An o-Toluidine Method for Body-Fluid

Glucose Determination

Kurt 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 after heating at 1000 for 10 mm., and the absorbance is determined at

630 or 635 mp. The reagent is stable for many months at room temperature, and the

reaction follows Beer’s Law over a very wide range of concentrations. The develop-

ment of the procedure is discussed, as is the specificity of the method for glucose.

\�MTH THE CURRENT EMPHASIS 0fl more frequent and more intensive

around-the- clock performance of clinical chemical determinations,

there is need for a simple, rapid, reliable, and economical method for

glucose determination that is equally adaptable to routine multiple or

single analyses of any body fluid and is capable of serving as a com-

patible manual stand-by and emergency test method in laboratories

using automatic glucose analysis. Preferably, such a method should

require only a single stable reagent, be adaptable to several of the

common methods of preparing protein-free filtrates which may al-

ready be employed in a given laboratory for other determinations, and

should possess considerable flexibility in its other operating charac-

teristics.

Colorimetric or spectrophotometric methods based upon the con-

densation of glucose with primary aromatic amines in glacial acetic

acid, which probably leads to formation of an equilibrium mixture of

From the Clinical Chemistry and Toxicology Laboratories, University of Florida Teaching

Hospital and Clinics, Gainesville, Fla.

Presented at the Thirteenth Annual Meeting of the American Association of Clinical Chem-

ists, New York, N. Y., August 1961.

The author is indebted to Natalie A. Green and Darcus A. Horsley for technical assistance.

Received for publication July 6, 1961.

*presellt address: Department of Biochemistry, University of Oklahoma School of Medicine,

Oklahoma City 4, OkIa.

215

216 DUBOWSKI CHnica� Chemistry

a glycosylarnine and the corresponding Schiff base, have been em-

ployed in the past with varying degrees of success. The benzidine

method of Jones and Pridham (1) yielded a nonlinear color at low

glucose concentrations and employed an unstable reagent. lJse of 2-

aminobiphenyl (C10H11N) was suggested by Timell et al. (2); and the

modified procedures of Athanail and Cabaud (3) and Forsell and

Palva (4) based upon this reaction met some of the above requirements

and have given us moderate satisfaction. However, the reagent proved

difficult to purify, was unstable, and required frequent recalibration.

Further, 2-aminobiphenyl is now believed to be markedly carcinogenic

(.5) and is consequently no longer readily available commercially.

Ortho-Toluidine (2-aminotoluene, C7H9N),whose structural formula is

CH3

7L/NHS

is structurally similar to 2-aminohiphenyl and might be expected to re-

act reasonably selectively with glucose. Its use in blood-glucose deter-

mination as a substitute for 2-aminobiphenyl was suggested to us by

0. M. Forsell (6) ; and the procedure we developed has beeii in routine

use in our laboratories for approximately 18 months and in several

other laboratories in OUI institution for lesser periods.

o-Toluidine, a primary aromatic amine, in acetic acid solution reacts

relatively selectively with glucose, yielding a stable green color which

adheres to the Beer-Lambert law over a wide range of concentration.

The reagent is compatible with several common protein-free filtrates

of blood and other body fluids, and the reaction conditions are readily

adjustable for the desired selisitivity, final reaction volume, concentra-

tion range, and the like.

Method

Reagents

1. o-Toluidine, 6.0% v/v in C. P. glacial acetic acid. Prepare

the reagent by dissolving the o-toluidine in the acid and allow it to

age for 1 week, durilig which time the initial yellow color changes

to amber. Store at room temperature in brown borosilicate glass bottle

or otherwise protected from light. J. T. Baker Chemical Co. reagent-

grade glacial acetic acid (Cat. No. 9506) and Eastman Organic Chemi-

Vol. 8, No. 3, 1962 BODY.FLUID GLUCOSE 217

cals o-toluidine (Eastman Grade, Cat. No. 253) have proved most suit-

able and can be used without further purification.

2. Trichloracetic acid, 3.0% w/v

Equipment

1. Automatic pipets. For efficiency, reproducibility, aiid con-

venience, the trichloracetic acid is dispensed from a 1.80-mi. automatic

pipet fitted with Teflon stopcock plug. (Cat. No. JP-6380 (Special),

Scientific Glass Apparatus Co., Inc., Bloomfield, N. J.) The o-toluidine

is delivered from a 3.00-ml. amber automatic pipet with Teflon stop-

cock plug, connected in series with a 1.00-ml. Thomas-Seligson auto-

matic pipet with Teflon stopcock plug (Cat. No. 8209-B2, Arthur H.

Thomas Co., Philadelphia 5, Pa.) which is used to measure the flitrate.

2. Fluid bath, 1000, thermostatically controlled. The bath liquid

may be a water-soluble polyalkalene glycol (Cat. No. 50-HB-280X

UCON Lubricant, Union Carbide Chemicals Co., New York 17, N. Y.).

3. Coleman Model 6D Junior Spectrophotometer (narrow band),

or equivalent

Calibration

Prepare aqueous standards containing 25-300 mg. of glucose per 100

ml. in steps of 25 mg., by appropriately diluting (with 0.2% benzoic

acid) a standard containing 1.00 gm. of anhydrous reagent-grade glu-

cose per 100 ml. in 0.2% ben�oic acid. Substitute 0.20 ml. of each stand-

ard for the body fluid sample in Step 1 of the procedure (see below)

and continue the analysis as outlined. Prepare a new calibration curve

for each new batch of reagent and check the curve periodically.

Procedure

1. Prepare a 1-in-lO protein-free filtrate of whole blood or other

body fluid by adding 0.20 ml. of well-mixed specimen to 1.80 ml. of 3.0%

trichloracetic acid; allow to stand for 5-10 mm. and filter through a

5.5-cm. Whatman No. 2 filter paper.

2. Into two 15 X 125-mm. borosilicate culture tubes with Teflon-

lined screw caps, place respectively 1.00 ml. of distilled water (Reagent

Blank) and 1.00 ml. of protein-free filtrate (Test). Add 3.00 ml. of

o-toluidine reagent to each tube, cap, and mix well by repeated inver-

sion.

3. Immerse tubes in a 100#{176}fluid bath for 10 miii.; remove and coo].

4. Measure the transmittance of the test specimen in a Coleman

218 DUBOWSKI Clinical Chemistry

Model 6D Jr. Spectrophotometer at 630 or 635 m� in 19-mm. cuvettes,

against the reagent blank adjusted to 100 per cent transmittance.

5. if the test specimen measures less than 10 per cent transmittance,

dilute both reagent blank and test specimen with equal volumes of gla-

cial acetic acid, mix well, and again measure as in Step 4 above.

6. Obtain the body-fluid-glucose level in milligrams per 100 ml. di-

rectly from the calibration table (multiplying the table values by the

dilution factor, if any).

Experimental

Development of Procedure and Determination of Optimal Conditions

For reasolls of laboratory convenience it was desired to employ a 1-

111-10 protein-free filtrate of all body fluid specimens, and to effect a

compromise between single-analysis range and sensitivity which al-

lowed the direct measurement of glucose concentrations of at least 250

l)ut not more than 300 mg./100 ml. of specimen. Preliminary tests of

reaction conditiolls were conducted to develop a temporary working

method adapted to use of the Coleman Model 6D Jr. Spectrophotome-

ter, and experimental findings were confirmed with use of the final pro-

cedure described above, where indicated. During this study, all volume

measurements were made manually, with the use of pipets and volu-

metric ware conforming to National Bureau of Standards Class A

specifications.

Effect of Protein-Free Filtrate Preparation

A pooled serum specimen, found to contain 93 mg. of glucose per

1.00 ml. by triplicate automatic glucose analysis with the AutoAna-

lyzer (7, 8), was deproteinized by six commonly used methods, and

the protein-free filtrates subjected to duplicate glucose determination

by the proposed o-toluidine method. The resulting absorbances were

converted to glucose levels with the use of individual calibration

curves established by diluting aqueous glucose standards with the re-

spective protein-precipitating reagents. The results, shown in Table

1, demonstrate interference with the proposed analysis by the per-

chioric acid filtrate, which preveiited formation of the usual green

color in the reaction mixture. The Abrahamson (9) tungstic acid fil-

trate yielded slightly higher serum glucose levels, and the Folin-Wu

(10) tungstic acid filtrate and the Somogyi (ii) barium-zinc filtrate

approximately the same levels, as the automatic reference method.

Vol. 8, No. 3 1962 BODY.FLUID GLUCOSE 219

Table 1. EFFECT OF PROTEIN-FREE FILTRATE PREPARATION ON GLUCOSE DETERMINATION BY

THE 0-TOLUIDINE METHOD

Teat No. Protein-free filtrate reageota

Serum glueoae fan fld�

(mg/I 00 ml.)

Filtrate ��rote in

(mg/I 00 ml.)

1 Abrahamson (9) 98 7.0

2 Folin-wu (10) 90 6.0

3 Perchloric acid, 10% -t -�

4 Somogyi (11) 92 7.0

5 Trichloracetic acid, 3% 94 4.0

6 Tiichloracetic acid, 10e/�� 93 -�

*Duplicate determinations by the o-toluidine method on a serum specimen found by the

reference automatic glucose method7 8 to contain 93 mg. glucose per 100 ml.

tAtypical pink color of equal absorbance in reagent blank and test developed.

�Biuret color destroyed by filtrate; protein determination not completed.

Both 3% w/v and 10% �v/v trichloracetic acid filtrates yielded the

same glucose level as the reference method; and deproteinization with

3% w/v trichioracetic acid, which yields an essentially protein-free

filtrate compatible with several other routine clinical chemistry deter-

millatiolls, was therefore selected as the method of choice.

Whole blood deproteillized with 3% w/v trichioracetic acid pro-

duced a filtrate having a pH of 1.3; and trichloracetic acid yielded

slightly greater volumes of protein-free filtrates than the tungstic aci(l

reagents, as also 1lOted by Sunderman et al. (12). The mixtures of

blood and protein precipitants were filtered through Whatman No. 2

filter paper; and control determinations with glucose-free materials

demonstrated that this filter paper did not contribute to the trichlor-

acetic acid filtrates any substances reacting with the o-toluidine re-

agent.

Determination of Spectral Characteristics of the Reaction Mixture

A series of aqueous standard glucose solutions (0-3 mg./ml. concen-

tration) was prepared from anhydrous National Bureau of Standards

certified standard glucose (Sample No. 41) and subjected to the entire

body-fluid-gluose procedure outlined above. Absorption spectra of the

resulting reaction mixtures were determined against reagent blanks

with a Beckman Model DU spectrophotometer and with a Coleman 61)

Jr. spectrophotometer. Figure 1 shows the data obtained for concen-

trations corresponding to body-fluid-glucose levels of 100 and 200

mg./100 ml., illustrating absorption maxima at 480 m� and 630 m� with

the Beckman instrument. The reaction mixture adheres to the Beer-

Lambert relation at both points, for body-fluid concentrations up to

400 425 450 475 500 525 550 575 600 625 650 675 700

220 DUBOWSKI Clinical Chemistry

‘.5

WAVE LENGTH, MILLIMICRONS

Fig. 1. Absorption spectra of o-toluidine reaction with glucose.

2000 mg./100 ml.; the absorption maximum at 630 m�is preferred for

the general procedure because of the greater sensitivity, the net ab-

sorbance being about 18 per cent greater than at 480 m�. On three of

our Coleman Model 6D spectrophotometers, maximal absorbance was

equal between 625 mj.t and 635 mp�, and the latter wavelength was

adopted for use with that instrument during the study.

Figure 2 shows a typical calibration curve for the procedure out-

lined above in which the Coleman 6D (“Narrow Band”) Jr. Spectro-

photometer was used at 635 mj�; compliance with the Beer-Lambert

relation over the range 0-300 mg. of glucose per 100 ml. of body fluid

is illustrated. Calibration curves prepared simultaneously for three

different Coleman Model 6D instruments in our laboratory were iden-

tical and corresponded to the equation

Body-Fluid-Glucose (mg./100 ml.) = Absorbance635 X 265.

In the preparation of the calibration curve, the aqueous glucose stand-

ards must be diluted with 3% trichloracetic acid as in deproteinization,

Vol. 8, No. 3, 1962 BODY.FLUID GLUCOSE 221

“.5

�0

GLUCOSE MG./IOO ML.

Fig. 2. Typical calibration curve for o-toluidine body-fluid-glucose determination with

Coleman Jr. Spectrophotometer.

since dilution with water produces a curve substantially different from

that applying to deproteinized body fluids.

Reagent and Reaction - Mixture Composition

Aqueous glucose standards containing 1, 2, and 3 mg./ml. were car-

ried through the entire Procedure, the o-toluidine reagent concentra-

tion 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 glucose concentration in the final reaction mixture

is maintained at increasing o-toluidine concentrations up to 8% v/v.

The sensitivity of the reaction can therefore be increased within limits

by employing a more concentrated reagent and/or by decreasing the

160

140

120

‘DO

G80

0.60

0.40

0.20

00

Fig. 3. Effect of o-toluidine reagent concentration on color development in o-toluidine-

glucose method.

0 I 2 3 4 5 6 7 8 9

o-TOLUIDINE CONCENTRATION, % v/v

222

‘C,5.5

DUBOWSKI Clinical Chemistry

dilution of the final reaction mixture by reducing the initial reagent

volume.

The 19-mm. round cuvette we wished to employ with a slightly modi-

fied adapter in our Coleman Model 6D Jr. spectrophotometers requires

a minimal sample volume of 3.2 ml. to cover the light path aperture, as

compared with a 5.8-ml. minimal volume for the unmodified instru-

ment. For convenience and accuracy in sample measurement, 1.0 ml.

of protein-free filtrate is used, and 3.0 ml. of o-toluidine reagent then

yield the desired minimal total reaction mixture volume. To achieve

the desired compromise between optimal single-analysis conicentra-

tion range and sensitivity and maximal precision and resolution, the

calibration curve had to cover the glucose range 25-250 mg./100 in the

photometric scale region of approximately 0.100-100 absorbance (�10-80 per cent transmittance), to minimize relative analysis errors per

unit photometric reading error, as pointed out by Ayres (13) and

Archibald (14). It is apparent in Fig. 3 that a 6% v/v o-toluidine re-

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 absorbance units, also corresponds to the most frequently

encountered glucose levels.

The 6.0% v/v o-toluidine reagent can react with sample glucose con-

centrations up to at least 2000 mg./100 ml., and it was determined that

the Beer-Lambert relation still applies to the reaction at this level.

Consequently, body-fluid-glucose concentrations up to 2000 mg./100

ml. can be determined by a single unknown analysis; for concentra-

tions greater than 300 mg./100 ml., both the reagent blank and the re-

action mixture are appropriately diluted with glacial acetic acid to

bring the final color intensity within the upper calibration limit, and

the calibration table values multiplied by the dilution factor. o-Tolui-

dime obtained from different sources shows some differences in reac-

tivity, the Eastman Organic Chemicals reagent being more sensitive

in our hands than a Matheson, Coleman & Bell reagent (Cat. No. 2714).

Various o-toluidine reagent batches prepared with the Eastman prod-

uct remained stable at room temperature, in the dark, for periods of

4.3-6 months without detectable changes in the calibration curves after

an initial 3- to 5-day ageing period. It was further unnecessary to re-

calibrate for reagent batches prepared from a single lot of Eastman

o-toluidine and a single lot of glacial acetic acid.

Time Effects: Color Intensity and Stability

The effect of heating time at 100#{176}on the color developed in the re-

action mixture was investigated with standard glucose solutions con-

taining 1, 2, and 3 mg./ml. and carried through the entire analysis. The

reaction mixtures and reagent blanks were heated for periods from 0

to 20 mm. inn steps of 2 mm., with the results shown in Fig. 4. Mixtures

of 1 ml. of glucose-containing fluid or filtrate and 3 ml. of 6% v/v o-

toluidine reached maximal absorbance in 12-14 miii., depending upon

the glucose concentration; heating for periods greater than 16 mm.

produced slightly lower absorbances. A compromise between reaction

sensitivity and minimal total analysis time was effected by choosing a

10-mm. period at 100#{176},which produces stable color intensities propor-

tional to glucose concentration. As illustrated in Fig. 4, the sensitivity

of the reaction can be increased approximately 12 per cent by prolong-

ing the heating time from 10 to 15 mm.Color stability in the final reaction mixtures was investigated in

224 DUBOWSKI Clinical Chemistry

1.60

1.40

120

‘4

� 1.00

080

0.60

0.40

0.20

010 12 14

TIME AT 100#{176}C., MINUTES

Fig. 4. Effect of heating time at 1000 on color development in o-toluidine-glueose method.

standard glucose solutions containing 1, 2, and 3 mg./ml., subjected to

the entire Procedure, rapidly cooled in a 4#{176}mechanical cooling bath to

room temperature (23#{176})after removal from the heating bath, and then

kept in ordinary room light (combined fluorescent illumination and

daylight) for periods up to 180 mm., with periodic photometric meas-

urement against reagent blanks similarly treated. The results are

shown in Fig. 5, illustrating that the color does not rapidly fade to an

appreciable extent. After 15 mm. of standing, approximately 98 per

cent of the original maximal absorbance remained; while 92-95 per

cent of maximal absorbances for various sample concentrations re-

mained 60 mm. after removal from the heating bath. Consequently, no

special precautions to prevent fading of the final reaction-mixture

colors appear necessary if photometric measurement is made reason-

ably promptly after termination of the heating period; and this factor

poses no problem in the analysis and photometric measurement of a

series of specimens.

1.40

1.20

too

080

060

0.40

0.20

0

Sample GlucoseMgIIOO ml.

�

300

�-_

�-

.

�-..

-�-

-__ 200- - -

PHOTOMETRY

Coleman 60 Sp.ctrophomet.r

Reagent Blank

19 mm. Round Cuvettes

6.0% V/V o-ToluidineI

0 30 60 90 120

TIME, MINUTES

ISO 180

Vol. 8, No. 3. 1962

“.5

5.5�0

‘41�‘.5

BODY.FLUID GLUCOSE 225

Fig. 5. Change in absorbance of o-toluidiime reaction mixture with time after heating.

Results

Reproducibility and Recovery Experiments

Precision and accuracy of the proposed method were investigated

by reproducibility and recovery studies. A human-blood-serum pool

assayed at 102 mg. of glucose per 100 ml. by the automatic ref ei’ence

method (7, 8) was independently analyzed 20 times by the proposed

procedure with the use of the same reagent lot and manual volume

measurements, all analyses being performed on the same day. The re-

sults, illustrating good precision, are shown in Table 2. The repro-

ducibility of the method is essentially limited only by the usual errors

of manipulation and measurement of volumes and absorbance; and

the precision shown in Table 2 can be significantly improved by con-

sistent employment of automatic or semiautomatic volume measure-

ment, e.g., with the equipment recommended under Method.

To investigate glucose recovery by the proposed method, pooled

heparinized human whole-blood specimens were analyzed in duplicate

by the proposed method and then reanalyzed after addition of 40-167

mg. of anhydrous glucose per 100 ml. blood, manual volume measure-

226 DUBOWSKI Clinical Chemistry

Table 2. RESULTS OF 20 CONSECUTIVE REPLICATE ANALYSES OF A SERUM POOL FOR GLUCOSE

BY THE O-TOLUIDINE METHOD

Serum glucose (mg./I00 ml.)

.4liquot No. Glucose found Difference from mean

1 103 +2.8

2 100 -0.2

3 100 -0.2

4 104 +3.8

5 97 -3.2

6 100 -0.2

7 102 +1.8

8 101 +0.8

9 101 +0.8

10 98 -2.2

11 98 -2.2

12 100 -0.2

13 103 +2.8

14 100 -0.2

15 97 -3.2

16 100 -0.2

17 100 -0.2

18 97 -3.2

19 100 -0.2

20 102 +1.8

Mean 100.2 ±1.5

S.D. ±2.0

*SD = , where d stands for difference from mean (15).

N-i

ments being employed throughout. The results, shown in Table 3, il-

lustrate good recovery.

Specificity Investigations

Most glucose-determination methods yield variable but significant

apparent glucose concentrations when applied to the analysis of blood

and some other body fluids from which glucose has been removed by

fermentation or enzymatic removal. The various copper-reduction

methods most commonly employed show such “saccharoid” values of

15-30 mg./100 ml. in glucose-free fasting blood specimens (12, 16-18);

the Folin-Wu method in which a tungstic acid filtrate is used (19)

when applied to blood is usually believed to include a fairly constant

nonglucose “saccharoid” concentration of about 20 mg./100 ml., al-

though occasional levels of 35 mg./100 ml. or more, calculated as glu-

*Duplicate determinations.

Vol. 8, No. 3, 1962 BODY-FLUID GLUCOSE 227

Table 3. RECOVERY Of ADDED GLUCOSE BY O-TOLUIDINE METHOD FROM BLOOD

Glucose (nuf 1100 sal.)

RecoveryInitial Glucose Total Glucose

Sample No. blood glucose* added glucase present found (%)

1 84 160 244 233 95.5

2 80 160 240 232 96.7

3 95 40 135 135 100.0

4 85 167 252 251 99.6

5 100 167 267 260 97.4

6 109 91 200 198 99.0

7f 100 40 140 142 101.4

Mean 98.5

�Duplicate determinations.

tAverage of 20 replicate recovery tests.

cose, are encountered (12, 16, 20). The reaction of nonglucose compo-

nents of blood, cerebrospinal fluid, and urine with the o-toluidine re-

agent was investigated by analysis of random fasting blood serum,

CSF, and urine specimens by the proposed method before and after re-

moval of glucose by yeast fermentation. One milliliter respectively of

serum, cerebrospinal fluid, and urine, each previously analyzed for

glucose by the o-toluidine procedure outlined, was incubated at 37#{176}for

45 mm. with 0.3 ml. of commercial dry granulated baker’s yeast which

had been repeatedly washed. The ability of the yeast to ferment glu-

cose was established by total removal of glucose from a 210 mg./100 ml.

aqueous standard under the same conditions. Following incubation,

the yeast was removed by centrifugation, and glucose determinations

were performed by subjecting the supernatant body fluid specimens to

the entire o-toluidine procedure outlined. A similar series of experi-

ments with the same specimens, with yeast incubation at 23#{176}for 45

mm., yielded identical findings. The results shown in Table 4 demon-

Table 4. BLANK VALUES FOR SERUM, CEREBROSPINAL FLUID, AND URINE BY O-TOLUIDINE

METHOD

Test No. Specimen

Glucose (mg/ZOO ml.)*

Rla,,k �

(osgluco�e, mg/ZOO ml.)IniZkit After fermentation

1 Yeast - 1 1

2 Glucose Std. 210 1 0

3 Serum 174 5 4

4 C.S.F. 118 2 1

S Urine 203 6 5

228 DUBOWSKI Clinical Chemistry

strate the relative lack of response of the o-toluidine method to the

nonglucose components of serum, cerebrospinal fluid, and urine and

the correspondingly low blank values for glucose-free body fluids.

The proposed o-toluidine method was further tested for specificity

by investigating the possibility of interference by certain physio-

logically occurring substances. Possible color production by some

relevant carbohydrates and polysaccharides was tested by subjecting

aqueous standard solutions of 8 substances, each containing 160 mg./

100 ml., to the complete procedure outlined and obtaining the glucose

equivalent corresponding to the color intensity developed. The fol-

lowing apparent glucose levels were obtained:

Substance tested Glucose equivalent (rng./100 ml.)

Arabinose 26

Chondroitin sulfuric acid A 1

Galactose 160

Glucosamine 3

ilyaluronidase 0

Lactose 52

Maltose 14

Mannose 158

A fiuial specificity investigation was concerned with the effect of high

added concentrations of some relevant compounds on the recovery of

glucose from blood. Twelve substances, including mono-, di-, and

polysaccharicles, aldopentoses, and aldo- and ketohexoses, were addedto a pooled whole blood specimen of known glucose level to yield con-

centrations of 100 mg./100 ml. for each compound. The resulting blood

specimens were analyzed by the entire procedure outlined and the ab-

sorbances converted to apparent glucose concentrations from the glu-

cose calibration curve. The results are shown in Table 5, which in-

cludes for each substance a quantitative “interference” estimate rep-

resenting the difference between the initial blood glucose level and the

apparent blood glucose level after addition of the compound being

tested. From these interferences and the known 100 mg./100 ml. con-

centration of each compound in the blood sample, a reaction ratio for

each substance as compared to glucose was calculated (Table 5). Of

the 12 substances tested, only galactose and mannose react with o-tolui-

dine equimolecularly with glucose, as is to be expected; and the pro-

posed method, therefore, cannot distinguish between these three stereo-

isomers. No significant interference should exist in this method from

physiologic concentrations of the other substances shown in Table 5.

Vol. 8. No. 3. 1962 BODY-FLUID GLUCOSE 229

Table 5. EFFECT OF SOME COMMON GuJoosE ANALYSIS INTERFERENTS UPON THE O-TOLUIDINE

METHOD�.

Test No. Substance added0

Appa rent glucose

found

(mg./100 ml.)f

Interference 08

glucose

(mg./100 ml.)

Reaction ratio

(substance/glucose)

1 Arahinose 90 17 0.17

2 Fructose 73 0 0

3 Galactose 173 100 1.00

4 Glucosainiiie HC1 72 0 0

5 Glucuronic acid 93 20 0.20

6 Glycogeit 75 2 0.02

7 Lactose 106 33 0.33

8 Maltose 78 5 0.05

9 Mannitol 73 0 0

10 Mannose 173 100 1.00

11 Urea 70 0 0

12 Xylose 92 19 0.19

8One hundred milligrams of each substance added per 100 ml. of blood, which initially con-

tained 73 mg. glucose per 100 ml.

tDuplieate determinations.

Comparison with Automatic Glucose Analysis

Our laboratory, in common with many others, routinely uses auto-

mated glucose analysis, predominantly for plasma or serum speci-

mens, by a modified Hoffman procedure (7, 8)-quantitative reduction

of alkaline ferricyanide to ferrocyanide-adapted to the AutoAnalyz-

er. The correlation of body fluid glucose analyses by the proposed

o-toluidine method with this automatic method is therefore important

and was extensively investigated. Routine human whole-blood, serum,

or plasma specimens, both fasting and nonfasting, received in the lab-

oratory for glucose determination were simultaneously analyzed by

the automated Hoffman method and by the o-toluidine procedure. The

specimens included 1)0th sera from unpreserved clotted blood and

whole-blood specimens treated with sodium heparin or with sodium

heparin and sodium fluoride, as well as plasma from the latter two

sources. All specimens were promptly processed and immediately ana-

lyzed, or, in a few instances, promptly processed and preserved by

freezing at -20#{176}until analysis could be performed.

The results of 100 consecutive simultaneous serum glucose deter-

minations by both methods are shown in Table 6 and illustrated in Fig.

6 and are typical of the correlation found in several hundred other un-

reported parallel analyses. The mean difference between tile glucose

levels found by the two methods, ±2.4 mg./100 ml. over a range from

Specimen

1

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

210�)

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

Table 6. COMPARISON OF 100 CONSECUTIVE S�aUM GLUCOSE ANALYSES BY AUTOANALYZEB

AND BY THE O-TOLUIDINE METHOD

Serum glucose (mg./iOO sal.) Serum gluco8e (mg./100 ml.)

AuloAnalyzer o-Taluidine Difference Specimen AutoAnalyzer o-Toluidine

178 176 2 52 #{149} 84 82

115 110 5 53 94 97

55 53 2 54 102 102

93 89 4 55 84 82

72 68 4 56 84 89

58 56 2 57 87 87

103 105 2 58 86 87

146 151 5 59 86 91

94 95 1 60 87 86

114 117 3 61 112 109

52 47 5 62 106 103

64 63 1 63 86 84

89 88 1 64 92 90

77 78 1 65 82 80

99 95 4 66 102 103

116 120 4 67 91 91

92 95 3 68 200 198

81 78 3 69 88 88

84 79 5 70 91 91

80 83 3 71 100 100

61 65 4 72 127 124

78 79 1 73 129 127

76 78 2 74 60 62

90 93 3 75 86 86

102 103 1 76 252 255

80 85 5 77 83 83

80 76 4 78 99 97

200 196 4 79 67 70

123 119 4 80 172 174

102 98 4 81 130 132

110 114 4 82 92 92

145 145 0 83 100 100

94 89 5 84 79 75

101 100 1 85 92 87

114 114 0 86 81 76

85 90 5 87 75 75

70 74 4 88 84 84

104 103 1 89 87 89

131 132 1 90 64 65

132 129 3 91 139 144

82 82 0 92 147 149

92 90 2 93 135 138

68 64 4 94 71 70

76 80 4 95 19 22

80 78 2 96 89 86

68 64 4 97 93 88

94 95 1 98 84 84

112 116 4 99 124 122

106 110 4 100 70 70

96 99 3 Mean

112 113 1 S.D.8

S.D. = , where d stands for difference between results by the two methods.N-i

Difference

2

3

0‘)

5

0

1

5

1

3

30

2

1

0

2

0

0

0

3

2

2

0

3

0

2

3

2

2

0

0

4

5

50

0

2

1

5

2

3

1

3

3

5

0

2

0

±2.4

±2.9

0 25 50 75 100 125 150 175 200

BLOOD GLUCOSE, MG./IOOML.-AUTOANALYZER

Vol. 8, No. 3, 1962 BODY-FLUID GLUCOSE 231

200

175

“U

150

125

-J� 100

� 75

50

0� 25

0

Fig. 6. Comparison of 100 consecutive routine blood-serum-glucose determinations by auto.

mated Hoffman and o-toluidine methods.

52 to 252 mg./100 ml., with a standard deviation of ±2.9 mg./100 ml.,

compares very favorably with other reported correlations between the

automated Hoffman method and manual glucose procedures (8, 21-

23), and with tile ±1.3 mg./100 ml. average difference from the mean

with a standard deviation of ±2.0 mg./100 ml. of replicate glucose

analyses by the o-toluidine method (Table 2). For all pi�actical pur-

poses, the proposed o-toluidine method can be regarded as fully com-

patible with the automated Hoffman procedure, and results obtained

by either procedure may be considered interchangeable.

Glucose Values Obtained with the o-Toluidine Method

After the development and complete investigation of the final o-

toluidine procedure described above, it was routinely employed in our

25

1330 �Fasting�

Blood Plasma Specimens

0

0 0 20 30 40 50 60 70 80 90 100 PlO 120 30 140 ISO 160 ITO 180 90 200 200300 400 500’500

BLOOD PLASMA GLUCOSE. MG/IOO ML

232 DUBOWSKI Clinical Chemistry

Fig. 7. Distribution of “fasting” blood plasma glucose levels in 1330 consecutive routine

hospital specimens analyzed by o-toluidine method.

laboratory for body-fluid-glucose determination for a period of ap-

proximately 18 months, to date. To illustrate the range and distribu-

tion of glucose levels encountered in blood and cerebrospinal fluid, a

portion of these routine laboratory data were examined further.

Figure 7 is a histogram of 1330 consecutive blood plasma glucose

levels from this group of data. The data were obtained on plasma

promptly separated upon receipt in the laboratory from whole-blood

samples treated with sodium heparin and designated as “fasting”

specimens and immediately analyzed or preserved by freezing at -20#{176}

until analyzed; specimens from an unselected general university hos-

pital patient population including adult and pediatric inpatients and

outpatients are included. Plasma glucose levels between 70 and 120

mg./100 ml. were found most frequently and represent 71.6 per cent of

all results. The histogram shows a skewed distribution toward the

higher levels as compared to the usual normal distribution for healthy

subjects, because of the substantial number of specimens from diabetic

patients included in our group. Normal plasma glucose levels are ap-

Vol. 8, No. 3, 1962 BODY-FLUID GLUCOSE 233

proximately 10-20 mg./100 ml. higher than whole-blood levels in the

same specimens (16, 20, 23) since the intracellular glucose concentra-

tions are lower than the extracellular ones. In view of this factoi’ and

the nature of the hospital population sampled, the most frequently

occurring range in this plasma series, 70-120 mg./100 ml., corresponds

well with the commonly cited 65-110 mg./100 ml. fasting whole-blood

glucose value for 95 per cent of “normal healthy subjects” by the

Nelson-Somogyi method (11, 24, 25).

A similar analysis of 210 consecutive cerebrospinal-fluid-glucose

levels in histogram form is shown in Fig. 8. Again, the specimens rep-

resent unpreserved routine samples from general university hospital

adult and pediatric inpatient services, processed and analyzed prompt-

ly upon receipt in the laboratory, or preserved by freezing at -20#{176}un-

til analyzed. The values occurring most frequently, 50-80 mg./100 ml.,

represent 74.3 per cent of all values in this group and correspond well

to the most frequently found plasma levels, which are usually 20-40

mg./100 ml. higher than simultaneous CSF concentrations in the same

patient (26, 27). Normal human cerebrospinal-fluid-glucose levels are

commonly given as 45-100 mg./100 ml. for adults (27,28), presumably

40

210 CSF.Specimens

Fig. 8. Distribution of cere- � 30

brospinal-fluid-glucose levels in

210 consecutive routine hospital �

specimens analyzed by o-tolui- � 25

dine method. :

K�2O

Lu IS0. 1/ 7

I /

�I0

0

0 10 20 30 40 50 60 70 80 90 100>100

CEREBROSPINAL FLUID GLUCOSE, MG/IOOML.

234 DUBOWSKI Clinical Chemistry

when determined by copper-reduction methods, and this range paral-

lels the values occurring most frequently in our series of CSF speci-

mens from hospitalized patients analyzed by the o-toluidine method.

Discussion

Arising out of a need for an adequate manual glucose determination

method compatible with the automated Hoffman procedure, a method

was developed which, in its final form, has these favorable major char-

acteristics: With respect to reliability, it has adequate accuracy, ade-

quate precision, good sensitivity and resolution, good selectivity for

glucose, with low biologic blank values, and freedom from interference

by the most common relevant substances. With respect to practicabili-

ty, it has simplicity and economy, reasonable rapidity of analysis, good

reagent stability, ready commercial availability in pure form of the

single reagent components, adherence to the Beer-Lambert law

throughout a wide range, compatibility with various filtrates, a good

range-and-sensitivity compromise, and comparability of results with

the automated glucose method.

In any analytical method, reliability considerations govern accept-

ability for a specific purpose and situation, while practicability con-

siderations affect the choice among acceptable procedures. The allow-

able error for manual body-fluid-glucose determinations has been pro-

posed variously as from 5.4 to 27.0 per cent by different investigators

(29-al). The proposed method amply meets these requirements, and

its performance appears adequate with respect to the other reliability

criteria listed.

Three of the practicability characteristics would seem to merit brief

discussion. The several adjustments in reagent and reaction mixture

composition and in reaction conditions resulted in a method combining

sufficient sensitivity and chemical resolution to permit� adequate dis-

crimination between closely adjacent glucose levels for special clinical

studies, with an adequately wide concentration range to cover approxi-

mately 95 per cent of all fasting blood glucose levels in a single un-

known analysis, without need for dilution of final reaction mixtures or

repetition of the analysis. The exceptionally wide glucose range over

which the Beer-Lambert law is followed further permits the determi-

nation of virtually all physiologic glucose levels in every body material

by simple dilution of the final reaction mixture for levels over 275 mg./

100 ml. Simplicity of test performance and concurrent economy are

combined with reasonable rapidity; and a single determination can be

completed within 20 mm., which makes the procedure practical for

casual and emergency single analyses. For most effective integration

into laboratories employing automated glucose determination by the

Vol. 8, No. 3, 1962 BODY-FLUID GLUCOSE 235

Hoffman method, a stand-by manual procedure should yield identical

results and be capable of operating on the same specimens normally

analyzed automatically to minimize problems of specimen procure-

ment and interpretation of results. The proposed o-toluidine proce-

dure fulfills this requirement and additionally employs a protein-free

filtrate which can be readily utilized for several other major clinical

chemical analyses, including urea nitrogen determination.

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