Colorimetric determination of free and total cholesterol by flow injection analysis with a fiber optic detector A. Krug*t, A. A. Suleimant, G. G. Guilbauit* and R. Kellner* * U n i v e r s i t y o f T e c h n o l o g y , I n s t t t u t e o f A n a l y t t c a l C h e m i s t r y , Vzenna, A u s t r i a ? U n l v e r s t t y o f N e w O r l e a n s , D e p a r t m e n t o f C h e m l s t r y , N e w Orleans, L o u l s t a n a
A flow mjectton method f o r the detelmmatton o f total and free cholesterol ts ppesented Cholesterol e~terase and cholesterol oxlda~e are tmmobthzed on ammoalkvl glass beads The beads are packed mto a tabular glass reactor The cholesterol esters traversmg through the esterase reactor are cleaved to cholesterol and fatty actds The ovtdase reactor converts cholesterol to ~holest-4-en-3-one and hydrogen pero~tde ts generated The sample stream t~ merged wtth reagent ~treams conststmg oJ a peroatda~e solution and a solutton o f 2,2'-azmo-bts-(3-ethvl-benzthtazohne-6-sulfomc acid) &aminomum salt, and a hsdrogen perortde-dependent color teactton taLe~ place m a short coded reactor The signal l'¢ momtored b3 means o f fiber optw mstrumentatton Cholesterol concentration c an be 1elated to the ab~orptton o f the oJtldtzed dye form at a wavelength oJ 425 nm The ~orl, mg range lS 0 5 - 0 8 mmol l l, and the sample throughputs are 60 and 30 h -I for free and total cholesterol, respectwely
Keywords Cholesterol,flow rejection fiber optic cholesteroloxldase cholesterolesterase
Introduction Although the number of pubhcatlons dedicated to the determination of cholesterol ts enormous, the effort of developing new methods continues unremittingly ~ This report outlines a method for the determination of free and total cholesterol utilizing flow injection analysis (FIA) and fiber opttc technology FIA has become a powerful analytical tool in the field of biochemistry and blotechnology 2 The conjunction of enzyme- or antibody-based reactions with FIA has proven its appllcabdlty to btologlcal samples In the last decades, countless methods have been developed for the determination of free and total cholesterol Enzymatic procedures have virtually replaced chemical methods in the clinical laboratory The enzymatic assay of total cholesterol employs cholesterol esterase (ChE) to cleave the cholesteryl esters to cholesterol and fatty acids (FA) In a second enzymatic step, cholesterol ts oxidized to cholest-4-en-3one by cholesterol oxldase (ChOD) in the presence of oxygen The typical feature of this oxldase-catalysed reaction is the production of hydrogen peroxide In
Address reprint requests to Dr Gullbaultat The Universityof New Orleans, Department of Chemistry, New Orleans, LA 70148 Recmved 3 July 1991, rewsed 8 October 1991
© 1992 Butterworth-Hememann
most cases, hydrogen peroxide is quantified spectrophotometrically or electrochemically In combination with FIA, several principles have been utilized for the detection of the enzymatlcally generated hydrogen peroxide The earher flow inJection methods for the determination of free and total cholesterol featured amperometrlc detecUon 3-6 Recently, several photometric flow injection approaches have been proposed for assaying cholesterol Petersson et al 7 and Malavoltl et al 8 developed flow lnjechon systems using lumlnol-H202 chemiluminescence, and Fernandez-Romero et al 9 proposed several photometnc and fluorlmetnc methods, either with dissolved or lmmobthzed enzymes The first fiber optic cholesterol blosensor, reported by Trettnak and Wolfbins, m is based on lmmoNhzed cholesterol ox~dase and an oxygen-quenched fluorescent reaction However, this optrode has a response time of 7 to 12 mm and is useful for the assay of free cholesterol only In this study, we investigated the feaslblhty of a photometric method utilizing a fiber optic detection approach for the determination of free and total cholesterol employing the dye 2,2'-azlno-bis-(3-ethylbenzthiazohne-6-sulfonlc acid) dlammonlum salt (ABTS) The procedure is based on the following reacUon scheme Cholesteryl ester + H20
ChE, cholesterol + FA
Enzyme Mmrob Technol, 1992, vol 14, April
:313
Papers Cholesterol + O~
~hO~ cholest-4-en-3-one + H202
H202 + "~ A B T S + "~ H +
]
'1
HRP 2H~O + 2ABTS +
ABTS forms a stable radical cation with H202 in the presence of horseradish peroxldase (HRP), which has a strong absorption band around 425 nm ABTS has p r o v e n useful for clinical application by Kahle et al J~ Majklc and Berkes ~2 used this dye for the determination of free and estenfied cholesterol by a kinetic method, and A b d e l - L a t l f et al t~ developed a fiber optic glucose sensor based on this color reacuon
Experimental Matermls Cholesterol esterase (ChE, EC 3 1 I 13) from P~eudomonas species, w~th a specific activity of 112 U mg solid, and cholesterol oxldase (ChOD, EC 1 13 6) from Stteptomyces species, with a specific actlwty of 23 5 U mg -~ sohd, were purchased from T o y o b o (Osaka, Japan) Cholesterol, cholesteryl-10-undecenoate, Triton X-100, blhrubln, 2,2'-azmo-bls-(3-ethylbenzthlazohne-6-sulfonlc acid) d l a m m o n l u m salt. and horseradish p e r o x l d a s e (HRP, EC I 11 1 7) with a ~peClfiC activity of 120 U mg ~ sohd were obtained from S~gma (St Louis, MO) The alkylamlne glass beads with a mesh s~ze 120/200 were a product of Cormng (Cornlng, NY) All other chemicals were of analytical reagent grade
A p p a t atu~ The hght source (quartz lamp), fiber optic bundle, m o n o c h r o m a t o r , photomultlpher, and detection umt, all from Oriel (Stratford, CT), were assembled as shown In Ftgute 1 The t~p of the fiber was incorporated Into a flow-through cell machined from a block of polytetrafluroethylene (PTFE) A c y h n d n c a i hole, which fit the tip of the fiber optic bundle tightly, was drilled Into th~s block The cylindrical c a w t y underneath the polished end of the fiber bundle measured 0 75 m m in height, with a diameter of 6 m m and was equipped with an inlet and outlet conduit To ensure absolute llght-t~ghtness, the whole flow cell was embedded in a housing made of gray PVC The flow scheme is depicted m Ftqute 2 The tubing used con-
Figure 2 FIA manifold for the determination of total cholesterol C, Carrier stream, R1, ABTS solution, R2, HRP solution, P, per~stalttc pump, S, sample m]ectton, ER~, enzymatic reactor w~th immobdlzed ChE, ER2, enzymatic reactor with ~mmobd~zed ChOD, CR, 30 cm coded reactor, FD, fiber optic flow detector, W, waste ER~ was omitted for the determination of free cholesterol
slsted of flexible P T F E with an inner diameter of 0 75 m m and was purchased from Small Parts Inc (Miami, FL) Tubing connectors were obtained from the same c o m p a n y For the enzymaUc reactors, glass tubes w~th an inner diameter of 1 5 m m were cut to the right length and the P T F E tubings were glued m with epoxyamine resin on both sides A Supelco (Bellefonte, PA) N-60 six-way valve served as a sample mjecto~ and the fluids were transported with a peristaltic p u m p (Ramm I n s t r u m e n t Co , Woburn, MA)
Preparation of c h o l e s t e t o l a n d reagent s olutton3 Cholestelol solutions in the concentration range of 0 5 to 5 mmol I J were prepared as described previously ~' Cholesterol was dissolved in 12 8 ml lsopropanol, 3 85 ml of Triton X-100 was added, and after homogenlzatlon, the volume was made up to 100 ml w~th a 0 [ M phosphate buffer, p H 7 0 The soluUons that ~ e r e needed for the assay of total cholesterol contained cholesterol and cholesteryl-10-undecenoate m such amounts that the ratio of free to estenfied cholesterol was 30 70 Due to the low solubility of cholesteryl-10undecenoate in l s o p r o p a n o l - b u f f e r mixtures, the solvent used was lsopropanol containing 4'Yc Triton X-100 for all concentrations from 1 to 8 mmol l ~ A 10 mM ABTS solution was prepared by dissolving 549 mg m 100 ml H~O An H R P solution with an activity of l0 U ml ~ was prepared by dissolving 8 3 mg sohd e n z y m e in 100 ml 0 1 M p h o s p h a t e buffer, pH 7 0 The buffer mixture needed as carrier stream was prepared by dissolving 38 5 ml Triton X-100 in 128 ml of lsopropanol and finally making the volume up to 1,000 ml ~ lth a 0 1 M phosphate buffer, p H 7 0
l m m o b d l z a t t o n oJ enzyme,s
Figure 1 Block diagram of fiber optic setup L, Light source, F, fiber optic bundle, M, monochromator, BF, bifurcated fiber optic bundle, FD, fiber optic flow detector, P, photomultlpher tube, R, photomulttpher readout, C, chart recorder
314
EnzymeMicrob Technol, 1992, vol 14, April
One hundred milligrams of a l k y l a m m e glass was weighed into a 25-ml E r l e n m e y e r flask, and 10 ml of 2 5% glutaraldehyde solution in 0 1 M phosphate buffer, p H 7 0, was added For the first 15 m m the flask was held under reduced pressure to degas the glass beads After a total of 60 rain the glutaraldehyde solution was r e m o v e d and the beads were washed with phosphate buffer The glass beads exhibited an orange color after glutaraldehyde activation The beads were suspended in 2 ml 0 I M phosphate buffer, pH 7 0,
Flow mlectton determmatton of cholesterol A Krug et al containing 5 mg C h E This mixture was allowed to stand for 60 mln at r o o m t e m p e r a t u r e , initially under reduced p r e s s u r e C h O D was immobilized on a second batch of alkylamine glass b e a d s exactly in the same way as C h E W h e n the l m m o b i h z e d e n z y m e s were not used, they w e r e kept m a refrigerator at 4°C For actual use in the F I A system, the immobilized e n z y m e preparations were p a c k e d into the glassy column reactors
350
/
300
/ /
250 ,/-
tf /
~ 200 ~2
Results and discussion
/*
E 150
Opnmtzanon of FIA mamfold
~
,
/*
To obtain the best results, mlhally several p a r a m e t e r s were varied to test the F I A manifold Carrier stream, A B T S solution, and H R P solution were p u m p e d at a flow rate of 4 5 ml rain -~ to ensure turbulence and p r o p e r mixing F o u r different sample volumes (10, 25, 50, and 90/~l) were considered for injection Finally, the injection of 50/~i turned out to have the best characterlshcs, l e the proportion of peak height to peak width was optimized The e n z y m a t i c reactor consisted of a tube 80 m m long packed with ChE immobilized glass beads o v e r a length of 40 m m and with C h O D beads o v e r the residual 40 m m F o r the F I A measurements of free cholesterol, a C h O D reactor with 30 m m was sufficient The coiled reactor after the confluence of the enzymatlcally degraded sample with the reagents was chosen to be 300 m m In the case of the F I A manifold for free cholesterol, the determined dlsp e r s m n was 3 1 and the sampling frequency ran up to 60 samples per hour The overall dispersion coefficient for the total cholesterol runs was 4 2 and 30 samples could be injected per hour The A B T S and the H R P had to be supplied in two separate reagent streams, because of the inhibitory effect of the dye on the enzyme
from 1 81 to 8 03 m m o l 1-j 14 Approximately 70% of the total cholesterol is esterlfied with fatty acids, and therefore the free cholesterol ranges from 0 54 to 2 41 mmol 1-1 H o w e v e r , the calibration could not be extended a b o v e 8 mmol 1 l total cholesterol, because at higher concentrations o f our standards the cholesterol started to precipitate in the enzymatic reactor after being mixed with the carrier stream The relative standard deviation for 10 replicate injections of cholesterol standards was found to be 2 5% for a 2 mM solution, 4 l%at5mM, and52%at8mM
Cahbratton for free and total cholesterol
Stabdttv
Table 1 shows the calibration data for free and total
The e n z y m a t i c reactors were used continuously to check the stability of the l m m o b l h z e d ChE and C h O D The C h E lost more than 20% of its activity within 3 weeks, whereas the C h O D exhibited 90% of its initial activity after I month
/
/
50
/ / /
/
/
0
I 50
0
_ I [ I I I 100 150 200 250 500 Paromox cholesterol reagent m g / d l
350
Figure 3 C o m p a r i s o n o f FIA m e t h o d w , t h an established cholesterol r e a g e n t used m chmcal l a b o r a t o r i e s
cholesterol The correlation coefficients for free and total cholesterol were 0 985 and 0 965, respectively L m e a r I t y m the lower concentration range and the detection limit were not investigated We only aimed at a sufficient linear range covering the reference ranges of free and total cholesterol in h u m a n blood serum samples The reference range of total cholesterol in serum or plasma of infants, adolescents, and adults extends
Table 1
/
Accuracy The p e r f o r m a n c e of the p r o p o s e d F I A a p p r o a c h was c o m p a r e d with the procedure established in clinical
C a h b r a t m n data f o r free and t o t a l c h o l e s t e r o l Cholesterol ( m m o l I 1)
Peak h m g h t a (poA) Peak h e , g h t b (/~A)
05
1
2
3
4
5
6
7
8
5 6 -
9 6 84
13 6 14 3
20 6 22 5
27 3 26 0
30 5 30 4
34 3
37 3
40 0
a Free c h o l e s t e r o l / ~ A = 6 0 1 × m m o l l 1 + 201 b Total c h o l e s t e r o l /~A = 4 9 1 × m m o l l 1 + 4 0 5
Enzyme M,crob Technol, 1992, vol 14, April
315
Papers use We analyzed 26 serum samples and correlated our values to those obtained using the P a r a m a x cholesterol reagent, as shown in the correlation graph (Ftgure 3) The values were c o n v e r t e d to the more clinically relevant mg dl J units The graph has a slope of 0 88 and a correlation coefficient of 0 949 It ~s concluded that our method leads to lower levels, especially at higher cholesterol concentrations This could be attributed to our calibration method, which e m p l o y s h o m o g e n e o u s cholesterol solutions H e n c e the total a m o u n t of cholesterol is available for enzymatic degradation in the C h E and C h O D reactors In the case of the serum samples, some of the hpoproteln particles are not accessible to the enzymatic reaction, b e c a u s e they are not dissolved fast enough by the surfactant This d r a w b a c k could be avoided either by preconditioning of the serum samples by addition of surfactant or by calibration w~th serum samples
Interference studte~ We investigated the interference of blhrubln, which is considered to be one of the most troublesome factors in cholesterol assays ~ N o interference was detected from bihrubm concentrations up to 20 mg dl ~ which is a p p r o x i m a t e l y 20 times the normally occurring blllrubln levels
Acknowledgements This w o r k was supported by a grant from the Louisiana Education Quality Support Fund ( L E Q S F - R D - B 17) and the Austrian Fond zur Forderung der Wlssenschafthchen F o r s c h u n g (Project 7181-CH E) Dr W T Wu, Director of the Clinical L a b o r a t o r y of the Charity Hospital of N e w Orleans, is gratefully acknowledged for providing h u m a n blood serum samples, Including the total cholesterol values assayed with the P a r a m a x cholesterol reagent system
Brief This work represents the first successful fiber optic sensor for the determination of total cholesterol due to colmmoblhzatlon of cholesterol esterase and cholesterol oxldase The method is fast, repeatable, and accurate in the desired clinical range In comparison to a clinical method based on the Trlnder reaction, the correlation coefficient is 0 949
References I 2 3 4 5 6 7
Conclusion
8
This study d e m o n s t r a t e s the feaslbdlty of a photometric F I A for cholesterol with a fiber optic detector To the best of our knowledge, this is the first reported continuous determination of both free and total cholesterol using an optrode transducer The proposed approach was successfully used for the analysis of serum samples M o r e o v e r , it clearly shows that fiber optic sensor technology IS a credible and attractive alternative to electrochemical sensors
9
316
Enzyme Microb Technol, 1992, vol 14, April
10 I1 12 13 14
Zak B and Artlss J D M t c t o c h e m J 1990, 41,251 Schmld R D and K u n n e c k e , W J Bu~te~hnol 1990 14, 3 Karube, l Hara J M a t s u o k a H ,and Suzuki S Anal Chtm A~ta 1982, 139, 127 Yao, T and K o b a y a s h l Y Ban~el~t AagaLu 1983, 32, 25~ M a s o o m M and T o w n s h e n d A Anal Chin7 4~ta 198s 174, 293 Yao, T and W a s a T Anal Chtrn A~ta 1988 207, 319 Petersson, B A H a n s e n E H and Ruzlcka J Anal L~tt 1986, 19, 649 Malavoltl, N L Pdosot D and N l e m a n n T ~ Anal Chtm 4~ ta 1988, 170, 199-207 F e r n a n d e z - R o m e r o J M L u q u e de Castro M D and Valcarel M Chn Chtm A~ta 1987 167, 97 Trettnak W and Wolfbels O S Anal Bto~hem 1990 184, 124 Kahle K , Weiss, L Klarweln, M and Wleland O Z 4 h a / Chem 1970 252, 228 Majklc, N and Berkes l Chn Ctum A~ta 1977 80, 121 Abdel-Latlt M S , Sulelman, A A and Gudbault G G Anal Lett 1988, 2 1 , 9 3 4 Tletz N W Fundamental~ oJ Chm~al Chemtstt~ 3rd ed W B Saunders C o m p a n y Phdadelphla, 1987 p 948
A flow mjectton method f o r the detelmmatton o f total and free cholesterol ts ppesented Cholesterol e~terase and cholesterol oxlda~e are tmmobthzed on ammoalkvl glass beads The beads are packed mto a tabular glass reactor The cholesterol esters traversmg through the esterase reactor are cleaved to cholesterol and fatty actds The ovtdase reactor converts cholesterol to ~holest-4-en-3-one and hydrogen pero~tde ts generated The sample stream t~ merged wtth reagent ~treams conststmg oJ a peroatda~e solution and a solutton o f 2,2'-azmo-bts-(3-ethvl-benzthtazohne-6-sulfomc acid) &aminomum salt, and a hsdrogen perortde-dependent color teactton taLe~ place m a short coded reactor The signal l'¢ momtored b3 means o f fiber optw mstrumentatton Cholesterol concentration c an be 1elated to the ab~orptton o f the oJtldtzed dye form at a wavelength oJ 425 nm The ~orl, mg range lS 0 5 - 0 8 mmol l l, and the sample throughputs are 60 and 30 h -I for free and total cholesterol, respectwely
Keywords Cholesterol,flow rejection fiber optic cholesteroloxldase cholesterolesterase
Introduction Although the number of pubhcatlons dedicated to the determination of cholesterol ts enormous, the effort of developing new methods continues unremittingly ~ This report outlines a method for the determination of free and total cholesterol utilizing flow injection analysis (FIA) and fiber opttc technology FIA has become a powerful analytical tool in the field of biochemistry and blotechnology 2 The conjunction of enzyme- or antibody-based reactions with FIA has proven its appllcabdlty to btologlcal samples In the last decades, countless methods have been developed for the determination of free and total cholesterol Enzymatic procedures have virtually replaced chemical methods in the clinical laboratory The enzymatic assay of total cholesterol employs cholesterol esterase (ChE) to cleave the cholesteryl esters to cholesterol and fatty acids (FA) In a second enzymatic step, cholesterol ts oxidized to cholest-4-en-3one by cholesterol oxldase (ChOD) in the presence of oxygen The typical feature of this oxldase-catalysed reaction is the production of hydrogen peroxide In
Address reprint requests to Dr Gullbaultat The Universityof New Orleans, Department of Chemistry, New Orleans, LA 70148 Recmved 3 July 1991, rewsed 8 October 1991
© 1992 Butterworth-Hememann
most cases, hydrogen peroxide is quantified spectrophotometrically or electrochemically In combination with FIA, several principles have been utilized for the detection of the enzymatlcally generated hydrogen peroxide The earher flow inJection methods for the determination of free and total cholesterol featured amperometrlc detecUon 3-6 Recently, several photometric flow injection approaches have been proposed for assaying cholesterol Petersson et al 7 and Malavoltl et al 8 developed flow lnjechon systems using lumlnol-H202 chemiluminescence, and Fernandez-Romero et al 9 proposed several photometnc and fluorlmetnc methods, either with dissolved or lmmobthzed enzymes The first fiber optic cholesterol blosensor, reported by Trettnak and Wolfbins, m is based on lmmoNhzed cholesterol ox~dase and an oxygen-quenched fluorescent reaction However, this optrode has a response time of 7 to 12 mm and is useful for the assay of free cholesterol only In this study, we investigated the feaslblhty of a photometric method utilizing a fiber optic detection approach for the determination of free and total cholesterol employing the dye 2,2'-azlno-bis-(3-ethylbenzthiazohne-6-sulfonlc acid) dlammonlum salt (ABTS) The procedure is based on the following reacUon scheme Cholesteryl ester + H20
ChE, cholesterol + FA
Enzyme Mmrob Technol, 1992, vol 14, April
:313
Papers Cholesterol + O~
~hO~ cholest-4-en-3-one + H202
H202 + "~ A B T S + "~ H +
]
'1
HRP 2H~O + 2ABTS +
ABTS forms a stable radical cation with H202 in the presence of horseradish peroxldase (HRP), which has a strong absorption band around 425 nm ABTS has p r o v e n useful for clinical application by Kahle et al J~ Majklc and Berkes ~2 used this dye for the determination of free and estenfied cholesterol by a kinetic method, and A b d e l - L a t l f et al t~ developed a fiber optic glucose sensor based on this color reacuon
Experimental Matermls Cholesterol esterase (ChE, EC 3 1 I 13) from P~eudomonas species, w~th a specific activity of 112 U mg solid, and cholesterol oxldase (ChOD, EC 1 13 6) from Stteptomyces species, with a specific actlwty of 23 5 U mg -~ sohd, were purchased from T o y o b o (Osaka, Japan) Cholesterol, cholesteryl-10-undecenoate, Triton X-100, blhrubln, 2,2'-azmo-bls-(3-ethylbenzthlazohne-6-sulfonlc acid) d l a m m o n l u m salt. and horseradish p e r o x l d a s e (HRP, EC I 11 1 7) with a ~peClfiC activity of 120 U mg ~ sohd were obtained from S~gma (St Louis, MO) The alkylamlne glass beads with a mesh s~ze 120/200 were a product of Cormng (Cornlng, NY) All other chemicals were of analytical reagent grade
A p p a t atu~ The hght source (quartz lamp), fiber optic bundle, m o n o c h r o m a t o r , photomultlpher, and detection umt, all from Oriel (Stratford, CT), were assembled as shown In Ftgute 1 The t~p of the fiber was incorporated Into a flow-through cell machined from a block of polytetrafluroethylene (PTFE) A c y h n d n c a i hole, which fit the tip of the fiber optic bundle tightly, was drilled Into th~s block The cylindrical c a w t y underneath the polished end of the fiber bundle measured 0 75 m m in height, with a diameter of 6 m m and was equipped with an inlet and outlet conduit To ensure absolute llght-t~ghtness, the whole flow cell was embedded in a housing made of gray PVC The flow scheme is depicted m Ftqute 2 The tubing used con-
Figure 2 FIA manifold for the determination of total cholesterol C, Carrier stream, R1, ABTS solution, R2, HRP solution, P, per~stalttc pump, S, sample m]ectton, ER~, enzymatic reactor w~th immobdlzed ChE, ER2, enzymatic reactor with ~mmobd~zed ChOD, CR, 30 cm coded reactor, FD, fiber optic flow detector, W, waste ER~ was omitted for the determination of free cholesterol
slsted of flexible P T F E with an inner diameter of 0 75 m m and was purchased from Small Parts Inc (Miami, FL) Tubing connectors were obtained from the same c o m p a n y For the enzymaUc reactors, glass tubes w~th an inner diameter of 1 5 m m were cut to the right length and the P T F E tubings were glued m with epoxyamine resin on both sides A Supelco (Bellefonte, PA) N-60 six-way valve served as a sample mjecto~ and the fluids were transported with a peristaltic p u m p (Ramm I n s t r u m e n t Co , Woburn, MA)
Preparation of c h o l e s t e t o l a n d reagent s olutton3 Cholestelol solutions in the concentration range of 0 5 to 5 mmol I J were prepared as described previously ~' Cholesterol was dissolved in 12 8 ml lsopropanol, 3 85 ml of Triton X-100 was added, and after homogenlzatlon, the volume was made up to 100 ml w~th a 0 [ M phosphate buffer, p H 7 0 The soluUons that ~ e r e needed for the assay of total cholesterol contained cholesterol and cholesteryl-10-undecenoate m such amounts that the ratio of free to estenfied cholesterol was 30 70 Due to the low solubility of cholesteryl-10undecenoate in l s o p r o p a n o l - b u f f e r mixtures, the solvent used was lsopropanol containing 4'Yc Triton X-100 for all concentrations from 1 to 8 mmol l ~ A 10 mM ABTS solution was prepared by dissolving 549 mg m 100 ml H~O An H R P solution with an activity of l0 U ml ~ was prepared by dissolving 8 3 mg sohd e n z y m e in 100 ml 0 1 M p h o s p h a t e buffer, pH 7 0 The buffer mixture needed as carrier stream was prepared by dissolving 38 5 ml Triton X-100 in 128 ml of lsopropanol and finally making the volume up to 1,000 ml ~ lth a 0 1 M phosphate buffer, p H 7 0
l m m o b d l z a t t o n oJ enzyme,s
Figure 1 Block diagram of fiber optic setup L, Light source, F, fiber optic bundle, M, monochromator, BF, bifurcated fiber optic bundle, FD, fiber optic flow detector, P, photomultlpher tube, R, photomulttpher readout, C, chart recorder
314
EnzymeMicrob Technol, 1992, vol 14, April
One hundred milligrams of a l k y l a m m e glass was weighed into a 25-ml E r l e n m e y e r flask, and 10 ml of 2 5% glutaraldehyde solution in 0 1 M phosphate buffer, p H 7 0, was added For the first 15 m m the flask was held under reduced pressure to degas the glass beads After a total of 60 rain the glutaraldehyde solution was r e m o v e d and the beads were washed with phosphate buffer The glass beads exhibited an orange color after glutaraldehyde activation The beads were suspended in 2 ml 0 I M phosphate buffer, pH 7 0,
Flow mlectton determmatton of cholesterol A Krug et al containing 5 mg C h E This mixture was allowed to stand for 60 mln at r o o m t e m p e r a t u r e , initially under reduced p r e s s u r e C h O D was immobilized on a second batch of alkylamine glass b e a d s exactly in the same way as C h E W h e n the l m m o b i h z e d e n z y m e s were not used, they w e r e kept m a refrigerator at 4°C For actual use in the F I A system, the immobilized e n z y m e preparations were p a c k e d into the glassy column reactors
350
/
300
/ /
250 ,/-
tf /
~ 200 ~2
Results and discussion
/*
E 150
Opnmtzanon of FIA mamfold
~
,
/*
To obtain the best results, mlhally several p a r a m e t e r s were varied to test the F I A manifold Carrier stream, A B T S solution, and H R P solution were p u m p e d at a flow rate of 4 5 ml rain -~ to ensure turbulence and p r o p e r mixing F o u r different sample volumes (10, 25, 50, and 90/~l) were considered for injection Finally, the injection of 50/~i turned out to have the best characterlshcs, l e the proportion of peak height to peak width was optimized The e n z y m a t i c reactor consisted of a tube 80 m m long packed with ChE immobilized glass beads o v e r a length of 40 m m and with C h O D beads o v e r the residual 40 m m F o r the F I A measurements of free cholesterol, a C h O D reactor with 30 m m was sufficient The coiled reactor after the confluence of the enzymatlcally degraded sample with the reagents was chosen to be 300 m m In the case of the F I A manifold for free cholesterol, the determined dlsp e r s m n was 3 1 and the sampling frequency ran up to 60 samples per hour The overall dispersion coefficient for the total cholesterol runs was 4 2 and 30 samples could be injected per hour The A B T S and the H R P had to be supplied in two separate reagent streams, because of the inhibitory effect of the dye on the enzyme
from 1 81 to 8 03 m m o l 1-j 14 Approximately 70% of the total cholesterol is esterlfied with fatty acids, and therefore the free cholesterol ranges from 0 54 to 2 41 mmol 1-1 H o w e v e r , the calibration could not be extended a b o v e 8 mmol 1 l total cholesterol, because at higher concentrations o f our standards the cholesterol started to precipitate in the enzymatic reactor after being mixed with the carrier stream The relative standard deviation for 10 replicate injections of cholesterol standards was found to be 2 5% for a 2 mM solution, 4 l%at5mM, and52%at8mM
Cahbratton for free and total cholesterol
Stabdttv
Table 1 shows the calibration data for free and total
The e n z y m a t i c reactors were used continuously to check the stability of the l m m o b l h z e d ChE and C h O D The C h E lost more than 20% of its activity within 3 weeks, whereas the C h O D exhibited 90% of its initial activity after I month
/
/
50
/ / /
/
/
0
I 50
0
_ I [ I I I 100 150 200 250 500 Paromox cholesterol reagent m g / d l
350
Figure 3 C o m p a r i s o n o f FIA m e t h o d w , t h an established cholesterol r e a g e n t used m chmcal l a b o r a t o r i e s
cholesterol The correlation coefficients for free and total cholesterol were 0 985 and 0 965, respectively L m e a r I t y m the lower concentration range and the detection limit were not investigated We only aimed at a sufficient linear range covering the reference ranges of free and total cholesterol in h u m a n blood serum samples The reference range of total cholesterol in serum or plasma of infants, adolescents, and adults extends
Table 1
/
Accuracy The p e r f o r m a n c e of the p r o p o s e d F I A a p p r o a c h was c o m p a r e d with the procedure established in clinical
C a h b r a t m n data f o r free and t o t a l c h o l e s t e r o l Cholesterol ( m m o l I 1)
Peak h m g h t a (poA) Peak h e , g h t b (/~A)
05
1
2
3
4
5
6
7
8
5 6 -
9 6 84
13 6 14 3
20 6 22 5
27 3 26 0
30 5 30 4
34 3
37 3
40 0
a Free c h o l e s t e r o l / ~ A = 6 0 1 × m m o l l 1 + 201 b Total c h o l e s t e r o l /~A = 4 9 1 × m m o l l 1 + 4 0 5
Enzyme M,crob Technol, 1992, vol 14, April
315
Papers use We analyzed 26 serum samples and correlated our values to those obtained using the P a r a m a x cholesterol reagent, as shown in the correlation graph (Ftgure 3) The values were c o n v e r t e d to the more clinically relevant mg dl J units The graph has a slope of 0 88 and a correlation coefficient of 0 949 It ~s concluded that our method leads to lower levels, especially at higher cholesterol concentrations This could be attributed to our calibration method, which e m p l o y s h o m o g e n e o u s cholesterol solutions H e n c e the total a m o u n t of cholesterol is available for enzymatic degradation in the C h E and C h O D reactors In the case of the serum samples, some of the hpoproteln particles are not accessible to the enzymatic reaction, b e c a u s e they are not dissolved fast enough by the surfactant This d r a w b a c k could be avoided either by preconditioning of the serum samples by addition of surfactant or by calibration w~th serum samples
Interference studte~ We investigated the interference of blhrubln, which is considered to be one of the most troublesome factors in cholesterol assays ~ N o interference was detected from bihrubm concentrations up to 20 mg dl ~ which is a p p r o x i m a t e l y 20 times the normally occurring blllrubln levels
Acknowledgements This w o r k was supported by a grant from the Louisiana Education Quality Support Fund ( L E Q S F - R D - B 17) and the Austrian Fond zur Forderung der Wlssenschafthchen F o r s c h u n g (Project 7181-CH E) Dr W T Wu, Director of the Clinical L a b o r a t o r y of the Charity Hospital of N e w Orleans, is gratefully acknowledged for providing h u m a n blood serum samples, Including the total cholesterol values assayed with the P a r a m a x cholesterol reagent system
Brief This work represents the first successful fiber optic sensor for the determination of total cholesterol due to colmmoblhzatlon of cholesterol esterase and cholesterol oxldase The method is fast, repeatable, and accurate in the desired clinical range In comparison to a clinical method based on the Trlnder reaction, the correlation coefficient is 0 949
References I 2 3 4 5 6 7
Conclusion
8
This study d e m o n s t r a t e s the feaslbdlty of a photometric F I A for cholesterol with a fiber optic detector To the best of our knowledge, this is the first reported continuous determination of both free and total cholesterol using an optrode transducer The proposed approach was successfully used for the analysis of serum samples M o r e o v e r , it clearly shows that fiber optic sensor technology IS a credible and attractive alternative to electrochemical sensors
9
316
Enzyme Microb Technol, 1992, vol 14, April
10 I1 12 13 14
Zak B and Artlss J D M t c t o c h e m J 1990, 41,251 Schmld R D and K u n n e c k e , W J Bu~te~hnol 1990 14, 3 Karube, l Hara J M a t s u o k a H ,and Suzuki S Anal Chtm A~ta 1982, 139, 127 Yao, T and K o b a y a s h l Y Ban~el~t AagaLu 1983, 32, 25~ M a s o o m M and T o w n s h e n d A Anal Chin7 4~ta 198s 174, 293 Yao, T and W a s a T Anal Chtrn A~ta 1988 207, 319 Petersson, B A H a n s e n E H and Ruzlcka J Anal L~tt 1986, 19, 649 Malavoltl, N L Pdosot D and N l e m a n n T ~ Anal Chtm 4~ ta 1988, 170, 199-207 F e r n a n d e z - R o m e r o J M L u q u e de Castro M D and Valcarel M Chn Chtm A~ta 1987 167, 97 Trettnak W and Wolfbels O S Anal Bto~hem 1990 184, 124 Kahle K , Weiss, L Klarweln, M and Wleland O Z 4 h a / Chem 1970 252, 228 Majklc, N and Berkes l Chn Ctum A~ta 1977 80, 121 Abdel-Latlt M S , Sulelman, A A and Gudbault G G Anal Lett 1988, 2 1 , 9 3 4 Tletz N W Fundamental~ oJ Chm~al Chemtstt~ 3rd ed W B Saunders C o m p a n y Phdadelphla, 1987 p 948
