Partial Characterization of an Abnormal Lactate

CLIN. CHEM. 35/5, 844-848 (1989)

Partial Characterization of an Abnormal Lactate Dehydrogenase lsoenzyme, LDH-1ex, in Serum from a Patient with Hepatocellular Carcinoma Dlmitrios L Kalpaxls1 and Elefterla E. GlannouIakF

Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1 ex, that on electrophoresis on 10g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1 ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypep-

tide chains (monomers), each having a molecular mass of about 32000 Da. LDH-lex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-lex showed no activity when lactate was omitted from the substrate

solution or replaced by ethanol. Additional Keyphrases: cancer tumor markers activity electrophoresis, polyacrylamide gel

enzyme

Lactate dehydrogenase (LDH; EC 1.1.1.27) in serum consists mainly of five isoenzymes of identical molecular mass but different charge. The LDH isoenzymes represent tetramers composed of four single polypeptide chains. These chains are of two kinds, the H-monomer (predominantly in cardiac muscle) and the M-monomer (predominantly in skeletal muscle and liver). Biochemical and genetic studies have elucidated the basis of multiplicity of this enzyme: two major genetic loci in the human genome each encode for a distinct subunit of LDH (1). A third genetic locus has been shown to be specifically active in relation to human spermatozoa (1-3). Several investigators have reported abnormal electrophoretic patterns of serum LDH isoenzymes (4-9) and additional bands (4, 7, 10-14) that correlate with cancers found in humans. Recently, we observed (15) an extra LDH isoenzyme, designated LDH-lex, in serum from patients with malignant diseases, that on electrophoresis on 10 g/L agarose gel at pH 8.6 migrated anodally to LDH-1; this extra isoenzyme was statistically correlated with malignancy. Here we report some physical and biochemical characteristics of LDH-lex isoenzyme, isolated from the serum of a cancer patient.

Materials and Methods Serum Samples As a source of LDH-lex, we used serum from a 65-year-old woman admitted to the General State Hospital “Agios Andreas” with hepatocellular carcinoma extending through ‘Department of Biochemistry,School of Medicine, University of Patras, Patras-26110, Greece. Present address: Institut fllr Biochemie/Med. Fak., Universitat Erlangen-Nurnberg, Fahrstrat3e 17, D8520 Erlangen, F.R.G. 2 Department of Internal Medicine, General State Hospital “Agios Andreas,” Patras-26110, Greece. Received December 30, 1988; accepted February 10, 1989.

844 CLINICALCHEMISTRY, Vol. 35, No. 5, 1989

a large part of the liver. Although metastases to other tissues were not observed, her clinical status worsened after a temporary response to chemotherapy, and she died from hepatic deficiency six months later. Venous blood was sampled in the morning before chemotherapy and during follow-up, and serum was separated from the clot as soon as possible by centrifugation (20 mm, 3000 x g, 20#{176}C). Serum was analyzed for LDH, HBDH (2hydroxybutyrate dehydrogenase) activities, and a-fetoprotein on the same day or kept at -70 #{176}C for further purification procedures. Materials Sephadex G-100, Sephadex G-200 (Superfine), and Blue Dextran 2000 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. a+)-Lactic acid, NAD, iodonitrotetrazolium chloride, phenazine methosulfate, Amido Black lOB, marker proteins of known molecular mass, and agarose were purchased from Sigma Chemical Co., St Louis, MO 63178. Agarose Universal Electrophoresis Film (cat. no. 470100), diethylbarbital buffer (pH 8.6), and LDH staining kit (cat. no.470014) were purchased from Corning Medical Co., Palo Alto, CA 94306. We assayed total LDH and HBDH activities at 25#{176}C with a Model 250 spectrophotometer (Gilford Instrument Labs. Inc., Oberlin, OH), using reagents and methods for the LDH kit (cat. no. 124885) and the HBDH kit (cat. no. 124800) from Boehringer Mannheim GmbH, Mannheim, F.R.G. AFP-EIA Monoclonal kit (cat. no. 7224) for enzyme immunoassay of a-fetoprotein was provided by Abbott Laboratories, North Chicago, IL 60064. Coomassie Brilliant Blue R-280 was from BDH Chemicals, Toronto, Ontario, Canada. All routine chemicals were of analytical grade (Sigma Chemical Co.). Analytical

Procedures

To determine protein concentration, we used the method of Lowry et al. (16), with bovine serum albumin as standard. Agarose electrophoresis. We electrophoresed 1-p.L serum samples on commercially available 10 g/L agarose gels and quantified each LDH isoenzyme as previously described (15). To test the heat stability of LDH isoenzymes, we preincubated the serum samples at various temperatures (060#{176}C) for 30 win and cooled them to 5 #{176}C just before electrophoresis. Partial purification of LDH-lex isoenzyme. We prepared partly purified LDH-lex isoenzyme by using the “Linca” preparative electrophoresis system (Lemon Instrumentation Co., Ltd., Tel Aviv, Israel). We diluted 1 mL of patient’s serum or normal serum to a total volume of 5 mL with 65 mmol/L diethylbarbital buffer (pH 8.6) and electrophoresed the samples with the same buffer on 150 mL of 10 g/L agarose gel containing 50 g of sucrose and 350 mg of Na2EDTA per liter. Electrophoresis was at 4#{176}C for 30 nun at 20 mA, followed by 40 mA for 16 h. After electrophoresis, we cut a 1-cm-wide strip from the gel and incubated it for 45 win at 37 #{176}C in 50 mL of 0.1 mol/L

Tris HC1 (pH 8.5) containing 100 mg of L( + )-lactic acid, 60 mg of NAD, 10 mg of mtroblue tetrazolium, 2 mg of phenazine methosulfate, and 1.5 mL of pure ethanol per 50 mL. We compared the unstained strip gel with the stained one and removed from the former the appropriate segment containing the LDH-lex isoenzyme. We gently pulverized this segment with 70 mL of the diethylbarbital buffer, using a small hand-operated homogenizer equipped with a Teflon pestle, let it stand for 6 h in an ice bath, then filtered this suspension through a sintered-glass ifiter by applying gentie suction. We repeated the extraction once, and reduced the volume of the combined extracts to 1 mL by ultrafiltration through an Amicon PM-1O membrane. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis. To separate proteins of different molecular masses, we used discontinuous polyacrylamide gel electrophoresis as described by Laemmli (17). The running and the stacking gels were 80 and 40 g/L acrylamide, respectively. We stained the gels with Coomassie Brilliant Blue R-250, 2.5 g/L in methanol/acetic acid/water (43:7:50, by vol) and destained them by gentle agitation in methanol/acetic acid/water (40:7:53, by vol). A solution containing molecular-mass markers (see legend to Figure 1) was electrophoresed in the same way. We used a log plot of the Mr values of these marker proteins vs their respective gel migration to establish the Mr of the LDH-lex monomer. Gel chromatography. Gel chromatography on Sephadex G-100 and Sephadex G-200 (Superfine) was carried out at 4#{176}C with 180 x 1.5 cm and 147 x 0.5 cm columns, respectively. We determined the void volume of the columns by using dye-labeled high-Mr dextran. We applied 0.5 mL of partly purified LDH-lex isoenzyme to the Sephadex G-100 column that had previously been equilibrated with diethylbarbital buffer (65 mmoIJL, pH 8.6), which also served as the eluent. We collected 3-mL fractions at a flow rate of 16.5 mL/h and analyzed them for protein and LDH Subsequently, we pooled the I II activity. NI IV eluted fraction in four groups (as indicated by the arrows in Figure 1) and reduced their volume to 0.5 mL by ultrafiltration through an Amicon PM-b membrane. We then examined the concentrated pools by sodium dodecyl sulfate/ polyacrylaniide gel electrophoresis. We also applied another 0.5-mL aliquot of purified LDHlex isoenzyme to the Sephadex G-200 Superfine column and

eluted it as previously but with a flow rate of 1.5 mL/h. We collected 0.5-mL fractions and diluted them sixfold with the diethylbarbital buffer. After assaying the diluted fractions for protein and LDH activity, we pooled them (as indicated by the arrows in Figure 2), reduced their volume by ultrafiltration, then electrophoresed them on sodium dodecyl sulfate/polyacrylanude gels. To establish the Mr of the LDHlex isoenzyme, we used a log plot of the values for the marker proteins vs their respective migration rates on the same chromatography column. Results Biochemical Analysis of the Patient’s Serum The total LDH activity (581 U/L) in the serum of our patient at the time of admission was remarkably increased. The a-fetoprotein concentration, 752 ng/mL, was also much above normal. This patient’s a-fetoproteindid not decrease significantly after chemotherapy. Serwn LDH isoenzymes, as evaluated by 10 g/L agarose gel electrophoresis (Figure 3), showed a shift toward M-type isoenzymes. LDH-4 (14% of the total activity) and LDH-5 (14.7% of the total activity) bands were more distinct and clearly increased as compared with the normal electrophoretic pattern. The electrophoretogram was also character-

ized by the presence of an extra band (LDH-lex), migrating anodally to the LDH-1 band and accounting for 13.2% of the total LDH activity. The proportion of LDH-lex decreased dramatically with monthly chemotherapy. After three months of chemotherapy, it completely disappeared and the isoerizyme pattern of LDH returned to normal, but again increased just before the relapse. As expected, the omission of lactate from the substrate solution or the replacement by ethanol resulted in the disappearance of the extra band, suggesting that the LDHlex isoenzyme is a true lactate dehydrogenase. Thermal Stability of LDH lsoenzymes To investigate the stability of LDH isoenzymes, we proincubated the patient’s serum for 30 miii at different temperatures before electrophoresis. Figure 4 gives a survey of the results. It is interesting to note that the LDH-lex isoenzyme retained its activity, evenwhen heated at 60#{176}C. G,oop

(a)

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Fig. 1. Gelfiltrationofpartlypuflf led LDH-1ex isoenzyme,elutedfroma 180 x 1.5cm SephadexG-100 columnwith65 mmol/L diethylbarbital

buffer(pH 8.6), at a flowrate of 16.5 mLih and 4#{176}C Fractions(3 mL) werecollectedand analyzedfor protein(0) and

LDH activity (#{149}). Right electrophoretogramsof thefirst pociof fractions(35 to 45), (a) in the absenceof 2-mercaptoethanoland(b) in the presence of 2-mercaptoethanol.The arrowsat leftindicatethe positionand P4 of standardproteinselectrophoresedin the same way: tnfpsinogen(P4 24000); egg albumin (45 000); bovine albumin (66 000); cross-linked bovine albumin (dimer: 132000; thmer:198000;tetramer: 264000).The arrowsat right indicatethe position of the A, B, and x proteins

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20 F,10leo,

30

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50

60

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Fig.2. Estimationof the M of LDH-1ex isoenzyme bygel filtrationona 147 x 0.5cm SephadexG-200 (Superfine)column,elutedas in Fig. 1 but witha flowrateof 1.5 mL/h Fractions(0.5 mL) were collectedand analyzedfor proteIn(0) andLDH activity (#{149}). Inset:the standardproteinsand theirP4valuesare:lysozyme(‘s), 13000; pepsin (p), 34000; bovine albumin (a/b); cross-linkedbovine albumindimer (a/b x , 132000; thrner(alb x , 198000. The x-pointon the vertical axis indicatesthe I-value for the LDH-lex isoenzyme

CLINICALCHEMISTRY, Vol. 35, No. 5, 1989 845

Molecular-Mass

Determination

We examined the macromolecular structure of LDH-bex origin

LDH-1.

(-)

(±)

Fig.3. LDH isoenzyme patternin serum from the patientwith hepatocellular carcinoma after agarose gel (10 g/L) electrophoresis,and densitometrictracingof the electrophoretogram Electrophoresiswas performedwith65 mmot/Ldiethylbartxtalbuffer(pH 8.6),for 37 mmat 90 V

The order of LDH isoenzyme stability at 60#{176}C was: LDHlex LDH-1 > LDH-2> LDH-3 > LDH-5 > LDH-4. No bands were visible when we used temperatures >60 #{176}C. As shown in Figure 4, the activity of the rest of the isoenzymes retained stability up to 20#{176}C. However, at higher temperatures there was an onset of inactivity, starting with LDH-4, the most heat-labile isoenzyme. Partial Purification of LDH-1 ex Isoenzyme As stated in the Methods section, we isolated partly purified LDH-bex isoenzyme by preparative electrophoresis and ultrafiltration. Analytical recovery of LDH-bex activity was 95%. The electrophoretic pattern of the purified isoenzyme on the agarose gel showed only one band at the initial position of LDH-lex isoenzyme. The LDH activity of the purified LDH-lex isoenzyme was 73.2 U/L, representing about 12.6% of the total LDH activity. The LDH-bex isoenzyme also reacted readily with 2-hydroxybutyrate. The HBDH activity of the purified isoenzyme was 52 U/L, representing about 50% of the total HBDH activity in serum and giving a ratio of HBDH activity/LDH activity equal to 0.71. 100 0l

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80

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isoenzyme by gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When the partly purified LDH-lex isoenzyme was chromatographed on a column of Sephadex G-bOO, LDH activity was eluted in the first pool of fractions, corresponding to the void volume (Figure 1). By sodium dodecyl sulfate/polyacrylamide gel electrophoresis, we found that the first poo1 of fractions contains mainly three protein bands of strong intensity and of mobility corresponding to Mr 132 000, 66 000, and 32 000, respectively (electrophoretogram a in Figure 1). When normal serum or patient’s serum after chemotherapy treatment were tested in the same way, the X band (Mr: 32000) was the only one missing. This band did not appear in electrophoretograms of the other eluate frac-

tions either. In contrast, the A band (Mr: 66000) was present in all fractions between fractions 35 and 60. Moreover, the B band was present only in the first pool of fractions.

out in the that this band was missing, whereas a new one appeared near the A band (electrophoretogram b in Figure 1). We estimated the relative molecular mass of the LDH-bex isoenzyme from its elution properties on gel ifitration (Sephadex G-200, Superfine). LDH activity was eluted as a single peak (fraction 24 to 30 in Figure 2). When we electrophoresed one aliquot of these pooled fractions on 10 g/L agarose gel, we observed that this pool also contained the LDH-bex band. The partition coefficient of the LDH-bex isoenzyme, chromatographed as described above, corresponds to an approximate mean Mr of 138000 (inset of Figure 2). Moreover, we pooled the eluates as indicated by the arrows in Figure 2, and after reduction of their volume by ultrailltration we electrophoresed them on sodium dodecyl sulfate! polyacrylamide gel. Electrophoretograms of these pooled fractions, shown in Figure 5, demonstrate that the X band coincided essentially with the position of the LDH-lex peak. presence

When electrophoresis was carried

of 6 mmoIJL 2-mercaptoethanol,

Discussion Increased

enzymes in serum as well as changes in isoen-

zyme patterns and newly developed enzyme variants have been found to reflect growth and regression of various malignant neoplasms (18-20). Reports of finding extra LDH isoenzymes in the serum of patients with liver malignancy are numerous (11, 12, 18, 21). Recently we observed, in serum from patients with various malignant diseases, an ABC

60

we observed

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Temperature

40

50

60

(#{176}C

Fig. 4. Stability of LDH-lex (0), LDH-1 (#{149}), LDH-2 (D), LDH-3 (#{149}), (+) LDH-4 (Li), and LDH-5 (A) isoenzymes at varioustemperatures Samplesfrom the patients serum were incubatedat varioustemperatures(0 to Fig.5. Proteinpatternsin pooled fractions eluted from the Sephadex 660#{176}C) in a waterbath for 30 mi then were cooled at 5#{176}C and were 200 (Superfine) column after SOS/PAGE electrophoresed.Each value of the percent LDHactivityis the averageof two (A)fractions25to29;(ons30to33;(C)fractions34to37;and(L determinations.PercentLDH activity at 25#{176}C in freshly drawn serum was fractions38 to 52 consideredas 100% 846 CLINICAL CHEMISTRY,Vol. 35, No. 5, 1989

extra

LDH isoenzyme, LDH-lex, migrating anodally to band on electrophoresis on 10 gIL agarose gel (15). The variety of malignant diseases occurring in these patients excludes the possibility of any disease specificity of LDH-1

this isoenzyme.

As a source of LDH-lex in the present study, we used serum from a patient with hepatocellular carcinoma, who presented with this isoenzyme increased in her serum (13.2% of the total LDH activity). To elucidate the macromolecular structure and some of LDH-lex biochemical properties, we partly purified the extra isoenzyme by preparative electrophoresis and ultrafiltration through an Amicon PM10 membrane. The electrophoretic pattern of the resulting LDH-lex preparation on agarose gel confirmed that sufficient separation was obtained between LDH-lex and the rest of the LDH isoenzymes. When partly purified LDH-lex isoenzyme was chromatographed on a Sephadex G-100 column, LDH activity was eluted in the void volume. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of this region revealed three protein bands, B, A, and X, corresponding to Mr values of 132 000, 66000, and 32 000, respectively. Based on the observation that the X band is absent in electrophoretograms of normal serum or patient’s serum after chemotherapy treatment, we concluded that the X band is related to LDH-lex isoenzyme and probably represents its monomer. The other two proteins (A and B) co-eluted with LDH-lex isoenzyme seem not to be related to LDH-lex activity, because they were also present in normal serum. We estimated the mean molecular mass of LDH-lex isoenzyme by gel electrophoresis on a Sephadex G-200 column. The difference between the expected (4 x 32000 Da = 128 000 Da) and estimated (138 000 Da) values is probably within the error of this technique. The molecular mass of LDH-lex isoenzyme precludes the possibility that this extra band is the result of an immunoglobulin or f3lipoprotein complex with some of the usual LDH isoenzymes. Such a complex should show a molecular mass >200 000 Da. LDH-immunoglobulin complexes as well as LDH-3-lipoprotein complexes have been detected by several workers in serum of patients with various malignancies. These macromolecular structures often result in additional LDH isoenzymes, which migrate between the normal location of LDH-1 and that of LDH-5 (7, 8,22-27). All of these complexes have molecular masses >200 000 Da. Different LDH isoenzymes have been shown to vary in several properties other than chromatographic behavior and electrophoretic mobility (20). Prompted by these observations, we examined the stability of LDH-lex isoenzyme at elevated temperatures and its activity on 2-hydroxybutyrate. The heat stability of LDH-lex appeared indistinguishable from that of LDH-1, but very different from the cationic isoenzymes, the predominant LDH isoenzymes in adult liver tissue and in hepatocellular carcinoma (19,21). Wroblewski and Gregory (28) distinguished isoenzymes according to thermostability, the M-type being more labile than the Htype. Moreover, we found that the ratio HBDH activity/LDH activity for the LDH-lex isoenzyme was about 0.71, which is considered to be more than for M-type isoenzymes (29). From the combination of the above properties with the demonstration of an increase in the patient’s serum afetoprotein, it would be supposed that the liver tissue undergoing malignancy is regressed, producing a more embryonic form of LDH showing characteristics of the Htype. However, such a hypothesis is not compatible with our

previous study (15) showing LDH-lex to be present nonspecifically in 53% of a wide variety of cancers, rarely dependent on a-fetoprotein. We believe that further studies on several properties of LDH-lex isoenzyme-e.g., immunochemical specificity, substrate affinities, effects of inhibitors, and coenzyme analog utilization-will complete the characterization of this isoenzyme and interpret its occurrence in serum of patients with malignancies. For clinical purposes, the examination of serum of patients with other diseases for probable LDH-lex activity will be also very useful. We are grateful manuscript.

to Dr. D. Synetos for critically

reading the

References 1. Markert CL, Shaklee JB, Whitt GS. Evolution of a gene. Multiple genesfor LDH isoenzymes provide a model of the evolution of gene structure, function, and regulation. Science 1975;189:102-14. 2. Blanco A, Zinkham WH. Lactate dehydrogenases in human testes.Science1963;139:601-2. 3. Goldberg E. Lactic and malic dehydrogenases in human spermatozoa. Science 1963;139:602-3. 4. Biewenga J. Complexes of lactatedehydrogenase and immunoglobulin G in human serum. Clin Chim Acts 1973;47:139-47. 5. Lippert M, Papadopoulos N, Javadpour N. Role of lactate dehydrogenase isoenzymes in testicular cancer. Urology 1981;18:50-3. 6. Vergnon JM, Guidollet J, Gateau 0, et al. Lactic dehydrogenase isoenzyme electrophoretic patterns in diagnosis of pleural effusion. Cancer 1984;54:507-11. 7. Rijke D, Trienekens PH. Variant expression of lactate dehydrogenase complexes, interfering with isoenzyme analysis. Clin Chim Acts 1985;146:135-.45. 8. SudoK, Maekawa M, Watanabe H, et al. A case of imniunoglobulin G conjugated with lactate dehydrogenase, producing both loss of enzyme activity and an abnormal isoenzyme pattern. Clin Chem 1986;32:1420-2. 9. Ng RH, Ethirajan S, O’Neil M, Statland BE. Increased activities of creatine kinase and lactate dehydrogenase isoenzymea in a patient with metastatic ovarian tumor. Clin Chem 1987;33:1484-5. 10. Tanaka F, Amino N, Hayashi C, Miyai K, Kumahara Y. Abnormal serum lactate dehydrogenaseisoenzyme in a case of laryngeal carcinoma and thyrotoxicosis. Clin Chim Acta 1976;68:235-40. 11. Anderson CR, Kovacik Jr WP. LDHk: an unusual oxygensensitive lactate dehydrogenase expressed in human cancer. Proc Natl Acad Sci USA 1981;78:3209-13. 12. Ketchum CH, Robinson CA, Hall LM, et a!. Clinical significance and partial characterization of lactate dehydrogenase isoenzyme 6. Clin Chem 1984;30:46-9. 13. Otsu N, Hirata M, Miyazawa K, Tuboi S. Abnormal lactate dehydrogenase isoenzyme in serum and tumor tissue of a patient with neuroblastoma. Clin Chem 1985;31:318-20. 14. Siciliano MJ, Bordelon-Riser ME, Freedman RS, Kohler P0. A human trophoblastic isozyme (lactate dehydrogenase-Z) associated with choriocarcinoma. Cancer Res 1980;40:283-7. 15. Giannoulaki EE, Kalpaxis DL, Tentas C, Fessas F. Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases. Clm Chem 1989;35:396-9. 16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.

17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;277:680-5. 18. Lubrano T, Tietz AA, Rubinstein HM. Extra lactate dehydrogenase isoenzyme band in serum of patients with severe liver disease. Clin Chem 1971;17:882-5. 19. Stefanim M. Enzymes, isoenzymes, and enzyme variants in the CLINICAL CHEMISTRY, Vol. 35, No. 5, 1989

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diagnosis of cancer [Review]. Cancer 1985;55:1931-6. 20. Schapira F. Isoenzymes and cancer [Review]. Adv Cancer Res 1973;18:77-153. 21. Podlasek SJ, McPherson RA, Threatte GA. Characterization of apparent lactate dehydrogenase isoenzyme 6. A lactate independent dehydrogenase. Clin Chem 1984;30:266-70. 22. Ganrot P0. Lupoid cirrhosis with serum lactic acid dehydrogenase linked to an 7A immunoglobulin. Experientia 1967;23:593. 23. Trocha PJ. Lactate dehydrogenase isoenzymes linked to betalipoproteins and inununoglobulin A. Clin Chem 1977;23:1780-3. 24. Gorus F, Aelbrecht W, van Camp B. Circulating IgG-LD complex dissociable by addition of NAD . Clin Chem 1982;28:2369. 25. Pudek ME, Jacobson BE. Falsely negative laboratory diagnosis for myocardial infarction owing to the concurrent presence of macro creatine kinase and macro lactate dehydrogenase. Cliii Chem

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1982;28:2434-7. 26. Weijers RNM, Mulder J, Kruijswijk H. Partial characterization, properties, and clinical significance of a lactate dehydrogenase-immunoglobulin Ak complex in serum. Clin Chem 1983;29:272-8. 27. Fujita K, Takeya C, Saito T, Sakurabayashi I. Macro lactate dehydrogenase: an LDH-immunoglobulin M complex that inhibits lactate dehydrogenase activity in a patient’s serum. Clin Chim Acts 1984;140:183-95.

28. Wroblewski F, Gregory KF. Lactic dehydrogenase isozymes and their distribution in normal tissue and plasma and in disease states. Ann NY Acad Sci 1961;94:912-32. 29. Plunumer DT, Elliot BA, Cooke KB, Wilkinson JH. Organ specificity of lactate dehydrogenase activity. The relative activities with pyruvate and 2-oxobutyrate of electrophoretically separated fractions. Biochem J 1963;87:416-22.

(1989)

A Simplified and Rapid Test for Acetylator Phenotyping by Use of the Peak Height Ratio of Two Urinary Caffeine Metabolites Adnan EI-YazlgI,1 Kutalba Chaleby,2 and Cazemiro R. Martin’

We describe a simplified liquid-chromatographic test in which acetylator phenotype is determined by measuring the peak height ratio of two urinary caffeine metabolites, 5-acetylamino-6-formylamino-3-methyluraciI and 1-methylxanthine. We applied this test to determine the acetylator phenotypes of 52 subjects who regularly drink coffee, tea, or caffeinated beverages. Also, we determined the acetylator phenotypes of these subjects according to a well-established sulfasalazine test, which yielded identical results. We established the reproducibility of the described test by determining the acetylator phenotypes of 10 additional subjects on two different days separated by a period of two to five weeks. Of the 52 subjects examined by both tests, 40 (76.9%) were classified as slow acetylators, which agrees well with the percentage reported elsewhere for 297 similar subjects from the Saudi population. N-Acetyl transferase is an enzyme responsible for biotransformation of several clinically important drugs, including procainamide, isoniazid, pheneizine, dapsone, hydralazine, and sulfonamides. The production of this enzyme in the liver and gastrointestinal mucosa is genetically controlled and hence the acetylating capacity of individuals within a population is subject to polymorphism with a bimodal or trimodal distribution pattern. Thus, a subject is classified as either a slow or rapid acetylator, with the latter being homozygote or heterozygote for this dominant character (1). In addition to its marked benefits in therapeutics, acetylator status has been linked to several disease states such as bladder cancer, diabetes mellitus, systemic lupus erythematosus, and Gilbert’s syndrome (2).

‘Biological and Medical Research Department and2 the Department of Medicine, King Faisal Specialist Hospital & Research Centre, Riyadh 11211, Saudi Arabia. Received December 28, 1988; accepted January 31, 1989. 848

CLINICAL CHEMISTRY, Vol. 35, No. 5, 1989

Several screening tests have been used for determining phenotype. These tests are based on the use of one of the above-mentioned drugs. Isoniazid has been the agent traditionally used (3) for these studies, but it has a complex metabolism and requires serial blood samples, which makes it inconvenient for routine, widespread use. Other commonly used tests involve a sulfonamide such as sulfadimidine, sulfapyridine, sulfasalazine, or sulfamethazine. These methods are based on the classical Bratton-Marshall procedure (4), and require the administration of a single oral dose of the drug and collection of multiple samples (5) or a single sample (6-9) of serum or urine. Dapsone was also used for acetylator phenotyping, a single sample of plasma being collected 2-72 h after administration of a single dose of this drug and the concentration ratio of monoacetyldapsone to dapsone determined by high-performance liquid chromatography (10, 11). Similarly, the concentration ratio of Nacetylprocainamide to procainamide in plasma was used to determine the acetylator phenotype, and the results obtained were similar to those acquired with dapsone (12). Recently, Grant et a!. (13) demonstrated that the production of 5-acetylamino-6-formylamino-3-methyluracil (AFMU), a caffeine metabolite, is mediated by N-acetyltransferase and that the molar ratio of AFMU to 1-methylxanthine (1MX), another metabolite of caffeine, in urine collected 2 to 6 h after consumption of a caffeinated beverage (coffee, tea, or soft drink) can be used for a large-scale assessment of acetylator phenotype (14). Because of the ubiquitous use and relative safety of caffeine and the ease and simplicity of the protocol used, this test may offer some advantages over other methods for population studies (2). In this report, we further simplify the test by using the peak height ratio of AFMU/1MX in lieu of molar concentration ratio of these metabolites for determination of the acetylator status. This allows elimination of the internal standard and calibration curves that would be needed to estimate the concentrations of these metabolites. We compared the results obtained by this test with those obtained acetylator