An

o-Toluidine Glucose
Kurt

Method

for

Body-Fluid

Determination
M. Dubowski*

o-Toluidine, 6% (v/v) in glacial acetic acid, is used to determine glucose in biologic material after deproteinization with 3% (w/v) trichloracetic acid. A stable green color develops 630 or 635 mp. reaction follows after heating at 1000 is stable for 10 mm., and the absorbance for many months range of concentrations. of the method is determined The developat The reagent at room temperature, and the

Beers Law over a very wide is discussed,

ment of the procedure

as is the specificity

for glucose.

\MTH

THE

CURRENT

EMPHASIS

0fl

more clinical reliable, and

frequent chemical and is capable test be

and economical to routine

more

intensive

around-thethere glucose
single

clock for
of

performance a simple, that

any

of rapid, fluid is equally and analysis.

determinations, method multiple as in to laboratories should of may characthe mixture

of Florida of technical School Clinical Teaching Chem-

is need
analyses

for or a com-

determination manual automatic only methods be employed possess stand-by glucose a single of

adaptable

body

of serving method such a method several which operating based amines equilibrium
University Association for

patible using require common ready should teristics.

emergency Preferably, reagent, laboratory flexibility

stable preparing

adaptable filtrates for other other

the aland

protein-free in its methods aromatic of an

Laboratories, of and the American

in a given considerable or

determinations,

Colorimetric densation acid, which

spectrophotometric with leads primary to formation

upon in glacial

conacetic of

of glucose probably

From the Clinical Chemistry Hospital and Clinics, Gainesville, Presented at the Thirteenth ists, New York, N. Y., The author is indebted

and Toxicology Fla. Annual Meeting A. Green

August 1961. to Natalie

Darcus University

A. Horsley of Oklahoma

assistance. of Medicine,

Received *presellt Oklahoma

for publication July address: Department City 4, OkIa.

6, 1961. of Biochemistry,

215

216

DUBOWSKI

CHnica

Chemistry

a glycosylarnine and the corresponding Schiff base, ployed in the past with varying degrees of success. method of Jones and Pridham (1) yielded a nonlinear glucose modified concentrations (C10H11N) procedures of upon this us moderate and employed was Athanail suggested and an unstable by Timell Cabaud some of the However, (3) aminobiphenyl Palva (4) based and have given

have been emThe benzidine color at low lJse

(2);

reagent. et al. and

of 2the and

and

Forsell

reaction met satisfaction.

above requirements the reagent proved recalibration. carcinogenic commercially. formula is

difficult to purify, was Further, 2-aminobiphenyl (.5) and is consequently Ortho-Toluidine

unstable, and required frequent is now believed to be markedly no longer readily available C7H9N),whose CH3
7L/NHS

(2-aminotoluene,

structural

is structurally

similar

to 2-aminohiphenyl

and

might

be expected

to re-

act reasonably selectively with glucose. Its use in blood-glucose mination as a substitute for 2-aminobiphenyl was suggested 0. M. Forsell (6) ; and the procedure we developed has beeii use other relatively adheres The of blood adjustable tion range, reagent and for and in our laboratories in a primary selectively to the with Beer-Lambert is compatible other the the body desired like. with fluids,
OUI

deterto us by in routine in several reacts which filtrates are readily concentra-

for

approximately for amine, yielding over several and the a wide lesser

18 months periods. acid in acetic a stable range common reaction final reaction

and solution

laboratories

institution aromatic glucose, law

o-Toluidine,

green protein-free

color

of concentration.

conditions volume,

selisitivity,

Method
Reagents 1.

o-Toluidine, reagent for by 1 week,

6.0%

v/v the which

in

C. time

P.

glacial in the initial

acetic the acid yellow

acid. and color

Prepare allow it to changes

the age

dissolving durilig

o-toluidine

to amber. Store at room temperature or otherwise protected from light. grade glacial acetic acid (Cat. No.

in brown borosilicate glass bottle J. T. Baker Chemical Co. reagent9506) and Eastman Organic Chemi-

Vol.

8, No.

3,

1962

BODY.FLUID

GLUCOSE

217

cals able
2.

o-toluidine and can Trichloracetic

(Eastman be used without acid,

Grade, further
3.0%

Cat. w/v

No.

253)

have

proved

most

suit-

purification.

Equipment 1.

Automatic the fitted with

pipets. trichloracetic Teflon

For acid stopcock

efficiency, is dispensed plug. (Cat. Bloomfield, automatic

reproducibility, from No. a 1.80-mi. JP-6380

aiid automatic (Special),

con-

venience, pipet Scientific is delivered cock matic Thomas

2.

Glass Apparatus from a 3.00-ml.

Co., Inc., amber

N. J.) The o-toluidine pipet with Teflon stopThomas-Seligson No. 8209-B2, Arthur autoH.

plug, connected in series with a 1.00-ml. pipet with Teflon stopcock plug (Cat. Co., Fluid be Philadelphia bath, 1000, 5, Pa.) which thermostatically polyalkalene

is used to measure controlled. The glycol (Cat. New No.

the flitrate. bath liquid 50-HB-280X

may UCON
3.

a water-soluble

Lubricant, Coleman

Union Carbide Model 6D Junior

Chemicals Co., Spectrophotometer

York 17, N. Y.). (narrow band),

or equivalent
Calibration

Prepare ml. acid) cose ard in steps

aqueous

standards by

containing appropriately

25-300 diluting of anhydrous Substitute 1 of the

mg.

of glucose (with 0.2%

per

100

of 25 mg.,

benzoic glustandbelow) curve

a standard per 100 ml. for the body

containing 1.00 gm. in 0.2% benoic acid. fluid sample in Step

reagent-grade 0.20 ml. of each procedure (see

and continue for each new

Procedure

the analysis as outlined. Prepare batch of reagent and check the

a new calibration curve periodically.

1. body

Prepare a 1-in-lO protein-free filtrate of whole blood or other fluid by adding 0.20 ml. of well-mixed specimen to 1.80 ml. of 3.0% 5-10

trichloracetic acid; allow to stand for 5.5-cm. Whatman No. 2 filter paper. 2. Into two 15 X 125-mm. borosilicate lined Blank) o-toluidine sion. 3. 4. Immerse Measure screw and caps, 1.00 reagent tubes the place ml. of respectively protein-free tube, cap, bath of the

mm.
culture

and tubes

filter

through with 3.00 Teflon(Reagent ml. inverand coo].

1.00 ml. filtrate and for

of distilled (Test). mix well by

water Add repeated

of

to each

in a 100#{176} fluid transmittance

10 miii.; specimen

remove in

test

a Coleman

218

DUBOWSKI

Clinical

Chemistry

Model against 5. dilute cial

6D Jr. Spectrophotometer the reagent blank adjusted if the both test reagent specimen blank measures and test again table

at 630 or 635 m to 100 per cent less specimen than 10 per with

in 19-mm. transmittance. cent equal volumes

cuvettes,

transmittance, of glaml. by dithe 4 above. per 100 values

acetic acid, mix well, and 6. Obtain the body-fluid-glucose from factor, the calibration if any).

measure as in Step level in milligrams (multiplying the table

rectly dilution

Experimental
Development of Procedure and Determination of Optimal Conditions

For

reasolls

of laboratory filtrate

convenience of all body fluid

it was

desired

to employ and to effect

a 1a al250 of

111-10 protein-free compromise lowed the l)ut not reaction

specimens,

between single-analysis range and sensitivity which direct measurement of glucose concentrations of at least than 300 mg./100 ml. of specimen. to develop Preliminary a temporary tests working conditiolls were conducted

more

method adapted to use of the ter, and experimental findings cedure described above, where measurements metric ware specifications.
Effect of Protein-Free Filtrate

Coleman Model 6D Jr. Spectrophotomewere confirmed with use of the final proindicated. During this study, all volume with the Bureau use of of pipets Standards and voluClass A

were made conforming

manually, to National

Preparation

A pooled serum specimen, 1.00 ml. by triplicate automatic lyzer (7, 8), was deproteinized the by protein-free the proposed to established filtrates o-toluidine glucose levels by diluting interference filtrate, reaction which mixture.

found to contain glucose analysis by six commonly to duplicate The the use glucose The proposed resulting of method. with aqueous reagents. with preveiited The the

93 mg. with used glucose individual standards results, analysis

of glucose per the AutoAnamethods, and determination were calibration with shown by the the reperin Table

subjected

absorbances

converted curves spective chioric color trate

(10)

protein-precipitating acid in the yielded tungstic

1, demonstrate

formation Abrahamson glucose Somogyi levels,

(ii)

of the usual green (9) tungstic acid filand the barium-zinc reference Folin-Wu filtrate method.

slightly higher serum acid filtrate and the the same levels, as

approximately

the

automatic

Vol.

8, No.

1962

BODY.FLUID

GLUCOSE

219
ON GLUCOSE DETERMINATION BY

Table

1.

EFFECT

OF

PROTEIN-FREE THE

FILTRATE 0-TOLUIDINE

PREPARATION METHOD

Serum Teat No. Protein-free filtrate reageota (mg/I

glueoae 00

fan ml.)

fld

Filtrate (mg/I

rote 00 ml.)

in

1 2 3 4 5 6 *Duplicate reference tAtypical Biuret

Abrahamson Folin-wu Perchloric Somogyi Trichloracetic Tiichloracetic determinations automatic glucose

(10)

(9)
acid,
(11)

98
90 10% -t 92 3% 10e/ the o-toluidine
8

7.0 6.0
-

7.0 4.0
-

acid, acid, by

94 93 method 93 mg. on a serum per specimen 100 ml. found

by

the

method7

to

contain

glucose and not

pink color color destroyed

of equal absorbance in reagent blank by filtrate; protein determination

test developed. completed.

Both

3%

w/v

and

10%

v/v

trichloracetic method; yields routine as the an

acid and

filtrates

yielded

the

same glucose level 3% w/v trichioracetic filtrate millatiolls, compatible was

as the with

reference acid, which several selected other

deproteinization with essentially protein-free chemistry deterof choice. acid proacid yielded tungstic aci(l mixtures of 2

clinical

therefore

method

Whole blood duced a filtrate slightly greater reagents, blood filter acetic agent.
Determination

deproteillized with 3% w/v trichioracetic having a pH of 1.3; and trichloracetic volumes of protein-free filtrates than the also 1lOted by Sunderman et al. filtered did not
(12).

as

The

and protein paper; and that filtrates acid

precipitants were control determinations this any filter paper substances

through Whatman No. with glucose-free materials contribute with the to the trichlorreo-toluidine

demonstrated

reacting

of Spectral

Characteristics

of the Reaction

Mixture

A series tration) certified was

of aqueous prepared standard

standard from glucose (Sample

glucose anhydrous No.

solutions National 41) and

(0-3 Bureau subjected

mg./ml. to the

concenentire

of Standards

body-fluid-gluose resulting reaction with a Beckman Jr. spectrophotometer. trations mg./100

procedure outlined above. mixtures were determined Model DU spectrophotometer Figure 1 shows the to body-fluid-glucose absorption maxima The reaction points, for

Absorption spectra of the against reagent blanks and with a Coleman 61) data obtained for concenlevels at 480 m of 100 and 200 and 630 m with Beerup to

corresponding ml., illustrating

the Beckman instrument. Lambert relation at both

mixture body-fluid

adheres to the concentrations

220

DUBOWSKI

Clinical

Chemistry

.5

400

425

450

475

500

525

550

575

600

625

650

675

700

WAVE

LENGTH, of

MILLIMICRONS reaction with glucose.

Fig.

1.

Absorption

spectra

o-toluidine

2000

mg./100

ml.;

the

absorption

maximum

at 630

mis

preferred the net On three

for abof was was

the general procedure sorbance being about our Coleman equal between adopted for Model 625 use with

because of the greater 18 per cent greater than 6D spectrophotometers, mj.t and 635 mp, that instrument and during the

sensitivity, at 480 m. maximal latter the study. for

absorbance wavelength

Figure 2 shows a typical calibration curve lined above in which the Coleman 6D (Narrow photometer was used at 635 mj; compliance relation over is illustrated. different tical and the range Calibration Model 0-300 mg. curves of glucose prepared

the procedure outBand) Jr. Spectrowith the Beer-Lambert per 100 ml. simultaneously of body for were fluid three iden-

Coleman corresponded

6D instruments to the equation

(mg./100
ml.)
=

in our
Absorbance635

laboratory X 265.

Body-Fluid-Glucose

In the preparation ards must be diluted

of the with

calibration curve, 3% trichloracetic

the aqueous glucose standacid as in deproteinization,

Vol.

8,

No.

3,

1962

BODY.FLUID

GLUCOSE

221

.5 0

GLUCOSE Fig.
Coleman

MG./IOO

ML.
determination with

2.
Jr.

Typical

calibration

curve

for

o-toluidine

body-fluid-glucose

Spectrophotometer.

since that

dilution applying

with

water

produces body
Composition

a curve fluids.

substantially

different

from

to deproteinized
-

Reagent

and Reaction

Mixture

Aqueous

glucose

standards

containing

1, 2, and

3 mg./ml.

were

car-

ried through the entire Procedure, the o-toluidine reagent concentration being varied from 0 to 10% v/v in steps of 1 or 2%. The effect of these changes in reagent concentration upon the color developed in the reaction mixture is shown in Fig. 3, which indicates that linearity of color intensity with at glucose concentration in the final reaction up mixture is maintained The sensitivity by employing increasing of the reaction o-toluidine concentrations can therefore be increased reagent and/or by to 8% v/v. within limits the

a more

concentrated

decreasing

222

DUBOWSKI

Clinical

Chemistry

160

140

120

C,

5.5

DO

G80

0.60

0.40

0.20

0
0 I 2 3 o-TOLUIDINE 4 5 CONCENTRATION, concentration on color 6 7 % v/v in o-toluidine8 9

Fig.
glucose

3.

Effect

of

o-toluidine

reagent

development

method.

dilution

of the

final

reaction

mixture

by

reducing

the

initial a slightly

reagent modirequires

volume. The 19-mm. round cuvette fied adapter in our Coleman

we wished to employ with Model 6D Jr. spectrophotometers

a minimal compared ment. For

sample volume with a 5.8-ml. convenience

of 3.2 ml. to cover the light path aperture, as minimal volume for the unmodified instruand accuracy in sample measurement, 1.0 ml. and 3.0 ml. of o-toluidine reaction mixture volume. optimal single-analysis precision range and 25-250 reagent then To achieve conicentraresolution, mg./100 the in the

of protein-free yield the desired the desired tion range calibration photometric 10-80 per

filtrate is used, minimal total between

compromise

and sensitivity and maximal curve had to cover the glucose cent scale region transmittance), of approximately to minimize

0.100-100 absorbance relative analysis errors out by Ayres (13) a 6% v/v o-toluidine

(
per and re-

unit photometric Archibald (14).

reading error, as pointed It is apparent in Fig. 3 that

Vol.

8, No.

3,

1962

BODY.FLUID

GLUCOSE

223

agent best meets these several requirements in the proposed analysis scheme. On the calibration curve for this reagent concentration (Fig. 2), the region of least relative analysis error caused by photometry, 0.30-0.50 encountered The centrations the Consequently, 6.0% absorbance glucose v/v units, levels. 2000 still also reagent mg./100 applies corresponds can react ml., to and the to with the most frequently glucose at to 2000 for blank this conthat level. mg./100 re-

o-toluidine relation body-fluid-glucose

sample reaction up

up to at least

it was

determined

Beer-Lambert

concentrations unknown both the

ml. can be determined tions greater than 300 action bring mixture the final are color table from

by a single mg./100 ml.,

analysis; reagent

concentraand the

appropriately intensity values different

diluted within the multiplied sources Chemicals Coleman batches temperature, changes period. prepared acetic

with upper

glacial calibration

acetic

acid to limit, and o-Toluiin reacsensitive No. 2714). prod-

the calibration dime obtained tivity, in our Various

by the dilution factor. shows some differences reagent being & Bell reagent prepared with the in the dark, in the calibration It was from acid. further a single more (Cat. Eastman for

the Eastman hands than o-toluidine stable without

Organic a Matheson, reagent at room detectable ageing batches

uct remained 4.3-6 months an initial calibrate o-toluidine

Time Effects:

periods of curves after to reEastman of

3- to 5-day for and

Color

unnecessary lot

reagent a single

lot of glacial
and Stability

Intensity

The action taining reaction

effect mixture

of heating was 3 mg./ml. and

time investigated and reagent

at

100#{176} the on with standard through were

color the heated

developed glucose entire for

in the solutions analysis.

reconThe

1, 2, and mixtures

carried blanks with

periods

from

0 o-

to 20 mm. inn steps of 2 mm., of 1 ml. of glucose-containing

the results shown fluid or filtrate and

in Fig. 4. Mixtures 3 ml. of 6% v/v upon 16 mm. reaction

toluidine reached maximal absorbance the glucose concentration; heating produced slightly lower absorbances. sensitivity 10-mm. tional of the ing the Color and period to glucose reaction heating stability minimal total analysis at 100#{176}, which concentration. can time in be increased from the final

in 12-14 miii., depending for periods greater than A compromise between time was effected color intensities in Fig. 4, the 12 per mixtures was cent

by choosing proporsensitivity by prolong-

produces stable As illustrated approximately

10 to 15

mm.
investigated in

reaction

224
1.60

DUBOWSKI

Clinical

Chemistry

1.40

120

1.00

080

0.60

0.40

0.20

0 10 TIME AT 12 14

100#{176}C., MINUTES development in o-toluidine-glueose method.

Fig.

4.

Effect

of

heating

time

at

1000

on color

standard

glucose

solutions

containing

1, 2, and

3 mg./ml.,

subjected cooling bath,

to

the entire Procedure, room temperature kept in daylight) urement shown

rapidly (23#{176})fter a

cooled in a 4#{176} mechanical removal from the heating

bath to and then and measare to an 98 per per reno reasonthis factor of a

ordinary room light (combined fluorescent for periods up to 180 mm., with periodic against in Fig. extent. original reagent 5, illustrating blanks that similarly the color treated. does not

illumination photometric The rapidly results fade 92-95

appreciable cent of the

After 15 mm. of standing, approximately maximal absorbance remained; while for from various sample the heating bath. fading of the and of heating photometric the final period; measurement

cent of maximal absorbances mained 60 mm. after removal special colors ably poses series precautions appear promptly no problem necessary after in the to prevent

concentrations Consequently, reaction-mixture is made and measurement

if photometric termination analysis

of specimens.

Vol.

8, No.

3.

1962

BODY.FLUID

GLUCOSE

225

1.40

Sample

1.20
-..

MgIIOO
300
-_
.

Glucose ml.

too
.5 5.5 0

080
41 .5
--__ -

200
-

060

0.40
PHOTOMETRY Coleman 60 Blank Round V/V Cuvettes Sp.ctrophomet.r

0.20

Reagent 19 mm. 6.0%

o-Toluidine

0 0

I 30

60 TIME,

90 MINUTES reaction

120

ISO

180

Fig.

5.

Change

in

absorbance

of

o-toluidiime

mixture

with

time

after

heating.

Results
Reproducibility and Recovery Experiments

Precision by reproducibility assayed method procedure measurements,

and at 102
(7, 8)

accuracy and mg. was the all

of recovery

the per

proposed studies. 100 ml. analyzed reagent are performed

method by

were

investigated pool ref eience proposed volume The rereproerrors and by con-

A human-blood-serum the automatic 20 times by the lot and manual same day. on the

of glucose independently use analyses of the

with

same

being

sults, illustrating ducibility of the of manipulation the sistent ment, precision

good precision, method is essentially and measurement in Table of automatic equipment 2 can

shown limited of volumes

in Table 2. The only by the usual and absorbance; improved volume Method.

shown

be

significantly

employment e.g., with the

or semiautomatic recommended under

measure-

To investigate heparinized human by the proposed mg. of anhydrous

glucose recovery by the proposed method, pooled whole-blood specimens were analyzed in duplicate method glucose and then reanalyzed per 100 ml. blood, after manual addition volume of 40-167 measure-

226
Table 2.
RESULTS OF

DUBOWSKI

Clinical

Chemistry

20

CONSECUTIVE BY THE

REPLICATE O-TOLUIDINE

ANALYSES METHOD Serum glucose

OF

A SERUM

POOL

FOR

GLUCOSE

(mg./I00

ml.)

.4liquot

No.

Glucose

found

Difference

from

mean

1
2

103
100 100 104 97 100 102 101 101 98 98 100 103 100 97 100 100 97 100 102 100.2

+2.8
-0.2 -0.2 +3.8 -3.2 -0.2 +1.8 +0.8 +0.8 -2.2 -2.2 -0.2 +2.8 -0.2 -3.2 -0.2 -0.2 -3.2 -0.2 +1.8 1.5 2.0

3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean S.D.

*SD

where

d stands

for

difference

from

mean

(15).

N-i

ments lustrate
Specificity

being employed good recovery.

Investigations

throughout.

The

results,

shown

in Table

3, il-

Most apparent and some fermentation methods 15-30

glucose-determination glucose other most concentrations body fluids or enzymatic commonly ml. method to blood from

methods

yield

variable

but

significant

when applied which glucose

to the analysis of blood has been removed by of

removal. The various copper-reduction employed show such saccharoid values fasting a tungstic believed ml. blood specimens
(12, 16-18);

mg./100

in glucose-free in which is usually

the Folin-Wu when applied

acid filtrate is to include a fairly of about or more, 20 mg./100 calculated

used (19) constant ml., alas glu-

nonglucose saccharoid though occasional levels

concentration of 35 mg./100

Vol.

8,

No.

3,

1962

BODY-FLUID

GLUCOSE

227
METHOD FROM BLOOD

Table

3.

RECOVERY

Of

ADDED

GLUCOSE

BY

O-TOLUIDINE

Glucose Initial Sample No. blood glucose* Glucose added

(nuf

1100

sal.) Total Glucose found Recovery

glucase

present

(%)
95.5 96.7 100.0 99.6 97.4 99.0 101.4 98.5

1 2 3 4 5 6 7f Mean
Duplicate determinations.

84 80 95 85 100 109 100

160 160 40 167 167 91 40

244 240 135 252 267 200 140

233 232 135 251 260 198 142

tAverage

of 20

replicate

recovery

tests.

cose, nents agent CSF, moval serum,

are was and

encountered investigated urine specimens

(12,

16,

20).

The and of proposed urine, dry

reaction urine random method One each

of nonglucose with the before o-toluidine blood and respectively analyzed fasting milliliter previously incubated bakers yeast

comporeserum, after reof for

of blood,

cerebrospinal by

fluid, analysis by the fermentation. and

of glucose cerebrospinal

by yeast fluid,

glucose 45 mm. had been cose was aqueous

by the o-toluidine procedure with 0.3 ml. of commercial repeatedly established standard washed. The by total removal under the same

outlined, was granulated of the

at 37#{176} for yeast which glu-

ability

to ferment

of glucose conditions.

from a 210 mg./100 ml. Following incubation, glucose determinations body fluid specimens series at in Table
AND URINE BY

the yeast was were performed the ments entire with yielded
4.
BLANK

removed by centrifugation, and by subjecting the supernatant procedure specimens, findings.

FOR SERUM,

to 45

o-toluidine the same identical

VALUES

outlined. with The yeast results

A similar incubation shown

FLUID,

of experi23#{176} for 4 demonO-TOLUIDINE

mm.,
Table

CEREBROSPINAL METHOD

Glucose Test No. IniZkit

(mg/ZOO After

ml.)* Rla,,k fermentation (osglucoe, mg/ZOO ml.)

Specimen

1 2 3 4 S *Duplicate

Yeast Glucose Serum C.S.F.

Urine

1 1 5 2 6

1 0 4 1 5

Std.

210 174 118 203

determinations.

228 strate the nonglucose the relative lack components low o-toluidine the

DUBOWSKI

Clinical

Chemistry

of response of serum, blank

of the o-toluidine cerebrospinal fluid,

method to the and urine and body for fluids. specificity physioby some subjecting 160 the mg./ glucose The fol-

correspondingly The proposed

values for glucose-free method was further tested of interference Possible color polysaccharides of 8 substances, was each

by investigating logically occurring relevant aqueous 100 ml., equivalent lowing carbohydrates standard

possibility substances. and

by certain production tested containing obtaining developed. by

solutions

to the complete corresponding apparent

Substance
Arabinose

procedure to the levels

outlined and color intensity were obtained:

equivalent 26

glucose
tested

Glucose

(rng./100

ml.)

Chondroitin Galactose Glucosamine ilyaluronidase Lactose Maltose Mannose

sulfuric

acid

A
160

1 3 0
52 14

158

A fiuial added glucose

specificity

investigation of some Twelve

was relevant substances, and

concerned

with

the

effect

of high of and

concentrations from blood. whole were converted curve. substance difference

compounds including

on the recovery mono-, di-,

polysaccharicles, to a pooled centrations specimens sorbances

aldopentoses, blood specimen analyzed by the

aldoand ketohexoses, of known glucose level each compound. procedure concentrations shown in interference blood Table are initial The outlined

were added to yield conresulting and from the blood the abgluinrepthe being confor Of

of 100 mg./100

ml. for

entire glucose

to apparent

cose calibration cludes for each resenting apparent tested. centration each the the

The results a quantitative between the after

5, which estimate level and ml. ratio

glucose

blood glucose level From these interferences of each compound as compared tested, with only

addition of and the known in the blood sample, to glucose galactose was and mannose

the compound 100 mg./100 a reaction (Table react with

substance 12 substances

calculated

5). o-tolui-

dine equimolecularly posed method, therefore, isomers. No significant physiologic concentrations

glucose, as is to be expected; cannot distinguish between these interference should exist in this of the other substances shown

and the prothree stereomethod from in Table 5.

Vol.

8. No.

3.

1962

BODY-FLUID

GLUCOSE

229
INTERFERENTS UPON THE O-TOLUIDINE

Table

5.

EFFECT

OF

SOME

COMMON .

GuJoosE

ANALYSIS METHOD

Appa
Test No. Substance added0

rent found

glucose ml.)f

Interference glucose (mg./100 ml.)

08

Reaction (substance/glucose)

ratio

(mg./100

1 2 3 4 5 6 7 8 9 10 11 12 8One tained hundred 73 mg.

Arahinose Fructose Galactose Glucosainiiie Glucuronic Glycogeit Lactose Maltose Mannitol Mannose Urea Xylose milligrams glucose per 100 determinations. of each ml. substance HC1 acid

90 73 173 72 93 75 106 78 73 173 70 92 added per 100 ml.

17 0 100 0 20 2 33 5 0 100 0 19 of blood, which

0.17 0 1.00 0 0.20 0.02 0.33 0.05 0 1.00 0 0.19 initially con-

tDuplieate

Comparison

with

Automatic

Glucose

Analysis

Our mated mens, er.

laboratory, in common with many others, routinely glucose analysis, predominantly for plasma or by a modified Hoffman procedure (7, 8)-quantitative ferricyanide correlation method extensively specimens, of to ferrocyanide-adapted body fluid glucose analyses to the by

uses autoserum specireduction AutoAnalyzthe proposed important serum, in the labby The

of alkaline The o-toluidine and was or plasma oratory for the automated specimens whole-blood heparin sources. lyzed, freezing The

with this automatic method is therefore investigated. Routine human whole-blood, both fasting and from with and nonfasting, received were simultaneously by the o-toluidine unpreserved sodium heparin

glucose determination Hoffman method included specimens 1)0th sera treated

analyzed procedure. clotted or

blood and with sodium two anaby deterin Fig. other un-

and sodium fluoride, as well as plasma All specimens were promptly processed or, in a few instances, promptly processed at -20#{176}until analysis could be performed. results of 100 consecutive simultaneous

from the latter and immediately and preserved serum glucose

minations 6 and are

by both methods are shown in Table 6 and typical of the correlation found in several mean 2.4 difference mg./100 ml.

illustrated hundred

reported parallel analyses. The levels found by the two methods,

between over

tile glucose a range from

Table

6.

COMPARISON

OF

100

CONSECUTIVE AND BY THE

SaUM O-TOLUIDINE

GLUCOSE METHOD

ANALYSES

BY

AUTOANALYZEB

Serum

glucose

(mg./iOO

sal.)
Difference Specimen

Serum
AutoAnalyzer

gluco8e

(mg./100

ml.)

Specimen

AuloAnalyzer

o-Taluidine

o-Toluidine

Difference

1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
0)

178 115 55 93 72 58 103 146 94 114 52 64 89 77 99 116 92 81 84 80 61 78 76 90 102 80 80 200 123 102 110 145 94 101 114 85 70 104 131 132 82 92 68 76 80 68 94 112 106 96 112

176 110 53 89 68 56 105 151 95 117 47 63 88 78 95 120 95 78 79 83 65 79 78 93 103 85 76 196 119 98 114 145 89 100 114 90 74 103 132 129 82 90 64 80 78 64 95 116 110 99 113

2 5 2 4 4 2 2 5 1 3 5 1 1 1 4 4 3 3 5 3 4 1 2 3 1 5 4 4 4 4 4 0 5 1 0 5 4 1 1 3 0 2 4 4 2 4 1 4 4 3 1

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Mean S.D.8

#{149}

84 94 102 84 84 87 86 86 87 112 106 86 92 82 102 91 200 88 91 100 127 129 60 86 252 83 99 67 172 130 92 100 79 92 81 75 84 87 64 139 147 135 71 19 89 93 84 124 70

82 97 102 82 89 87 87 91 86 109 103 84 90 80 103 91 198 88 91 100 124 127 62 86 255 83 97 70 174 132 92 100 75 87 76 75 84 89 65 144 149 138 70 22 86 88 84 122 70

2 3 0
)

5 0 1 5 1 3 3
0

2 1 0 2 0 0 0 3 2 2 0 3 0 2 3 2 2 0 0 4 5

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

5
0 0 2 1 5 2 3 1 3 3 5 0 2 0 2.4 2.9

S.D.

where

d stands

for

difference

between

results

by

the

two

methods.

N-i

Vol.

8, No.

3,

1962

BODY-FLUID

GLUCOSE

231

200

175

150

125

-J

100

75

50

0 25

0 0 25 BLOOD Fig. mated 6. Comparison and of 100 50 75 GLUCOSE, consecutive methods. routine 100 125 150 175 200 MG./IOOML.-AUTOANALYZER blood-serum-glucose determinations by auto.

Hoffman

o-toluidine

52 to 252 compares automated

23),

mg./100

ml.,

with

a standard

deviation

of

2.9

mg./100

ml., the
21-

very favorably with Hoffman method with by

tile

other reported correlations between and manual glucose procedures (8, ml. of 2.0 method average mg./100 (Table 2). difference ml. of For all from replicate piactical as results the

and

1.3 deviation o-toluidine

mg./100

mean glucose pur-

with analyses poses, patible

a standard the

the proposed o-toluidine method with the automated Hoffman procedure

Values Obtained

can be regarded procedure, and interchangeable.

Method

fully comobtained

by either
Glucose

may
with

be considered
the o-Toluidine

After toluidine

the

development described

and

complete above,

investigation it was routinely

of

the

final

o-

procedure

employed

in our

232

DUBOWSKI

Clinical

Chemistry

laboratory for body-fluid-glucose proximately 18 months, to date. tion of glucose levels encountered portion Figure levels promptly samples of these routine 7 is a histogram from this separated treated with immediately specimens population are included. were found The histogram group upon

To

determination for a period of apillustrate the range and distribuin blood and cerebrospinal fluid, a data were consecutive examined blood further. plasma glucose on as plasma whole-blood fasting

laboratory of 1330 of sodium data. receipt analyzed from an including most

The heparin

data and

were

obtained from

in the

laboratory designated

specimens and until analyzed; pital patient ml. outpatients mg./100 all results. higher subjects, patients
25

or preserved by freezing at -20#{176} unselected general university hosadult and pediatric inpatients and 70 and 120 per cent of toward for healthy from diabetic levels are apthe

Plasma glucose levels between frequently and represent 71.6 shows a skewed distribution usual normal distribution number of specimens Normal plasma glucose

levels as compared because of the included in our

to the substantial group.

1330 Blood

Fasting Specimens

Plasma

0 0 0 20 30 40 50 60 70 80 90 100 PlO 120 PLASMA 30 140 ISO 160 ITO 180 MG/IOO ML 90 200 200300 400 500500 BLOOD GLUCOSE.

Fig. hospital

7.

Distribution specimens

of analyzed

fasting by

blood o-toluidine

plasma method.

glucose

levels

in

1330

consecutive

routine

Vol.

8, No.

3,

1962

BODY-FLUID

GLUCOSE

233

proximately same tions specimens are lower

10-20
(16,

mg./100
20,

ml.
23) since extracellular

higher the

than

whole-blood view glucose of this

levels

in the

than

the

intracellular ones. In

concentrafactoi and

the nature occurring well with

of the hospital population range in this plasma series, the commonly for cited 65-110

sampled, the 70-120 mg./100 mg./100 ml. healthy

most frequently ml., corresponds whole-blood by the subjects

fasting

glucose value Nelson-Somogyi A levels resent adult ly upon similar in histogram unpreserved and

95 per cent of normal method (11, 24, 25). of 210 consecutive

analysis

cerebrospinal-fluid-glucose the specimens repuniversity hospital and analyzed promptat -20#{176}unml., well 20-40 same are

form is shown in Fig. 8. Again, routine samples from general inpatient in the laboratory, services, processed or preserved

pediatric receipt

by freezing

til analyzed. The values represent 74.3 per cent to the most frequently mg./100 patient commonly ml. higher
(26, 27).

occurring most frequently, 50-80 mg./100 of all values in this group and correspond found plasma levels, which are usually simultaneous CSF concentrations in the human cerebrospinal-fluid-glucose mg./100 ml. for adults (27,28),
40

than Normal as 45-100

given

levels presumably

210 Fig. 210 dine 8. Distribution routine analyzed by of levels hospital o-tolui25 cerein 30

CSF.

Specimens

brospinal-fluid-glucose consecutive method. specimens

:
K

2O

Lu
0.

IS
1/

I0

0 0 10 20 30 40 50 FLUID 60 70 80 90 100>100 MG/IOOML.

CEREBROSPINAL

GLUCOSE,

234 when determined lels the values mens from

DUBOWSKI

Clinical

Chemistry

by copper-reduction occurring most frequently patients analyzed

methods, and this range in our series of CSF by the o-toluidine

paralspeci-

hospitalized

method.

Discussion
Arising method was acteristics: developed With out compatible which, respect of a need with for the an adequate automated form, manual Hoffman has these it has glucose procedure, favorable adequate determination a method major accuracy, charadefor

in its final

to reliability,

quate precision, good sensitivity and glucose, with low biologic blank values, by the most common relevant substances. ty, it has reagent single throughout range-and-sensitivity the simplicity stability, reagent a wide and ready components, range, compromise, economy, commercial adherence compatibility and

resolution, good selectivity and freedom from interference With respect to practicabilirapidity to with the various of analysis, in pure form Beer-Lambert filtrates, of results govern practicability The has requirements, other reliability been investigators availability

reasonable

good of the law a good with acceptconallowproand

comparability considerations

automated glucose method. In any analytical method, reliability for error a specific affect for The listed. of the The variously the manual as from proposed appears practicability several purpose choice and among

ability siderations able posed

(29-al).

situation, acceptable per with cent meets respect

while determinations by

procedures. different these to the would seem and

body-fluid-glucose 5.4 to 27.0 method adequate amply

its

performance

criteria Three discussion.

characteristics adjustments in reagent

to merit

brief mixture

reaction

composition and in reaction conditions resulted sufficient sensitivity and chemical resolution crimination between closely adjacent glucose studies, with an adequately wide concentration mately known repetition which nation 100 ml. combined completed 95 per cent of all fasting blood glucose analysis, without of the analysis. need for dilution The exceptionally

in a method combining to permit adequate dislevels for special clinical range to cover approxilevels in a single mixtures range the body over unor over

of final reaction wide glucose further levels permits in every for levels

the Beer-Lambert law is followed of virtually all physiologic glucose dilution of the final reaction Simplicity of test with reasonable within 20 performance rapidity; which analyses. automated

determimaterial 275 mg./

by simple

mixture and makes

and concurrent economy are a single determination can be the procedure effective determination practical integration by for the For most glucose

mm.,

casual and emergency single into laboratories employing

Vol.

8, No.

3,

1962

BODY-FLUID

GLUCOSE

235

Hoffman results analyzed ment dure filtrate chemical

method, and

a stand-by

manual

procedure

should

yield

identical normally procure-

be capable automatically

of operating to minimize of

on the same specimens problems of specimen

and interpretation fulfills this requirement which can be readily including analyses,

results. The proposed o-toluidine proceand additionally employs a protein-free utilized urea for nitrogen several other major clinical determination.

References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Jones, Timell, Athanail, Forsell, J. T. K. N., and Pridham, E., Glaudemans, J. B., C. P. J., Biochem. J. 58, 288 (1954). and Currie, A. L., Anal. Chem. (1958). 11, 28, 409 1916 (1959).
Products

(1956).

0., and Cabaud, 0. M., and Palva,

P. 0., J. Lab. Ctin. Med. 51, 321 I. P., Scand. J. Gun. 4 Lab. Invest.

Fuess, J. T., Manager, Industries Division, Forsell, 0. M., personal Hoffman, W. Grady, H. J., Abrahamson, Fohin, 0., and Somogyi, M., Sunderman, 21, 901

Eastman Organic Chemicals Department, Eastman Kodak Co., personal communication, communication, Mar. 13, 1960.

Distillation

Mar.

13,

1959.

S., J. Biol. Chem. 120, 51 (1937). and Lamar, M. A., Clin. Chem. 5, 542 (1959). E. M., Am. J. Clin. Path. (Tech Suppl.) 4, Wu, H., J. Biol. CViem. 38, 81(1919). J. Biol. Chem. 160, 69 (1945). MacFate, R. P., Evans, G. T., and

75

(1940).

F. W., (1951).

Fuller,

J.

B.,

Am.

J.

Gun.

Path.

Ayres, G. H., Anal. Chem. 21, 652 (1949). Archibald, R. M., Anal. C7iem. 22, 639 (1950). Youden, W. J., Statistical Methods for Chemists. Wiley, Ackerman, I. P., Diseases of carbohydrate metabolism, Examinations in Clinical

New York, 1951. in A Syllabus

of

Laboratory Culver.

17. 18. 19. 20. 21.

Diagnosis, rev. ed., edited by L. B. Page and P. J. Harvard, Cambridge, Mass., 1960, pp. 466-474. Mosenthal, H. 0., Ann. Internal Med. 33, 1175 (1950). Young, N. F., Glucose, in Standard Methods of Clinical Chemistry, edited by M. Academic Press, New York, 1953, vol. 1, pp. 60-64. Folin, 0., and Wu, H., J. Biol. Chem. 41, 367 (1920). Kugelmass, I. N., Biochemistry of Blood in Health and Disease. Thomas, Springfield, 1959. Anon., Blood sugar results (Hand ern Div., Albert Einstein Medical Instruments Corp., Chauncey, N. Anon., Comparison bia-Presbyterian sruinents Corp., method: Center, Y. Nelson-Somogyi) Philadelphia,
;

Ruiner,

Ill.,

information

from

1957;

distributed

by

NorthTechnicon

22.

of Folin-Wu sugar with AutoAnalyzer; Medical Center, New York, undated; Chauncey, N. Y.

information distributed by

from ColumTechnicon In.

23. 24. 25. 26.

Hill, J. B., and Kessler, G., J. Lab. Clin. Med. 57, 970 (1961). Nelson, N., J. Biol. Chem. 153, 375 (1944). Reinhold, J. G., Glucose, in Standard Methods of Clinical Reiner. Academic Press, New York, 1953, vol. 1, pp. 65-70. Shafer, W. Laboratory Harvard, H., Adams, Examinations R. P., and Calkins, E., in Clinwal Diagnosis, pp. 265-291. Parenteral Saunders, M., Am. Extravascular edited by

Chemistry, fluids, L. B. Page Body

edited

by

M.

in A Syllabus of and P. J. Culver. Cavities. Grune, 1956, Path. 23, p. 285

Cambridge,

1960,

27. 28. 29. 30. 31.

Hoeprich, P. D., and Ward, J. R., Jhe Fluids of the New York, 1959. Handbook of Biological Data, edited by W. S. Spector, 57. Henry, R. J., Bcrkman, S., Golub, 0. J., and Segalove, (1953). Kingsley, G. R., The Filter 18, 1-9 (1956). Freier, E. F., and Rausch, V. L., Univ. of Minn. Med.

Philadelphia, J. Gun.

Bull.

24,

190

(1958).