23 Comparative evaluation of the enzymatic assay, the immunoassay and a gas-chromatographic procedure for the determination of serum theophylline

P-6 chloroform under acid and alkaline conditions. The extracts were dried, redlssolved in methanol and developed on aluminium backed silica gel G plates (Merck) in benzene/diethylamine end ethylacetate/methnnol/as~onla solvents for the alkaline extract, and chloroform/acetone for the acid extract. To detect the spots, a sequence of spray reagents was used. Appropriate standards prepared from pharmaceutical preparations were run alongside the extracts to aid identification of the parent drugs. Data concerning position, shape, colour and reaction to sprays end pattern of spots were recorded. Good quality photographic reproductions of the TLC plates were compiled for use in the laboratory. Staff could compare patient sample plates directly with the photographs. The compendium has improved the quality of drug screening. Presumptive identification of spots other then the parent drug as metabolites or another compound could be made. Greater standardization of the interpretation of TLC plates resulted as it became less dependent on the previous experience and/or recall ability of the technical staff. As TLC remains the most practical method of overdose screening, this approach is r e c o ~ e n d e d for laboratories contemplating such a program.

I 23 J COMPARATIVE EVALUATION OF THE ENZYMATIC ASSAY, THE JI~(UNOASSAY AND A GAS-CHROMATOGRAPHIC PROCEDURE FOR THE DETERMINATION OF SERUM THEOPHYLLINE. B. Vinet, L. Zizian, D~pt de Biochimie, H6p. Notre-Dame, Montreal, Qua., Canada H2L 4MI A comparative evaluation of three methods for the determination of serum theophylline by three different principles of analysis is described. The enzymatic assay consists in the extraction of 0.4 ml of serum by chloroform-isopropanol, back-extractlon in 1 m M N a O H and measurement of the inhibition of alkaline phosphatase by this alkaline fraction with p-nitrophenylphosphate as substrate in AMP-buffer. The GC procedure requires on-column butylation and a NPD (Vlnet B. and Zizian L., Clio. Chem. 25, 156 (1979)). The immuooassay is with the E~E[T reagents. The precision of the three methods is good 42.0% for the enzymatic assay, 4.5% for EMIT and 5.2% for the GC procedure). Caffeine (50 mg/L) increases the EMIT values by 1 mg/L while 3-methylxanthlne 44 mg/L) increases the-values of the enzymatic assay by no more than 1 mg/L. Other xanthlne derivative~ theophylline metabolites and drugs concomitantly administered with theophylline do not interfere. The GC procedure is free of interference. The EMIT assay is the fastest with an analysis time of 2 minutes compared to 7 and 6 minutes respectively for the GC and the enzymatic assay. The methods correlate well with each other. The major advantages of the enzymatic assay are in its low cost, stability of the reagents. The GC procedure requires special instrumen=ation not always available in clinical laboratory. The three methods are suitable for emergency analysis and their low sample volumes make them suitable for pediatric cases.

I 24 I THE DETERMINATION OF SERUMTHEOPHYLLINE: A COMPARISON OF THREE ASSAYPROCEDURES. I.S. Kampa, L.K. Dunikoski Jr., J . I . Jarzabek, and D. Grubesich, Department of Pathology, The Valley Hospital, Ridgewood, New Jersey 07451, and Department of Clinical Chemistry, Perth Amboy General Hospital, Perth Amboy, New Jersey 08861. Theophylline (1,3-dimethylxanthine) is widely used as a bronchodilator in the treatment of reversible airway obstruction and as an adjunct in the therapy of acute l e f t ventricufar failure. The accurate measurement of serum theophylline levels is important because effective and toxic levels are related to serum concentrations rather than to administered dosage. We examined f i f t y serum samples, submitted to our laboratories for theophylline levels,, by the "Enzyme Multiplied Immune Technique" (EMIT), by a radioimmunoassay procedure, and by a high pressure liquid chromatographic (HPLC) assay. We compared validity of data, relative cost per assay, and rate of throughput for each. All three procedures classified patients similarly into subtherapeutic, therapeutic and toxic levels. Precision and recovery values showed no significant differences between the three procedures. Precision ranged from 3.24 to 13.66 percent, while recovery values were from 97.0 to 110.0 percent. Significant differences were observed in the cost per assay and the rate of throughput. The EMIT procedure was the most costly and required the greatest amount of time. The HPLC method was less expensive and required the least amount of time. The excellent correlation between the Syva EMIT assay and the other two procedures make this a desirable method for those laboratories which lack the more expensive instruments

required for the other two procedures. Although we used a Gilford Stasar Ill spectrophotometer equipped with a thermo-

regulated cuvette for the EMIT determination, any spectrophotometer with a temperature controlled cuvette can be used.

J 25 IVISUAL (U.V.) EVALUATION OF CREATINE KINASE (CK) ISOENZYMES. HOW "SAFE" IS IT IN COMPARISON WITHFLUOROMETRY? P. Fan, D.A. Progay, M.E. Chilcote, Dept. of Biochemistry, SUNY and Erie County Laboratory, Buffalo r N. Y . 14215. Visual evaluation of CK isoenzymes is practiced in 20% of Western N . Y . laboratories because it is inexpensive and simple. Conditions of this type of evaluation were not assessed. The literature considers approximately 3-5 International Units ( I . U . ) a s a cut-off base-llne level for the MB fraction in normal human sera. We used an ACI Coming electrophoresls system (agarose) and visual evaluation of the isoenzymes with 3 different U.V. lamps. In comparison, a Turner ill Fluorometer was used for the densitometry of electrophoretic strips. Specimens from 188 patients (suspected myocardial infarction ( M . I . ) and open heart surgery)were collected and examined from the Intensive Care units of 3 local hospitals. No difficulty was found to observe peaks equivalent to 3 [oU. or above with fluorometry. Resuhs t however e showed that faint or questionable fluorescence could only be "seen" at 3-6 [.U. levels. Detection depended on sensitivity of the individuaPs eye ("trained eyes" were preferable). Vhual evaluotionr thus e was subjective and peaks could be overlooked by rotating inexperienced technicians. No one overlooked MB peaks above 8-10 I.U. On the descending line of the CK-MB time curve small peaks (3-7 I . U . ) could be missed. Confirmation of MI with EKG t LDH isoenzymes or AST would be essential in such cases. Sometimes unexplained, peaks were observed between MM and MB; therefore, the use of proper control serum (human source) with 3 isoenzymes is mandatory. For acceptable results, the electruphoretlc films must be fresh (no salt precipitates, molds); reagents and specimens must be fresh. Reading of electropherograms should be done as soon as poss~ble. In conclusion, it appears that inexperienced techn~clans could miss an MB peak positive for MI at 5-7 I.U. levels of CK-MB.

I I 26 I ~ PITFALL IN L~POB~TORY DIAGNOSIS OF MACPO~NYLASEMI~. ~kbmrali H. Todai and D. ~cheamcnn¢-Mensah, Division of Biochemistry, Humber Memorial Hospital, 200 Church St., Weston, Ontario, Canada, M9N IM8. Laboratory sereenin~ methods for d i a ~ o s i s of macroamylasemia are ~enerally based on ~el filtration. (Frldhandler et. el., Clio. Chem. /.7_ ~23, 1971; Lon~ and Kowlessar, Gsstroenterol. ~.~ ~64, 1972; Peeters and Vantranpen. Clio. Chim. ~eta ~ ~37. 107~). The thin layer eel filtration method of Peeters and Vantrappen employs Phosphate-Penzoip acid buffer pH 7.0 for elution. Using this method we observed dissoelation of a ?S macroamylase. A 11S maeroamylase was not affected. Further investiaations were carried out on Sephadex columns. Partial dissociation of the 7S maerosmylase oecured on minrocolumn of Sephadex 0 10o w~th Phosphate-Penzoate buffer as the eluant. On a mseroeolumn of Sechadex G 2on, the 7S maeroamylase was completely dissociated whereas the 11S was not affected bY the buffer. When Phosphate buffer DH 7.0 or Trls-HCl buffer nH 7.2 was used for elution durin= mleroeolumn ~el filtration, no dissoelation was observed. However with Glyeine-HCl buffer DH 3.~ both the 7S and 11S maeroamylases completely dissociated on the maeroeolumn. The results suggest that the Denzoate component of the Phosphate-Benzoate buffer is responsible for the dissociation of the 7S maerosmylase. The variable response of 7S and 11S maeroamylases to Phosphate-Penzoate buffer, are consistent with the heterogeneous nature of macroamvlases. The phenomenon could result in false negative results by the screenin~ method of Peeters and Vantrsppen.

I 27 [LYSOSOMAL ENZYMES AND RIBONUCLEASE IN THE SERUM OF HOSPITAL PATIENTS. LEVY t S.W. end MARIERp A.W. Queen Elizabeth Hospital, Montreal, Quebec. H4A 3L6. Following a study into optimum conditions for their assay, methods were established for the determination of five lysosomal enzymes in serum as follows: acld~-glyeerophosphatase (A), ~-glucuronldase (B),~-galactosldase (C), N-acetyl-~ glucosaminidase (D), and rlhonuclease (RN-ese) (E). The enzymes were then measured in the blood of hospital patients. Some of F