the substrate, f a i n t staining bands may be demonstrable at sites of LD activity on the support medium. This phenomenon is frequently named the "nothing dehydrogenase" reaction, and NAD is necessary for its demonstration. Its cause is unknown but lactate, or a lactatelike substance, bound to the separation medium is its likely explanation 23.

ISOENZYME ASSAYS

35

A less likely explanation, t h a t it represents enzyme-bound lactate, has also been suggested 19, and "nothing dehydrogenase" activity resulting from sample alcohol dehydrogenase reacting with ethanol-contaminated NAD or protein additive is also a possibility 24. F a i n t staining of albumin in advance of LD is frequently observed on stained LD isoenzyme preparations, most probably due to tetrazolium reduction by protein sulphydryl groups. The concentrations of the constituents of the LD isoenzyme staining mixture show remarkable variation in the published literature. The extent of this is illustrated in Fig. 1. It is true t h a t the concentration of some components, e.g. PMS, may be widely varied, but this is not so for all components, and certainly not to the extent found in published work, Optimal concentrations of reactants may be determined by variation of individual components of the staining mixture, and suitable concentrations will be suggested later. The concentration of L-lactate which yields optimal activity for fastmoving LD isoenzymes is somewhat lower than t h a t required for optimal activity by slow-moving. At 37 °, and at a pH approximating to 8, a final concentration of 50 mmol/1 is a suitable compromise for both fractions13. ' With NAD, whilst a reagent excess is required, this should not be of such a degree t h a t NAD inhibitors of LD become a problem. STANDARDISATION OF STAINING CONDITIONS

Following electrophoretic separation, stain may be applied as a solution, as a gel or as a cellulose acetate overlay. These latter may be preferable to staining solutions because of reduced diffusion of dye from the separation (enzyme) medium. With cellulose acetate separation, layering the cellulose acetate on to a substrate gel 2~ has proved a most convenient technique, and results in compact staining bands. With staining gels, there may be a differential partition of staining between the enzyme and substrate layers, and it has been suggested t h a t the staining of high activity bands in the enzyme layer becomes preferentially reduced by diffusion, with concomitant preferential enhancement of such bands in the staining layer '-'1. However, this has not been generally confirmed. With cellulose acetate overlay technique, identical relative isoenzyme staining in both enzyme and substrate layers appears to be the usual finding 1~, 19 although overall staining intensity of the two layers may differ. The staining temperature must be controlled, and 37 ° is suitable for LD isoenzymes. Temperatures above this can result in inactivation of slowmoving LD isoenzymes, and increased background staining. At temperatures below 35 ° the enzyme reaction proceeds at an unsuitably low rate 26.

36

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The duration of staining should be such as to give adequate demonstration of isoenzyme bands of suitable activity levels. T h i r t y minutes to one hour is generally satisfactory. This staining time should not be exceeded in order to enhance the staining of bands of low activity, since this m a y result in over-staining of more active bands, with loss of linear staining intensity/isoenzyme activity relationship, diffusion of staining bands, and enhanced background staining. Limitation of the enzyme activity of the sample separated, as discussed previously, prevents the unduly rapid development of staining. A variety of procedures have been utilised for stain fixation and preservation. Suitable fixation may vary with the dye and staining conditions chosen and the proposed method of quantitation. Formadehyde19, dilute nitric acid 25 and dilute acetic acid 19, 2z have all been utilised for stain fixation. Five minutes fixation in 5% v / v aqueous acetic acid solution followed by five minutes washing in distilled" water and air-drying between blotters has proved suitable for cellulose acetate membrane stained by NBT or INT. MTT staining, however, frequently shows fading on storage after such treatment.

ISOENZYME ASSAYS

37

STANDARDISATION OF STAINING QUANTITATION

P r i o r to any form of quantitation it is essential t h a t visual inspection of the stained electrophoretic strip be carried out. This ensures t h a t separation and staining are satisfactory, t h a t staining bands are compact and undistorted, t h a t background staining is at a low level, and t h a t the separation medium is free from artefacts. Because visual inspection is poorly quantifiable, some more suitable form of staining quantitation must be achieved. Two differing types of procedures may permit this: elution of stained fractions or densitometric scanning of stained strips. Both these procedures may give satisfactory results, but densitometry is more rapid and convenient and is more suitable for the routine laboratory. It m a y be carried out by the reflectance method on uncleared material, or by transmission of cleared samples. With cellulose acetate, reflectanr~ densitometry is the more convenient. Standardisation of densitometry requires satisfactory instrument performance and wave-length calibration. The wave-length chosen for scanning should be at the absorption maximum of the reduced tetrazolium salt, and linearity with increasing concentration of formazan must be demonstrable. When this has been confirmed, a linear relationship between absorbance and isoenzyme activity on individual stained isoenzyme fractions 15 will demonstrate both instrumental scanning performance and the adequacy of the staining procedure. Quantitation of the absorption peaks obtained from scanning may be based on peak height o r peak-.a~ea, hut. t h a l a t t e r i a pre£exable 15, particularly in the presence of any peak asymmetry. It is the values obtained by measurement of peak areas t h a t shouid therefore relate to enzyme activity. Replicate scanning of individual isoenzyme separations must also be carried out to assess the reproducibility of machine performance. STANDARDISATION OF QUALITY CONTROL PROCEDURES

The problems of reagent quality, and the need to check scanner linearity, reproducibility, and the linear relationship of staining intensity with isoenzyme activity have been previously mentioned. However, additional quality control procedures are required. Within-day precision of patient samples of v a r y i n g isoenzyme composition and of reference samples can be calculated from an adequate number of duplicate or replicate determinations. More important, however, is the calculation of between-day (between-batch) precision. This may be conveniently determined from carry-over repeat determinations on patient samples suitably stored, or may be calculated from data obtained from

ROSALKI

88

Table Suggestions for a Standardised LD Isoenzyme Separation and Staining Technique I. Fasting, resting sample, collected without stasis into glass tubes. 2. Serum harvested aseptically within 4 h of clotting after 10 min. centrifugation at

3000g. 3. Examined within 24 hours with storage at 10 °, together with freshly reconstituted control serum. 4. Total LD activity determined by standardised L --* P. method at 37 °. 5. Batch-standardised cellulose acetate for separation. 6. Standardised separation of sample together with control: e.g. 200 V in l~eckman Microzone Central sample application Bromophenol blue-marked albumin migration of 2 cm Cooling to 4 °. 7. Sample volume appropriate to enzyme and isoenzyme activity (1 ~l with Microzone and total L D less than 250 U / l ) 8. Sample dilution in 1% w / v albuminsaline if necessary. 9. Tank buffer: chilled to 4 ° Vetonal 0.025 mol/1 Tris 0.050 mol/1 Citrate 0.012 tool/1 pH 8.2 at 37 ° i0. Membrane buffers: as tank buffer, plus bovine gamma globulin (alcohol-free) 1 mg/ml buffer. 11. Freshly prepared stain, with PMS added last, warmed to 37 ° and added to cooled (56 ° ) melted Agar-Noble. Final agar concentration I % w/v. 12. Stain: High q u a l i t y - L q- lactate, lithium salt NBT PMS (stored in dark)* NAD (alcohol and inhibitor free) Gamma globulin (alcohol free)

13. Toyield final gel concentration : L-lactate 50 mmol/1 NBT 0.5 mg/ml (0.61 mmol/1) PMS 0.025 mg/ml (0.08 mmol/1) NAD 1.0 mg/ml (1.5 mmol/1) Gamma-globulin 1.0 mg/ml in: Tris 0.10 mol/1 Pyrophosphate 0.015 mol/1 E D T A 0.12 mmol/1 Final pH 7.8 at 37 ° 14. Membrane layered onto staining gel and incubated in dark at 37 ° in moist chamber for 30 minutes. 15. Membrane removed from gel to 5% v / v aqueous acetic acid; surface wiped; fixed for 5 minutes with three changes of acetic acid within this period. 16. Washed 5 minutes in distilled water with 3 changes of water within this period. 17. Dried under pressure between filter paper. 18. Visual inspection. If satisfactory, scanned by reflectance densitometry at 540 nm by scanner with linear response. 19. Peak area quantitated. 20. Result expressed in activity units. 21. Result reported if control values satisfactory.

*Meldola's Blue (9-methylaminobenzo-=-phenazonium chloride) can be substituted at equal weight concentration for PMS (Burd J. F., Usategui-Gomez, M., Clin. Chim. Acta, 46(1973) 223-227). It is more Stable and background staining is reduced.

ISOENZYME ASSAYS

39

freshly reconstituted lyophilised reference sera included in each separation run. The check. ensure under-

use of such reference sera is also recommended, as an accuracy They can facilitate interlaboratory comparison of results and so that a technique, even though precise, does not result in spurious o r over-estimation of isoenzyme fractions. STANDARDISATION OF RESULT DISPLAY

Stained isoenzyme separations may be displayed visually or isoenzyme values expressed in digital or graphical form. With the latter, isoenzyme fractions may be calculated as a percentage of the total enzyme activity of the sample, or each isoenzyme fraction may be expressed in activity units. The disadvantage of the percentage mode of expression is t h a t enhancem e n t of the percentage level of one fraction implies t h a t the percentage of other fractions will be diminished, even though their activities may be elevated. Visualisation of the strip permits assessment of all isoenzyme fractions, but visual display is poorly quantifiable. The most desirable mode of expression must therefore be in activity units. For clinical appreciation of results, a graphic display of isoenzyme values in activity units may be particularly helpful2L Isoenzyme nomenclature should be in accord with the convention whereby the five isoenzymes of normal sera are numbered consecutively, with the electrophoretically fastest moving fraction (the one with the greatest mobility towards the anode) numbered as Fraction 1 and the slowest moving as Fraction 5. Where isoenzyme patterns are displayed for visual inspection the anode should be at the top or on the r i g h t hand side 2. SUGGESTIONS FOR A STANDARDISED LD ISOENZYME STAINING PROCEDURE FOLLOWING ELECTROPHORESIS

Having detailed so many factors requiring consideration in LD isoenzyme demonstration by staining following electrophoresis, it would appear appropriate to make suggestions for a standardised procedure. Some experimentally based proposals are outlined in the Table. These are, however, to be regarded as guide-lines rather than rigid final rcommendations.

ACKNOWLEDGEMENTS

The author wishes to acknowledge the assistance of D. Tarlow, BSc, in the evaluation of the lactate dehydrogenase isoenzyme staining procedure suggested in the text.

40

ROSALKI REFERENCES

1. Recommendations of the German Society for Clinical Chemistry: (1972). Z. Ktin. Chem. Kiln. Biochem. 10, 281-291. 2. The Nomenclature of Multiple Forms of Enzymes: IUPAC-IUB Commission on Biochemical Nomenclature Recommendations (1971); (1971). Eur. J. Biochem. 24, 1-3. 3. Langrnan, M.'J. S., Leuthold, E., Robson, E. B., Harris, J., Luffman, J. E. and Harris, H. (1966). N a t u r e (Lond.) 212, 41-43. 4. Halonen, P. I. and Konttinen, A. (1962). N a t u r e (Lond.) 193, 942-944. 5. Starkweather, W. N., Spencer, H. H., Schwartz, E. L. and Schoch, H. K. (1966). J. Lab. Clin. Med. 67, 329-343. 6. Starkweather, W. H., Green, R. A., Spencer, H. H. and Schoch, H. K. (1966). J. Lab. Clin. Med. 68, 314-323. 7. Cohen, L. and Larson, L. (1966). N e w Engl. J. Med. 275, 465-470. 8. Kreutzer, H. H. and Fennis, W. H. S. (1964). Clin. Chim. A e t a 9, 64-68. 9. Kreutzer, H. H. and Eggels, P. H (1965) Clin. Chim. Acta. 12, 80-88. 10. Wieme, R. J. (1966). Clin. Chim. A c t a 13, 138-139. 11. Hyldgaard-Jensen, J., Valenta, M., Jensen, S. E. and Moustgaard, J. (1968). Clin. C h i ~ A c t a 22, 497-509. 12. Rosalki, S. B. and Montgomery, A. (1967). Clin. Chim. A c t a 16, 440-441. 13. Babson, A. L. and Phillips, G. E. (1965). Clin. Chim. A c t a 12, 210-215. 14. Jordon, W. C. and White, W. (1967). Clin. Chim. Adta 15, 457-464. 15. Latner, A. L. and Turner, D. M. (1967). Clin. Chim. A e t a 15, 97-101. 16. Somer, H. and Konttinen, A. (1972). Clin. Chim. A c t a 40, 133-138. 17. Markert, C. L. and Moller, F. (1959). Proc. Nat. Aead. Sci. 45, 753-763. 18. Dewey, M. M. and Conklin, J. L. (1960). Proc. Soc. E x p . Biol. (NY) 105, 492-494. 19. Barnett, H. (1964). J. Clin. Path. 17, 567-570. 20. Rosalki, S. B. (1971). Anal. Lett. 4, 819-835. 21. Wieme, R. J., Van Sande, M., Karcher, D., Lowenthal, A. and Van Der Helm, H. J. (1962). Clin. Chim. A c t a 7, 750-754. 22. Wieme, R. J. (1970). Methoden der Enzymc~tisehen Analyse, (Bergmeyer, H.U. ed.) Verlag Chemie, 2nd Edit. Vol. 1 p. 557. 23. Falkenberg, F., Lehmann, F. G. and Pfleiderer, G. (1969). Clin. Chim. A c t a 23, 265-278. 24. Shaw, C. R. and Koen, A. L. (1965). J. Histochem. Cytoehem. 13, 431-433. 25. Myers, R. C. and Van Remortel, H. (1968). Clin. Chem. 14, 1131-1134. 26. Kreutzer, H. J. H. and Kreutzer, H. H. (1965). Clin. Chim. A c t a 11, 578-581. 27. Elevitch, F. R., Sweatman, T W. and Sampson, J. J. (1969). Clinical Lab. F o r u m 1, 8. 28. Homer, G., Yott, B. and Lim, J. G. (1969). A m e r . J. Clin. Path. 51, 287-292. 29. Matson, C. F. (1962). Lancet 2, 1278-1279. 30. Mull, J. D. and Starkweather, W. H. (1965). A m e r . J. Clin. Path. 44, 231-237.