A study of the direct o-toluidine blood glucose determination

Clznica Chimica Acta, 43 (1973) 105-1 I I J’! Elscvier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

105

CCA 5388

A STUDY

OF THE

DIRECT

o-TOLUIDINE

BLOOD

GLUCOSE

DETERMINATIOK

CHhRLBS

F. F.-\SCE, JR.,* ROBERT

RI:J**

APZDANTHOST

J. PIGNATrlRO

New York State Depavtnaent of Health, Division of Labovatories and lieseavch, Albany, N. Y. 122 OI (U.S.A.) (Received

June 13. 1972)

The acetic acid-o-toluidine method for measuring serum glucose concentration was studied. Changes in concentrations and conditions were performed manually and by continuous variation. Maximum absorbance at 630 nm was produced at concentrations less than 100% acetic acid solvent. The effects of increasing water concentration for a variety of samples and conditions are shown. A wide disparity of patterns was observed when patient and lyophilized control sera were studied. At water concentrations less than 10% (v/v) marked enhancement of color due to borate ion was observed; in glacial acetic acid, borate more than doubles color produced. Addition of trichloroacetic acid showed inhibition of the reaction. Absorbance was found to increase with increasing o-toluidine concentration over the range studied (I to II:/,). At concentrations of water greater than 15% significant serum blanks were observed. This blank was not due solely to protein turbidity, and is dependent upon the presence of both glucose and /?-lipoprotein. Effects of other reagent parameters, color development time, and sample constituents were investigated.

ISTRODUCTION

The o-toluidine reaction for the estimation of glucose concentration in serum has gained popularity in clinical laboratories, demonstrated by an increase in New York State from five laboratories in 1965 to over 170 in 1971. In a recent glucose proficiency testing program interlaboratory agreement using this technique was poor (coefficient of variation of 7%). Over this seven-year period investigators have modified the original method1 through changing the sample:reagent ratio, the reagent acetic acid:water ratio, and by addition of other substances2p9. A careful study of the otoluidine glucose reaction was undertaken in the hope of achieving a more reproducible assay. * Present address: City of Kingston Laboratory, **’ To whom inquiries should be addressed.

Kingston,

N.Y.

106

FASCFC, JR.

MATERIALS

ASD

METHODS

Reagents o-Toluidine”

was purified

by double

distillation

under reduced

chromogenic reagents contained 1.25 g/liter thiourea. Concentrations and acetic acid were varied as described in RESL-LTS. Glucose (dextrose) was obtained

L’t d.

from the National

Variations in reagent variation as described by sorbance as a function of flow diagram for the study

Bureau

of Standards,

M’ashington,

pressure.

All

of o-toluidine for standards

D.C.

concentrations were obtained by the method of continuous Ryland et uZ.l” which enables a continuous graph of abreagent concentration in the AutoAnalyzer’:‘~’ system. A of the o-toluidine glucose reaction is shown in Fig. I. Rc-

073 FLOW CELL

RECORDER

COLORIMETER 15MM

f/c (a) Fig.

PULSE I.

SUPPRESSOR

TUBULAR

FILTERS630”m 0.005

“I.D.

Flow diagram for continuous

variation study of the o-toluidinc

~lucosc reaction.

agent line A pumps the varying chromogenic reagent from a mixing reaction manifold, while Reagent line B constantly pumps the limiting of the reagent. The variation in concentration is described by

flask into the concentration

where Ct is the concentration at time t, C, the concentration of solution being fed (Reagent B), Y is the pumping rate, and v the liquid volume in the mixing flask. Reagent A and B pump tubing are standardized as described previouslyll. The sample line continuously pumps specimen into the manifold. The standard AutoAnalyzer heating bath was modified by insulating glass coils with asbestos sheets on sides and bottom. Without such insulation chart recordings show a regular sinusoidal response due to the fluctuating temperature produced by thcrmoregulator cycling. * Eastman Organic Chemicals, Rochester, New York 14650. ** Tcchnicon lnstrumcnts Corporation, Tarrytown, Y\‘owYork

ro,jgr

0-TOLUIDINE

For

DETERMINATION

assessment

107

of color formation

with time, both manual and semi-automated

techniques were utilized. In the latter, reagent was pre-heated to desired temperature in a heating block, specimen added, mixed, and continuously sampled through colorimeter flow cell. P-Lipoprotein

was prepared

from human serum by precipitation

with dextran

sulfateI”. RESULTS

Variations iu acetic acid concentration The continuous variation of acetic acid: water ratio with a constant concentration of 6”/, o-toluidine was studied. These variations, expressed as percent water, are shown for several specimens in Figs z and 3. A maximum absorbance at 630 nm is 5

4 I-

.oe 30

20 %

IO

15

H20

(V:V)

Fig. 2. The effect of variations reagent blank.

“-

%

Hz0

(V:V)

in acetic acid: water ratio. Plots 1-4 individual

patient

sera. Plot .j,

Fig. 3. The effect of variations in acetic acid : water ratio. Plot I, Versatol A ( x 2). This commercial control material was reconstituted with one-half the volume recommended in order to increase any protein effects; 2, protein-free filtrate of a human serum; 3, Hyland hbnormal; 1, roe mg/dl glucose standard; 5, reagent blank.

obtained at about 10% water in the reagent for aqueous standards. A pronounced variability in curves was observed with the various patient sera studied. A curious phenomenon is a pronounced irregular increase in absorbance at a water concentration in the 17~20~/~ (v:v) range. This “blip” was observed in nearly every patient serum tested. These variations are not due to turbidity effects of serum proteins in varying concentrations of water-acetic acid. When patient sera were dialyzed extensively against isotonic saline, variations were similar to the reagent blank in the oo2504 water range (Figs 2 and 3). Addition of glucose reinstituted the original patterns. Protein-free filtrates, prepared by boiling human serum, showed a pattern identical to that of an aqueous standard (Fig. 3). We have noticed this “blip” with some commer-

FASCE,

108

JR. et d.

cial serum controls” but it was not seen in controls in which P-lipoprotein is minimal~:~: (Fig. 3). Similar patterns were obtained with a glucose+-lipoprotein solution. It appears that with the direct method serum protein levels and composition play a11 important factor in the color formation at any water-acetic acid mixture. Serum free from glucose was prepared by thorough dialysis against saline and known amounts of glucose added. These showed a marked variation in measured glucose concentration when acetic acid : water ratio was varied. Increasingly variable results are seen with lower concentrations of acetic acid. AAt concentrations of water more than 20% (v:v) significant serum blanks were observed. Serum blanks in tllc glacial acetic acid system were almost negligible. Supplementation of serum wit11 bilirubin or hemoglobin produced no qualitative changes in the patterns obsc~rvcd. ITfects

of borate Increasing borate concentration chromophore development. The effects aqueous system diminished this

Heating

are shown enhancement

to I g/liter of increasing

in I:ig. 4. Increasing of color (Fig. 5).

time variations Color formation as a function

of heating

H,B@, produced an increase in BO,“p concentration in a non

the amount

time

is shown

of water

in the

S!_StC!!il

in Figs b and 7, Qualita-

t

010

030

BORATE

043

068

43

IO

I26 17

27

33

% H,O (V.VJ

CONCENTRATION

Pig. 4. Absorbance at 630 nm in the o-toluidinc-glucose reaction with increasing tration in g/liter. The reaction mixture contained no exogenous water; the sample glucose standard. :i’ borate

Fig. 5. Asno in the o-toluidine glucose reaction, containin content. The sample was a normal human strum. * Hyland Normal and .\bnormal (Hyland nia, 92626). Monitrol I and 11 (Dade Division, 33152). * * Versatol Xbnolmal (General Plains, New Jersey (07950). Labtrol (Dade)

Diagnostics

Division American

Travenol Hospital

Division,

borate conccw was a IOO mg/tll

(I sg/litcr) as a function

Laboratories,

Supply

WarnerpChilcott

Costa

Corporation,

Mesa,

of water

Califor-

Miami,

Laboratories,

Pk. Morris

109

O-TOLIJIDINE DETERMINATION

tively, patterns obtained are alike. Notably, in the presence of borate maximum color occurs and fades rapidly. Table I shows the time at which maximum color is formed with representative materials. An estimate of color stability, the duration of maximum ( 31 3%) A 6301is shown in Table 11;fading of developed color is estimated in Table III. Other tsfects Increasing

concentrations

causes a slight depression

of A,,,.

of trichloroacetic acid (TCA) in the reaction mixture We were unable to confirm the finding that citrate

I

I 5

0

‘0

15

20

25

30

I

5

0

M!NUTES

IO

15

20

25

MINUTES

Fig. 6. Development of absorbance at 630 nm as a function of time in the o-toluidine glucose reaction. Curves I and 2 were obtained using the reagent conditions of Cooper and McDaniel (I, L’crsatol A, a lyophilized abnormal control serum; 2, aqueous glucose standard, IOO mg/dl). Curves 3 and 4 were obtained by modifying the above conditions by the addition of I g/liter H,BO, to the chromogenic reagent and decreasing the sample: reagent ratio to I : 300 (3, Versatol A; 4. aqueous glucose standard, 100 mg/dl). Temperature for all data was goO. Fig. 7. Development of absorbance at 630 nm in the o-toluidine glucose reaction as a function ol time and temperature in the presence of borate. All curves were obtained using 6Ob (v: v) o-toluidine in glacial acetic acid containing I g/liter H,BO, and a I : 300 sample: reagent ratio. Curves I and 2 were established using a reaction temperature of 60 ’ (I, aqueous glucose standard, 300 mg/dl; 2, aqueous glucose standard, zoo mg/dl). Curves 3 and 4 were obtained at a reaction temperature of 90~ (3, aqueous glucose standard, zoo mg/dl; 4, Labtrol, a commercial control serum). TABLE

I

Sample

Twnr in minutes *Wethod I .~

100 mg/dl

standard mg/dl standard 300 mg/dl standard Vcrsatol Abnormal Labtrol Hyland .\bnormal 200

105’ ~

90’

13.6 IS.4

22,s

14.0

20.3

14.6 14 o

20.6

19.8

20.4

Method 2 _~ ~_~~ 90’ 6.4

6.3 64 h.6 6.5

6.6

60’ 19.0

19.4 r9.r ‘9.3 ‘9.1

Method I: 69,; o-toluidine in glacial acetic acid. Sample to reagent ratio I : 75. Method 2 : 6% o-toluidine in glacial acetic acid, I g/liter H&1,. Sample to reagent ratio I : 300.

110

FASCE,

100 mg/dl standard zoo q/d1 standard 300 mg/dl standard \‘ersatol Abnormal Labtrol Hyland Abnormal

M&hod Method

I : 6”,, o-toluidine L: G”,, o-toluidinc

J.0

7.6

.i.*

g.0

5 1 .i 1 5.1

‘) s

Hyland

Abnormal

Method Method

I 2

: 6”, : 6qb

o-toluidine o-toluidine

‘) s

I .‘)

Ns

2.2 2.2

s. 2 H.0

.?,.3

Sr

1.1

in glacial acctlc acid. Sample to reagent ratio I : 7.5. in glacial acetic acid, I g/liter H,KO,. Sample to rea:cnt

IO.s> 1oo mg/dl standarcl LOO mg/dl standard 300 mg/dl standard l’ersatol Abnormal Labtrol

7.2

1 ..5

IO.1

2.4

I .‘) I.($

-2.2 ~~ 1.6

JR. Pt al.

qo

‘)o’>

I.2 1.2 m-O.7

-1.1 0. G

in glacial acetic acid. Sample in glacial acetic acid, I g/liter

-6.2

-6.2

T : LOO.

60 -1.3

-6.5

I .o 1.3

-5.3

I-3

--6.2

ratio

7.7

5.Y to reagent ratio I : 7.5. H,BO,. Sample to reagent

ratio

I :3oo

increases color formation4. Increasing o-toluidine concentration shows a roughly linear increase in color formed from 3 to 11% (v:v) o-toluidine. This increment of increase is about 0.05 A/y/o o-toluidine/Ioo

mg/dl glucose.

DISCUSSION

The o-toluidine direct method for glucose determinations has increased in use, primarily due to its specificity and apparent simplicity. This increased popularity and discrepancies between the direct and filtrate methods in our laboratory prompted the current study. From data presented (Figs z and 3) water concentration in the assay is a critical factor in the direct method. Those direct procedures using water concentrations greater than ZOO/~ (v:v) would be expected to give rise to values influenced by the protein composition of the sample. The presence of /Llipoprotein in the specimen has been shown markedly to affect color development. Our studies utilizing dialyzed serum show this artifact to be glucose-dependent and not correctable by a specimen-acetic acid blank. While recoveries were extremely variable at reagent : water concentrations greater than 20% (v : v) no data could be found supporting any one particular water concentration in the 0-15~/~ range. The enhancement of absorbance by BO, 3- is extremely dependent upon water concentration (Fig. 5). This, in itself, should not vitiate the use of borate-glacial acetic

III

O-TOLUIDINE

DETERMINATION

acid methods

in that the water content

of the reagent should be constant

and random

errors in sample volume change insignificantly the final water concentration. A far more important deterrent to inclusion of borate in glacial acetic

acid

reagents is the low stability of color formed at 90’ (Figs 6 and 7 ; Tables II and III). In contrast, borate stabilization of color at higher water concentrations has been retemperature to 60” increases the chromophore ported2y8. Lowering the incubation stability in the borate system to values comparable to the non-borate system at 90’ (Fig. 7; Tables II and III). The above data indicate that the manual direct o-toluidine glucose assay is best accomplished using the procedure of Cooper and iMcDaniel13. This method utilizes glacial acetic acid, the reagent which most consistently yielded best recoveries. Chromophore stability is also high. While use of a glacial acetic acid solution has been considered a disadvantage, these findings indicate that the so-called “aqueous” o-toluidine reagents cannot be used satisfactorily in the direct method due to large, non-correctable blanks due, in part, to the protein composition of the specimen. Manual procedures including borate and incubation temperatures of 90-95’, while showing high sensitivity, necessitate rigorous control over timing due to the rapid increase and subsequent decrease in d 63,,. However, for an automated direct assay the inclusion of borate, to increase sensitivity, is possible. Automation with its highly . reproducible timing obviates fade rate influence.

We gratefully project

as a Summer

acknowledge

the help of Robert

Fellow through

the assistance

D. Sax, who participated of a Brown-Hazen

in this

fellowship.

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