Analytical applications for routine use with immobilized enzyme nylon tube reactors

Analytical applications for routine use with immobilized enzyme nylon tube reactors P. V. S U N D A R A M Abteilung Klinische Chemie, Medizinische Poliklinik, D-3400 Gdttingen, West Germany

Summary. Analytical systems have been developed for the automated assay o f urea, uric acid, glucose, pyruvate, lactate, creatinine, creatine, glycerol, triglycerides and cholesterol. These analytical systems consist o f hollow tubes o f polymeric nylon to the insides o f which specific enzymes are covalently immobilized and analysis is performed by perfusion o f the sample through the reactors at a rapid rate o f 5 0 - 6 0 tests per hour. The design and development o f these reactors that show high specificity and operational dependability, as demonstrated by detailed clinical trials, is discussed. All the metabolites mentioned above can be detected by the linear range clinically relevant in these reactors, which are very stable during use in continual analysis and storage. Keywords: Analysis;clinical chemistry; immobilized enzymes;

metabolites; nylon tube reactors; automated assay.

samples and drugs used in therapy impose more exacting limitations. Thus, the reliable analysis of biological samples is a far more difficult task than detecting samples in pure aqueous solutions. The concept of immobilizing enzymes onto the inner surfaces of thin nylon tubes for analysis was explored by Sundaram and Hornby.1 Since then, this idea has been further developed in our laboratories during the last ten years and brought to a stage where routine application of these reactors in conjunction with autoanalysers of the Teclmicon type has become possible. These nylon tubesupported enzymes are referred to as Immobilized Enzyme Nylon Tube Reactors for brevity; i;:dividual reactors are often called urease reactors, a glucose dehydrogenase reactor or, in the case of a coupled enzyme system, as a pyruvate kinase-lactate dehydrogenase (PK-LDH) reactor.2,3

Objectives and practical considerations Introduction The demand for highly specific routine analytical methods utilizing naturally occurring catalysts or macromolecules such as enzymes, antigens and antibodies has increased dramatically in the last two decades. Today, these routine analyses are the norm; indeed, some of the recent advances in therapy would have been impossible without the current sophistication in clinical chemistry. Because of the ease of access to these sophisticated tests, the minimum number of tests automatically requested by the physician, even for a patient who attends for a periodic check-up, has increased so much that the cost of medical care is escalating. Consequently, a more recent development in this area has been the application of immobilized enzymes in place of enzyme solutions in analysis because the former can be used repeatedly, with considerable cost savings. However, any new method must be capable of a rapid turnover of at least 5 0 - 6 0 tests per hour. Routine analysis at a rapid rate using the flow-through principle seems to be the best way to approach the problem because automatic analysers of this type are already available. In developing systems for analysis of metabolites such as urea, glucose, uric acid, creatinine, triglycerides and cholesterol rigid requirements have to be met not only with respect to overall sensitivity but very often this high sensitivity is required in a specific concentration range. Interferences from natural substances present in biological

290

Enzyme Microb. Technol., 1982, Vol. 4, September

Some of the following objectives have to be satisfied in developing immobilized enzyme systems for routine use in analysis: (a) sufficient activity of the enzyme must be obtained upon immobilization; (b) the enzyme should be stable in operation and storage; (c) the conditions of operation should be made favourable by exploiting the possibilities of changing kinetic properties after immobilization; (d) ultimately, repeated use in routine analysis should be the aim so that (1) the cost of operation is actually reduced, (2) cumbersome procedures are eliminated and the method does not call for more expensive equipment, (3) the new method should improve existing precision and, (4) the method must be reliable and reproducible. A vast number of polymers of various physical forms, such as powder, flakes, gels, beads, films, fabric and hollow tubes, are available as solid supports for the immobilization of enzymes. When it comes to analysis using rapid sampling flow-through a hollow tube is recognized to be the best physical form. Among polymers such as polyethylene, polypropylene, polystyrene, polycarbonate and nylon, all of which are available in the form of thin tubes, nylon is the most 0141 --02291821050290--09$03.00 o 1982 Butterworth & Co. (Publishers) Ltd

Immobilized enzyme nylon tube reactors: P. V. Sundaram

suitable support because it has good flow characteristics, it is compact, has good wetting properties, is easy to derivatize, and is inexpensive and easily available.

-C = NHI

0 -

Methods of activation of nylon Approaches to devising methods for covalent coupling of enzymes to nylon tubes, or any other polymer intended for routine production, should alwaYs be guided by the following considerations: ease of operation and minimum operating time; low cost; safety of the method; ease of availability of chemicals used in activation; and the method should have a high enough capacity for fixing enzymes. Recognizing these criteria, two different approaches are possible: coupling to partially hydrolysed nylon, and direct activation of native nylon to which an enzyme may be coupled with or without an additional step, the latter facilitating'further derivatization of the polymer via addition of a spacer molecule. The first method consists of controlled partial hydrolysis of nylon by acid,1 which releases the NH,~ and COOgroups, one for each amide bond cleaved. These two groups may be further utilized in coupling reactions. 1'4 The second method, that of direct activation, consists of O-alkylation of native nylon with reagents such as dimethyl sulphate (DMS) (Sundaram s) or triethyloxonium tetrafluoroborate (TTFB) as in Morris et al. 6 and Sundaram.7 This O-alkylation of nylon yields an imidoester derivative which upon treatment with an amino group yields an amidine or substituted amidine in the case of an enzyme. A detailed investigation of this procedure and the properties of the O-alkylated product, namely the imidoester of nylon reported by Sundaram,s has demonstrated the reversible nature of the coupling procedure. This does not, however, preclude the successful use of this method for producing reactors for prolonged use as an analytical tool. Other methods of activation of nylon for coupling enzymes and other ligands are discussed in an earlier report by Sundaram. 7 Coupling o f e n z y m e s Enzymes may be crosslinked to the amino groups of partially hydrolysed nylon using bifunctional reagents such as glutaraldehyde, terephthalaldehyde or bisimidates.4 Similarly, the COO- groups released by hydrolysis may be activated by reagents such as carbodiimide or N- eth oxy carb onyl-2- ethoxy- 1,3- dihydroquin oline (EEDQ). 9 Coupling yields by these methods vary, and by far the easiest method is crosslinking with a dialdehyde. Direct O-alkylation of native nylon has advantages in that it prevents physical alteration or damage to the nylon surface which may be caused by hydrolysis and every amide bond produces a pair of opposite charges which, after crosslinking with a dialdehyde, leaves behind negative charges (of C 0 0 - groups remaining) whereas positive charges will remain if CO0- groups are activated for coupling enzymes. This does not occur when enzymes are coupled directly to O-alkylated nylon, although a residual positive charge remains on the amide N that is alkylated; the charge density in this case is probably less than in the case of hydrolysed nylon. Polyanionic, polycationic or neutral derivatives of nylon can also be produced by linking spacer molecules of defined characteristics to alkylated nylon. Thus, enzymes may be attached directly

ee

@ Uricase =

= ,~H-

-C =,'~H-

;C-~H 5 NIHz "(CH2 -CH-CH-CH)~

-C= NH~.~ HN- CH

IqN-uricese

PEI

I

H C - NH z Nylon- PF_.[ cepolymer

CH 2 ...p

OHC(CHz)3 CHO e -C=NH-

Uricase

(~) =,

HN-CH I

HCI

• -C=NH

-

HN - C H I

H

HC-N = ClCHzl3CHO

HC

g H H Hill - N = C(CHz)3C = N - uricose

CHz

-.p

Nylon - P E I - uricose

Figure 1 Chemical reaction schemes for attaching an enzyme (uricase) directly to an O-alkylated nylon and indirectly to n y l o n PEI copolymer [Reproduced from Sundaram, P. V., Igloi, M. P.,

Wassermann, R. and Hinsch, W. C/in. Chem. 1978, 24, 1813--1817 by permission of the American Association for Clinical Chemistry © ]

to the alkylated nylon tube by filling it with an enzyme solution, or to the spacer molecule by a suitable method as detailed in Sundaram and Apps 1° (Figure 1).

Characterization of reactors and optimization of conditions During characterization the reactors are connected to a peristaltic pump and substrate(s) are pumped through at specific flow rates. The ,product in the effluent is analysed manually either by direct spectral measurement, in the case where substrates (including cofactors) show a spectral shift after the enzymic reaction, or the product is analysed by a specific colour reaction. Such characterization becomes necessary since immobilization invariably affects not only the specific activity of the enzyme but many of the kinetic properties often change along predictable lines. (i) Specific activity is invariably reduced. 0i) The pH optimum may change, either slightly or dramatically. Such a shift can be used beneficially (Figure 2), as in the case of the glycerol dehydrogenase reactor catalysing the breakdown of glycerol. (iii) Binding constants such as K m are likely to change in both directions. The magnitude of the change can vary from a small, insignificant increase or decrease to a sizeable one that may affect the efficiency of catalysis. Factors (i) and (iii) are somehow interrelated: specific activity is reduced if a proportion of the active sites are not available for catalysis because they are covered or hidden; and the activity of available active sites may be affected by altered binding constants, with the possibility that the latter could be further complicated by diffusional effects. Although at first sight a reduction in activity due to poor binding o f enzyme and substrate, as reflected by an increase in/(ampp, may appear to be a disadvantage, there are instances

Enzyme Microb. Technol., 1982, Vol. 4, September

291

Reviews

1.0

g 0.5 :g E3 (.9

0

1 8,0

I I0.0

1

9.0

I I I.O

I 12 0

pH

Figure 2 pH dependence of glycerol dehvdrogenase activity in solution (e) and in the immobilized form (o) [Reproduced from Hinsch, W. and Sundaram, P. V. Clin. Chim. Acta 1980, 104,

87--94 by permission of Elsevier North-Holland Biomedical Press© ]

where a diminished rate can be turned into an advantage with respect to designing an analytical application using an immobilized enzyme (e.g., a progress curve of (o) vs. (s) may show a curvature within a small concentration range of 1 to 10 mM substrate because the order of the reaction changes from second order to zero order within this range). Thus, it is difficult to achieve operable linearity within these two limits. However, because of a reduction in rates, the same 1 to 10 mM concentration range may still be part of the initial second order segment of the progress curve in the case of the immobilized enzyme. In many cases, problems such as these have been encountered in our systems. Diffusion effects may become dominant at specific substrate concentrations and flow rates. Thus, kinetic analysis of a series of reactions may yield double-reciprocal plots (Figures 3a and 3b) which can be biphasic, or even worse. The example shown here is that of glycerol breakdown by a glycerol dehydrogenase reactor. In such a system, one has to chose carefully the operational conditions, such as the potential substrate concentration and flow rate, so that the reaction occurs within a known phase which is linear. For the same reason, analysis of kinetic data along conventional fines may not be possible since more than one value for/(ampp and Vmax may be obtained. Sometimes, meaningless values are obtained because of a negative intercept upon extrapolation. It is thus necessary to investigate the kinetics of each system thorouglfly before they are incorporated into a flow system for automated analysis.

_...124A

}

R o u t i n e analysis b y i n c o r p o r a t i o n w i t h an a u t o a n a l y s e r

~ 62.2

Normally, 5 0 - 6 0 analyses per hour are expected of a system that uses the flow-through principle in automated analysis. In all the systems developed for autoanalysis using immobilized enzyme nylon tube reactors we have used either the AutoAnalyzer I (AA I) or II (AA II) system manufactured by Technicon Instruments Corp., Tarrytown, NY, USA. These reactors are integrated into the flow system, and are always connected to the circuit after the dialyser, so that the dialysed sample is free of all high molecular weight compounds such as proteins. The sample then enters the reactor and the effluent is automatically analysed for the product. At the end of each day, the reactors are washed and stored, filled with a suitable buffer at 4°C. Further details, including flow diagrams for all the systems, have been described previously.2' n-2o

0

02

O.15

F e a t u r e s o f each s y s t e m

0"05I

OIb 0

5

I0

15

20

v/S (s) Figure 3 (a) kineweaver--Burk plot of glycerol breakdown in the concentration range 0.002--1.0 mM by a Gly-DH reactor at flow rates 0.16--3.9 ml min -~. (b) Eadie--Hofstee plot of the data in

(a) (see Figure 3 and Table 1 of ref. 15)

292

Enzyme Microb. Technol., 1982, Vol. 4, September

Blood urea determinations can be made quite satisfactorily with reactors consisting of minimally purified urease (3500 Sigma Units or 28 kU g~l) or with a highly purified urease S (45 kU g-l). Performance characteristics are shown in Table 1. Urinary urea may also be determined, but the sample is well diluted before analysis. A novel approach to the assay of citrulline using an urease reactor has been devised. This method depends on the differential assay technique, n taking advantage of the fact that Ehrlich reagent reacts with both urea and citrulline, but not with ammonia. Thus, a serum sample after a sufficiently long residence time in a urease reactor has all its urea converted to ammonia, and subsequent colorimetry

Immobilized enzyme nylon tube reactors: P. V. Sundaram Table 1 Performance characteristics of various immobilized enzyme nylon tube reactors Optimum pH Substance analysed

Enzyme system

Urea Citrulline

Urease Urease

Uric acid

Uricase

Pyruvate Lactate Glucose

Glycerol Triglycerides ATP, PEP, NADH Creatinine Creatine Adenosine Adenine Adenine nucleotides Malathion Parathion

Uricase/ALDH Uricase/ NADH-Perox LDH LDH/A LT Gluc-DH GOD/ALDH GOD/ NADH-Perox Gly-DH Gly-DH PK/LDH Creatininase/ CK-PK/LDH CK/PK--LDH Ad. deaminase Alkaline phosphatase/ Ad. deaminase Parathion hydrolase

Method of analysis

Free enzyme

Immobilized enzyme

Average no. of tests

Concn range

Test of analyser

7.0 7.0

7.0 7.0

2000

- 3 3 mM

AA I

20--120 mg1-1

AAII

Berthelot reaction Ehrlich reagent Diff. assay Peroxidase Pap test NAD a NADH

7.5--9.5

7.0

4000

7.0/9.0 7.0/6.O

8.6 7.0

5000

NADH NAD NAD NAD a NADH

7.0/ 8.0 7.0--7.6 5.6/9.0 5.6/6.0

8.0 9.4 6.8-7.6 8.0 7.0

4000 (min.) 2000 3500

--1 mM --10 mM --4000 mg 1-1

AA I/Eppnd 4412 AA I/Appnd 4412 A A II

NAD NAD b NADH

8.5

3500 2000

--240 mg 1-1 --4000 mg I-1

AA I I AA I I

9.0/7.0

NADH

8.0/9.0

NADH 264 nm

9.0 7.0-7.4

10.0 10.0 8.0

AA I I

8.0 8.5-9.0 8.5--9.0 7.0

259nm

10.0/7.0--7.4

8.0

440 nm

10.0

8.0--10.5

a Catalase and ethanol added in solution b Lipase and esterase added in solutions

measures only citrulline, whereas analysis of an untreated serum gives a value for urea plus citrulline. Analytical recovery of citrulline in the concentration range 0.1 to 1.0 m~t is very good by this method. Uric acid estimation using a reactor containing microbial uricase 12 and peroxidase solution is a good method with excellent precision and recovery. The reactor is stable for at least 5000 tests. Peroxidase was not immobilized because it is difficult to stabilize, but fortunately it is also relatively inexpensive. Glucose estimation using a microbial glucose dehydrogenase (EC 1.1.1.47), supplied by E. Merck GmbH, immobilized to make a reactor has been employed using 1.0 mM NAD as the cofactor. This method (flow-diagram in Figure 4) proved to be excellent, specific and inexpensive.23 Low levels of cofactor are required for this test, in contrast to commercial test-kits. Methods have been developed for the routine determination of pyruvate and lactate, the former with the aid of a lactate dehydrogenase reactor and the latter with a reactor made by coimmobilization of lactate dehydrogenase and alanine aminotransferase (known also as glutamate-pyruvate transaminase). There has recently been an increased need for lactate determination due to the dangers of high lactate levels in diabetic patients undergoing biguanide therapy. The flow diagram using this coupled enzyme system 13 is given in Figure 5. Again, low levels of the cofactors NADH (for pyruvate estimation) and NAD (for lactate estimation) are used in these tests, 0.66 m~ in each case. Detection of creatinine and creatine using a four-enzyme and a three-enzyme system immobilized in nylon tubes and catalysing a sequence of reactions in which the last

OranQe-

~

5011

Woste Sumple White 0.60ml/min ~0.9% NaCt Orange-

white0.23 mt/min -'~ Air Red 0.80 mr/rain ~,Buffer Orangewhite 0.23 mL/min ~ A i r

lOT

25"C INyJon tube reoctor

](Gluc-DH) J

Waste~ Waste

White 0.60 mL/min

T Red 0.60 mL/min ~Water

340nm

Figure 4 Flow diagram of automated analysis of serum glucose with a Gluc-DH reactor [Reproduced from Sundaram, P. V., Blumenberg, B. and Hinsch, W. Clin. Chem. 1979, 25, 1436-1439 by permission of the American Association for Clinical Chemistry © ]

two enzymes are the pyruvate kinase-lactate dehydrogenase (PK-LDH) indicator system that measures ADP produced from within the system is a challenging approach to analysis. Creatininase Creatinine . ~ creatine creatine kinase Creatine + ATP - creatine phosphate + ADP Pyruvate kinase PEP + ADP = pyruvate + ATP Lactate dehydrogenase Pyruvate + NADH ~ lactate + NAD Provided that enough activity can be obtained of all the enzymes, the system can be optimized for routine analysis.

E n z y m e Microb. Technol., 1982, V o l . 4, S e p t e m b e r

293

Reviews

Tables 2 and 3 give details of all preparative methods of these multienzyme reactors and their kinetic properties. 14 Clinical trials have not been conducted with either of these systems that detect creatinine or creatine because reactor lengths upwards of 2 m are required to produce enough enzymic activity, which would involve a delay in the delivery time of the sample. Since the aim is to achieve: at least 50 tests per hour with flow-through analysers, further work would require more active preparations of the enzymes used in our system, or the availability of a single enzyme that cleaves either of these substrates (see Figures 6 and 7). Demand is increasing for an economical, fast, reliable and specific method for triglyceride estimation. Until recently, there was no enzyme-based method available for Woste

l

~ir 0 16 mr/rain Sompler Sornple 0.10r n t / r n i n ~

Diotyser {17.inch)

~uffer (soln.I]

3.32 rntlmin 40 sornples/h Sornple : wo~

2:1 ~*ir0.16mllmin ISoln.I[)O.42 mr/rain :Soln.n'r)0,10rnL/min 5 turns I LDH- GPTreoctor Woter

t.4OrnL/rnin

IO turns (ostel

.

10.32 m t / m i n - - - -

3:54nm~ _

Figure 5 Flow diagram of the analysis of lactate using an

L D H - G P T reactor linked to a modified Technicon AutoAnalyzer I [Reproduced from Sundaram, P. V. and Hinsch, W. C/in. Chem. 1979, 2 5 , 2 8 5 - 2 8 8 by permission of the American Society for Clinical Chemistry©]

this metabolite. Even the more recent method that employs glycerokinase requires a total of five enzymes, including the lipolytic enzymes and the P K - L D H detector system, to estimate the ADP produced in the reaction. Our method relies on the use of glycerol dehydrogenase, which is now available. Glycerol released from the fatty acid esters is directly accounted for by this enzyme, which requires high NAD concentrations and alkaline pH values for reactions conducted in solution. Immobilization on nylon shifts the pH optimum from 8.5 to 11.5 (Figure 2). This reactor is quite stable for repeated use at pH 10 for glycerol analysis. The high pH has to be maintained to shift the unfavourable reaction equilibrium for the conversion of glycerol to dihydroxyacetone. Also, the glycerol concentration has to be kept low since the product accumulated ~ ' o 20E .E E -~ 0152 E ~g olo~ E 0051 ~g 0L 0

& I

7

I

8 pH

I

9

E

b 01106

l

G053

t I

10

~

u

0

0

O 4

8 12 Time (rain)

16

Figure 6 (a) pH dependence of activities of a creatininase reactor assayed with: o, creatinase reactor + CK, PK and LDH in solution; ~7, separate reactors of creatinase and creatine kinase linked together and effluent assayed with PK and LDH in solution. (b) Effects of different buffers on creatine estimation with a creatininase reactor and CK, PK and LDH in solution, and N A D H concentration on the analysis of effluent from a creatininase reactor, o, Glycylglycine buffer, pH 8, 1 mM creatinine, 0.3 mM NADH; ~7, glycylglycine buffer, pH 8, 5 mM creatinine, 1 mM NADH; x, phosphate buffer, pH 8, 1 mM creatinine, 0.3 mM NADH; o, Tris buffer, pH 8, 5 mM creatinine, 1 mM N A D H (effluent diluted five-fold and reacted with PK--LDH solution); e, Tris buffer, pH 8, 5 mM creatinine, 1 mM N A D H (effluent treated directly with PK--LDH solution) [Reproduced from Sundaram, P. V. and Igloi, M. P. Clin. Chim. Acta 1979, 94, 295- - 307 by permission of Elsevier North-Holland Biomedical Press© ]

Table 2 Preparative methods and activities of immobilized enzyme nylon tube reactors. HPO4, phosphate buffer (0.1 M). Runs 7 and 8 separate activities of creatininase and creatine kinese for creatinine and creatine are abbreviated as C and CK, respectively. In Run no. 3 with a 3 metre creatininase (a) was assayed with CK reactor from Run no. 4 and (b) with CK reactor from Run no. 6. PK--LDH reactors were made with a nylon--PEI copolymer tube. All experiments were carried out at 25 ± 1°C and 0.33 ml min -~ flow rate of substrate. Activity measurements were made with 10mM substrate concentrations. [Reproduced from Sundaram, P. V. and Igloi, M. P. Clin. Chim. Acta 1979, 94, 295--307 by permission of Elsevier North-Holland Biomedical Press©} Polymer, coupling method

Run no.

Enzyme reactor

Reactor length

1

C

1m

2

C

1m

3

C

3m (thick)

Nylon--PEI

Nylon, direct Nylon--PEI

4

CK

1m

5

CK

1m

6

CK

7

C+CK

2m (thick) 3m

8

C+CK

3m

294

Nylon, direct Nylon--PEI

Nylon-PEI Nylon, direct Nylon, direct

E n z y m e M i c r o b . T e c h n o l . , 1 9 8 2 , V o l . 4, S e p t e m b e r

Enzyme used in coupling 2mg/1.5ml pH 8, HPO 4 1 mg/ml pH 8, HPO4 2.5 mg/6.5 ml pH 8, HPO4 recirculated 2 mg/ml pH 8, HPO4 2 rag/m! pH B, HPO, 2 mg/ml pH 8, HPO 4 lmgC+2mg CK/ml pH 8, HPO4, Mg 2+ 2.Stag C + 7 mg CK pH 8, HPO 4 (0.05 M)

Assayed with

Initial activity (#mol rain -1 m -] )

CK, PK and LDH solution CK and PK--LDH reactors P K - L D H reactor (a) 1 m CK (nylon) (b) 2 m CK (nylon- PEI) PK--LDH solution

0.0127

0.0106 0.0122 C.9223

PK--LDH reactor

0.0764

PK--LDH reactor

0.051

PK, L D H s o l u t i o n

C 0.014 CK 0.024

PK, LDH solution

C 0.0115 CK 0.0116

0.0191

Immobilized enzyme nylon tube reactors: P. V. Sundaram Table 3 Kinetic parameters of creatininase and creatine kinase reactors under optimized conditions. All reactors were made by crosslinking enzymes to a nylon--PEI copolymer tube on the same day: a 3 m long creatininase reactor was used in conjunction with a 1 m long creatine kinase reactor and a 2 m long P K - L D H reactor. Activities expressed as #tool rain -~ m -1 of creatininase or creatine kinase reactor. The length of the combined enzyme indicator ( P K - L D H ) reactor is not taken into consideration in activity expression. All measurements were made at 25 -+ 1°C and 0.33 ml min -I flow rate. Kinetic plots for various concentrations of ATP, PEP and N A D H (Run 4a) are shown in Figure 6a. [Reproduced from Sundaram, P. V. and Igloi, M. P. Clin. Chim. Acta 1979, 94, 295--307 by permission of Elsevier North-Holland Biomedical Press©]

Initial activity (/zmol min -~ m -1 )

K,~ p of different substrates (mM)

Run no.

Reactor specification

Assayed with

1 2 3

PK--LDH reactor CK reactor CK reactor

0.146 0.041 0.045

Pyruvate

0.95

4a

C + CK reactors

-PK--LDH reactor PK and LDH solution P K - L D H reactor

0.029

ATP 0.38 PEP 0.05 N A D H 0.061

Creatinine 4.14 Creatine 4.31

4b

C + CK reactor

0.033

4c

C reactor

PK and LDH solution CK, PK and LDH solution

5

C reactor (hydrolysed, crosslinked)

Creatinine 3.3

0.0726

CK, PK--LDH

N A D H 0.66

Creatinine various values - diffusion effect

0.0021

4.C

t.o I7

3£

0£

2.~= 0.6 ,<~ 2.C

.o <3 0.4

~.5,

0.2 05 0 o

a

J

I

2

I

I

~

I

i

I

4 6 8 f.Creatinine] or [.Creatine] (mu)

I

I

I0

0

-0.4

-0.2

0

0.2 0.4 06 0.8 I/[Creatinine] 0r [Creatine](ram-I)

1.0

Figure 7 Kinetic plots of (a) v vs. s and (b) Lineweaver--Burk plot f o r creatinine ( v ) and creatine (o) estimation with all four enzymes immo-

bilized [Reproduced from Sundaram, P. V. and Igloi, M. P. Clin. Chim. Acta 1979, 94, 2 9 5 - 3 0 7 by permission of Elsevier North-Holland Biomedical Press ©

inhibits the reaction. On the other hand, the sensitivity of detection by the autoanalyser does not permit the use of glycerol concentrations below a certain level. These factors were taken into consideration in devising a flow diagram. If the sample volume is low enough, tinearity is obtained up to a concentration of 0.8 raM, which is about four times the upper limit of glycerol concentration in normal human sera. Figure 8 shows the effect of the concentration of NAD, esterase and lipase on this automated method. Precision data are given in Table 4. Excellent correlation (r = 0.9949) when compared with the giycerokinase method was obtained for our new method tested on pure glycerol standards. Figure 9 is a regression plot of performance data for this method) s'16 Since glycerol estimation in food analysis is of interest, we also tested various beverages for glycerol using a modified

methodY which also performed well (r = 0.9988) and reactor stability was not affected by this application.

A novel heterogeneous multienzyme system for

H202 analysis For the first time, we have demonstrated t8-2° the feasibility of working with two immobilized enzymes separated by an enzyme in solution. In a three-step reaction, the first and third steps are catalysed by immobilized enzymes whereas the second step is catalysed by the enzyme in solution (Figure 10). This implies that H202, the product of the first step, has to diffuse into solution for ethanol to be converted to CH3CHO by catalase, which in turn has to be transported back to the solid phase for attack by ALDH. Despite this transport step which intervenes in

E n z y m e M i c r o b . T e c h n o l . , 1982, V o l . 4, S e p t e m b e r

295

Reviews

performed with each reactor before the activity drops to 25% of the original and autoanalyser sensitivity becomes limiting .20 Systems capable of measuring adenine nucleosides and nucleotides are the reactors containing coimmobilized alkaline phosphatase and adenine deaminase (Table 1 ). Pesticide detection in waste waters will be a great advantage in ecological monitoring. Parathion and allied organophosphorous pesticides may be detected using a reactor containing parathion hydrolase isolated from a mixed culture of Pseudomonas. 21 The product may be analysed spectrally at 440 nm.

IOC o T 9c

E o~

NAD final concentration (mmol I"1)

O p e r a t i o n a l a n d storage stability

I00 .'g

7

9(;

o; 0 0

I

IO I

I

20 I K)O0

I

I

I

50 40 Esterose (U ml-q I I 2000 3000 Lipose (U ml4)

30

I

60 I

Table 1 presents the data on the average number of tests that can be performed with the various types of reactors. Although the half-life of these reactors may differ, it is safe to assume that an average of 4000 tests may be performed with most of them. The reactors may be stored at 4°C for several months, but operational stability depends on the total number of tests carried out rather than the length of time. In other words, as explained in detail in ref. 8, the unusual reversible nature of coupling of enzymes to alkylated nylon is such that the coupled enzyme is

Figure 8 Optimal reaction conditions for automated determination of triglycerides using a Gly-DH reactor: (a) NAD + requirement; (b) requirement for esterase (o) and lipase (=) [Reproduced from Hinsch, W., Ebersbach, W.-D. and Sundaram, P. V. C/in. Chim, Acta 1980, 104, 95--100 by permission of Elsevier North-Holland Biomedical Press © ] Table 4 Precision data for triglycericde determination with the glycerol dehydrogenase method using control sera of different concentrations. [Reproduced from Hinsch, W., Antonijevic, A. and Sundaram, P. V. C/in. Chem. 1980, 26, 1 6 5 2 - 1 6 5 5 by permission of the American Association for Clinical Chemistry©]

4000 =% oo ""

3000

E

cv(%)

o

2ooo

Avarage concn (mg 1-1)

Day-to-day (n = 20)

Within a day (n = 20)

1040 115o 1190 1330 1730 a 3400 a

4.9 3.6 4A 3.8 3.7 3.0

3.2 1.8 1.8 3.1 1.5 1.6

E

I000

Z a

catalysis, the whole system functions unhindered. Glucose and uric acid may be determined by a GOD-ALDH or a uricase-ALDH reactor, catalase and ethanol being taken in excess along with the sample, to which NAD has been added. The appearance of NADH in the effluent gives a measure of the original H202-producing substrate. Cholesterol, which can also be estimated by this method, is determined using an ALDH reactor alone since cholesterol oxidase and esterase can not be immobilized in a stable form. First, the sample is treated with a mixture of cholesterol oxidase and esterase and runs through a channel that is incubated at 37°C long enough.for the enzyme mixture to produce H202 from the free and esterified cholesterol, which then passes through the dialyser block. A mixture of ethanol, catalase and NAD fed through another channel meets the sample and then enters the ALDH reactor before being read at 340 rim. An average of 4500 tests can be

E n z y m e M i c r o b . - T e c h n o l . , 1982, V o l . 4, S e p t e m b e r

I 1000 Cony.

apatient plasma-pool, kept at --20°C for day-to-day precision

296

Q o

8

I 2000

I 3000

I 4000

rneth, t r i g l . (rag ~'=)

F i g u r e 9 Statistical regression plot of the performance of a glycerol dehydrogenase reactor in the estimation of triglycerides [Reproduced from Hinsch, W., Ebersbach, W.-D. and Sundaram, P. V. C/in. Chim. Acta 1980, 104, 95--100 by permission of Elsevier North-Holland Biomedical Press© ]

Cholesterol esterase and oxidose

S

( Esterified and Glucose I free cholesterol uricoracid

~

~ e

h~de~.

= H I O ' z / ~ t o l o ~ NAD+ • ~thonol ~NADH + H* acetate

Ambient medium 10 Reaction scheme of heterogeneous multienzyrne systems containing immobilized aldehyde dehydrogenase Figure

Immobilized enzyme nylon tube reactors: P. V. Sundaram Table 5 Statistical parameters o f the performance o f various reactors

c v (%) Metabolite

Enzyme reactor

Urea

Urease reactor

Uric acid

Uricase reactor

Method compared to

A L D H reactor + uricase soln Uricase--ALDH reactor Gluc-DH reactor GOD--ALDH reactor GOD soln A LDH reactor LDH reactor L D H - - A LT reactor Gly-DH reactor Esterase-lipase (soln) Gly-DH reactor Cholesterol esterase/ cholesterol oxidase soln + A LDH reactor

Glucose

Pyruvate Lactate Glycerol Triglycerides

Cholesterol

Y

r

Within day

Day-to-day

1;3

3.3

2.3-8.9 a

3.1-6.4 b

1.5

4.0

DAM Urease soln Uricase/Pap soln Uricase/MBTH soln Technicon SMA 12 U r i c a s e / D M A - M BTH Technicon SMA12 Uricase/DMA-MBTH H K--G6PDH soln

0 . 9 6 X + 1.65 0 . 9 9 X + 2.08 0 . 9 8 X + 0.15 0 . 8 7 X + 0.69 0 . 9 9 2 X - 1.78

0.998 0.996 0.978 0.972 0.99

6 . 9 5 X + 2.17 1.067X + 2.22 0 . 9 8 X + 1.99

0.985 0.998 0.996

1.6--2.9 c

2.2--3.9 d

3.2--2.1 e

3.9-2.8 f

HK--G6PDH soln

1.0X-

0.997

0.6-1.4 g

2.4-3.3 h

H K--G6PDH soln

1.02X - 2.45

0.997

1.0-1.1 i

1.9-3.3J

LDH soln

1.081X - 2.5

0.997

3.3

5.0

L D H - - A L T soln

0 . 9 7 1 X -- 0.04

0.993

3.1

4.9

G K - - P K - L D H soln

1.02X + 0.017

0.996

1.7--2.7 k

3.3-5.5 •

G K - P K - - L D H soln

1.02X + 74.2

0.993

3.2-1.6 m

4.9-3.0 n

Technicon SMAC Cholesterol o x i d a s e PAP

0 . 9 9 X + 0.05

0.993

1.4-2.6 °

1.7--3.0 °

2.03

Uric acid: a62 to 23 mg r 1; b91 to 43 mg I-a ; c 1.6 to 2.9 mg 1-1 ; d2.2 to 3.9 mg 1-1 Glucose: e 750 to 2100 mg I- i ; t 750 to 2100 mg I- i ; g 1030, 2320 and 840 mg I- I ; h 2580 to 840 mg I-1 ; i1030 to 2320 mg I-1 ;J2940 to 1030 mg I- 1 Glycerol: k 0 . 1 6 to 0.7raM Triglycerides: m 1040 to 3400 mg 1-1 ; n 1040 t o 3400 mg 1-1 Cholesterol: o 1.4 to 2.6 mM ;P 1.7 to 7.0 rnM

I00(

~- 80- \x

8 :~ 6° N

"~" ~

-

o

=iO

" " ° - - - °x

20

6

o,

l 1 I I I 1500 2000 2500 3000 3500 Number of tests Figure 11 Typical activity decay curve o f an immobilized enzyme nylon tube reactor. Plots show behaviour patterns o f an A L D H reactor (e) and a G O D - A L D H reactor (o) [Reproduced from Hinsch, W., Antonijevic, A. and Sundaram, P. V. Clin. Chem. 1980, 26, 1 6 5 2 - 1 6 5 5 by permission o f the American Association for Clinical Chemistry © ] I 500

since cost will continue to be a major consideration, immobilized enzymes will be increasingly utilized in future. Gloger22 has reported that an average of 120 000 tests may be performed with 35 g of enzyme in solution. On the other hand, it is our experience that at least 1000 times more than this number of tests may be possible if the same amount of enzyme is immobilized. 23 Another type of analysis that may gain popularity is in vivo monitoring, similar to glucose monitoring in diabetic patients. Immobilized enzymes are ideally suited for this purpose.

I IOO0

Conclusions

slowly replaced by small nucleophilic substances in the prepared sample. This does not, however, preclude the profitable application of the reactors, as our experience shows. Figure 11 is a typical decay curve.

This review has been consciously restricted to the work carried out in our laboratories over the last decade. Hornby and Horvath have worked on similar systems, but our work currently emphasizes the clinical application of immobilized enzyme nylon tube reactors in large hospitals on several continents. The earlier part of the developmental work concentrated upon the fundamental aspects of polymer derivatization, immobilization techniques and kinetics of these reactors. The detailed technology is now available for the methods to be adopted in routine analysis.

Cost and the future o f analysis

Acknowledgements

The future of analysis in clinical chemistry, at least in the affluent areas of the world, will probably be directed towards further innovation to achieve faster analysis with even smaller sample volumes. Nevertheless, enzymes will continue to be used because of their high specificity. And

The work covered in this review was supported by DFVLR (Deutsche Forschungs-und Versuchsanstalt Eir Luft-und Raumfahrt) and DFG (Deutsche Forschungsgemeinschaft). This article was written when the author was on a sabbatical at the Indian Institute of Technology, Madras, India.

Enzyme Microb. Technol., 1982, Vol. 4, September

297

Reviews 6

Abbreviations Gluc-DH GOD LDH PK ALT

C]y-I~ NADH-Perox ALDH PAP test DMS TTFB DAM method

Glucose dehydrogenase Glucose oxidase Lactate dehydrogenase Pyruvate kinase Alanine aminotransferase (previously referred to as glutamate-pyruvate transaminase, GPT) Glycerol dehydrogenase NADH peroxidase Aldehyde dehydrogenase Peroxidase-aminophenzaone test Dimethyl sulphate Triethyloxonium tetrafluoroborate Diethylmonoxime method

References 1

Sundaram, P. V. and Hornby, W. E. FEBS Lett. 1970, 10, 325-327 2 Sundaram, P. V., Blumenberg, B. and Hinsch, W. Clin. Chem. 1979, 25, 1436-1439 3 Sundaram, P. V.J. Solid-Phase Biochem. 1978, 3,185-197 4 Sundaram, P. V. in Enzyme Labelled Immunoassays o f Hormones and Drugs (Pal, S. B. ed.) Walter de Gruyter & Co., Berlin-New York, 1978, pp. 107-127 5 Sundaram, P.V.NucleicAcidsRes. 1974,1,1587-1599

298

EnzymeMicrob. Technol., 1982, Vol. 4, September

Morris,D. L., Campbell~J. and Hornby, W. E. Biochem. J. 1975, 147,593-603 7 Sundaram, P. V. in Biomedical Applications o f Immobilized Enzymes and Proteins (Chang, T. M. S., ed.) Plenum Press, New York, 1977, vol. 1,pp. 317-340 8 Sundaram, P . V . Biochem. J. 1979,183,445-451 9 Sundaram,P. V. Bioehem. Biophys. Res. Commun. 1974, 61,717-722 10 Sundaram, P. V. and Apps, D. K. Bioehem. J. 1977, 161, 441- 443 11 Sundaram, P. V., Igloi, M. P., Wassermann, R., Hinsch, W. and Knoke, K.-J. Clin. Chem. 1978, 24,234-239 t 2 Sundaram, P. V., Igloi, M. P., Wasserman, R. and Hinsch, W. Clin. Chem. 1978, 24, 1813-1817 13 Sundaram, P. V. and Hinsch, W. Clin. Chem. 1979, 25,285288 t4 Sundaram, P. V. and Igloi, M. P. Clin. Chim. Acta 1979, 94, 295 -307 15 Hinsch, W. and Sundaram, P. V. Clin. Chim. Acta 1980, 104, 87 -94 16 Hinsch, W., Ebersbach, W.-D. and Sundaram, P. V, Clin. Chim. Acta 1980, 104, 95-100 17 Hinsch, W., Antonijevie, A, and Sundaram, P. V. Z. Lebensm. Unters. Forsch. 1980, 171,449-450 18 Hinsch, W., Antonijevic, A. and Sundaram, P. V. Clin. Chem. 1980, 26, 1652-1655 19 Hinsch, W., Antonijevie, A. and Sundaram, P. V.J. Clin. Chem, Clin. Biochem. 1981, 19, 307-308 20 Hinseh, W., Antonijevic, A. and Sundarama, P. V. Fresenius Z. Anal. Chem. 1981, 309, 25-29 21 Sundaram, P. V: and Munnecke, D. G. unpublished 22 Gloger, M. in Enzyme Engineering (Wingard, L. B. Jr, Chibata, I., Fukui, S., eds) Plenum Press, New York, 1982, vol. 6, in press 23 Sundaram, P. V. Ibid in press