ring of aniline is o-anisidine. Heretofore, in carbohydrate identification this
compound and its para analog have been used extensively in various
formulations of.
CLIN. CHEM. 19/6, 597-601 (1973)
Measurement of Hexoses and Pentoses, Singly or in Combination,with o-Anisidine Jesse F. Goodwin and Hugh Y. Yee
Hexoses and pentoses can be measured ly or in combination, with an o-anisidine
either singreagent. It
has the advantages of a fastreactionrateand relativelylittle interference from hexoses (Amax = 660 nm) in the measurement
of pentoses. A simple sub-
traction of absorbance attributable to glucose at the maximum for pentoses (465 nm) is sufficient for satisfactory pentose estimation in urine. The method has been automated, and resultscompare favorably with those from an automated procedure for the measurement of serum glucose inwhich hexokinase is used, and also with those from a colorimetric aniline method for measurement of pentoses in urine. Interferences and procedural variables are discussed. Urinary pentose and hexose values for normal infants are reported. AddItional Keyphrases: AutoAnalyzer #{149} values for carbohydrate in urine from infants #{149}pentoses in urine Several substituted aniline compounds react with monosaccharides in acid media to produce chromogens with maximum absorption at or near 480 and 630 nm (1-4). The latter wavelength is commonly used for quantification of hexoses. The pentoseamine reaction products usually exhibit maxima at lower wavelengths. The compound in which a methoxy group is substituted at the ortho position of the benzene ring of aniline is o-anisidine. Heretofore, in carbohydrate identification this compound and its para analog have been used extensively in various formulations of chromatographic sprays (5). The reaction product of o-anisidine with hexoses produces a green solution similar to that produced with o-toluidine. However, the color of the o-anisidine-hexose reaction product tends to decrease in intensity with an increase of temperature and heating time, and its absorption maximum is at a slightly higher wavelength. the General Clinical Research Center of Children’s Hosof Michigan, 3901 Beaubien, Detroit, Mich. 48201, and Wayne State University School of Medicine (J.F.G.); and the Department of Pathology, Hutzel Hospital, Detroit, Mich. 48201 From
pital
(H.Y.Y.). Received
Feb. 19, 1973; accepted
April 2, 1973.
The purposes of this investigation were (a) to examine more extensively the reaction of hexoses and pentoses with o-anisidine, with the possibility of introducing means of quantifying both simultaneously, with minor spectral interference; and (b) to devise means of automating the o-anisidine procedure, principally for use with urine and other physiological fluids in which hexoses and pentoses are found in much lower concentrations than in blood. A simple automated procedure for estimating urinary hexoses and pentoses would be useful in identifying those patients for whom more extensive metabolic studies are appropriate.
Material
and Methods
Reagents Unless otherwise specified, all chemicals are reagent grade and all solutions aqueous. Acetic acid-thiourea-borate reagent. Dissolve 1.5 g of thiourea and 10.0 g of boric acid in glacial acetic acid and dilute to 950 ml. o-Anisidine reagent. Mix one volume of o-anisidine and nine volumes of acetic acid-thiourea-borate reagent. Store in an amber-colored bottle. Reagent is stable for one month at room temperature. Stock glucose standard, 5 mg/mI. Dissolve 5.0 g of D-glucose in a solution of benzoic acid (10 g/liter), and dilute to 1 liter. When stored in a glass container, this standard is stable for six months. Working glucose standards. Prepare dilutions of the stock standard with the benzoic acid solution to give working standards of 5, 10, 20, 40, and 80 mg of glucose per deciliter. Stock ribose standard, 5 mg/mI. Dissolve 5.0 g of D-ribose in benzoic acid solution (10 g/liter), and dilute to 1 liter. This solution is stable for one year when stored at room temperature in a glass container. Working ribose standards. Prepare dilutions of the stock ribose standard with the benzoic acid solution to give working standards of 5, 10, 20, 40, and 80 mg of ribose per deciliter. CLINICAL CHEMISTRY, Vol. 19, No.6, 1973
597
Instrumentation
(Urine Analysis)
id.
“AutoAnalyzer” modules (Technicon Instruments Corp., Tarrytown, N. V. 10591) are arranged according to the schematic flow-diagram shown in Figure 1. They include Sampler II (50/h, 1:1 cam), Pump I or II, 90#{176}C heating bath with 1.6 mm i.d. X 12.4 m (40 ft) coil, 15-mm tubular flow-cells, 660-nm and 465nm filters, and recorder. Measurements were also made with a Model 300-N spectrophotometer (Gilford Instrument Laboratories Inc., Oberlin, Ohio 44074) at 465 nm, with a 10-mm flow cell.
5% 11
Procedures Automated procedure. The flow diagram for the simultaneous estimation of pentoses and hexoses by automation (Figure 1) describes a procedure with a sampling rate of 50 per hour with a 1-to-i samplewash ratio. The sample is mixed with reagent in the ratio of approximately 1 to 11 and incubated in a 90#{176}C heating bath for about 5 mm. To stop the reaction, the reaction mixture is cooled by circulation through an ice-bath. The sample stream is then allowed to flow through two colorimeters. One of the colors is measured at 660 nm with an AutoAnalyzer colorimeter with a 15-mm light path; the other is measured with a Gilford Model 300-N spectrophotometer at 465 nm with a 10-mm flow-cell. The latter measurement may also be carried out with a regular AutoAnalyzer colorimeter fitted with the proper filters and semilogarithmic chart paper for the recorder. Measurement of hexoses (manual). Pipet 0.1 ml of carbohydrate sample into a 19 x 105 mm test tube followed by 6.0 ml of o-anisidine reagent. Place in an aluminum heating block and heat for exactly 5 mm at 90#{176}C. Cool and measure the absorbance at 670 nm against a blank. Measurement of pentoses (manual). Pipet 0.1 ml of the carbohydrate sample into a 19 x 105 mm test tube, followed by 6.0 ml of o-anisidine reagent. Place in an aluminum heating block and heat at 90#{176}C for exactly 5 or 10 mm. Cool by immersion in cold water and measure the absorbance at 465 nm against a blank. Simultaneous measurement of hexoses and pent ases. Proceed as outlined in the manual procedure for hexose measurement. For pentoses, measure the absorbance at 465 nm corresponding to the concentration of known hexose standards whose absorbance readings at 465 nm have been determined. Subtract this absorbance from the absorbance of the sample at 465 nm to obtain a net absorbance: A5
nm sampIe1
minus
A465 nm (hexose conc.)
A465 nm
=
lnet
pentose
conc.)
Use the net pentose absorbance at 465 nm to calculate the pentose content of the sample by means of a concentration vs. absorbance calibration plot. Standardization. Treat working standards of hexose (glucose) and pentose (ribose) as outlined under 598
CLINICAL CHEMISTRY, Vol. 19, No.6, 1973
t.r
6 *
_
415..
Fig. 1. Schematic flow-diagram for automated ment of hexoses and pentoses with o-anisidine
measure-
the manual or automated method. Measure hexoses at 670 nm and pentoses at 465 nm. From these data, construct the appropriate concentration vs. absorbance calibration plot. For the simultaneous measurement of hexose and pentose, refer to the formula listed under “Simultaneous measurement of hexoses and pentoses.”
Results and Discussion Analytical variables. A direct method (6) has been reported for simultaneous quantification of glucose and xylose in serum by use of o-toluidine. The glucose-o-toluidine reaction product has a principal maximum at 630 nm with additional maxima at 480 and 380 nm. Xylose and other pentoses react with o-toludine to yield spectra having a maximum near 460 nm. However, there is overlap in the 630-nm absorption region. The o-anisidine-glucose reaction products have maxima at 670 and 430 nm (Figure 2); the ribose reaction product has a single maximum at 465 nm. Of importance here is the almost total lack of absorption by the pentose-o-anisidine reaction product at 670 nm, in contrast to that of the o-toluidine reaction product, for which there is considerable spectral overlap between the reaction products of hexoses and those of pentoses. The effect of temperature on color development in the o-anisidine-glucose reaction for a 5- or 10-mm heating period is shown (Figure 3). Maximum color develops in 5 mm at 90#{176}C. After this time, color formation is greatly decreased. At 80#{176}C, color intensity is greatest in 10 mm and decreases with a prolongation of heating time. Heating at 100#{176}C results in a less intense color after either 5 or 10 mm. This temperature dependence has not been observed in other ortho-substituted aniline derivatives that have been studied for their reaction with hexoses and pentoses (2, 6). In these studies, we found that once optimal temperature conditions for color formation had been
Os 04
I.J
a2
z
4 15
I,
340
,_..._.__j
,I,I,I,I
380420
460
500
540
580
620
15 4
I
,
660
700
nm Fig.2.Absorptionspectraof reactionproducts of glucose and ribosewitho-anisidine Glucose, 100 mg/dl (-), with o-anisidine
by heating
and ribose, 50 mg/dl (- - - -), were reacted for 5 mm at 90#{176}C. Spectrum scanned with a
5
DB spectrophotometer equipped with recorder (Beckman Instruments calIf. 92634)
10
15 20
Co.. Fullerton,
30 10 IS TINE IN MINUTES
20
25
30
Fig.4. Color development of xylose and glucose with 0-
anisidine. at90#{176}C Manual procedure used. Measurements
made with a Coleman II spec-
trophotometer
Ui
z
0.1 Q
(10mm)
90 ec
#{149}l0 mm reaction S 5 mm reaction 100 C,
Fig. 3. The effectof temperature on color formation
in
theglucose-o-anisidine reaction Color measured with a Model II spectrophotometer kin-Elmer Corp., Maywood, III. 60153).
(coleman Dlv., PerSu.e
established, prolonging heating time had relatively little effect on the intensity of the color. However, the colors developed with other ortho-substituted anilines, including o-anisidine, are stable at room temperature for at least 4 to 5 h. The rate of color development of a hexose (glucose) or a pentose (xylose) with o-anisidine at 90#{176}C is shown in Figure 4. In the case of glucose, the maximum at 430 nm increases as the maximum at 670 nm decreases. This is also true for measurements made at 465 urn, the maximum for the pentose-oanisidine product. Maximum color formation for hexoses when measured at 465 nrn is obtained in 20 mm. Hexose interference with pentose measurement is negligible with a 5-mm heating time. For xylose, absorbance at 465 nm is greatest in 20 mm at 90#{176}C. An added feature of the color developed with xylose and other pentoses is an increased absorbance, which is approximately four times greater at its maximum than that of hexoses at 670 nm. The color reaction ratio (using glucose as a refer#{149}ence) of various carbohydrates with o-anisidine with absorbances measured at 670 urn, is shown in Figure 5 for a 5- and 10-mm heating period. Both galactose and mannose exhibit more sensitivity after a 10-mm heating, the relative color intensity being greater than that developed with glucose. The same is true
OH MeFurftvol
Fig. 5. Color developed by glucose in the o-anisidine reactioncompared with thatdeveloped by othercarbohydrates The ‘glucose ratio” was calculated by dividing the absorbance reading for the reacfion product of 50 zg of glucose reacted with o-anisldine Into the absorbance similarly obtained with 50 zg of the specified carbohydrate. (Measured with a Beckman DB Spectrophotometer)
of the disaccharides, maltose and sucrose. Interference from the pentoses, arabinose and ribose, is low. The absorbances of the reaction products of fucose and 5-hydroxymethylfurfural are negligible at this wavelength. Other materials tested and exhibiting ratios below 0.03 were glyceraldehyde, erythrose, and furfural. Owing to the effect of temperature control on the manual method, the Beer-Lambert relationship is not followed in concentrations above 60 mg/dl. For this reason, the use of a calibration graph is required. However, this shortcoming is offset by excellent sensitivity, which is similar to that obtained by the reaction of aniline with pentoses. Because of the sensitivity to temperature and the necessity for strict adherence to time factors, the reaction with o-anisidine is ideal for automation. A calibration curve and tracing for the automated quantification of glucose is linear to 25 mg/dl (Figure 6). The lack of interference of xylose in the reacCLINICAL
CHEMISTRY,
Vol. 19, No. 6, 1973
599
Table 1. Recovery of Glucose or Xylose Added to Urine Samples, as Measured by an Automated o-Anisidine Method
a
ConcentratIon
a III
-
Added
calculated
Found
Difference
mg/dl
Glucosea
-
along
with
16.2
16.6
+0.4
5.0 10.0 20.0 30.0
19.2 24.2 34.2 44.2
18.9 25.2 36.9 45.9
-0.3 +1.0 +2.7 +1.7
2.0 5.0 10.0 20.0 30.0
17.0
17.0
20.0
20.7
25.0 35.0 45.0
25.5 37.0 46.0
0 +0.7 +0.5 +2.0 +1.0
Xylose8
Fig.6. Calibration graph forthe automated quantification ofglucosewith the AutoAnalyzer Three different samples were assayed in triplicate, (AutoAnalyzer recorder used)
2.0
standards.
All results are av of five replicate analyses. Added to a urine specimen containing 14.2 mg of glucose per deciliter. b Added to a urine sample containing 15.0mg of xylose per deciliter. C
‘LArv1kr\IJ:\. /1
a
a
Ik
,.
aa
St.d
Table 2. Comparison of Total Urinary Pentose Values (Measured as Ribose) Obtained by an Aniline and o-Anisidine Method for a Group of 21 Infants(6 to 18 Months Old) Pentose, total mIllIgrams per 24-hour urine
Fig. 7. Calibration
graph
for the automated
quantification o-Anlsldlne method (x)
ofxylosewiththe AutoAnalyzer Results shown for samples (left-hand peaks) and standards (right-hand peaks). (Absorbance measured with a Gilford 300N Spectrophotometer attached to a Coleman 165 recorder)
Mean ± SD Range CV,% Estimating
tion of glucose with o-anisidine is depicted in this AutoAnalyzer tracing, which was produced without additional electronic amplification. Both the intensity of the color formed and the sample resolution are excellent. A calibration curve and tracing for the automated quantification of xylose exhibits linearity up to 40 mg/dl (Figure 7). The measurements (at 465 nm) were made with a Technicon AutoAnalyzer system, but the AutoAnalyzer colorimeter was replaced with a Gilford 300-N spectrophotometer fitted with a flow cell and attached to a Coleman Model 165 recorder (Coleman Div. of Perkin-Elmer Corp., Maywood, Ill. 60153) to obtain superior resolution. The absorbances from urine samples and xylose standards are shown (Figure 7, right). The color produced by the reaction of glucose in additive when measured at 465 nm. Both sensitivity of the color reaction and the resolution are excellent. Urinary Carbohydrate
Measurements
Data on recovery of glucose and xylose added to a urine sample are given in Table 1. The recovery ranged from 98 to 108%. Xylose added to urine (2-30 mg/dl) was 100 to 106% recovered. 600
CLINICAL CHEMISTRY, Vol. 19, No.6,1973
Aniline method (y)
56.60 ± 18.7 17.10 92.40
54.78 ± 21.5
33.05
39.15
15.20
-
equation:
y
8.06 + 1.11x;Sy.
=
±10.98;
94.50
-
r
=
0.885.
Urinary carbohydrate concentrations are very low, and published information concerning these values is lacking. Twenty-five urine samples from a group of infants, 6 to 18 months of age, were analyzed for hexoses and pentoses by the automated procedure. The amount of hexose, expressed as glucose, ranged from 4.0 to 64 mg/dl (mean, 21.1 mg/dl). Pentose measurements ranged from 3.0 to 16.8 mg/dl (mean, 11.1 mg/dl). These values for hexoses are comparable to those reported by Jolley et al. (7), by use of high-resolution column-chromatography. For estimating urinary glucose only, the automated hexokinase procedure (8) would appear to be more suitable and specific. Total urinary pentose values obtained by the automated method with o-anisidine and a manual method for pentose analysis (9) with aniline are compared in Table 2. Results with the aniline method compare very favorably with those for a procedure for pentose analysis in which glucose is removed by reaction with glucose oxidase before pentoses are assayed (2). Urinary hexoses measured manually on the same samples by the o-anisidine procedure yielded a mean of 46.7 mg/24 h (SD, ± 19.06).
Blood Carbohydrate Measurements We compared 35 serum glucose values obtained with a Technicon SMA 12/60 automated system and with the present tnethod. The mean values were identical for both methods, and the ranges showed excellent agreement (o-anisidine method (y): mean, 80.2 mg/dl; range, 55-130 mg/dl; and SMA 12/60 method (x): mean, 80.2 mg/dl; range 10-122 mg/dl; y = 11.98 + 1.15x; r = 0.98, = 3.43). Pentoses were estimated in the same serum samples with aniline and o-anisidine, before and after treatment with glucose oxidase. Both amines were proven to be unsuitable for measurement of pentose in serum without glucose oxidase treatment. In the specimens treated with glucose oxidase, the mean pentose values (expressed as ribose) ranged from 4.5 to 10 mg/dl, when the aniline reagent was used, compared to 2.0 to 9.0 mg/dl when o-anisidine was used. The mean values for pentoses obtained with the o-anisidine method were higher (X = 6.7 mg/dl) than the mean obtained with the aniline method (X = 3.4 mg/dl). The increased intensity of the color formed from the reaction of aniline with ribose may account for the difference in mean values. In view of this difference, we do not recommend that o-anisidine be used to measure pentoses in serum or other samples in which the hexose (glucose) concentration is more than eight times the concentration of pentose. The presence of other carbohydrates such as disaccharides may also account for the discrepancies between the two procedures. The effect of bilirubin on the manual method was studied by adding purified bilirubin, dissolved in a minimum of sodium carbonate, to albumin contain-
ing 100 mg of glucose per deciliter. Our results suggest that bilirubin, in concentrations up to 19 mg/dl, does not interfere significantly with the measurement of glucose with o-anisidine. Our studies also indicate that when cell-free hemoglobin is added to albumin containing glucose, interference occurs at hemoglobin concentrations greater than 40 mg/dl. For these reasons, we recommend that glucose not be directly measured in grossly icteric or hemolyzed samples. This work was supported in the General Clinical Research search Resources NIH.
part
by USPHS Grant RR-74 from Center Program; Division of Re-
References 1. Deckert, T., Method for determining glucose in plasma, cerebrospinal fluid, and urine by means of p-bromoaniline. Scand. J. Clin. Lab. Invest. 20, 217 (1967).
2. Goodwin, J. F., Reaction of aniline and its alkyl derivatives with hexoses and pentoses. Anal. Biochem. 48, 120(1972). 3. Gras,
body
M.,
fluid
and
Smrekar,
glucose
M.,
An aniline-acetic
determination.
acid
Clin. Chim.
method 17,
Acta
for 518
(1967). 4. Hultman, E., Rapid specific method for determination saccharides in body fluids. Nature 183, 108(1959). 5. Scott, R. M., Clinical Analysis Techniques, Ann Arbor-Humphrey Mich., 1969, p 64. 6. Goodwin, J. F., Method glucose and xylose in serum.
of aldo-
by Thin-layer Chromatography Science PubI., Ann Arbor,
for simultaneous direct estimation Clin. Chem. 16,85(1970).
of
7. Jolley, R. L, Warren, K. S., Scott, C. D., Jainchill, J. L., and Freeman, M. L., carbohydrates in normal urine and blood serum as determined by high-resolution column chromatography. Amer. J. Clin. Pat hol. 53, 793 (1970). 8. Yee, H. Y., Automated hexokinase procedure for assaying cose in urine, serum or plasma. Clin. Chem. 18, 1416(1912).
9. Goodwin, an aniline
J. F., Micromethod reagent. Clin. Chem.
for measuring 17, 397 (1971).
CLINICALCHEMISTRY,
pentoses
Vol. 19, No.6,
glu-
by use of
1973
601
