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CLINICALCHEMISTRY,Vol. 28, No. 12, 1982 2405

OLIN. CHEM. 28/12, 2405-2407 (1982)

Four Methods for Glucose Assay Compared for Various GlucoseConcentrations and under Different Clinical ConditionsOttavio Giampietro, Alessandro P110,Giuseppe Buzzigoli, Claudlo Boni, and Renzo Navalesi

Glucose was measured by the ferricyanide, the Beckmanglucose oxidase, and the hexokinase procedures in 228plasma samples taken during standard oral glucose-tol-erance tests in 17 normal subjects and in 21 chemicaldiabetics. The neocuproine method was also used tomeasure glucose concentration in 156 samples (78 beforeand 78 after dialysis) collected from six diabetic and uremicpatients who were on maintenance hemodialysis. Fern-cyanide in all conditions and neocuproine in uremic pa-tients overestimated glucose concentrations over theentire experimental range as compared with either enzy-mic method. This bias or systematic error of the reducingvs the enzymic procedures, due to nonglucose reducingsubstances (saccharoids), becomes considerablygreater when their concentration is increased as in chronicuremia. Also, the inverse relation between glucose con-centration and overestimation of glucose by the reducingmethods has been detected. With respect to the hexoki-nase method, a mild but significant underestimate of glu-cose oxidase readings has been observed for higher glu-cose concentrations. We find neocuproine to be the mostimprecise of these procedures.

Addftlonal Keyphrases: glucose oxidase Beckman GlucoseAnalyzer ferricyanide hexokinase neocuproineuremia #{149}diabetesmellitus

Methods forglucosemeasurement based on the reducingpropertiesof glucose,becauseof interferenceby so-calledsaccharoids (nonglucose reducing substances) (1), sys-tematically overestimate the true plasma glucose concentra-tion, as compared with enzymic procedures (2). This sys-tematic error, or bias, is especially relevant in chronic uremia,in which these interfering substances are present in concen-trations much higher than normal (1, 3). Here we report re-sults of a comparison between two reducing (ferricyanide,neocuproine) and two enzymic (glucose oxidase, hexokinase)procedures for glucose analysis under various clinical condi-tions.

Materials and MethodsGlucosewas measured in228 plasmasamplestakenduring

standard oral glucose-tolerance tests in 17 normal subjects andin 21 with diabetes mellitus. Each sample was assayed by theferricyanide procedure, automated on a Technicon AutoAn-alyzer(4);the hexokinase procedure, automated on a LKB8600 Reaction Rate Analyzer (5); and the glucose oxidaseprocedure, with a Beckman Glucose Analyzer (6).

Clinica Medica II and Cattedra di Malattie del Ricambio dellU-niversit#{224}di Pisa; Istituto di Fisiologia Clinica del C.N.R. di Pisa,Italy.

Address correspondence to O.G., at the Istituto di Fisiologia ClinicsdelC.N.R.,Via Savi 8, 56100 Pisa, Italy.

Received Sept. 21, 1981; accepted July 13, 1982.

Next, we measured glucose by these same methods and alsoby the neocuproine method, automated on a Technicon Au-toAnalyzer (7), in 156 samples (78 before and 78 after dialysis)collected from six diabetic and uremic patients who were onmaintenance hemodialysis; the dialysate was free of glucose.The venous blood samples were all collected during onemonth, a sample being withdrawn before dialysis, and theothersample from thesame patientafter4 to5 h of dialysis.Each blood sample was collected, separated, and stored asreported elsewhere (8). Creatinine and nonprotein nitrogen(azotemia) were measured in uremic sera by usual methods.

ResultsBecause results by the various analytical procedures for

normal individuals and the diabetics were superimposable,we show in Figure 1 only those for the diabetics, for whom therange of glucose concentrations is broader. Ferricyanide sig-nificantly overestimates glucose concentrations over the entireexperimental range as compared with either enzymic method.Values by the latter methods tend mostly to concordance,though glucose oxidase gives generally higher readings than

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Fig. 1. Mean (and SE) plasma glucose concentrations during anoral glucose-tolerance test in subjects with diabetes, assayedwith the glucose-oxidase (Gox), ferricyanide (FC), and hexoki-nase (HK) methodsComparison by Students paired t-test

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Fig. 2. Mean (and SE) plasma glucose concentrations measured in six diabetic uremics before(a)and after(b)dialysis, by the FCneocuproine (NC), Gox, and HK methodsEach series of 78 specimens was divided on the basis of the glucose concentration Into six groups:

(+7.8%)HK 0.99

(-0.5%)Uremicsbeforedialysis(B)

FC 1.298 2.298(+28.6%)

HK 0.98(-1.8%)

NC 1.138(+ 13.4%)

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(+2 9%) a narrow range of glucose concentrations (most of them were1 89a around 0.80-1.20g/L) and so theirconclusionsmay not bejustifiedforhigherbloodglucoseconcentrations.In the other

(5.3%) paper (3), from the experimentaldata (see Table 3 in thepaper of Powell and Djuh) it is clear that the percent bias

3.31 a shown by fernicyanide over the o-toluidinereadingsisin-verselyrelatedto glucoseconcentration,althoughthe biasincreasesasan absolutevalue(g/L) with increase in glucose(3).

. Resultsby thehexokinasemethod aregenerallyverynearto those of glucose oxidase, but tend to be a littlelowerinall(+ 11.2%) (+ 10.5%) clinical conditions-and become significantlyloweraround

1.8-2 g/L. However, hexokinase, like glucose oxidase, mea-3 15a sures no uremic interferences, as may be seen from the nearly

(+8.8%) (+5.2%) absolute agreement between glucose measurements before1.948 2.848 and after dialysis.

Uremicsafterdialysis(C)FC 1.198 2.17a

(+19.5%)HK 1.04

NC(+3.8%)

1.08(+8.3%)

(-3.1%)2.10

(+5.2%)

(-5.4%)3 12 b In conclusion, our data confirm that reducing proceduresforglucoseassayshouldbe discontinued(3, 9, 10), because

(+4.1 #{176}) they are strongly affected by interferingsubstancesand (toa lesser degree) also by glucose concentration excursions. Thusthesemethods may involveerrorsin the glucosereadingsmainly in conditionsof borderlinecarbohydrateabnor-mality.

CLINICAL CHEMISTRY, Vol. 28, No. 12, 1982 2407

Table 1. Mean Plasma Glucose Concentrations asMeasured by the Ferricyanide (FC), Hexokinase(HK), and Neocuproine (NC) Methods, Compared

with the Glucose Oxldase (Gox) Method1.00 2.00 3.00

Glucose, g/L.

Normalsand diabetics(A)FC 1.08 2.06a

These data have been computed from the regression lines for each method vsglucose oxidase values in correspondence to the concentrations of 1.00, 2.00,and 3.00 g/L. The percent differences from the glucose oxidase readings arealso reported. The equationsof the regression lines, the respective correlationcoefficients (,), and the standard errors of estimate (SEE) are as follows:

A. FC = 0.098 + 0.98 Gox; r = 0.99, SEE = 0.051.HK = 0.096 + 0.90 Gox; r = 0.99, SEE = 0.064.

B. FC = 0.276 + 1.01 Gox; r = 0.99, SEE = 0.084.HK = 0.012 + 0.97 Gox; r = 0.99, SEE = 0.115.NC = 0.044 + 1.09 Gox r = 0.96, SEE = 0.257.

C. FC =0.215 + 0.98 Gox; r = 0.98, SEE = 0.127.Ilk = 0.137 + 0.90 Gox; r = 0.98, SEE = 0.131.NC 0.063 + 1.02 Gox; r 0.95, SEE = 0.283.

The significance of the differences of each value from the glucose oxidasevalue shown in the heading of the columns has been computed from the conf I-dence limits of the respective regression lines. >0p