A study of the direct o-toluidine blood glucose determination

A study of the direct o-toluidine blood glucose determination
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  Reprinted fi·om Clinica Chimica Acta Elsevier Publishing Company, Amsterdam - Printed in The Netherlands CCA 5388 A STUDY OF THE DIRECT a-TOLUIDINE BLOOD GLUCOSE DETERMINATION CHARLES F. FASCE, JR.,* ROBERT REJ ND ANTHONY J. PIGNATARO I05 New York State Department of Health, Division of Laboratories and Research, Albany, N.Y. I22 I U.S.A.) (Received June 13, 1972) SUMMARY The acetic acid-a-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 concentrations less than IOo% 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 con;trol sera were studied. At water concentrations less than IO% (v Jv) marked enhancement of color due to borate ion was observed; in glacial acetic acid, borate more than doubles color produced. Addition of trichloroacetic acid showed inhibition of the reaction, Absorbance was found to increase with increasing a-toluidine concentration over the range studied {I to n%). At concentrations of water greater than I5% significant serum blanks were observed. This blank was not due solely to protein turbidity, and is dependent upon the presence of both glucose and P-lipoprotein. Effects of other reagent parameters, color development time, and sample constituents were investigated. INTRODUCTION The a-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 Ig65 to over I70 in I97L 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 srcinal method 1 through changing the sample:reagent ratio, the reagent acetic acid:water ratio, and by addition of other substances 2-9• A careful study of the a- toluidine glucose reaction was undertaken in the hope of achieving a more reproducible assay. *Present address: City of Kingston Laboratory,· Kingston, N.Y. **To whom inquiries should be addressed.  ro6 FASCE, JR. et al. MATERIALS AND METHODS Reagents a-Toluidine* was purified by double distillation under reduced pressure. All chromogenic reagents contained r.25 gjliter thiourea. Concentrations of a-toluidine and acetic acid were varied as described in RESULTS. Glucose (dextrose) for standards was obtained from the National Bureau of Standards, Washington, D.C. Method Variations in reagent concentrations were obtained by the method of continuous variation as described by Ryland et al. 10 which enables a continuous graph of absorbance as a function of reagent concentration in the AutoAnalyzer** system. A flow diagram for the study of the a-toluidine glucose reaction is shown in Fig. r. Re- PCI .090 REAGENT A セ .045 AIR '---------0--- .010 SAMPLE D COLORIMETER. RECORDER 15M M TUBULAR f/c FILTERS 630 nm .(a) PULSE SUPPRESSOR 0.005 "1.0. Fig. 1. Flow diagram for continuous variation study of the a-toluidine glucose reaction. agent line A pumps the varying chromogenic reagent from a mixing flask into the reaction manifold, while Reagent line B constantly pumps the limiting concentration of the reagent. The variation in concentration is described by C 1 = C 1 r-e-rjvt) where Ct is the concentration at time t, C 1 the concentration of solution being fed (Reagent B), ris the pumping rate, and v the liquid volume in the mixing flask. Reagent A and B pump tubing are standardized as described previously 11 • The sample line continuously pumps specimen into the manifold. The standard AutoAnalyzer he<J,ting 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 te:t;nperature produced by thermoregulator cycling. *Eastman Organic Chemicals, Rochester, New York q6so. ** Technicon Instruments Corporation, Tarrytown, New York 10591.  a-TOLUIDINE DETERMINATION 107 For assessment 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 colori meter flow cell. P-Lipoprotein was prepared from human serum by precipitation with dextran sulfate 12 • RESULTS Variations in acetic acid concentration The continuous variation of acetic acid:water ratio with a constant concentration of 6% a-toluidine was studied. These variations, expressed as percent water, are shown for several specimens in Figs 2 and 3· A maximum absorbance at 630 nm is w 0 z < CD 0::: 0 j) CD < .5 .4 0 o H20 (V :V) w .) z X en a:: 0 (/) en X .7 .6 .5 .2 .o H 2 0 (V:V) Fig. 2. The effect of variations in acetic acid: water ratio. Plots r -4 individual patient sera. Plot 5, reagent blank. Fig. 3. The effect of variations in acetic acid: water ratio. Plot 1, 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 Abnormal; 4, roo mgfdl glucose standard; 5, reagent blank. obtained at about Io% 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 o-25% water range (Figs 2 and 3). Addition of glucose reinstituted the srcinal patterns. Protein-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-  ro8 FASCE, JR. et al. cial serum controls* but it was not seen in controls in which {1-lipoprotein is minimal': :' (Fig. 3). Similar patterns were obtained with a glucose-{1-lipoprotein solution. It appears that with the direct method serum protein levels and composition play an 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. At concentrations of water more than zo% (v: v) significant serum blanks were observed. Serum blanks in the glacial acetic acid system were almost negligible. Supplementation of serum with bilirubin or hemoglobin produced no qualitative changes in the patterns observed. Effects of borate Increasing borate concentration to r gfliter H 3 B0 3 produced an increase in chromophore development. The effects of increasing B0 33- concentration in a nonaqueous system are shown in Fig. 4· Increasing the amount of water in the system diminished this enhancement of color (Fig. 5). Heating time variations Color formation as a function of heating time is shown in Figs 6 and 7· Qualita- w 0 :'i CD a:: 0 /) CD <{ .5 .2 0.10 0.30 0.43 0.68 BORATE CONCENTRATION .5 z <{ g:? .4 0 セ .3 .2 N 4.3 12.6 17 27 33 Fig. 4· Absorbance at 630 nm in the a-toluidine-glucose reaction with increasing borate concen tration in gfliter. The reaction mixture contained no exogenous water; the sample was a roo mgfdl glucose standard. Fig. 5· A 630 in the a-toluidine glucose reaction, containing borate (r gfliter) as a function of water content. The sample was a normal human serum. * Hyland Normal and Abnormal (Hyland Division Travenol Laboratories, Costa Mesa, Califor nia, 92626). Monitrol I and II (Dade Division, American Hospital Supply Corporation, Miami, Fla. 33152). ** Versatol Abnormal (General Diagnostics Division, Warner-Chilcott Laboratories, Morris Plains, New Jersey (07950). Labtrol (Dade).  a-TOLUIDINE DETERMINATION I09 tively, patterns obtained are alike. Notably, iri 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 (± 3 ) A 630 , is shown in Table II; fading of developed color is estimated in Table III. Other セ Increasing concentrations of trichloroacetic acid (TCA) in the reaction mixture causes a slight depression of A 630 • We were unable to confirm the finding that citrate w u z < QJ ' 2 10 15 20 25 MINUTES w (.) .5 .4 .3 .2 j) 0 5 セ 10 15 MINUTES 20 25 Fig. 6. Development of absorbance at 630 nm as a function of time in the a-toluidine glucose reaction. Curves I and 2 were obtained using the reagent conditions of Cooper and McDaniel (r, Versatol A, a lyophilized abnormal control serum; 2, aqueous glucose standard, roo mgfdl). Curves 3 and 4 were obtained by modifying the above conditions by the addition of r gfliter H 3 B0 3 to the chromogenic reagent and decreasing the sample: reagent ratio to I: 300 (3, Versatol A; 4· aqueous glucose standard, IOO mgfdl). Temperature for all data was go 0• Fig. 7· Development of absorbance at 630 nm in the a-toluidine glucose reaction as a function of time and temperature in the presence of borate. All curves were obtained using 6% (v: v) a-toluidi ne in glacial acetic acid containing r gfliter H 3 B0 3 and a I: 300 sample: reagent ratio. Curves I and 2 were established using a reaction temperature of 6o 0 (I, aqueous glucose standard, 300 mgfdl; 2, aqueous glucose standard, 200 mgfdl). Curves 3 and 4 were obtained at a reaction temperature of goo (3, aqueous glucose standard, 200 mgfdl; 4, Labtrol, a commercial control serum). TABLE I INCUBATION TIME FOR MAXIMUM ABSORBANCE AT 630 nm AS A FUNCTION OF TEMPERATURE AND PRESENCE OF BORATE Sample Time in minutes Method I Method 2 IOSO goo goo 60° roo mgfdl standard 13.6 22.8 6.4 Ig.o 200 mgfdl standard 15·4 Ig.8 6.3 rg.4 300 mgfdl standard 6.4 Ig.2 Versatol Abnormal q.o 20.3 6.6 rg.3 Labtrol I4.6 20.4 6.5 Ig.4 Hyland Abnormal 14.0 20.6 6.6 Method r: 6% a-toluidine in glacial acetic acid. Sample to reagent ratio I: 75· Method 2: 6% a-toluidine in glacial acetic acid, I gfliter H 3 B0 3. Sample to reagent ratio I: 300.

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