Enzymatic Determination of Cholesterol and Triglyceride with the Abbott Bichromatic Analyser

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1 Ann. din. Biohem. 14 (1977) nzymati Determination of Cholesterol and Triglyeride with the Abbott Bihromati Analyser HATHR M. BARBOUR Diagnosti ChemialPathology, St. Mary's Hospital, Praed Street, London W.2 nzymati methods for holesterol and triglyerides on the Abbott Bihromati Analyser are ompared with standard non-enzymati assays. Fators affetingthe performane of the ABA 100 and preision of the enzymati assays on this instrument are disussed. Beause onventional lipid assays are nonspeifi and utilise solvent extration and orrosive reagents, they are being replaed by the more speifi, aurate, and onvenient enzymati assays. The aim of this study was to ompare the determination of holesterol and triglyerides by nonenzymati and enzymati methods and to examine the use and performane of the Abbott Bihromati Analyser" employed for the enzymati assays. Cholesterol determined by the Liebermann-Burhard method on the Tehnionf AutoAnalyzer was ompared with two enzymati assays using the Hantzsh reation (Roshlau, 1974) and the phenol-aminophenazone method (Trinder, 1969; Allain et al., 1974; Witte et al., 1974) to quantitate the hydrogen peroxide liberated from the holesterol oxidase reation. Triglyerides were measured on the Tehnion AutoAnalyzer by the fluoresene of the Hantzsh ondensation produt derived from glyerol (Nash, 1953; Belman, 1963) and by two enzymati proedures on the ABA 100. MTHODS A. Automatedproedure for the simultaneous hemial determination ofholesterol and triglyerides Cholesterol and triglyerides were simultaneously determined on the Tehnion AutoAnalyzer (Leon et al., 1970). The holesterol method was based on the Liebermann-Burhard reation, and the triglyeride assay was a modifiation of the method by Kessler and Lederer (1965). After isopropanol extration and zeolite adsorption, triglyerides are saponified to glyerol, whih is oxidised by periodate to formaldehyde, whih then yields the f1uorimetri end produt by the ondensation between aetylaetone and ammonium ion. B. Automated enzymati determinations ofholesterol and triglyerides holesterol Commerial kits produed by Boehringer.] Calbiohem, and Abbott were evaluated. The Boehringer kit No was adapted for the ABA 100 by the manufaturers, Table 1 (a); the method measures the rate of olour development in the Hantzsh ondensation reation. This method employs the reation of atalase with hydrogen peroxide and methanol to produe formaldehyde, whih ondenses with ammonium ion and aetyl aetone to yield 3,5-diaetyl-l,4-dihydrolutidine, whih is measured at 410 nm. The phenol-aminophenazone reation employed in Calbiohem kit No and Abbott kit No. 6095, Table 1 (b), uses the reation of peroxidase with hydrogen peroxide, and the oloured produt phenazone-quinoneimine is measured at 550 nm. The enzymati holesterol assays were alibrated with aqueous holesterol standard (Boehringer Preiset holesterol No ). Triglyerides The proedure was based on the method desribed by ggstein (1966) and Wahlefeld (1974). The Smith Kline Instrument] kit No and Boehringer kit No were similarly modified for operation on the Abbott Analyser, Table 1 (). The enzymati determination of serum triglyerides is as follows: Triglyerides lipase +~ glyerol + free fatty aids glyerol + ATpglyerol kinase> glyerol- I-phosphate + ADP ADP + PP pyruvate kinase> pyruvate + ATP pyruvate + NADH + H+ LDH ---+ latate + NAD+ The one-stage enzymati method employs a single *Abbott Laboratories. Diagnostis Division, 820 Mission reagent (Sampson et al., 1975), whereas glyerol Street, So Pasadena, California, 91030, U.S.A. ttehnion Instrument Corporation. Tarrytown, New York, kinase is omitted from the reagent in the two-stage U.S.A. assay. With the Abbott Analyser enzymati hydrolysis of triglyerides to glyerol proeeds during the tboehringer Corporation (London) Ltd., Bell Lane,L ewes, ast Sussex. first two arousel revolutions in the two-stage Calbiohem Ltd., Thorpe House, King Street. Hereford. IISmi!h Kline Instruments In., 440 Page Mill Road, Palo method. Glyerol kinase is then added manually to Alto, California, U.S.A. individual uvettes after the first photometri reading. 22

2 nzymati determination of holesterol and triglyeride with the Abbott Bihromati Analyser 23 Table 1. Instrument settings for the assay ofholesterol and triglyerides on the ABA 100 (a) Cholesterol method (b) Cholesterol method () Triglyeride method Instrument setting Boehringer No Calbiohem No skalab No , Abbott No Boehringer No Temp.oC nm No. revs Rev. time Deimal Reation diretion Down Up Down ndpointrate Rate nd point Rate Syringe plate 1:201 1:201 1:51 Sample volume <J.<I) Table 2. Performane and fators affeting preision on the ABA 100 Item or parameter assessed :v, (%)* Method used for preision study I Optial system (1.12 A solution of 10 mg NADH in 100 ml Tris buffer ph 7.4 plaed in multiuvette 2 I :51 syringe plate (5 p.1 sample) 0.60 A solution of 5 mg NADH in 1 rnl Tris buffer 3 I :201 syringe plate (2.5 p.1 sample) 1.10 plaed in sample ups 4 (i) Before addition of sera seal to samples 2.60 tassay of 20 repliate serum samples, (ii) After addition of sera seal mmol holesteroll 5 (i) Before probe height adjustmentfor dispensing 5.80 tassay of 20 repliate serum samples, (ii) After probe height adjustment mrnol holesteroli ON = 20. tboehringer holesterol kit. In the last arousel revolution glyerol kinase reats with glyerol. The amount of NADH onsumed during the subsequent reations is equivalent to the amount of glyerol originally present. Calibration with triolein standards in isopropanol demonstrated a onstant alibration fator of RSULTS 1. Performane of the ABA 100 Preision of the optial system and features of the sample dispensing unit appear in Table 2. The oeffiients of variation for parameters 2 to 5 inlude the variane due to the photometer. 2. Cholesterol study Non-enzymati determination Linearity to at least 12 mrnoll was demonstrated with the Libermann-Burhard reation. Carry-over between speimens of 2.5 and 9.7 mrnll was 5.8%. Within- and between-bath preision data is shown in Table 3. nzymati determination HANTZSCH RACTION With Preiset aqueous holesterol standards it was shown that the reation was not linear above 8 mmoll (Fig. 1). Non-linear alibration plots were also produed with dilutions of sera with elevated holesterol levels (Fig. 1). Sensitivity of this method was disappointing. A serum holesterol of 7.8 mmoll gave an absorbane differene of only in this kineti assay. Attempts to inrease sensitivity by using the 1:101 syringe plate, or doubling the reation time merely exaggerated the non-linearity. There was no detetable arry-over between speimens with 2.5 and 9.7 mmol holesteroli. Within- and between-bath preision data are shown in Table 3.

3 24 Heather M. Barbour Table 3. Within- and between-bath preision Constituent *Within-bath preision tbetwn-bath preision Assay and analytial instrument Mean S.D. Coeff, Mean S.D. CoiI. onn. var, (%) onen. var, (%) Cholesterol Liebermann-Burhard Thnion AutoAnalyzr II Abbott Boehringer Kit No Abbott Calbiohem Kit No Triglyerides Fluorimetry Thnion AutoAnalyzer II tnzymati Abbott skalab Kit No tnzymati Abbott Boehringer Kit No nzymati Abbott Boehringer Kit No nzymati Abbott Calbiohem Kit No *For 20 observations. tfor 10 sets of observations. ~Two stage reation. One-stage reation. Correlation with the Liebermann-Burhard reation (Fig. 2) demonstrated lower results for the enzymati assay at levels below 5 mmoll and equivalent or higher results above 7.5 mmoli, The interept of 1.22 is greater than reports using the end-point Hantzh reation or other hromogen oxygen aeptors, and more than expeted for the onentration of irulating sterols, besides holesterol that reat with the Liebermann-Burhard reagent. TRINDR'S RACTION Determination of serum holesterol with the Calbiohem or Abbott kit was more sensitive, linear (Fig. 3) and preise (Table 3) than the enzymati reation employing the Hantzh reation. The majorityof holesterol values by this enzymati tehnique were onsistently lower than the Liebermann Burhard results (Fig. 4), in agreement with other authors. However, the distribution of results is suh that the interept rosses the y axis. A similar orrelation resulted from a omparison of holesterols determined by Liebermann-Burhard (x) with the Abbott holesterol kit (y). The orrelation oeffiient was , and regression equation y = l.00x Triglyeride study Non-enzymati determination The method was linear to at least 6 mmoll, Carryover between 0.5 and 3.5 mmoll was 2.8 %. Within- and between-bath preision data is shown in Table 3.

4 nzymati determination 0holesterol and triglyeride with the Abbott Bihromati Analyser 25...J <, (5-0 l... ::J 1Il a ol Il o s: u 10 ::J "0 5 > Fig. I.-Linearity of the kineti holesterol determination using the Hantzsh reation. Preiset aqueous holesterol standards. J 6. Two sera with elevated holesterols serially diluted Denotes the deviation from linearity. mmol holesteroll 10 : 0 13 D ~."! D 5 >, N :.JJ 5 10 mmolholesteroll Liebermann - Burhard reation Fig. 2.-Correlation of holesterol results: the Boehringer enzymati holesterol kit v. the automated Liebermann Burhard reation. Regression equation y = 1.16 x N = = regression line. 5 7 B nzymati determination Cholesterol 10 4 mmol L Preiset 1-0 serum dilution skalab KIT Study of the reation on a reation rate analyser indiated that, although triglyeride hydrolysis was omplete within 10 minutes and the subsequent reations in 5 minutes, the test and blank reations ontinued to show the same signifiant derease in absorbane 20 minutes after the addition of glyerol kinase. The reagent blank was also noted to be high and variable on the Abbott and reation rate analyser. It is essential to mix when glyerol kinase is added during the fourth arousel revolution. This improved the preision from 6.8 %to 2.3 %(C.Y.) with a serum triglyeride of 1.30 mmoli, Providing the mixing rod was drained or wiped, no detetable arry-over ourred between 0.5 and 3.5 mmoll. The method was linear to at least 6 mmol triglyeridei, Within- and between-bath preision results appear in Table 3. Comparison between the fully enzymati determination of serum triglyeride (y) and the fluorimetri method (x) gave a orrelation oeffiient of and regression equation y =

5 26 Heather M. Barbour SOO-500nm B rnrnol holesteroll BOHRINGR KIT Studies on a rate analyser showed that the test and blank reations were omplete within 5 minutes with no subsequent hanges in absorbane. The reagent blank reation was lower and more reproduible than the skalab kit. With triolein standards in isopropanol linearity was shown to 8 mmoll (Fig. 5). Within-bath preision was similar to the skalab kit (Table 3), but between-bath preision was superior. Comparison of triglyeride results with the nonenzymati method is shown in Fig. 6. The regression equation was similar to the orrelation with the skalab kit. The enzymati method again inludes serum glyerol as well as triglyeride. Fig. 3.-Demonstration of the linearity of the phenolaminophenazone enzymati assay for holesterol, using a serially diluted serum with an elevated holesterol onentration. mmol holesteroll TRIGLYCIRID DTRMINATION BY A ON-STAG ASSAY In this tehnique the glyerol kinase was ombined with the main reagent, used immediately, and a single absorbane reading made after 10 minutes (Sampson et al., 1975). Fully enzymati triglyeride kits manufatured by Boehringer, Calbiohem, and skalab were tested. The skalab and Calbiohem 10..t" 9.2 U ~ l,! -0 Ẹ>, N w.::. 5 10rrrrdhdesterolL Liebermann - Burhard reation Fig. 4.-Correlation of holesterol results: the Calbiohem enzymati holesterol kit for holesterol modified for the Abbott Analyser (y) v, the automated Liebermann Burhard reation (x), Regression equation y = 0.95 x N = = regression line. 1.08x The enzymati method determines free glyerol together with glyerol derived from triglyerides. An aurate enzymati determination of triglyerides should inlude a separate determination of free glyerol sine serum levels vary in healthy individuals from 0.05 to 0.18 rnmoll ! ~ ::J 4 Vl g 3 C!J ::J a > 2 C!J 0 ~ D Dilution of a 10mmol triolein standard Fig. 5.-Demonstration of the linearity of the two-step triglyeride method on the Abbott Analyser.

6 nzymati determination of holesterol and triglyeride with the Abbott Bihromati Analyser 27 rrmd triglyeerides.a- 6 Table 4. Running osts for the analysis ofholesterol and triglyerides (Otober, 1976) Cost for 9 weeks (pounds (989 Tests 127 QC) Q U ~ \! a 2 '" Abbott Cholesterol kits Triglyeride kits Multiuvettes 7.50 Sample ups 8.00 Sera-seal N C W Automote fluorimetri assay 6moo trlglyeridejl AutoAnalyzer Cholesterol reagents Triglyeridereagents Pump tubes xtration reagents and tubes 4.48 Fig. 6.-Correlation of triglyeride results, the fully enzymati triglyeride assay, Boehringer (y) v. the automated f1uorimetri assay on the Tehnion Auto Analyser (x), Regression equation y = 1.19 x N = = regression line. reations took 10 minutes for ompletion at 37 C, but the Boehringer reation remained inomplete even after 20 minutes. Preision of the one-stage reation was poor (Table 3). Preision with the onestage skalab kit was also disappointing, but an aeptable orrelation was obtained between the one-stage(x) and two-stage (y) reations, orrelation oeffiient , regression equation y = 0.94x DISCUSSION nzymati lipid analysis on the Abbott Analyser requires the minimum of reagent preparation, and assays an begin soon after the instrument has been swithed on. In ontrast the non-enzymati determination of lipids requires substantial sample preparation and the fluorimeter may require up to 30minutes to reah a steady baseline. However, the dual hannel AutoAnalyzer simultaneously analyses holesterol and triglyeride, whereas these are determined sequentially on the Abbott with a hange of reagent, filter, and alibration fator. Any throughput omparison between ontinuous and disrete analysis must be onsidered as only partial, but it is estimated that 60 holesterol and triglyeride determinations would take approximately 70 minutes on the Abbott and 130 minutes on the AutoAnalyzer. A omparison of running osts for the determination of holesterol and triglyerides on the Tehnion AutoAnalyzer and Abbott Bihromati Analyser, exlusive oflabour osts, appears in Table 4. A diret ost omparison is diffiult between ontinuous and disrete methods of analysis due to variation in bath sizes, wastage ofreagent, and the ost of equipment maintenane and onsumables, besides reagents. It was therefore deided to assess the running osts on both analytial systems over a period of two months to provide more useful information. Preision of these enzymati assays is good. Care must be taken to optimise the probe height for sampling and dispensing, and silione overlay of samples also improves analytial performane, probably by reduing evaporation and ontamination through arry-over. It was onsidered that a one-stage assay for the determination of triglyeride should be faster, less laborious, and more preise than the two-stage, sine evaporation losses should be redued. However, preision was poor and the one-stage reation with the Boehringer kit was slower. The ombined reagent is also reported to deteriorate rapidly (Sampson et al., 1975). The one-stage method was therefore found to be unsatisfatory. The poor between-bath preision ofthe two-stage skalab triglyeride assay ould be due to the lak of a satisfatory reation ompletion within the reommended measuring time and a high and variable blank. The latter is automatially subtrated from the test results on the Abbott. The Boehringer triglyeride method was hosen for routine use in our laboratory owing to the presene of a low reagent blank and good preision. It is useful to ompare the hemistry of the olori-

7 28 Heather M. Barbour metri stage of the two enzymati proedures used for holesterol determination, sine this appears to influene the omparison of results with the Liebermann-Burhard reation (Figs. 2, 4). There are several favourable features of the Hantzsh reation used in the Boehringer enzymati holesterol kit. The kineti holesterol assay does not require a separate blank determination, and the Hantzsh reation is reported to be unaffeted by bilirubin, haemolysis, and asorbate (Kageyama, 1971). However in this study the method was insensitive, and linearity ould not be demonstrated to 10.4 mmoljl, ontrary to the manufaturers' advie. While the omparison with the Liebermann-Burhard reation gave an aeptable orrelation oeffiient, the slope indiated that the differene between enzymati and non-enzymati holesterol results was not onsistent. Sine this slope was not demonstrated by Roshlau (1974) using an end point Hantzsh reation, the results possibly indiate that, although the rate of olour prodution is linear with aqueous holesterol standards to 7.8 mmoll, this may not apply to all sera where rates of olour prodution ould be subjet to interferene. The phenol-aminophenazone reation used in the Calbiohem and Abbott holesterol kits is more sensitive, preise, and linear to at least 16mmoljl but the method is subjet to some interferene by bilirubin (Allain et ai., 1974). Also, as it is an end point reation, a blank determination is neessary for haemolysed speimens as the bihromati filter does not fully ompensate for this. Besides these disadvantages the reation was more reliable than the Hantzsh reation, demonstrated a onsistent differene between the Liebermann-Burhard results, and therefore was onsidered the most satisfatory method for holesterol determination on the Abbott. The Abbott Bihromati Analyser ombines speed with preision, reagent and sample eonomy, and versatility, and is a suitable instrument for lipid investigations. Although the equipment is designed for assays initiated by single reagent addition it was readily adapted for the triglyeride assay involving the addition of a seond reagent. I thank Dr. S. Rosalki for his advie in the preparation of the manusript. RFRNCS Allain, C. C., Poon, L. S., Chan, C. S. G., Rihmond, W., Fu, P. C. nzymati determination of total serum holesterol. Clin. Chem. 20 (1974) 470. Belman, S. The fluorimetri determination of formaldehyde. Ann. him. Ata 29 (1963) 120. ggstein, M. ine neue Bestimmungder Neutralfette in Blutserum und Gewebe. Klin. Wshr. 44 (1966) 267. F1egg, H. M. An investigation of the determination of serum holesterol by an enzymati method. Ann. din. Biohem. 10 (1973) 79. Kageyarna, N. A. Diret olorimetri determination of uri aid in serum and urine with uriase-atalase system. Clin. Chem. 31 (1971)421. Kessler, G., Lederer, H. Fluormetri measurement of triglyerides, Tehnion Symposium, Automation in Analytial Chemistry, New York (1965) 341. Leon, L. P., Rosh, R. L., Turrell, J. Advanes in automated analysis. Tehnion International Congress (1970). Nash, T. The olorimetri estimation of formaldehyde by means of the Hantzsh reation. Biohem. J. 55 (1953) 416. Roshlau, P. et al. nzymati determination of total holesterol serum. Z. klin. Chem. 12 (1974)403. Sampson,. J., Demers, L. M., Drieg, A. F. Faster enzymati proedure for serum triglyerides. Clin. Chem.21 (13) (1975) Trinder, P. Determination of gluose in blood using gluose oxidase with an alternative oxygen aeptor. Ann. din. Biohem. 6 (1969) 24. Wahlefeld, A. W. Triglyerides: Determination after enzymati hydrolysis, in Bergrneyer (ed.), Methods of nzymati Analysis, 2nd nglish edition. New York and London, Verlag Chemie Weinheim and Aademi Press (1974)1831 if. Witte, D. L., Barrett, D. A., Wyott, D. A. valuation of an enzymati proedure for determination of serum holesterol with the Abbott ABA 100. Clin. Chem. 20 (1974) 1282.