-
dIN. CHEM. 21/12, 1754-1760 (1975)
1754 CLINICAL CHEMISTRY, Vol. 21, No. 12, 1975
Evaluation of Trinders Glucose Oxidase Method
for Measuring Glucose in Serum and Urine
John A. Lott and Kathie Turner
Trinders method for glucose has nearly all the attrib-utes of an
ideal automated colorimetric glucose oxidaseprocedure. The
chemicals used in the color reactionwith peroxidase are readily
available, the solutions arestable and can be prepared by the user,
the method ishighly specific and largely free of interferences,
thesensitivity can. be adjusted by the user to cover a widerange of
glucose concentrations, and the reagents arenot hazardous. We found
very good agreement be-tween results by this method and by the
hexokinase andBeckman Glucose Analyzer methods. The method hasbeen
modified and adapted to the AutoAnalyzer I andSMA 6/60 (Technicon)
with manifolds that give very lit-tle interaction between
specimens. A study of the meth-od by the simplex technique revealed
that the glucoseoxidase activity in the reagent is the most
critical vari-able.
AddItIonal Keyphrases: continuous-flow analysis #{149}glu-cose
and reagent preservatives #{149}intermethod comparison#{149}optimum
analytical conditions #{149}normal (reference)values #{149}sample
stability
The glucose oxidase/peroxidase (EC 1.1.3.4/EC1.11.1.7) method
for glucose described by Trinder in1969 is very attractive (1). The
method is specific forglucose and the reagents are all readily
available. So-lutions of the reagents are stable. The Trinder
re-agents are less of an occupational hazard than re-agents used in
other methods-o-toluidine, o-dianisi-dine, or N,N-dimethylaniline,
which are all quitetoxic.
Here, we describe automated methods for serumand urinary glucose
with use of the AutoAnalyzer I orSMA 6/60 (Technicon Instruments
Corp., Tarry-town, N. V. 10591) and of reagents that the user
canprepare. We also describe optimization of the methodwith the
simplex technique, method interferences,normal values, and other
data.
Materials and MethodsReagents and Standards
Stock glucose oxidase, 106 U/liter, Type V, No.G-6500, Sigma
Chemical Co., St. Louis, Mo. 63178.
Division of Clinical Chemistry, Department of Pathology,
OhioState University, 410 W. 10th Ave., Columbus, Ohio 43210.
Presented in part at the Ninth International Congress on
Clini-cal Chemistry, Toronto, Ont., 1975 (Clin. Chem. 21, 978
(1975),abstract).
Received July 21, 1975; accepted Aug. 18, 1975.
Peroxidase, Type II, No. P-8250, Sigma. Note thatthis peroxidase
is as satisfactory as the much moreexpensive Type VI, No. P-8375
from Sigma.
4-Aminoantipyrine, No. A-4382, Sigma (alsoknown as
4-aminophenazone).
Peroxidase/buffer reagent, pH 7.0. Dissolve 8.5 gof anhydrous
reagent-grade Na2HPO4 and 5.3 g ofanhydrous reagent-grade KH2PO4 in
about 800 ml ofdistilled water, and adjust the pH to 7.0 0.1 with
1mol/liter HC1 or NaOH if needed. Add 4 mg of perox-idase and 300
mg of 4-aminoantipyrine and dissolve,and dilute to 1 liter with
distilled water. The final re-agent contains 0.1 mol of phosphate
per liter, and isstable for as long as four weeks when stored
inamber-colored glass bottles at 4 #{176}C.
Glucose oxidase/peroxidase reagent. Add 12 ml ofstock glucose
oxidase (or 12 000 U) to 1 liter of theperoxidase/buffer reagent.
The complete reagent isstable for 1 week at 4 #{176}C.
Phenol solution, 1.5 g/liter. Dissolve 1.5 g of phe-nol,
analytical-reagent grade, in enough distilledwater to make 1 liter
of solution. Stable for at leastsix months when stored in
amber-colored glass bot-tles at room temperature.
Saline, 9 g/liter. Dissolve 9 g of sodium chloride inenough
distilled water to make 1 liter of solution.Add 1 ml of Tween 20
surfactant (Technicon) perliter just before use.
Stock glucose standard, 10 g/liter. Dissolve 10.000g of
primary-standard grade dextrose in enough dis-tilled water that is
saturated with benzoic acid (about3 g/liter) to make 1 liter of
solution.
Working glucose standards. Prepare dilutions ofthe stock glucose
standard with a saturated aqueousbenzoic acid solution to give
standards containing250, 500, 1000, 1500, 2000, 2500, 3000, and
4000 mg ofglucose per liter,
ProcedureThe flow diagrams for the AutoAnalyzer and SMA
6/60 are shown in Figures 1 and 2. Freshly separatedserum or
urine can be analyzed directly. In the caseof the AutoAnalyzer I,
the standards are analyzed inthe order listed above and a
calibration curve is pre-pared on semilog paper. The glucose in
patients sam-ples and in controls is estimated from the curve.
Thestandards should be run at the beginning of each
-
6 Dialyzer__________8-turnI.
I AirSdine/Tween .051[ AirGluc.Ox./Perox.
.035.035
0.420.42LU1709
2,9-1 ---- Lturn 37#{149}coils Both
________ PhosingCoilColor- Recordermeter
505 nm ______
Phenol .030 0.32Flowcell Retum.05l 1.0
Sampler IV
Fig. 2. Flow diagram for the SMA 6/60
Recovery,mg/liter %
SAIl results are means of three determinations.
CUNICAL CHEMISTRY, Vol. 21, No. 12, 1975 1755
Fig. 1. Flow diagram for the AutoAnalyzer I
Tube Flow,id.in. rt/min
Tube Flow,id.in. mI/mm
Samole .020 0.16[ Saline/Twn 0l 10.035 0.42
I.0
Table 1. Analytical Recovery of Glucose fromBovine Albumin
Solutions
Added Founda
5001000200030004000
5101000200030404020
102100100101101
third tray of 40 samples. At least two controls shouldbe
analyzed on each tray.
The glucose concentration of the serum-based cali-brating
material used to set the SMA 6/60 should beestablished by analysis
with the method proposedhere. The insert or label values must not
be useduncritically, because they may have been establishedby a
less-specific method. Standards and at least twocontrols should be
analyzed on every tray of 40 sam-ples on the SMA 6/60.
At the end of the day, 1 mol/liter NaOH is pumpedthrough all
lines, including the dialyzer, for 15-30mm, followed by a 30-mm
wash with distilled water.This washing effectively prevents
shifting baselines,drift, clogged tubing, etc. A sodium
hypochlorite so-lution must not be used, because it is difficult
towash out completely, and the hypochlorite reacts toform a color
with the glucose oxidase/peroxidase re-
agent. In the case of either the AutoAnalyzer I or theSMA 6/60,
replace the manifold tubing after no morethan 140 h of running
time.
ResuftsAnalytical Variables
Analytical recovery. Bovine albumin (No. 905-10,Sigma) contained
no glucose detectable by the Au-toAnalyzer I method described here
or by the Calbio-chem hexokinase procedure (No. 869204;
Calbio-chem, San Diego, Calif. 92112). Solutions were pre-pared to
contain, per liter, 70 g of albumin and 500,1000, 2000, 3000, and
4000 mg of glucose, and theywere then assayed with the AutoAnalyzer
I. Becausethe analytical recoveries (Table 1) were all withinabout
2% of the expected values, we concluded thataqueous standards can
be used to calibrate the Au-toAnalyzer I and that protein does not
interfere inthe analysis for glucose.
Precision. The method described has been in rou-tine use here
since October 1974. Between November1, 1974 and March 31, 1975, we
did about 44 000serum glucose determinations. Blind controls
(2)from the same lot numbers were randomly distribut-ed between
patients samples during that time, andthe results are listed in
Table 2. An equal number ofblind controls were analyzed with the
AutoAnalyzer Iand SMA 6/60. We think that the precision of
themethod over this time span is satisfactory.
Comparison studies. We compared our results forthis method to
those obtained by the Calbiochem
Month
Table 2. Summary of Quality-Control Data for Glucose in Samples
Analyzed asVersatola Versatol A0 Versatol A-AItarnate
n Mean CV n Mean CV n Mean CV n
BlindsPooled serum
Mean CVmg/liter % mg/liter % mg/liter % mg/liter %
Nov. 74 20 820 2.4 20 2020 2,9 30 2990 2.0 30 840 3.1Dec. 74 21
820 3.0 21 1980 2.1 31 2980 1.8 31 840 2.3Jan. 75 22 830 3.0 22
1990 1.7 31 2990 2,8 31 840 3.1Feb. 75 20 820 4.3 20 1980 1.6 28
2980 2.2 28 810 2.7Mar. 75 21 820 2.7 21 1980 2.6 31 2940 1.3 31
810 4.1
a General D iagnostics, Morris Plains, N.J. 07950. Lot numbers,
left to right, were 2406103. 2262043, and 1176112.
-
glucose, mg/liter
1758 CLINICAL CHEMISTRY, Vol. 21, No. 12, 1975
Table 3. Results by Three Methods forSerum Glucose Compared
(Mean of Duplicate Results)Auto-
Material Analyzer I SMA 6/60 Hexokinase
VersatolaVersatol AVersatol A AlternateCalibrateaVersatoI
Automated LoSerum ReferencebScale lbScale IlbPool IC (lipemic)Pool
IICPool IlICPool lVPool VC (lipemic)Pool VICPool VIIC
O General Diagnostics.bTechniconC Freshly pooled human
serum.
760 780 7701840 1790 18402960 2820 27901830 1840 1810
770 800 8402350 2310 2300
860 870 8703710 3550 3590
470 470 320560 550 540850 850 850940 940 910
1100 1050 8401410 1380 13803000 2860 2930
hexokinase procedure for various lyophilized controlsera and
pooled fresh sera. Two serum pools weremade up from lipemic
samples. Agreement was good(Table 3) except for the lipemic pools
(pools I and V),for which the hexokinase procedure gave
somewhatlower results.
We also assayed 54 freshly collected patients seracontaining 540
to 4760 mg of glucose per liter, withthe Glucose Analyzer (Beckman
Instruments, Inc.,Fullerton, Calif. 92634) and with the SMA 6/60.
Re-sults obtained with the two instruments agreed well.The means
and standard deviations for the SMA andBeckman were 1460 930 and
1470 910 mg of glu-cose per liter, respectively, the correlation
coefficientwas 0.9994, and the slope and intercept were: Beck-man =
0.972 (SMA) + 4.5 (3). To be certain that wehad no bias between the
AutoAnalyzer I and SMA6/60, we assayed 73 fresh patients sera with
both in-struments. The range of values was 350 to 7890 mg/liter,
the means and SD were 1260 970 (AutoAna-lyzer I), and 1260 960
mg/liter (SMA 6/60). Thecorrelation coefficient was 0.9993, and the
slope andintercept were: SMA = 0.98.4(AutoAnalyzer I) + 2.51.
Interferences. Various anticoagulants, drugs, me-tabolites,
sugars and other compounds were testedfor potential interferences
with the method (Table4). For the first group the same glucose
concentrationwas observed when either saline or a solution of
thecompound was added to pooled fresh serum. Theconcentration of
anticoagulants that we tested ismuch higher than is commonly used,
but none inter-fered. The serum drug concentrations that we
stud-ied are much higher than would be expected after atherapeutic
dose. It is significant that none of the
commonly used oral hypoglycemic agents interfered.The
concentrations of metabolites are far above thenormal range and
generally exceed those seen for cre-atinine, urea, and uric acid
even in patients with se-vere azotemia. For all of these, we
observed no inter-ference.
Uric acid was examined in more detail. Pooledserum was diluted
with saline or a stock uric acid so-lution to give pools with 100,
250, and 500 mg of uricacid per liter. The addition of uric acid
did notchange the observed glucose concentration of 630mg/liter as
compared to the same pool diluted withsaline (Table 4). Likewise,
when uric acid was addedto three other poois to give a
concentration of 200 mgof uric acid/liter, the glucose
concentration of 510,970, and 1940 mg/liter were the same as was
observedwhen saline was added to the poois.
That the sugars listed in Table 4 do not interferereflects the
specificity of the method, maltose beingan exception. The
interference from maltose was dueto the presence of maltase in the
glucose oxidase. He-moglobin did not interfere, as was also
reported byGochman et al. (4). Ascorbic acid produced
dramaticdecreases in the observed glucose value, in contrast tothe
findings of others (5) who observed no effect onresults by Trinders
method (1) of ascorbic acid, 1000mg/liter.
In vivo concentrations of ascorbic acid are too lowto
significantly interfere. In a study by Schrauzer andRhead, the
maximum ascorbic acid concentration inplasma or erythrocytes never
exceeded 27.5 6.5(SD) and 15.1 3.6 mg/liter, respectively, in 17
vol-unteers who had taken 2 g of the drug daily for ninedays (6). A
large fraction of ingested ascorbic acid isexcreted unchanged in
the urine (7), hence a poten-tial interference exists in cases of
renal failure.
Gentisic acid, a metabolite of salicylic acid, inter-feres with
the method. However, only a small frac-tion [of salicylic acid] is
converted to this metabolite(8), so that, in vivo, gentisic acid is
probably not asource of interference.
Reduced glutathione is present in whole blood at aconcentration
of 280-340 mg/liter (9). When weadded reduced glutathione to serum,
we observedfalsely low glucose values with the method. But
whenwhole blood was intentionally hemolyzed to produceplasma with
20 g of hemoglobin per liter, we observedno interference. The
intentional hemolysis may havedestroyed the reduced glutathione so
it remains a po-tential interferant in hemolyzed blood.
Levodopa (L-DOPA) in the concentrations indicat-ed in Table 4
seriously interferes. These concentra-tions of L-DOPA are much
higher than have been ob-served in vivo. Muenter and Tyce (10)
observed peakconcentrations of 0.40 to 7.3 mg/liter of plasma
after0.25- to 2-g doses of L-DOPA, in a study involving 26patients.
In another study with 15 patients (11), peakconcentrations of 1.0
to 4.0 mg/liter of plasma wereobserved after doses of 0.25 to 1.9
g. Whether the me-tabolites of L-DOPA interfere is an open
question; it
-
Glucose concn
1080
1020
1070
990
130012201110790
0
101010801010110010801070
1030830760630570
Glucose concnConcns in pooled serum, in poo1, Concns in pooled
serum, in pool,
Substance mg/liter or as stated#{176} mg/liter Substance
mg/liter or as stated0 mg/literCompounds that do not change the
obser,.ed glucose Compounds that interferea
Anticoagulants Ascorbic acid 0Heparin 75000and 150000 50
units/liter 100Sodium citrate 5000, 10 000, and
20 000200
500Sodium fluoride 10 000, 20 000,
40000 Gentisic acid 0
Sodium oxalate 2000, 4000, 8000 100200
Drugs 400Acetohexamideb 50, 100, 200 800ChlorpropamideC 50, 100,
200Phenformind 20, 40, 80 Glutathione 0Tolbutamidee 50, 100, 200,
400 (reduced) 250Tolazamidee 50, 100, 200 500Sodium 100, 200, 500,
1000 woo
salicylate
Metabo/ites L4)QPAI 0Bilirubin 50, 100, 200Creatinine 500, 1000,
2000Hemoglobin 5000, 10 000, 20 000
standardUrea 500, 1000, 2000 MaltoseUric acid 100, 250, 500Uric
acid 200
SugarsFructose 1000 2000 5000 1030 a Either saline or the
indicated compound was added to a serumGalactose 1000, 2000, 5000
1020Lactose 1000, 2000, 5000 1010
pool. For any compound, the actual concentration of glucose in
theparticular pool was constant. For the uric acid study, see
text.
bEli Lilly and Co., Indianapolis, md. 46206.Mannose 1000,2000,
5000 1030Sucrose 1000, 2000, 5000 1070
C Pfizer Inc., New York, N.Y. 10017.dGeigy Pharmaceuticals,
Ardsley, N.Y. 10502.e The Upjohn Co., Kalamazoo, Mich. 49001.
d-Xylose 1000, 2000, 5000 1080 f Eaton Laboratories, Norwich,
N.Y. 13815.
124011001000
840
11601060440
1110100200400
1090630
510, 970, 1940
820620530
0100020005000
1070123013801830
Table 4. Interferences Study
CUNICAL CHEMSTRY, Vol. 21, No. 12, 1975 1757
is metabolized by at least three major pathways (12).The major
components excreted in urine are the un-changed drug, dopamine, and
homovanillic acid (13).
Serum Normal ValuesSerum from 72 presumed healthy adult
volunteers
who had fasted for at least 12 h was examined for glu-cose by
this method (Table 5). Our median and meanwere both 840 mg of
glucose per liter and the histo-gram was reasonably gaussian. For
comparison, wehave also listed normal values for several methods
inwhich glucose oxidase but different chromophoreswere used. In his
review, Free lists normal (reference)values for glucose in serum as
measured by glucoseoxidase methods published before 1962 (14).
Glucose Estimation in UrineTrinders method is also suitable for
quantitating
glucose in urine. We added glucose to four differentglucose-free
urines to give glucose concentrations of
2500, 5000, and 10000 mg/liter. The analytical recov-ery was
98-104% (average, 100%). The urinary glu-cose estimations were done
with the AutoAnalyzer I.They can be done with the SMA 6/60, but
then aque-ous glucose solutions must be used to calibrate
theinstrument.
As much as 1.6 g of uric acid in per liter of urine, orboric
acid at concentrations of 0.4, 0.8, and 1.6 g/liter,or a saturated
aqueous solution of thymol do not in-terfere with the method.
Stability of glucose in urine. Four patients urinesamples were
chosen for study on the basis that theywere free of glucose and
they contained more than100 000 organisms per milliliter. Glucose
was addedto each urine to prepare samples of each to containabout
5000 and 10000 mg of glucose per liter. Onegram of boric acid, or
about 200 mg of thymol, ornothing was added to three 100-ml
aliquots of eachsample. These 24 samples were analyzed for
glucosewith the AutoAnalyzer I immediately after prepara-
-
1.2
1.0
0.80
.0. 0.60
.00.4
0.2
0
Sc00o 00
; .!o = ,/mg/liter0 10 /
o . ,
0 / 20000000
N -0 0 0 w .// $000,/,, .-,
,-
- -:-
1758 CLINICAL CHEMISTRY, Vol. 21, No. 12. 75
Table 5. Normal (Fasting) Values for SerumGlucose, by Various
Glucose Oxidase Methods
Mean Mean 2S0mg/liter No. Comment
830 750-1080 32 AutoAnalyzer I,MBTH-D MAO
880 700-1070 32 AutoAnalyzer II,M BTH-D MA0
920 720-1160 151,age SMA12/60,20-49 MBTH-DMA0
(fasting?)1110 840-1280 185,age SMA12/60,
over 50 MBTH-DMA0(fasting?)
890 670-1120 58 AutoAnalyzer II,glucose oxidase/neocuproine
880 660-1110 58 Glucose oxidase/dianisidine
920 700-1150 58 Glucoseoxidase/M BTH-D MA0
840 670-1010 72b This method,AutoAnalyzer I
a Chromophore linked to peroxidase described in ref. 18.b 61
women, 11 men; age range 20-57 yr (mean, 29).
tion and again after 1, 2, 3, 7, and 14 days of storageat room
temperature. Boric acid is somewhat superi-or to thymol as a
preservative, although neither isideal. The maximum loss in glucose
after 1, 2, 3, 7,and 14 days was 8, 14, 26, 27, and 34%,
respectively,for boric acid and 14, 28, 28, 36, and 54%,
respective-ly, for thymol. Unpreserved samples lost as much as40%
of their glucose in one day. From these limiteddata, we concluded
that analysis for glucose in urineis invalid after the urine has
stood for more than 1day at room temperature, even when thymol or
boricacid is present.
Simplex Optimization of Analytical ConditionsThere are many
variables in this method: pH, type
of buffer, incubation temperature, concentrations ofthe reagents
and sample in the final reaction mixture,sample-to-wash ratio, etc.
The simplex method foroptimizing analytical conditions has been
describedelsewhere (15-17). We chose to use a 0.1
mol/literphosphate buffer because it has been used successful-ly by
others (18), but our decision was really arbi-trary. A pH of 7.0
was chosen because it is close toboth the pK2 of phosphoric acid
(7.13) and the re-ported (19) optimal range for glucose oxidase (pH
4.0to 6.5). The variables we investigated by using thesimplex
method were the glucose oxidase and peroxi-dase activity and the
concentrations of 4-aminoan-tipyrine and phenol in the final
reaction mixture.One milliliter each of solutions of glucose
oxidase,peroxidase, and 4-aminoantipyrine were mixed and6.5 ml of a
phenol solution was added. The mixturewas incubated at 37
#{176}Cfor 10 mm, and the absorb-
ance was measured at 505 nm in a Model 2000 spec-trophotometer
(Gilford Instrument Laboratories,Inc., Oberlin, Ohio 44074) vs. a
water blank. The op-
Ref. timum sought was the maximum color intensity.The
concentrations of each of the reagents used in
the final reaction volume (9.6 ml) is given in Table 618 along
with the progress of the simplex. The simplexhas four dimensions
and five vertices and was there-
22 fore treated by Longs calculation technique (15).The starting
concentrations (vertex 1) were deliber-ately set far from the
presumed optimum, and a step
2 size of 80% of the starting concentrations was used.2 The
simplex study was stopped at 18 experiments,
even though we had not found the optimum. We didfind that
glucose oxidase activity is a primary deter-
23 minant of the final color intensity. Glucose oxidaseactivity
plotted vs. absorbance gives a straight linewith some scatter of
the points (correlation coeffi-
23 cient, r = 0.96). The final color intensity is less
sensi-tive to changes in the concentration of 4-aminoan-
23 tipyrine (r = 0.43) and still less sensitive to changesin the
peroxidase activity (r = 0.27) or the concentra-tion of phenol (r =
0.14).
The concentrations of the reagents in the solutionentering the
37 #{176}Cbath (see Figures 1 and 2) of theAutoAnalyzer I and SMA
6/60 are also listed in Table6. The peroxidase activity and the
concentrations of4-aminoantipyrine and phenol are somewhat in
ex-cess of what is needed. The solutions with concentra-tions
described at vertices 13, 14, and 15 (Table 6)give nearly the same
absorbancies and were obtainedby using about the same amount of
glucose oxidase.A large variation in the peroxidase activity
(vertex 13vs. 15), the concentration of 4-aminoantipyrine (ver-tex
13 vs. 15) or of phenol (vertex 13 vs. 14) had prac-tically no
effect on the absorbance,
In another experiment, the glucose oxidase activitywas varied,
and the concentrations of the other re-agents were the same as
described under Materialsand Methods. We found that the sensitivity
of the
4 5Log Glucose Oxidose Activity, U/liter
Fig. 3. Absorbance of the 1000, 2000, and 4000 mg per
literglucose standards after reaction with glucose
oxidase/peroxi-dase reagent containing increasing amounts of
glucose oxi-dase
-
Table 6. Data for Simplex Optimization of Glucose Oxidase
Reaction.Concentrations in Final Reaction Mixture
Peroxidase 4-aminoantipyrine Phenol
Vertex no.Glucose oxidase
U/literAbsorbance
observedVertices retained from
previous simplexmg/liter1 417 0.596 26.0 169.2 0.092 -2 750
0.596 26.0 169.2 0.138 -3 583 1.009 26.0 169.2 0.113 -4 583 0.734
43.0 169.2 0.104 -5 583 0.734 30.2 276.0 0.106 -6 833 0.941 36.6
223.0 0.139 2, 3,4, 57 792 0.906 16.4 250.0 0.129 2, 3, 5, 68 896
0.992 22.3 129.7 0.138 2, 3, 6, 79 1052 0.709 24.7 216.8 0.148 2,
6, 7, 810 973 0.713 38.4 119.4 0.151 2,6,8,920 750 0.596 26.0 169.2
0.1306 833 0.941 6.6 223.0 0.145 -11 1125 1.082 35.0 175.3 0.179
6a,8,9,1012 1097 0.731 45.1 237.6 0.187 6a, 9, 10, 1113 1291 0.677
35.0 151.6 0.179 9,10,11,1214 1308 0.887 31.5 271,3 0.192 9, 11,
12, 1390 1052 0.709 24.7 216.8 0.155 -15 1358 0.980 48.6 201.1
0.185 11, 12, 13, 14
Auto- 3200 1.07 80 267Analyzer 1a
SMA 6/600 2900 0.966 72.4 276
CUNICAL CHEMISTRY, Vol. 21, No. 12, 1975 1759
0 Concentrations in final reaction mixtures by proposed
method.
method could be altered by simply changing the glu-cose oxidase
activity, as is illustrated in Figure 3. At aglucose oxidase
activity of 48000 U/liter, the curvebegins to flatten. For the 2000
mg/liter glucose stan-dard, the absorbance increases by 0.142 when
the glu-cose oxidase activity is doubled (from 12 000 to24 000
U/liter). When the glucose oxidase activity isdoubled again (from
24 000 to 48 000 U/liter), the ab-sorbance increases by only 0.067
for the same stan-dard.
We chose to use 12000 U of glucose oxidase perliter of reagent
because this gave us a linear curve to4000 mg of glucose per liter
and good sensitivity inthe 0-1500 mg/liter region with a 0.2 ml
serum sam-ple.Limits of the Method
Glucose concentration and absorbance are linearlyrelated to at
least 4000 mg/liter on the AutoAnalyzerI and to 5000 mg/liter of
the SMA 6/60. The AutoAn-alyzer I method can be altered to permit
analysis ofsamples containing as much as 5000 mg of glucoseper
liter by reducing the sample line one size, to 0.10ml/min, but some
precision is lost in the low concen-tration range results.
With either instrument the method is suitable foranalysis of
samples with glucose concentrations of
-
II
I
I
I
$
S
$ II
I
$
$
I
S
I
I
r
iI/SteodVStole Stondords Interoction test ltgh control Low
Control
Fig. 4. Recorder tracing from the AutoAnalyzer ISteady-State
tracing obtained by constant sampling of the 4000 mg/literstandard
for 5 mu; Standards are the eight aqueous glucose standards
do-sctlbed In the text; Interaction test, tracings for the 1000,
4000, 1000, and1000 mg/liter standards; b ConVol and Low ConVol
replIcate analyses oftwo commercial control set-a
dase reagent should be added through the middleconnection of the
GO cactus (Figure 1), to minimizeinteraction.
Reagent stability. We examined several differentpreservatives
for the peroxidase/buffer reagent listedearlier. This reagent
developed a fine sediment afterone to two weeks at room
temperature, and afterthree to four weeks, sensitivity declined.
The reagentis stable for at least four weeks at 4 #{176}C.We
havefound the stability of phosphate buffers to be quitecapricious.
Some appear to be stable for months atroom temperature, while mold
is growing in otherlots soon after preparation. Apparently the
stabilityof the buffer is determined by what spores, dust,
etc.,fall into the solution at the time of preparation.Trig
(hydroxymethyl)aminomethane buffers alsoshowed mold growth.
We investigated three preservatives in some detail.Sodium azide
(4 g/liter) was unsatisfactory; the per-oxidase/buffer reagent
became yellow after one week,and linearity and sensitivity
deteriorated after twoweeks. According to Bergmeyer et al. (21),
azide in-hibits peroxidase. Thimerosal (Merthiolate), 20 mg/liter,
is unsatisfactory. Results for glucose with theSMA 6/60 were lower
when thimerosal was present inthe final reagent (glucose
oxidase/peroxidase re-agent) vs. the reagent without thimerosal.
This wasnot true of the AutoAnalyzer I; identical sera assayedwith
and without thimerosal in the reagent gave thesame results.
Cacodylic acid (dimethylarsinic acid) looked prom-ising as a
preservative because the peroxidase/bufferreagent containing only
10 mmol of cacodylate perliter was stable for five months at room
temperature.Unfortunately, reagent with cacodylate gave
consis-tently higher results on the AutoAnalyzer I with
freshpatients sera than did the reagent without cacody-late.
The 1.5 g/liter phenol solution was colorless and
1780 CLINICAL CHEMISTRY, Vol. 21, No. 12 75
free of sediment after six months of storage at roomtemperature
in amber-colored glass bottles, andcould not be distinguished from
a freshly preparedphenol solution when used in conjunction with
theother reagents for glucose.
We thank Drs. H.-D. Gruemer and G. F. Grannis for
helpfulcomments on the manuscript, and Rita Beal, B. W. Durham,
JoanMercier, Kathy Rieger, and Tim Walters for technical
assistance.
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