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.

References1. Trinder, P., Determination of glucose in blood using glucose ox-idase with an alternative oxygen acceptor. Ann. Clin Biochem. 6,24 (1969).2. Allen, J. R., Earp, R., Farrell, E. C., Jr., and Gruemer, H.-D.,Analytical bias in a quality control scheme. Clin. Chem. 15, 1039(1969).3. Moroney, M. J., Facts from Figures, Penguin Books, Baltimore,Md., 1951, p291.4. Gochman, N., Ryan, W. T., Sterling, It E., and Widdowson, G.M., Interlaboratory comparison of enzymatic methods for serumglucose determination. Clin. Chem. 21,359 (1975).5. Pennock, C. A., Murphy, D., Sellers, J., and Longdon, K. J., Acomparison of AutoAnalyser methods for the estimation of glucosein blood. Clin. Chim. Acta 48, 193 (1973).6. Schrauzer, G. N., and Rhead, W. J., Ascorbic acid abuse: Effectsof long term ingestion of excessive amounts on blood levels andurinary excretion. mt. J. Vitam. Nutr. Res. 43, 201 (1973).7. Mayersohn M., Ascorbic acid in man. Pharmo-kinetic implica-tions. Eur. J. Pharmacol. 19, 140 (1972).8. Goodman, L. S., and GiJman, A., The Pharmacologic Basis ofTherapeutics, 4th ed., Macmillan, 1970, p 234.9. Ibbott, F. A., Amino acids and related substances. In ClinicalChemistry: Principles and Technics, 2nd ed., R. J. Henry, D. C.Cannon, and J. W. Winkelman, Eds. Harper and Row, Hagers-town, Md., 1974, p 618.10. Muenter, M. D., and Tyce, G. M., L-DOPA therapy of Parkin-sons disease: Plasma L-DOPA concentration, therapeutic re-sponse, and side effects. Mayo Clin. Proc. 46, 231 (1971).11. Tyce, G. M., Muenter, M. D., and Owen, C. A., Dihydroxy-phenylalanine (DOPA) in plasma during DOPA treatment in pa-tients with Parkinsons disease. Mayo Clin. Proc. 45,438, (1970).12. Weiss, J. L., and Chase, T. N., Levodopa in parkinsonism.Drugs 2, 257 (1971).13. Abrams, W. B., Coutinho, C. B., Leon, A. S., and Spiegel, H.E., Absorption and metabolism of levodopa. J. Am. Med. Assoc.218, 1912 (1971).14. Free, A. H., Enzymatic determination of glucose. Adu. Clin.Chem. 6,84 (1963).15. Long, D. E., Simplex optimization of the response from chemi-cal systems. Anal. Chim. Acta 46, 193 (1969).16. Deming, S. N., and Morgan, S. L., Simplex optimization ofvariables in analytical chemistry. Anal. Chem. 45, 278A (1973).17. Krause, R. D., and Lott, J. A., Use of the simplex method tooptimize analytical conditions in clinical chemistry. Clin. Chem.20, 775 (1974).18. Gochman, N., and Schmitz, J. M., Application of new peroxideindicator reaction to the specific, automated determination of glu-cose with glucose oxidase. Clin. Chem. 18,943 (1972).19. Ref. 14, p 70.20. Thiers, R. E., Cole R. R., and Kirsch, W. J., Kinetic parame-ters of continuous flow analysis. Clin. Chem. 13,451(1967).21. Bergmeyer, H. -U., et al., Biochemical reagents. In Methods ofEnzymatic Analysis, Section D. H.-U. Bergmeyer, Ed. AcademicPress, New York, N. Y., 1965, p 991.22. Carey, R. N., Feldbruegge, D., and Westgard, J. 0., Evaluationof the adaptation of the glucose oxidase/peroxidase 3-methyl-2-benzothiazolinone hydrazone-N,N-dimethylaniline procedure tothe Technicon SMA 12/60, and comparison with other auto-mated methods for glucose. Clin. Chem. 20,595 (1974).23. Romano, A. T., Automated glucose methods: Evaluation of aglucose oxidase-peroxidase procedure. Clin. Chem. 19, 1152(1973).