Effects of Analgesic and Antirheumatic Drugs on the Assay of Serum Enzymes

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Jelio, Majkio-Singh, Spasio, Todorovio and 2ivanov-Stakio: Effects of drugs on the assay of serum enzymes

559

J. Clin. Chem. Clin. Biochem.

Vol. 22, 1984, pp. 559-563

Effects of Analgesic and Antirheumatic Drugs on the Assay of Seruni Enzymes

By Z. Jelic, N. Majkic-Singh, S. Spasic, P. Todorovic and D. Zivanov-Stakic Department of Biochemistry, Faculty of Pharmacy, Beigrade, Yugoslavia (Received November 28, 1983/March 23, 1984)

Summary: Interference by some commonly used analgesic and antirheumatic drugs in the spectrophotometric and colorimetric assays of serum enzymes was examined. None'of the investigated methods was significantly influenced by caffeine, phenacetin, ibuprofen or indometacin. Acetylsalicylic acid affected the continuous assays of creatine kinase and lactate dehydrogenase (with pyruvate äs Substrate), and the colorimetric assay of alanine aminotransferase. Aminophenazone interfered with the colorimetric method for the determination of aspartate aminotransferase and alanine aminotransferase, while phenobarbital interfered only with the con- tinuous method for lactate dehydrogenase (with L-lactate äs Substrate). Ketoprofen interfered with the colo- rimetric assays of lactate dehydrogenase and aspartate aminotransferase, while diclofenac affected the contin- uous assay of aspartate aminotransferase. None of the tested drugs interfered with the continuous methods for the determinätion of alkaline phosphatase and -hydroxybutyrate dehydrogenase.

Einfluß analgetischer und antirheumatischer Medikamente auf die Bestimmung von Enzymen im Serum Zusammenfassung: Die Interferenz einiger häufig gebrauchter Analgetica und Antirheumatica mit spektro- photometrischen und kolorimetrischen Bestimmungmethoden für Enzyme im Serum wurde geprüft. Coffein, Phenacetin, Ibuprofen oder Indometacin beeinflußten keine der untersuchten Methoden signifikant. Aceto- salicylsäure störte die kontinuierliche Bestimmung von Kreatinkinase und Lactatdehydrogenase (mit Pyruvat als Substrat) sowie die kolorimetrische Bestimmung von Alaninaminotransferase. Aminophenazon interfe- rierte mit der kolorimetrischen Bestimmungsmethode für Aspartat- und Alaninaminotransferase, während Phenobarbital nur die kontinuierliche Methode für Lactatdehydrogenase (mit L-Lactat als Substrat) beein- flußte. Ketoprofeä störte die kolorimetrische Bestimmung von Lactatdehydrogenase und Aspartatamino- transferase, Diclofenac die kontinuierliche Bestimmung von Aspartataminotransferase. Keines der geprüften Medikamente interferierte mit der kontinuierlichen Methode zur Bestimmung von alkalischer Phosphatase und a-Hydroxybutyratdehydrpgenase.

Introduction

Drugs may alter clinical laboratory test results either chemically (analytieal interference) or pharmacolog- ically (in vivo effects). Analytieal interference by nine analgesic and antirheumatic drugs in the assays of serum alkaline phosphatase (orthophosphoric- monoester phosphohydrolase, EC 3.1.3.1), lactate dehydrogenase and -hydroxybutyrate dehydroge- nase (L-lactäte: NAD* oxidoreductase, EC 1.1.1.27), creatine kinase (ATP: creatine N-phos- photransferase, EC 2.7.3.2), aspartate aminotrans- ferase (L-aspartate: 2-oxoglutarate aminotransfe-

J. Clin. Ghem. Clin. Biochem. / Vol. 22,1984 / No. 8

rase, EC 2.6.1.1) and alanine aminotransferase (L- alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2) was examined.

Diclofenac, indometacin, ibuprofen and ketoprofen

are widely used in the management of various rheu-

matic disorders. The readily available analgesics,

acetylsalicylic acid, phenacetin and aminophenazone

are also frequently taken without knowledge of the

physician. It is thus important to know whether these

drugs interfere with clinical laboratory assays. Caf-

feine and phenobarbital were also tested, being com-

mon constituents of various analgesic preparations.

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560

Jelic, Majkic-Singh, Spasio, Todorovio and 2ivanov-Staki6: Effects of drugs on the assay of serum enzymes

Materials and Methods

To investigate (he effects of drugs on the results of enzyme assays, the general protocol prepared by Siest et al. (1) was followed.

Commercial lyophilized human sera were reconstituted with aqueous Solutions of each drug tested. The drug concentrations (tab. 1) were approximately ten times the maximal therapeutic levels (2-5). For control sample preparation, the drug Solutions were replaced by distilled water.

For each method, six tests were performed ori control samples, and six on samples containing the drug (Ni = Na = 6); the statisti- cal parameters: mean value (x) and Standard deviation (s), were determined. Using Sludenfs t-test, the significance of the differ- ences between enzyme activities obtained in sera with and without drugs was assessed.

All the assays were performed with a spectürophotometer Pye Uni- cam SP 8-100.

Tab. 1. Concentrations of added drugs in reconstituted lyophilized human sera.

Drug Concen-

tration ( / ) Acetylsalicylic acid 8326 [2-(acetyloxy)benzoic acid]

Aminophenazone 540 (4-dimethylamino-

2,3-dimethyl-l-phenyl-3pyrazolin-5-one)

Phenacetin ' ' 112 [N-(4-ethoxyphenyl)acetamide]

Caffeine 772 (l ,3,7-trimethylxanthine)

Phenobarbital 1076 [5-ethyl,5-phenyl-2,4,6(lH,3H,5H)-pyrimidinetrione]

Ibuprofen 970 [a-methyl-4-(2-methylpropyl)-benzeneaceticacid]

Diclofenac 71 2-[(2,6-dichlorophenyl)-amino]-benzeneaceticacid

Indometacin 35 [l-(p-chlorobenzoyl)-

5-methoxy-2-methylindole-3-acetic acid]

Ketoprofen 236 [2-(3-benzoylphenyl)-propionicacid]

Alkaline phosphatase, lactate dehydrogenase, a-hydroxybutyrate dehydrogenase, creatine kinase, aspartate aminotransferase and alanine aminotransferase activities were determined. The princi- ples and reference data of methods used for the enzyme assays are shown in table2.

Results and Discüssion

No definite influence of indometacin, ibuprofen, caf- feine and phenacetin could be detected on any meth- od. The enzyme catalytic activities and their väriä- tions obtained in samples with and without the drugs are shown in table 3, äs well äs the corresponding p values. Day-to-day variations were foürid to be 4.3—

8.4% and 8.9—1L2% for continuous and colorimet- ric methods, respectively (tab. 3).

The apparent catalytic activity of creatine kinase in sera containing acetylsalieylic acid was decreased by 8%, lactate dehydrogenase by 6% (continuoüs method with pyruväte äs Substrate) and alanine ami- notransferase by 13% (colorimetric method).

Aminophenazone interfered with aspartate amino- transferase and alanine aminotransferase colorimet- ric assay, yielding 16% lower aspaftate aminotrans- ferase and 22% higher alanine aminotransferase ac- tivities. In samples containing phenobartital, 4%

higher values for lactate dehydrogenase activity were obtained using L-lactate äs Substrate. Decreased ac- tivities of three enzymes were.obtained in the pres- ence of ketoprofeii: lactate dehydrogenase (24%, colorimetric method), aspartate aminotransferase (8%) and alanine aminotransferase (10%) using continuous assays. The continuous test for aspartate aminotransferase catalytic activity determination gave higher results (4%) for samples containing dic- lofenac.

Tab. 2. The methods used for enzyme activity assay.

Enzyme Method Reference

Alkaline phosphatase Creatine kinase Lactate dehydrogenase

-Hydroxybutyrate dehydrogenase Aspartate aminotransferase Alanine aminotransferase

continuous, optimized continuous, optimized

continuous, with pyruväte äs Substrate continuous, with lactate äs Substrate colorimetric

continuous, optimized continuous, optimized colorimetric

continuous, optimized

colorimetric s *

6,7 8 6,7 910 6,7 6,11 12,13 6,11 12

J; Clin. Chem. Clin. Biochem. / Vol. 22,1984 / No. 8

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Jelio, Majkio-Singh, Spasio, Todorovio and 2ivanov-Staki6: Effects of drugs on the assay of serum enzymes

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562

Jelio, Majkio-Singh, Spasio, Todorovio and ivanov-Stakio: Effects of drugs on the assay of serum enzymes

The effects found were studied further using lower concentrations of drugs, down to the therapeutic lev- els. The interference of ketoprofen with the colori- metric determination of lactate dehydrogenase cata- lytic activity could still be seen in the presence of 94 μπιο1/1 (p < 0.001). Significantly decreased activity of aspartate aminotransferase (colorimetric method) was found in samples containing aminophenazone: p

< 0.01 with 216 μπιοΐ/ΐ and p < 0.05 with 108

μτηοΐ/ΐ. Figure l shows the dose dependence of these two interferences. Other drugs did not interfere in lower concentrations.

None of the enzyme assays was significantly altered by the drugs in the ranges of theif therapeutic con- centrations (tab. 4). The observed effects must therefore be taken into account only in cases of drug overdosage or poisoning.

80 120 K e t o p r o f e n

160 200 240 100 200 300 400

Aminophenazone [/umol/l]

500

Fig. l. Dose dependence of ketoprofen interference in the lactate dehydrogenase colorimetric assay (a) and aminophenazone inteffer- ence in the aspartate aminotransferase colorimetric assay (b).

Tab. 4. Effects of therapeutic concentrations of some analgesic and antirheumatic drugs on enzyme assays (N number of the degrees of freedom

6, 0 = 10) 0 =

Enzyme (U/l)

Method

Control without drug Standard deviation "

Acetylsalicylic acid (833 μπιοΐ/ΐ) Aminophenazone (54 μπιοΐ/ΐ) Phenobarbital (108 μπιοΐ/ΐ) Diclofenac (7.1 μπιοΐ/ΐ) Ketoprofen (23.6 μπιοΐ/ΐ)

Xs

X

s P

X

s P

X

s P

Xs P

X

s P

Lactate dehydrogenase (continuous method, with pyruvate

s Substrate) 117.4

5.442 116.8

1.492

> 0.8 117.1 2.346

> 0.9 116.7

3.059

> 0.8 117.1

4.315

> 0.9 117.1 1.867

> 0.6

Lactate dehydrogenase (continuous method, with L-lactate

s Substrate) 38.60

0.735 38.62

0.871

> 0.9 38.62

0.492

> 0.9 38.80

1.797

> 0.8 37.98

0.930

> 0.05 38.15

1.765

> 0.6

Lactate dehydrogenase (colorimetric method)

86.00 7.149 82.20

5.475

> 0.3 87.43

4.638

> 0.6 88.81

6.500

> 0.5 91.05

6.320

> 0.25 84.39 5.265

> 0.6

Creatine kinase (continuous method)

47.93 1.689 47.78

1.233

> 0.8 38.80

1.103

> 0.3 48.80

1.181

> 0.4 47.54

1.407

> 0.6 48.80

1.807

> 0.4

Aspartate aminotransferase (continuous method

17.73 0.468 17.65

1.241

> 0.8 18.05

1.243

> 0.6 17.90

0.276

> 0.5 17.73

1.268

^ 17.14

0.752

> 0.1

Aspartate aminotransferase (colorimetric method)

19.03 1.585 19.10

1.305

> 0.9 19.16

1.932

> 0.9 18.41

1.781

> 0.5 18.41

1.875

> 0.5 19.58

1.802

> 0.6

Alanine aminotransferase (continuous method)

9.31 0.397 9.150.420

>0.5 9.45 0.437

>0.6 9.27 0.562

>0.8 0.6429.15

>0.6 8.81

•0.584

«*0.1

Alanine aminotransferase (colorimetric method)

9.760.812 9.800.859

> 0.9 10,28

0.929

> 0.3 9.92 0.547

> 0.7 9.600.352

> 0.6 9.600.629

> 0.7

J. Clin, Chem. Clin, Bi chem. / Vol. 22, 1984 / No. 8

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Jelic, Majkic-Singh, Spasio, Todorovio and £ivanov-Stakio: Effects of drugs on the assay of serum enzymes

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Zorana Jelic

Department of Biochemistry Faculty of Pharmacy Dr Subotica 8, POB. 146 YU-11000 Beograd

J. Clin. Chem. Clin. Biochem. / Vol. 22,1984 / No. 8

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