Determination, by Radioimmunoassay, of the ... - Clinical Chemistry

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We report here the development of a sensitive RIA for measuring the mass concentrations of LD-1 in human serum. We have used this assay to determine half- ...

CLIN. CHEM. 33/10,

1863-1868

(1987)

Determination, by Radioimmunoassay, of the Mass of Lactate Dehydrogenase Isoenzyme 1 in Human Serum and of Its Rate of Removal from Serum after a Myocardial Infarction Deborah A. Smfth, Fred Y. Leung, George Jablonsky,1 and A. Ralph Henderson2

In this radloimmunoassay of lactate dehydrogenase-1 (LD-1; EC 1.1.1.27) in human serum we use a commercial LD-1selective assay system and a goat antiserum. We have determined the fractional rate of disappearance from serum and thehalf-life ofLD-1,interms ofbothenzyme activity and enzyme mass, in 21 myocardial infarction patients. Our evidence suggests that this isoenzyme is inactivated in serum. Furthermore,

our data suggest

that the conventionally

accepted half-life of about 110 h for serum LD-1 activity may grossly overestimate the actual LD-1 half-life in many postmyocardial

infarction

patients.

Analytical

Additional Keyphrases: enzyme kinetics biological half-life heart disease

.

(RIA) has been increasingly used to measure mass concentrations of enzymes or isoenzymes in human serum such as prostatic acid phosphatase (EC 3.1.3.2) (1), amylase (EC 3.2.1.1) (2), creatine kinase (EC 2.7.3.2) (3,4), trypsin (EC 3.4.21.4) (5), chymotrypsin (EC 3.4.21.1) (6), and aspartate aminotransferase (EC 2,6.1.1) (7). Immunological methods have also been developed for measuring, for example, aspartate aminotransferase isoenzymes (8, 9) and isoenzyme 1 of lactate dehydrogenase (EC 1.1.1.27; jAactate:NAD oxidoreductase; LD-1) (10, 11) by the use of precipitating or enzyme (activity)-inhibiting antibodies. We report here the development of a sensitive RIA for measuring the mass concentrations of LD-1 in human serum. We have used this assay to determine half-life values for LD-1 mass in serum of post-myocardial infarction patients.

Materialsand Methods Materials NAD, NADH, and sodium pyruvate were obtained from Boehringer Mannheim Canada Ltd., Dorval, Quebec. Coomassie Brilliant Blue R-250, sodium lauryl sulfate, silver stains, and all chemicals for polyacrylamide gel electrophoresis were from Bio-Rad Labs. (Canada) Ltd., Mississauga, Ontario. Diethylaminoethyl (DE)-Sephacel, DEAE-Sephadex A-50, and 5-AMP-Sepharose 4B [1V6-(6-aminohexyl)-5’AMP bound to Sepharose 4B] were from Pharmacia (Canada) Ltd., Dorval, Quebec. LKB Ampholines and Ampholine ‘PAGplates” were from Fisher Scientific Co. Ltd., Don Mills, Ontario. All otherchemicals,generallyof “AnalaR” grade, were from BDH Chemicals, Toronto, Ontario.

Departments of Clinical Biochemistry and 1 Medicine, University Hospital (University of Western Ontario), London, Ontario, Cana-

da. 2Address correspondence to this author, at the Department

Stn. A,

Biochemistry, University Hospital, London, Ontario, Canada N6A 5A5.

of

P.O. Box 5339, Postal

Received June 15, 1987; acceptedJuly 28, 1987.

Procedures

LD assays. We used the Scandinavian method (12), and the EPOS discrete analyzer (EM Diagnostics Inc., Gibbstown, NJ 08027) in kinetic, time-sharing, mode to deter-

enzyme activity

In the last decade, radioimmunoassay

Clinical

The Bolton and Hunter reagent for protein iodination (-74 x iO’ Bq/mol) was from Amersham, Oakville, Ontario. We used donkey antibody to goatIsheep IgG, coated on cellulose (“Sac-Cel”) as the solid-phase second antibody (Wellmark Diagnostics Ltd., Guelph, Ontario). All water was purified through the Millipore Milli-RO (reverse osmosis) and Milli-Q (activated carbon, de-ionization beds, and membrane filter) units (Millipore Ltd., Mississauga,Ontario) with a specific resistance greater than 18 MCi cm.

mine total LD activity. One unit (U) of enzyme activity is the amount of enzyme catalyzing the oxidation of 1 mol of NADH per minute at 37.0 #{176}C in the presence of 1.2 mmol of pyruvate per liter. To determine the LD isoenzymes, we used thin-layer agarose electrophoresis followed by fluorescent quantification of the isoenzymes (13). Individual isoenzymes of high enzymic activity were accurately quantified by suitable dilution of these preparations (14). The limit of detection for any one isoenzyme in this system was 10 UIL. We used the supernate from the “Isomune LD” assay (Roche Diagnostic Systems, Nutley, NJ 07110) for determination of LD-1 activity, and for immunological determination of LD-1 mass after checking the linearity limits of the

method. Preparation of purified human LI) isoenzymes. We used two units of outdated packed human erythrocytes supplied by the hospital’s blood bank. The preparation of LD-1, LD-2, and LD-3 was as previously described (15). Preparation and purification of antiserum. All antisera were raised for us by De Rose Immunochemicals, Downsview, Ontario. Purified human LD-1 (about 0.4 g/L) was emulsified with an equal volume of Freund’s complete adjuvant and given in four subcutaneous injections to rabbits at weekly intervals. After the fifth week, the rabbits were bled weekly and the antisera tested for antibody specificity. Because none of the rabbits showed any antibody response, goats were then given a similar mixture, in four subcutaneous injections at 15-day intervals. One goat gave a satisfactory antibody to human LD-1 and blood was sampled over a two-week period. We purified the goat antiserum by the caprylic (n-octoic) acid procedure (16), diluting the antiserum with two volumes of 60 mmoIJL sodium acetate buffer, pH 4.0. After adjusting the pH to 4.8 with 1 moIIL HC1, we added n-octoic acid dropwise, with constant stirring, 0.74 mL (5.9 mnioI for every 30 mL of diluted antiserum. Stirring was continued for 30 mm, after which we filtered the solution through Whatman no. 1 paper to remove precipitated proteins (all proteins but IgG, ceruloplasmin, and some IgA are precipitated). The clear filtrate was dialyzed for three days at 4 #{176}C against multiple changes of isotonic saline. We then concenOIMCAL

CHEMISTRY, Vol. 33, No. 10, 1987

1863

trated the antiserum solution and applied it to a 50 x 300 mm column of DEAE-Sephacel previously equilibrated with 15 mmol/L sodium acetate buffer, pH 5.7 (which also served as the eluent). The IgG, which was not absorbed onto the DEAE-Sephacel, was eluted in the solvent front. lodination of LD-1. We radioiodinated LD-1 with the Bolton and Hunter reagent as described elsewhere (17). The reagent (37 MBq), supplied in benzene, was evaporated under a stream of nitrogen. To the residue of dried iodinated ester we added approximately 5 pg of LD-1 in 10 L of 100 mmolJL borate buffer, pH 8.0 (at 4 #{176}C), and stirred the reaction mixture for 30 mm at 4 #{176}C. We then added 500 L of 400 mmol/L glycine solution in borate buffer (100 mmol/L, pH 8.0 at 4#{176}C), and incubated for 5 mm, to trap unreacted ester. To separate the ‘251-labeled isoenzyme from lowmolecular-mass labeled compounds, we passed the mixture through a Sephadex G-26s column previously equilibrated with 20 mmol/L Tris HC1 buffer, pH 7.4. Thus we recovered the enzymically active LD-1-labeled antigen. The labeled antigen was diluted with Tris HC1 buffer (20 minollL, pH 7.4, containing 500 mg each of bovine serum albumin and Triton X-100 per liter) to give about 11000 counts/mm per 100 p.L. This solution, stored at 4 #{176}C, was useable for up to four weeks. Determination of antiserum avidity. The antiserum to LD1 was titrated (in dilutions up to 64 000-fold) against 100 L of the radiolabeled LD-1. The free and bound lmIlabeled antigens were separated by precipitating the antigen-antibody complex with the Sac-Cel donkey anti-goatisheep IgG antiserum. We found that antisera diluted of 8000-, 16000-, or 32 000-fold showed a high percentage of binding: the optimum dilution, to bind 50% of the total radioactivity, was the 16 000-fold dilution. Accordingly, we used 100 L of this antiserum dilution in the RIA procedure. Radioimmunoassay. Table 1 summarizes the protocol used for radioimmunoassay of LD-1. The assay was linear for sample volumes ranging from 100 to 400 jiL. The diluting buffer contained, per liter, 20 mmol of Tris HC1

buffer (pH 7.4), 5 g of bovine serum albumin, and 5 g of Triton X-100. The added bovine serum albumin decreased the nonspecific binding to I-

0 -J

05

HOURS AFTER ADMISSION

300

0j

-

-J

40

FRACTION NUMBER C

Fig. 1. Purification of goat anti-LD-1 IgG by ion-exchange chromatography About100mL of goatantiserum,treatedwith noctoicacidand concentrated,was applied to a 50 x 300 mm column of DEAE-Sephacel previously equilibratedwith sodiumacetate buffer(15 mmoL/L,pH 5.7).The samebufferwasusedto elutethe lgG; fraction size,5 mL

(1)

a

--A

-

200 0

-J

l00#{149}

-J 50

ISO

100

HOURS AFTER ADMISSION 400

-.6

A. - - 300 -J / 0, 0)

ENZYMECONTRATION

.6’

(ng/L)

Fig.2. Dose-response standard curves for human LD-1

j

The standardcurvesare shown for humanW-1 (S) and for crossreactivitywith human LD-2 (0) and LD-3 (A). Errorbars show ±2 SDvalues

/

20C

0 >‘.

I

‘II-’

a myocardial wave inferior

infarction (Figure 3B), and for a small myocardial infarction (Figure 3C).

non-Q-

DC

Serum LD-1 Disappearance Rates

Values for kd and t05 were determined for both serum LD1 activity and mass in myocardial infarctions the paired 5 values significant (P

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