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CLINICAL CHEMISTRY, Vol. 17, No. 8, 1971 715
Feasibilityof Multiple Simultaneous Enzyme Assays,for
Diagnostic Purposes,with the GeMSAEC Fast Analyzer
Thomas 0. Tiffany,1 George F. Johnson,2 and Max E. Chilcote
The GeMSAEC fast analyzer provides the clinical chemistry
laboratory withan analytical instrument that can be used to perform
large numbers ofkinetic enzyme analyses. Precise enzyme-rate
analyses can be doneroutinely, on a large scale, and at a decreased
cost per test. Improvedprecision in analyses of enzymes should
provide more reliable data be-cause analytical variation is
lessened. We have asked how the fast analyzermight provide more
useful diagnostic information to the clinician. We haveselected the
ratio of SGOT to SGPT activity in serum as an example, andexamined
instrumental precision. The coefficients of variation of the
ratio,determined in the range of 50 and 140 Karmen units (which
represents slightlyelevated to clearly elevated values), are 4.8%
and 2.2%, respectively. Weexamined the feasibility of measuring two
or more enzyme activities simul.taneously in one sample, to produce
a diagnostic enzyme profile. Deter-mination of SGOT, SGPT, and GLDH
in parallel is presented as an example.In addition, we illustrate
spectrophotometric linearity at 340 nm and discussinstrumental
noise and an experimental approach to determining it by useof a
premix experiment.
Additional Keyphrases diagnostic aids . liver-function
assessment #{149} in-strumental noise #{149} SOOT, SGPT, GLDH, CPK
#{149} parallel (profile) assays ofseveral enzymes #{149}
CENTRIFICHEM
The introduction of the GeMsAEc3 high-speed,parallel-sample,
fast analyzer by Anderson (1) hasprovided research laboratories in
general and theclinical laboratory in particular with an
analyticalinstrument capable of performing high-volumeenzyme-rate
analyses. The ability of this instru-
From the Department of Clinical Chemistry, H. J. MeyerHospital
Division, Erie County Laboratories, 462 Grider St.,Buffalo,N. Y.
14215.
NIH ClinicalChemistry postdoctoral Fellow, Departmentof
Biochemistry, State University of New York at Buffalo,Buffalo,N. Y.
14214. Present address: MAN Program,Oak Ridge National
Laboratories,Oak Ridge, Tenn. 37830.Send reprintrequeststo this
address.
2 NIH ClinicalChemistry PostdoctoralFellow,Departmentof
Biochemistry,State University of New York at Buffalo,Buffalo,N. Y.
14215.
Nonstandard abbreviationsused: GeMSAEC, an acronym forGeneral
Medical Sciences Atomic Energy Commission. KU,Karmen unite (the
oxidationof NADH can be measured interms of the decreasein
absorbance at 340 nm. A decrea.seof0.001 is equal to 1 icu/ml/min);
sooT, serum glutamic-oxalo--acetic transaminase (i-aspartate:
2-oxoglutarate amino trans-ferase, EC 2.6.1.1.);SGPT, serum
glutamic-pyruvic transaminase(o-alanine: 2-oxoglutarate
aminotransferase, EC 2.6.1.2); GLDH,glutamate dehydrogenase
[1.-glutamate: NADP oxidoreductase(deaminating)]; CPK, creatine
phosphokinase (ATP: creatinephosphotransferase,EC 2.7.3.2); mA,
milliabsorbance units.
ment to perform a large number of tests in parallelallows its
operator to run the reaction long enoughto obtain a sufficient
absorbance change to assurereasonable precision in the normal range
and stillmake a large number of enzyme determinationsper hour. The
small sample and reagent volumerequired by the fast analyzer makes
it possible toperform kinetic rate enzyme analyses at a
greatlydecreased cost per test.
Pragmatically, the fast analyzer offers a solutionto a problem
facing the clinical chemistry labora-tory in that it provides
precision data at a lowercost per sample and is capable of handling
anever-expanding enzyme workload. Furthermore,multiple-sample,
parallel, fast analysis should makeit feasible to perform several
different enzymeassays simultaneously on one or several
samples,with the advantage that the resulting equality
oftemperature and sample handling should lead tomore reliable
comparisons of the various activitiesof the enzymes in the serum
sample. In this man-ner, a panel of enzymes can be assayed as a
pro-file for a certain disease state and provide moreinformation
for differential diagnosis. If the fastanalyzer can perform enzyme
assays with suffi-cient precision and provide a series of
enzyme
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716 CLINICAL CHEMISTRY, Vol. 17, No. 8, 1971
assays rapidly, at a reduced cost to the patient,and yield
useful diagnostic information to theclinician, it will have made a
significant contribu-tion to clinical enzymology.
In our initial evaluation of a commercial proto-type of the
GCMSAEC fast analyzer we have ex-plored the potential of the fast
analyzer to produceprecision enzyme data by measuring the
coefficientof variation of the ratio of SOOT activity to
SGPTactivity. Also we have investigated the feasibilityof running
different enzyme assays simultaneouslyon several serum samples, and
present data for sucha profile (for liver-function assessment),
con-sisting of SGOT, SGPT, and GLDH. Information con-cerning the
instrument evaluation pertinent tothis study is also presented.
Methods and Materials
ApparatusThe fast analyzer used in this work is a CEN-
TRIFICHEM (Union Carbide Research Institute,Tarrytown, N.Y.
10591), a prototype model pro-vided to us for initial evaluation.
The transferdisc on this instrument has a capacity of 29 sam-ples.
Samples were pipetted satisfactorily by usingvarious sizes of
Eppendorf micropipets. Reagentwas added to each of the 29 sample
compartmentsor sectors on the transfer disc with a
Biopetteautomatic pipet (Schwarz/Mann, Division ofBecton, Dickinson
& Co., Orangeburg, N.Y.10962).
Materials
Enzyme kits and reagents used in this studywere purchased from
the Boehringer MannheimCorp., New York, N.Y. 10017.
Methods
Enzyme determinations performed in this studywere done in
accordance with the manufacturersrecommendations except for
temperature, time ofreaction, and volume of reagents used. The
tem-perature used was 30#{176}C.The results for severalSGOT and
SGPT determinations on samples andcontrols in the normal and
moderately enhancedrange indicated the rate to be linear over a
A340change of 1.0 absorbance unit. We thus felt justi-fied in
running our SGOT, SGPT, and GLDH for 10mm, to obtain better
precision in the normal andmoderately elevated ranges. A 1.0-unit
absorbancechange would correspond to a sample activity of147 mU/ml
or 237 KU (at 25#{176}C).Under the above-described conditions most
samples would not haveto be diluted and 75 to 100 samples could be
as-sayed per hour, in addition to controls. A samplevolume of 50 d
was used for SGOT, and SGPT, 100jl for GLDH. Reagent volume was
400
Results
Initial Concerns
Most of the ultraviolet spectrophotometricenzyme assays now in
use are optimized eitheraccording to Henry et al. () or Bergmeyer
andBernt (3). For enzyme determinations involvingthe conversion of
NADH to NAD the optimized pro-cedures require an initial NADH
concentrationwithin the range 0.17 to 0.20 mmol/liter. SGOT,SGPT,
and GLDH procedures require larger serumvolumes in the reaction
mixture, because activityis less in normal sera. The additive
absorbance ofserum and NADH produces initial starting absor-bances
of 1.6 to 2.0 absorbance units. In systemswhere reference blanking
with an absorbing solu-tion is not practicable, good
spectrophotometriclinearity is required. The linearity obtained
forNADH solutions in the CENTRIFICHEM (Figure 1) isquite good to
2.0 absorbance units. The linearityof SOOT and SGPT with respect to
time is shown inFigure 2.
Contribution of Instrumental Noiseto Total Analytical
Variation
In the kinetic methods we used, a zero-orderreaction rate is
considered to be proportional toactivity of a particular serum
enzyme. Determina-tion of this zero-order rate frequently requires
thata small absorbance change with time be measuredat moderately
high absorbanees. In a measurementmade under these circumstances,
instrumentalnoise can contribute substantially to the
observedvariation between replicate samples.
If the standard deviations involved in measur-ing two different
absorbances are known, then thestandard deviation of the
measurement of the differ-ence between these two absorbance values
can becalculated. This is shown in the following expres-sion: $42 =
S2A, + S242, where S4, and 84 arethe standard deviations in the
separately measuredabsorbance values and &A is the standard
devia-tion in the measurement of the difference betweenthese
values. The coefficient of variation owing tonoise that would be
found upon replicate deter-minations of A can be expressed as
&A X 100/A.In the case where a small absorbance change athigh
absorbance values is measured, a useful ap-proximation is that $4,
= S42. The coefficient ofvariation due to noise can then be
expressed asj2(S4) /A. Because most kinetic enzyme assaysof
clinical interest exhibit zero-order kinetics over aconsiderable
absorbance change, the contributionof noise to the observed
analytical variation isminimized by maximizing the measured
iSA.
A useful method for studying instrumental noisewith CENTRIFICHEM
involves a premixed type ofexperiment. In these experiments, enzyme
reac-tions were started in batch by adding serum to
-
EC0
Ce
UiC,z4
0C,,
4
Fig. 1. Spectrophotometric linearity at 340 nm
1 2 3 4 5 6 7 8 9 10TIME Mm
Fig. 2. Linearity of SGOT and SOPT assay with respect totime (.
SOOT, 143 U/liter; #{149}sopT, 90 U/liter)
Table 1. CPK Premix Experiment to AssessInstrumental Noise
LV, mm * x 10 SAA X 10 CV, % SA//2 - 10
Av 1.0
CLINICAL CHEMISTRY, Vol. 17, No. 8, 1971 717
reagents, mixing well, and then pipetting the react-ing mixture
into the transfer disc. Data weretaken over a period of time after
the reacting mix-ture was spun into the individual cuvets. The
re-sults of such an experiment with CPK are shown inTable 1.
Absorbance at 340 nm is increasing withtime. As the reaction is
measured over longertimes, the coefficient of variation decreases,
withthe standard deviation in the measured EA remain-ing constant.
Since absorbance is changing at the
2 15.2 1.3 8.8 0.94 30.9 1.3 4.2 0.96 45.2 1.6 3.5 1.18 60.3 1.6
2.7 1.1
10 75.7 2.1 2.7 1.512 88.5 0.9 1.0 0.614 103.4 1.6 1.6 1.116
119.7 1.6 1.3 1.118 134.3 1.6 1.2 1.1
same rate in each cuvet, differences between theEA values
measured at the same time for eachcuvet reflect only the random
noise of the instru-ment. If there were significant differences
betweenthe reaction rates in the cuvets, the standard de-viation in
A would increase with A, which in apremix experiment would imply
contamination ofthe cuvets. The data from premix experiments
forvarious assays allow a time interval to be set foreach assay so
that acceptable precision can beobtained in the normal range. A
coefficient ofvariation attributable to noise of 1% to 2% at
theupper end of the normal range for the enzymes inthis study was
considered satisfactory. This allowsthe obtainable precision within
a run to be limitedmainly by pipetting errors.
The de Ritis Quotient
Our first approach to our attempt to obtainmore useful
diagnostic information and diagnosticenzyme profiles was to see
whether or not the in-strument can perform with sufficient
precision tomake current tests more reliable. SGOT and SGPTform the
basis of a very simple diagnostic enzymeprofile. De Ritis et al.
(4) demonstrated a largeincrease of the two serum transaminases in
patientssuffering from acute hepatitis, with the increase ofSGPT
activity being more pronounced than that inSOOT. This resulted in
their suggestion that the useof the ratio of SGOT to SGPT is
diagnostically usefulfor differentiating predominately
inflammatorylesions from necrotic processes in the liver.
Theutility and limitations of this approach have re-cently been
reviewed by Zimmerman and Seeff(5). Wilkinson (6), in a recent
review of theclinical significance of enzyme activity
measure-ments, pointed out that the data obtained in thismanner can
be equivocal because of the lack ofprecision in determining the
activity of theseserum enzymes, and thus the ratio has not
beenextensively applied in diagnosis.
The experimental approach was to select apooled serum control
and a commercial enzymecontrol that would provide enzyme activities
inthree ranges of interest: normal, slightly supra-normal, and
supranormal. We examined in-discvariation and the variation during
two weeks.Both SOOT and SGPT were run in parallel on onedisk. Table
2 summarizes these data. In-run varia-tion is 4.6% (1 SD) in the
normal range, and1.6% (1 SD) in the abnormally high range.
For a within-disc run, where temperature andreagent variations
should be the same, the majorcontribution to the overall error for
large per-minute changes in absorbance is due to pipetting.The
supranormal control absorbance changes at0.0603 absorbance units
per minute (90 U/liter) ora total change in absorbance of 0.603/A
per 10mm. The coefficient of variation, 1.6%, obtained
-
718 CLINICAL CHEMISTRY, Vol. 17, No. 8, 1971
Table 2. In-Disc and Two-Week AnalyticalVariation in Results for
SGOT and SGPT
In.dlsc variation: (N - jj)a
A. SGOT(Pooled serum) 48KUX = 20.7 0.7mA/mm; CV = 3.41%
B. SGPT (Pooled serum) 18WLUX = 8.1 0.3 mA/mm; CV = 4.58%
C. SGPT (Control A) 143 WLUX = 60.3 0.9 mA/mm; CV = 1.57%
Two-week variatIon
A. SGOT(Pooled serum) (N = 108)X = 20.9 1.1 mA/mm; CV =
5.20%
B. SGPT(Pooledserum)(N =94)6X = 8.1 0.6 mA/mm; CV = 7.71%
C. SGPT (Control A) (N = 85)6X = 60.1 1.3 mA/mm; CV = 2.12%
N = no.ofdiscsdetermined.
= no.of samples.
for this range is representative of the combinedpipetting error
for sample reagent.
The propagation of error for a simple functionu = x/y (where the
ratio of SGOT/SGPT is an ex-ample) is presented in the appendix.
The result isan expression for the coefficient of variation of
theratio, and is given by the equation cv = (cv2 +cv,2) 1/2 If the
coefficients of variation for SOOT(x) and SOPT (y) are assumed to
be the same (whichis reasonable for an in-disc run of this type),
thenthe coefficient of variation for the ratio will becomecv,, =
cv,V. With the coefficients of variationlisted in Table 2, the
coefficient of variation for theratio in the range of 50 KU at
25#{176}C(30 U/liter) is4.8% and decreases to 2.2% in the range of
140KU at 25#{176}C(90 U/liter) where differential diag-nostic
significance has been attributed to the ratio.Thus, as a first
approximation, we can cite 2.2%(1 SD) as the analytical variation
that we wouldexpect for the use of the ratio in this range.
We have compared the activities of severalSOOT and SGPT samples,
as measured on the CEN-TRIFICHEM in parallel, with results for
automatedprocedures currently being used in the laboratoryfor SGOT
and SOPT (Table 3). The automated SOOTmethod is an adaptation of
the method describedby Morgenstern et al. (7), and is designed to
cor-relate with spectrophotometric methods. Correla-tion
coefficients of 0.92 and 0.99 were obtained forthe SOOT comparison.
The standard error of esti-mation (s,,) is lower in both cases for
the sootdata than for the SGPT data, and indicates lessscatter
about the linear regression line. The SGPTdata are a correlation
with a modified automatedReitman-Frankel procedure (unpublished)
per-formed on a discrete analyzer. The correlation
coefficients are still good but the larger standarderror of
estimation indicates greater scatter aboutthe linear regression
line. Giusti et al. (8) have re-ported a correlation study between
the spectro-photometric and Reitman-Frankel methods. Theyhave
reported that correlation is poorer for theSOPT methods, but that
the variation coefficientis of about the same magnitude.
Feasibility of PerformingDiagnostic Enzyme Profiles
The necessity of collating the results of two ormore enzyme
determinations to form enzymepanels or diagnostic profiles for
differentialdiagnosis and for determining patterns of ab-normality
has been established (5, 9, 10). rFheCENTRIFICHEM can perform
several differentenzyme determinations in parallel. The
largesample-per-disc capacity, in conjunction withsmall reagent and
sample volumes, makes this areasonable approach. Most enzyme assays
ofclinical interest involve either coupled NAD/NADHassay systems or
NAD or NADH as a substrate, sothat wavelength is not a limiting
factor.
Henley et al. (11) have suggested that the mito-chondrial enzyme
ourn, found in high concentra-tion in the liver, is of value in
assessment of liver-cell necrosis. To test the feasibility of the
diagnosticenzyme profile approach, GLDH was set up anddetermined in
parallel with SOOT and SGPT. Sampleswere preincubated in the rotor
in the presence ofNADH and ammonium ion for 20 mm to
removeendogenous substrates. Nine sets of samples can bedetermined
simultaneously on the CENTRIFICHEMwhen performing a profile that
requires threedifferent enzyme determinations.
A replicate analysis of SOOT, SGPT, and GLDHsamples, run in
parallel, are shown in Table 4.GLDH activity was determined at
30#{176}C.The GLDHassay is less precise in the normal range because
ofits very low activity in the serum.
The rate curves obtained for SOOT, SOPT, GLDH,run in parallel on
a sample from a patient withacute hepatitis, are shown in Figure 3.
The SGPTspectrophotometric method used in this study wasessentially
that of Wroblewski-LaDue (1P2)and wasnot optimal (2). A higher
sGoT/sGPr ratio than 1 isfound in this range by some authors (5)
and proba-bly reflects the use of suboptimal assay systems(8).
Discussion
We have attempted to show that the multiple-sample, parallel,
fast analyzer is becoming a practi-cal reality. We have
demonstrated some acceptableprecision data for SOOT and SGPT run in
parallel,and have established that of confidence limit for the
-
In
0.95
0.84
0.60
0.89
Syx
3.4 0.92 0.86
10.9 0.99 0.98
11.7 0.91 0.83
-1.1 0.88 0.78
32631.2
14.8
65.2
375
44 50
223 252
I I
SAMPLR a / sCOOTII 205SK.U.// 75511.1.
55 OPT///7 7131/ #{163}OLOH -
RSI.U.
Sample 3 5SGOT(XU) SGPT(WLU) GLDHU/ml
3 5 3 S
PS 40 40 18 20 5 43 121 122 83 896 26 24
38 46 42 52 57 6 558 43 44 69 77 5 419 25 23 17 17 5 332 17 17 9
10 4 334 29 28 17 166 6 39 25 23 16 17 9 6
41 85 86 49 476 3 4
Range of errorfor the replicate samples:0%-2%.2%-4%.4%-6%.PS,
pooled serum samples.
CLINICAL CHEMISTRY, Vol. 17, No. 8, 1971 719
Table 3. Correlation of Data on SGOT and SGPT Activities with
Automated Laboratory Data
SGOTN = 111SGOTN = 31SGPTN = 111SGPT
Y = mX + b; Syx = standard errorof estimation.Samples lessthan
100 KU.Samples greaterthan 100 KU.
1 X (Sy-x/V)X1006.1 41 42 14.5%
9.6%
35.2%
29.2%
0
4I
S GOT. SGPT-GLDH(PARALLI!L mUN)
0.I
0.7
0.3
4 5 S 10 15 14
TI MS (MIt..)
Fig. 3. Simultaneous determination of sooT, SGPT, andOLDH on a
serum sample from a patient with acutehepatitis
ratio of SGOT to SOPT. We have attempted to showthat the
CENTRIFICHEM fast analyzer can produceprecision data and that the
use of a ratio deter-mined under carefully defined conditions
shouldnot be equivocal because of analytical variation.
Furthermore, we have demonstrated that threedifferent assays for
enzyme activities can be per-formed on several different samples
simultane-ously, and that the results can be used for a
specificdiagnostic profile. Thus, enzymes of interest canbe assayed
in one sample, at the same time, at thesame temperature, and with
the same (and verylittle) handling of the sample. This approach
mayassume a role in special chemistry or as an emer-gency function,
where simultaneous information onCPK, LDH, and SOOT activity could
be obtainedvery rapidly. The usefulness of this approach
iscurrently being evaluated in this laboratory.
We wish to express appreciation for technical assistance
pro-vided by Mary Howe, Colleen Heigl, Sandy Casey, and
SandyMarszalek. This study was supported in part by TrainingGrant
in Clinical Chemistry, 5 TOl GM 01960 (NIGMS).
Table 4. SGOT-SGPT-GLDH Parallel Run. Replicate Analysis of
Samples
-
References
720 CLINICAL CHEMISTRY, Vol. 17, No. 8, 1971
1. Anderson, N. (1., A multiple-cuvette rotor for a new
micro-analytical system. Anal. Biochem. 32, 59 (1969).2. Henry, H.
J.,Chiamori, N., Golub, 0. J.,and Berkman, S.,Revised
spectrophotometric determination of glutamic oxalo-acetic
transaminase, glutamic pyruvic transaminase and lacticacid
dehydrogenase. Amer. J. Clin. Pathol. 34, 381 (1960).3. Bergmeyer,
H. U., and Bernt, E., Glutamic-oxaloacetic trans-aminase.
Determination with malic dehydrogenase as indicatorenzyme with
optimum conditions for measurements. In Methodsof Enzymatic
Analysis, Academic Press, New York, N.Y., 1963,p 837.
4. De Ritis, it., Coltori, M., and Giusti, G., 1)iagnostic
valueand pathogenic significance of transaminase activity changes
inviral hepatitis, Minerva Med. 47, 167 (1956).5. Zimmerman, H.
J.,and Seeff, L. B., Enzymes in HepaticDisease. In Diagnostic Ens
ymology, Coodley, E. L., Ed. Lea andFebiger, Philadelphia, Pa.,
1970, p 1.6. Wilkinson, J. H. Clinical significance of enzyme
activitymeasurements. CLIN. CHEM. 16, 882 (1970).7. Morgenstern,
S., Okiander, M. Auerbach, J.,Kaufman, J.,and Klein, B., Automated
determination of serum glutamicoxaloacetic transaminase. CL1N.
CHEM. 12, 95 (1966).8. Giusti, G. Ruggiero, G., and Cacciatore, L.,
A comparativestudy of some spectrophotometric and colorimetric
proceduresfor the determination of serum glutamic oxaloacetic
trans-aminase and glutamic pyruvic transaminase in hepatic
disease.Enzyrn. Biol. Clin. 10,17 (1969).9. Schmidt, E.,Schmidt, F.
W., Horn, H. D., and Gerlach, U.,The importance of measurement of
enzyme activity in medicine.In Methods of Enzymatic Analysis,
Bergmeyer, H. U., Ed. Aca-demic Press, New York, N.Y. 1963, p
651.10. Bat.sakis, J. G., Briere, H. 0.,Enzymatic profile of
myo-cardial infarct. Amer. Heart J. 72, 274 (1966).
11. Henley, K. S.,Schmidt, E.,and Schmidt, F. W., Enzymesin
Serum. Their Use in Diagnosis. Charles C Thomas Co., Spring-field,
Ill. 1966, p 54.12.Wroblewski, F.,and LaDue, J.S.,Serum glutamic
pyruvictransaminase in cardiac and hepatic disease. Proc. Soc.
Exp.Biot. Med. 91,569 (1956).13.Parratt,G.,Probabilityand
Experimental Errors in Science.John Wiley and Sons, New York, N.Y.,
1961, p 114.
AppendixThe equation for the propagation of error for a
function
u = f(x,y) is given by
#{246}u2 #{246}u2S,,2 = (_) 3,,2 + () 52 (1)where S,,2, and 5,2
are the variance (13). For the equationu = x/y, #{246}u/#{246}x=
l/y and #{246}u/y= -x/y2 so that the ap-plication of Equation 1
after substitution and rearrangement,gives
S,.2/(x2/y)2 = + _ix2 y2 (2)
Given the following identities, x2/y2 u2, S,,2/,,2 = cv,,2-= cv2
and S,2/,2 = cv,2 (where cv,,, cv,, and cv
represent the coefficient of variation of u, x, and y),
theresulting equation for the propagation of error for a ratiox/y
is
cv,, = (cv2 X CV,2)h/2 (3)