CLINICA CHIRIICA ACTA 23

A GENERAL COLORIMETRIC PROCEDURE FOR THE ESTIMATlON 01;

ENZYMES WHICH ARE LINKED TO THE NADH/NAD+ SYSTEM

SUMMARY

I. Two colour reactions for determining the reduced coenzyme NADH are described.

(a) A coloured formazan is produced by the reduction of the tetrazolium salt INT with NADH, electron transfer being facilitated by PMS; the formazan is stable and the extinction is read at 500 m/f.

(b) The coloured ferrous dipyridyl complex is produced following the reduction of a ferric salt with NADH, in the presence of PMS; the coloured complex is stable for 30 min. The extinction is read at 520 m/r.

I qole of NADH, when reacted with the ferric salt, gives the same absorbance as z /moles of a ferrous salt, and it is possible to standardise the solution of NADH against a stable iron standard without the need for an ultraviolet spectrophotometer.

2. Details are given of the assay of HBD, LDH, SDH, MDH and GOT, GPT, using these colour reactions, micro-volumes of sample being used.

3. Details are given of standard deviation, cost, reliability, etc., of the methods described.

4. Present methods for the assay of GOT and GPT are discussed in the light of recent literature.

L-Aspartate: 2.oxoglutarate aminotransferasc, E.C. 2.6.1.1.

L-Alanine: 2.oxoglutarate aminotransfcrase, E.C. 2.6.1.2. Nicotinamide adcnine dinuclcotide. Nicotinamide a&nine dinucleotide, reduced form. r-~-Iodophenyl-~-p-nitrophenyl-~-phenylt~trazoliu~n chloride. L’hcnazine methosulphatc (S-methylphcnazonium mcthosulphatc). L-Lactate: NhD oxitloreductase, E.C. 1.1.1.27. L-1Ialate: NAD oxidorcductase, E.C. 1.1.1.37. Tris (hydroxymethyl) aminomethane. T,DH,-isoenzyme hydroxybutyrate dehydrogenase, E.C. I. T. I .27. L-lditol: SAD oxidoreductase, E.C. r.r.r.r4.

C/in. Chim. Acta, ‘.+ (1969) 23-37

WHIT.kKER

Tetrazoliurn salts in the presence of a suitable hydrogen acceptor, such as PMS, and reducing agents such as SXDH, are converted into highly coloured forrna- zans, whose colour obey Beer’s Law and can be stahilised. Such con~pounds Ilaw, been used to determine the activity of several enzymes, notably lactic dellydrogtnase, (refs. I, z), using the tetrazolium salt INT, thereby elinlinating tlrc need for ultra- violet equipent. The first of these n~ethods gi\w results in terlns of rnilligranns of forrnazan produced, while tlrc second is standardised by using a conlinercial scruni, the LDH activity of which had been assayed 17~ another nlethotl. In tlrc author’< opinion, neither of these niethods of stanclardisation is ideal. One tlran;lx~ck is that no t\vo lxtclics of tetrazolium salt will necessarily gi\re tlic sanie optical derlsit~~ with a standard amount of SAL)H. I~urtlicrmorc, standartlisin,q \vitli a (-oninicrcial scrunl n~akes the user depciident on the reliabilit!. of its \xlues. Instead, the author leas not di\orccd assay-, using INT, froni ultraviolet standal-disatioI1, hut this ncwl onI\, lx, done at lnonthly intervals.

13~ taking increasing anwunts of N.\I>H (I)!- ultra\iok+ spc,ctrol)hotonl~,tr~,) and reacting theni with wcess of IST, stabilising tllc colour and reading at $0 m/ , a graph of extinction against j-rxoles of SXDH can be prepared. As the I X1‘ reagent is fairI\- ihI& (lasting at least 1 montll when kept dark) standardisatioll of (w-11 new batch of reagent onlv is rccluirctl. Tllis graph can then lx: usctl [or all enzVni( assays described, and results calculated in international units (I.L7.), taking hto account tlrc, quantity of scruni used and the tinie of incuhtion (see I:ig. I).

IOO-

0.02 0.06 0.10 0.14 0.18 0.22 pmoles NADH

COLORIMETRIC ESTIMATION OF ENZYMES 25

The reactions involved are as follows: (a) NADH + PMS a NADf + reduced PMS. (1~) Reduced PMS + INT -i PMS -1 formazan (coloured). The formazan is held in solution by the use of Tween 20, and the colour is

stabilised by bringing tlie pH to betlveen 1.0 and 5.0 with phthalate buffer.

The substrates described below were kept at 4O in chemically~ clean ground glass-stoppered bottles (plastic bottles and stoppers would probably be quite satis- factory). For routine purposes smaller bottles were used and refilled at intervals from the stock. By taking these precautions the risk of bacterial and fungal contamination was minimised. All enzyme preparations were kept continually at 4’ and have been found to keep satisfactorily.

Colow reaged (a) INT 200 mg, PMS 50 mg, Tween 20 2.5 ml, deionised water to 250 ml. Store

at 4O in a gutta percha bottle. N.B. The reagent deteriorates rapidly in light, it should be bottled as soon as

possible in a completely opaque container. Gutta percha has been found suitable.

0.05 M Phthalate buff& (Clark-Lubs) fiH 3.0 Dissolve 10.21 g potassium hydrogen phthalate in approximately 700 ml of

deionised water, add 204 ml IL’/IO HCl and IO ml Tween 20. Adjust to pH 3.0 if necessary. Make up to I litre.

Standnrdisatiox of tetrazolitm salt INT using NADH Important: this procedure must be carried out with each new batch of colour

reagent. I. Prepare IO ml of an approx. 0.2 /mole/ml (i.e. 1.6 mg) solution of NADH

disodium salt in 0.1 :W phosphate buffer pH 7.4. 2. Dilute 2 ml of this solution with 2 ml phosphate buffer. (The dilution is neces-

sary to bring the extinction into an acceptable range.)

3. Measure the extinction of the diluted SADH at 340 m/s. Since for iSADH at 340 ny’ E = Gzoo, (~a).

fiyp,;“” V 2 I\

6.2 ~ = /moles NADH~ml

X standard curve is then prepared for the range O.OI--0.20 /~moles SXDHjnll, :I\ outlined in Table I and T;ig. I.

Colour rractim (0) The reaction described above still requires occasional access to an ultraviolet

spectrophotolneter, and therefore the author sought another reaction which \vould be stoichiometric for the amount of SADH present, with reagents not varying front batch to batch, and thus enabling the assay of enzymes to be carried out with simple equipment.

Blue tetrazolium was employed by Rosenkrantz” in a comparative study of the reaction rates and intensities of 27 compounds with the tetrazolium salt, and witll ferric chloride-z.z-dipyridyl reagent.

The author considered the possibility of using a reagent containing ferric ammonium sulphate, PMS and a,z-dipyridyl for the determination of NADH. Standard amounts of NADH (determined at 340 m/t) were reacted with ferric ammo- nium sulphate-z,z-dipyridyl reagent and the absorbance values were read at 520 In/!. Standard amounts of ferrous ammonium sulphate were reacted with 2,2-dipyridyl and were found to give half the extinction given by NADH, (see Fig. 2). It is, therc- fore, possible to standard& NADH by measuring the extent to which it reduces ferric ion to ferrous:

(a) NADH + Fe, (SO,), (NH,), SO, P-b NAD+ + 2 FeSO, (NH,):! SO, - H,SO, (b) Fez+ + z,z-dip. -+ Fez+ 2,z-dip. complex (coloured).

The colour obeys Beer’s Law at 520 m/r.

Solutions rcpivcd fw the jwcparntion of the colow reagent (h) (T) o.oooj M PMS. PMS--153.1 mg/litre of deionised water. Store in a dark

bottle, at 4’. Discard after 14 days.

COLORIhIETRIC ESTIMATION OF ENZYMES 27

(2) o.ooo_j AI Fcrvic ~~~~z~~zo~zzizz~z sulfihatc. Ferric ammonium sulphate (24 H,O)- .@2.1 mg, glacial acetic acid--1o.o ml, deionised water to I litre. Keeps indefinitel!,.

(3) 0.1” u z,a-Difiyridyl ix 3Ob acetic acid. z,z-Dipyridyl (:\R)---I g, glacial acetic acid---30 ml, deionised water to I litre. Store in dark bottle.

Mix equal quantities of a,z-dipyridyl, Pi% and ferric ammonium sulphate solutions in a dark bottle, shortly before use, since the reagent is light sensitixre.

Sodium fluoride-r.5 g. Dissolve in a litre of IO ~01s. hydrogen peroxide. Store at 4-O.

Solzdiom rquivcd fw fin~~avation of th stmtlard fcuous solution (I) Ferrous avzmnzizm sztl~hate 0.001 M. l;eSO,‘(XH,),SO,.6 H,O (AR)-

3q.z mg. Dissolve in deionised water, add a few drops of sulplmric acid and make up to 100 ml.

(2) 0.1 N sodium szdj&ite. (Prepare freshly.) Sodium sulphite (AR) anhydrous-- 1.26 g, deionised water to IOO ml.

Preparation of the stadard Xix the following together: 0.001 N ferrous ammonium sulpliate- 25 ml (i.e. 25 pmoles) 2,2-Dipyridyl -100 1111

2s \rxIT.Zw:I<

0.1 X sodium sulphite --I00 ml

Tkionised water 12-j 1111.

Heat to 00‘ , allow to reinain at tliis temperature for j iiiiii, to tlcvclop full colour, allow to cool and add a few crystals of mercuric acetate. Stow in a dark bottle. This standard keeps for nian~~ iuonths. Its cstinction can be read against ;I water blank, as the dipyridyl aud sodium sulphite should give a negligilk I~lanl; value.

It has been shown that I ,c.inole of ferrous anmoniuiii sulpliate, \vllen reactotl wit11 z,z-dipyridyl gives l&f the absorbance at 520 III!; that is gi\ren by I j mole of NADH when reacted wit11 the ferric-dipyridJ.1 colour reagent. Tllerefore, in nlaking up the standard 25 ~moles of ferrous arnmoniunl sulpllate \verc wacted with 2,~. dipyridyl and brought to a final volume of 350 ml, giving an absorlzmcc cquivalrnt to 12.5 /:nloles of KADH in 350 ml (0.125 ~moles in 3.5 ml). As all the enzyme test5 described finish with a final Twluule of 3.5 ml, in each of these tests the standard \yill be equivalent to 0.125 ,/ moles of NXI)H.

The methods described below were based on the determination of the amount of NLADH utilised during incubation rather than the production of NADH. It ~vas, therefore, necessar!. to have two tubes for each assa)., one to act as a “control” in which the enzyme under assay could not act through lack of substrate but an\’ side reaction could proceed, and a second tube (the “test”) in which enzyme activity would take place, together with the side reactions, for a known time at a standard tern- perature. The amount of KADH remaining in each tube was then determined 1)~. either of the colour reactions described earlier. The difference in /moles of NADH between “test” and “control” is the measure of enzyme activity. It was therefore essential that the dispensing of NADH into “coiitrol” and “test” tubes should liavc a high degree of precision.

(I) Estiwzatimz of GOT and GE’?“,5 (modified) The enzyme reactions are as follows:

GOT

I. I.-:\spartatc I- s-oxoglutarate ‘z/Y I,-glutamate + oxaloacetate. L__~_ oo-I‘

2. Osaloacetatc -I-- SADH !Y!’ 111 malate + SAD 1 MDH

GPT

I. I.-Al?nine + r-oxoglutarate ‘z?! pyruvate m1m I,-glutamate. (>PI

pH’ lactate + KXIV. 2. Pyruvate -7 NADH _ ~ l.l)H

0.1 dI L-Asjxzrtntc. r,-,Aspartic acid--13.3 g, L\T/I NaOHPm IOO ml. Make up to I 1 vvith 0.1 df phosphate buffer, pH 7.50. Adjust if necessary to pH 7.50, add a fem. drops of cliloroform and store at 4@,

COLORIMETRIC ESTIMATION OF ENZYMES 29

Malic de&vdrogenase (MDH) 500 h/g/ml. This can be purchased from Boehringer Corporation Ltd. in the form of a suspension in 2.2 111 ammonium sulphate. It can be stored at 4’ in this form for at least 6 months.

GOT substmtc. (Prepare immediately before use.) To every millilitre of L-aspar- tate add 50 ,~l of MDH and 5 ;rl of diluted LDH (500 pg/ml). These proportions must be adhered to. (See notes on Endogenous 0x0 acids.)

0.2 M uL-Ala&e in 0.1 ,V pH 7.50 phosphate buffer. or,-Alanine-17.6 g. Make up to I 1 with buffer. Adjust to pH 7.50 if necessary. Add a few drops of chloro- form and store at 4O.

Lactic delzydrogenase (LDH) 5 mg/ml. The enzyme suspension as purchased requires ten-fold dilution with 2.2 M ammonium sulphate before use. It was found that the diluted form showed no detectable loss of activity in 4 months at 4’.

GPT substrate (prepare immediately before use). To every millilitre of DL-

alanine add 50 ,~l of LDH suspension (500 /#g/ml). 0.0 25 nf a-Oxoglzttnrate. cr-Oxoglutaric acid--o.367 g, X/I NaOH-5 ml. Dilute

to IOO ml with 0.1 M pH 5.0 phosphate buffer. Add a few drops of chloroform and store at 4’.

KADH +t H,O &sodium salt, M.lfT. 709.46. It is preferable to use the hydrated coenzyme rather than the anhydrous form which tends to absorb variable amounts of water from the atmosphere.

FVorkiq solution of NADH ap$roximatel. o.5pmoleslml. Dissolve I mg NADH in 2.5 ml of 0.1 rll phosphate buffer pH 7.5 (correcting for y6 purity).

Procedure (All volumes are in ml)

Iimgrnt GOT

Test

Serum 0.05

sut,strate 0.p NAnH 0.25 Mix. Incubate for 3-5 min at 25”. wOxoglutarate 0.20 Mix. Incubate for 20 min at 25’.

Contvol

o.o=j

0.50 0.25

-

GPT

Test

0.05 0.50

0.2j

0.20

Control

0.05 0.50

0.25

Blmk*

0.50

0.20

* Either GOT or GPT substrate can be used in the Blank tube.

Either colour reaction can be carried out as described below.

Reactiolt (a)

Tetrazolium colour reagent 0.50 0.50 0.50 O.jO

oc-Oxoglutarate 0.20 0.20

Mix. Allow I min. I’hthalatc buffer 2.00 2.00 2.00 2 .oo

Phosphate buffer pH 7.5 Mix. Iieatl within ,j h at 500 m,u or with Ilfortl 603 and 623 filter.

0.50 --

2.00

0.30

AIte~natively Reaction (b)

Ferric-dipyridyl reagent 2.00 2.00 2.00 2.00 2.00

d)xoglutarate 0.20 0.20 -

I’hosphatc buffer pH 7.5 0.3 Mix. .Ulow I min. Stabilizer O.jO 0.50 0.50 0.50 O.jO

Mix and read within 20 min at 520 rnp or with an Ilford 624 filter (see Fig. 3). Also measure the extinction of the “Standard” (ferrous dipyridyl-sulphite mixture) against a water blank.

Vv’HITAKER

Calcdntioiz fey rractim (II) Determine /Tmoles of NADH utilisecl 1)~ deducting test extinction from control

extinction and referring to graph. Then :

,tmoles of N4DH utilisecl 1000 \b’

incubation time (min) = l.IF./litre of wruni

’ volunw of serum used (nil)

Calcdutio~~ fw vcactioll [I)) The “standard” value as stated above corrcsponcls to 0.125 ~m~~lcs of YrlDH,

and for 20 inin incubation, using 0.05 ml sanlple,

Extinction of c_ontrol~estiIz~tion of test

mtinction of standard ,’ 125 -= 1.I.. ,‘litre of scrun1

The enzyme catalyses the conversion of pyruvate to lactate at 11tI 7.4 witI1 resultant removal of NAI>H from the system. Tile quantity of PI;ADH rclnaining is deterniincd 1,~ dour reaction (a) or (I)).

I. 0.1 .\I phosphate buffer pH 7.4. z. Soclium pyruvate III IngJ in pH 7.4 buffer. Prepare freslll!-. 3. NADH. 0.5 ,~mloles/ml in pH 7.4 buffer. Prepare fresllly.

4. Tetrazoliuin or iron dipyridyl reagent.

5, Phtlialate buffer for tetrazoliuni reaction.

COLORIMETRIC ESTIMATION OF ENZYMES 31

Reaction (a)

Tetrazolium colour reagent 0.50 0.50 0.50 Mix. Allow I min.

l’yruvate 0.75

I’hosphate buffer 0.30 I’hthalate buffer 2.0 2." 2.0

Mix. licatl within 3 h at 500 m,u or with an Ilfortl 603 or 623 filter.

Altevnatixly reactiou (b)

Ferric dipyridyl reagent 2.0 2.0 2.0

Mix. Alloy I min. l’y’“\‘“tc 0.75 l’hosphate buffer 0.3” Stabilizer 0.j 0.5 0.5 Mix and read within LO min at 520 rn@ or with an Ilford 624 filter.

Cahlatioiz fov reaction (a)

As for GOT and GPT

Calczdatiox SOY vcaction (6)

The standard is E 0.125 pmoles of NADH. Therefore, for 5 min incubation and 0.05 ml serum:

Extinction of control-extinction of test

extinction of standard X 500 = I.U./litre of serum.

LDH isocm~wzcs7 (modified) Xfter heat fractionation by the method of Wroblewski and Gregory7, the

remaining enzyme activity is determined as above for total LDH.

(I) Mis-allow all tubes to stand at room temperature for 20 min. (2) Incubate tubes at appropriate temperatures for exactly 30 min. Cool in

iced water. Proceed as for total LDH.

32 WHITAKliIt

(3) Estimation of Hl3D (modified) The enzyme catalyses the reduction of cc-oxobutyric acid to cc-lrydroxybutyric

acid at pH 7.4 with the resultant removal of N_%DH from the s>,stem. The cluantit!. of NADH remaining is determined by colour reaction (a) or (I~).

Keagexts

I. 0.1 ,lJ phosphate buffer pH 7.4. 2. 0.01 M sodium a-oxobutyrate (British Drug Houses Ltd., England) in pH 7.4 buffer. 3. XADH. 0.5 qoles/ml in pH 7.4 buffer. Prepare freshly. 4. Tetrazolium or ferric dipyridyl colour reagent. 5. Phthalate buffer or stabilizer.

The test is carried out as for LDH except that the incubation time is IO nrin. Enzyme activity calculated as follows :

Calcdation fey reaction (a)

As for GOT and GPT

Calculation for reaction (b) The standard is E 0.125 i/moles of NADH.

Therefore, for IO min incubation and 0.05 ml serum:

Extinction of control&extinction of test

extinction of standard ~~~ x 250 = I.Ii./litre of serum.

(4) Estimation of MDH9 (modified) GOT is used to generate substrate for MDH, by converting L-aspartate to

oxaloacetate, which is converted to malate in the presence of NADH. The quantit? of NADH remaining is determined by colour reaction (a) or (1~).

Reagents I. 0.1 M phosphate buffer pH 7.4. 2. 0.1 M r_-Aspartate in phosphate buffer pH 7.4. 3. 0.025 &f. cc-oxoglutarate in phosphate buffer pH 7.4, 4. GOT suspension z mg/ml (Boehringer). This must be diluted with the following diluent, the molarities being final

concentrations in the diluent : 3.0 M ammonium sulphateeo.og M maleate (adjust to pH 6.o)-o.oo25 M oc-oxoglutarate. To the z mg/ml suspension add 12.3 volumes of this diluent, giving 150 ,q/ml. Keep at 4O.

5. cc-Oxoglutarate-GOT. To every ml of x-oxoglutarate add 50 Hal of dilute GOT suspension immediately before use. 6. NADH 0.5 /!moles/ml in pH 7.4 buffer. Prepare freshly. 7. Tetrazolium (or ferric dipvridyl reagent). 8. Phthalate buffer (or stabilizer).

COLORIMETRIC ESTIMATIOS OF ENZYMES 33

Iiraplt Test CWLtYOl Blank

Serum 0.05 0.05 _

S,\DH 0.2j 0.25

L-.kpartate 0.j" 0.5" "..5"

Incubate for 5 min at 25’-. cc-ox”glutarate/~~oT 0.2” 0.2” Incubate for I” tnin at 25’. 1Sither colour reaction can now be carried out as described below.

Reactiolz (a)

Tetrazolium colour reagent ".jO 0.5" 0.5"

or-Oxoglutarate/GOT 0.2” -

Mix. .-\llo~ I min. I’hthalate buffer 2.“” 2.“” 2.“” Phosphate buffer pH 7.5 0.3” Mix and read within 3 h at 5”” m/c or with an Ilford 603 or 623 filter.

Alternatively reaction (b)

ITerric dipyridyl reagent 2.0” 2.“” 2.“” a-Oxoglutarate GOT 0.2” -

Mix. Allow 1 min. Phosphate buffer pH 7.5 0.3” Stabilizer 0.5” 0.5” 0.j"

Xx and read within 20 min at 52” m/* or with an Ilford 624 filter.

Calculatio~z ,for reaction (a)

As for GOT and GPT.

Calculation fey reaction (b)

The standard value as stated above is E 0.125 /lmoles NADH. Therefore for IO min incubation and 0.05 ml serum:

Extinction of control-extinction of test

extinction of standard ~-~-~~ X 250 = I.U./litre of serum.

(5) Estimation of SDH”’ (modified) The enzyme catalyses the following reaction:

n-Sorbitol + NADr 2 D-~7rUCtOSe + NADH

The assay reaction is most conveniently carried out from left to right at pH 8.8. As tetrazolium salts decompose at this high pH, reaction (b) must be employed.

Reap& I. 0.25 M Tris buffer pH 8.8. 2. 0.5 M Sorbitol in Tris buffer. 3. NADf 5 mg/ml in Tris buffer. 1. Ferric dipyridyl reagent. 5. Stabilizer.

Clin. Chim. =1&z, ‘4 (1969) 23-3,

34 WHITAKER

Method

Calculation

The standard value as stated above is z 0.125 /moles of NADH, but when used with this method the standard should be diluted with 4 volumes of deionisctl water, and is therefore z 0.025 /moles of NXDH. Therefore for zo min incubation and 0.20 ml serum :

Extinction of test-extinction of control _~ extinction of standard

>i 6.25 = I.C./litre of serum.

EXPERIMESTAL

(I) 1Vormal vanges

The enzyme reactions, concentrations of substrates, etc., were as in the original methods and their normal ranges still apply, i.c. for human serum

COLORIMETRIC ESTIMATION OF ENZYMES 3.5

I. U/l&e of serum GOT, Karmen - 4-20 GPT, Wroblewski, La Due - 3-15 LDH, Wrbblewski, La Due - 966240 HBD, Elliott and Wilkinson - 56-125 MDH, Wacker, Ulmer and Valee- 12-48 SDH, Sevela and Tovarek - less than I I.U.

(z) Interference in GOT and GPT estimations due to endogenous keto acids

Some abnormal sera contain appreciable amounts of pyruvate and other keto acids. In the GPT assay system, there is sufficient NADH and added LDH to remove any pyruvate during the pre-incubation. However, the GOT system, removal of pyruvate occurs very slowly, due to the low value of endogenous LDH, unless LDH is added.

Sera containing 5 mg pyruvate/roo ml were tested, using the GOT-containing LDH substrate described above, and this amount of pyruvate was found to be re- moved in I min (Fig. 4). It is considered, therefore, that 3-5 min pre-incubation is adequate for the removal of quantities of pyruvate likely to be encountered in clinical samples.

The “lag phase” has not been detectedI

(4) Zero order kinetics Under the conditions of the tests described, zero order kinetics were obeyed

to the complete utilisation of the NADH. With greatly elevated values, therefore, it is suggested that the incubation time be shortened in preference to making dilu- tions of serum.

(5) Types of sficcimclt Haemolysed specimens should be avoided, and serum is preferable to plasma ;

reaction (a) is unsuitable for heparinised samples as a turbidity develops, while se- questrene samples are unsuitable for assay using reaction (b).

(6) Corvclntion with ,ultvaviolet methods

There was good correlation with the ultraviolet reference methods for GOT and GPT over a wide range of values (Fig. 5).

(7) Standard dcviatim

A commercial serum whose GOT value was stated to be 31 I.U. was tested thirty times, taking each individual sampling through the complete procedure; the standard deviation was found to be 0.3 I.U.

(8) Price fOY test

Below are details of the approximate cost of materials in the United Kingdom for IOO tests using colour reaction (a). If reaction (b) is used the cost is approximately 2,‘- less in each case.

36 WHITAKIIR

20 30 40 50 60

Ultra -violet meth60d 80 90

l’ig. 5, Correlation of COT and GPT values obtained by the colorimctric method (using co10~1~~ reaction (a) with those obtained by the ultraviolet methods of Iiarmcn, Wroblewski and La I)u,.I respecti\-ely. ’ = GOT activity, Y = GPT activity (T.Iv.).

ColorimetriP-19 methods for GOT and GPT assay have come in for considerablt criticism. The experience of the author and his colleagues in Lincoln, with z,+dini- trophenylhydrazine methods has also led to the conclusion that these methods arc of doubtful accuracy. It was for this reason that the above calorimetric methods were developed, and it has been shown for GOT and GPT that they correlate well with ultraviolet methods of assay. The methods are considerably cheaper than the standard ultraviolet procedures, and are adaptable to batch analysis. The amount of serum required is small, and the good colour stability in the formazan reaction allows more latitude in timing for the technician. No expensive equipment is required if the second colour reaction is used, and a number of other assays could doubtless be adapted to these reactions.

All the methods described have been in operation for eighteen months and have proved extremely reliable.

COLORIMETRIC ESTIMATION OF ENZYMES 37

ACKNOWLEDGEMENT

I wish to thank Dr. Naftalin and my laboratory colleagues for their assistance in the preparation of this paper.

I RI. 11. NACKLAS, S. I. MARGULIES, J, D. GOLDBERG AND X. bl. SELIGMA~-, .4nal. Riochrrn., I

(1960) 317. 2 A. L. BABSOX AND G. E. PHILLIPS, Clin. Chim. Acta, 12 (1965) 210. m B. L. HORECKER AND 4. KORNBERG, J.Hiol. Chew., 175 (1948) 385. 3 H. ROSENKRAETZ, Arch. Biochem. Biophys., HI (1959) ‘94. 4 h. KARMEN. J. Clin. Invest., 34 (1955) 131. 5 F. WROBLEWSKI AND J. S. LA DUE, Ann. ~VZ&?%Zl Med., 43 (1955) 345. 6 F. \VROBLEWSKI AND J. S. LA DUE, Proc. sot. Exptl.Bzol. :Wed., 90 (1955) 210. 7 F. \VROBLEWSKI AND K. F. GREGORY, Ann. N.Y. Acad. Sci., 9~ (1962) 912. >; B. A. ELLIOTT AND J. H. ~VILKINSON, Lancet, i (1961) 69X. 9 C. 1:. BOEHRIXGER AP;D SOEHNE, YVlodQiedjmm test kit TC-L. IO M. SEVEL.~ AND TOVAREK, .I, Euvop. Symp. Med. Enzymol.. (1960) 147. I I J. KIKG, Practical Clinical Ezymology Van Nostrand, I,ondon, 1965. 12 1:. AMADOR, K. FRAP~EY AND BI. MASSOD, Clin. Clzem., IL (1966) 475. 13 E. ~\~x~DoR, &I. MASSOD AND R. FRAXEY, Anz.J. Clin. Patkol., _17 (1967) 419. 14 P.CABAUD, R. LEEPERAXD 1:. WROBLE~SKI, Am.J.Clin.PathoI., 26(1956) 1101. 15 F. L. HUMOLLER, J. RI. HOLTHAUS AED J. R. WALSH, Clin.Chmz., 3 (1957) 703. 16 m;:. \f:. UMBREIT, X. J. ROH~VYA, G. K. KINGSLEY, K. R. SCHAFFERT AEIJ H. SIPLET, J, Lab.

Clip. :Ilcd., 49 (1957) 454. 17 F. ~VROBLEWSKI, in H. SOBOTKA AKD C. P. STEWART(~%~S.), _4dz~ancrs ix Clinxal Chmnlstvy,

hcademic Press, NC\Y York, 1958, p. 311. IS J, \I:. BOYD, Biochem. J,, 81 (1961) 434, 19 J. \y. HOYD, Clin. Chim. Acta, 7 (1962) 424.

Clim. Chim. Acta, 24 (1969) 23-37