ANALYTICAL
BIOCHEMISTRY
Fluorometric
94, l-5 (1979)
Determination
YOSHIHIRO Faculty
ASABE,
of Trioses
with o-Phenylphenoll
SHIGEYOSHI MOMOSE, AND SHOJI TAKITANI
of Pharmaceutical Ichigaya-funagawara-machi,
Sciences.
Science Shinjuku.
MASAO
SUZUKI,
University of Tokyo, Tokyo 162, Japan
12,
Received December 14, 1977 It was found that trioses, such as glyceraldehyde and dihydroxyacetone, could be determined by using the assay for acetaldehyde reported earlier, on the basis of the fluorescence reaction between acetaldehyde and o-phenylphenol in sulfuric acid. The calibration curves for the standard solutions of glyceraldehyde and dihydroxyacetone showed straight lines passing through the origin with the same slope for 1.8-72 nmoU200 ~1. Then, the standard method for the assay of trioses, acetaldehyde, and lactic acid in mixed sample solutions was established by a combination of separation of lactic acid from the other two components with an anion-exchange resin treatment and simultaneous determination of trioses and acetaldehyde based upon the differences in the reaction condition between the both compounds. Finally, almost 100% recovery was obtained for each of the components in the examination of the recovery of these three compounds added to the chemistry control serum.
In previous papers (2,3), the authors have reported the microdeterminations of lactic acid and muramic acid based upon a fluorescence reaction of acetaldehyde with o-phenylphenol (OPP)* in sulfuric acid. Because of its simplicity, rapidity and high sensitivity, this method seemed applicable to rapid analysis of other samples which liberate acetaldehyde. In fact, it was found that methylglyoxal and trioses (glyceraldehyde and dihydroxyacetone) liberate acetaldehyde when heated with sulfuric acid, which then gives blue fluorescence by the reaction with OPP. The present study aimed at establishing a fluorometric determination of trioses on the basis of this reaction. Previously published methods for the assay of trioses include the spectrophotometric method (4), the polarographic method (5), and also the enzymatic methods (6,7). On the other hand, their fluorescence reactions with chromo-
tropic acid (8) and Shydroxy-1-tetralone (9) are reported but these reactions are interfered with formaldehyde and common sugars, and hence no application of these reactions to quantitative determination of trioses is reported. Our method should be useful for the assay of biological samples if it allows simultaneous determination of trioses, acetaldehyde, and lactic acid in a mixture because acetaldehyde, and lactic acid naturally occur in usual biological fluids. Therefore, the standard procedure for the assay of the three components in the mixture was established by a combination of separation of lactic acid from the other two components with an anion-exchange resin treatment and simultaneous determination of trioses and acetaldehyde based upon the differences in reaction condition in the fluorescence reactions of both compounds. Finally, recovery of the added trioses, acetaldehyde, and lactic acid to the chemistry control serum was examined, as a preliminary attempt, in order to apply the proposed method to biological samples.
’ This paper constitutes Part V of a series entitled “Color Reactions of Carbonyl Compounds with Substituted-phenols”; Part IV in the series is Ref. (1). * Abbreviations used: OPP, o-phenylphenol. 1
0003-2697/79/050001-05$02.00/O Copynght 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
2
ASABE ET AL.
MATERIALS
AND METHODS
taining 1J-72 nmol of trioses (or 1- 10 nmol of acetaldehyde or 1.1-22.5 nmol of lactic Materials. The standard samples used acid) in a glass-stoppered test tube was were 9-360 nmol/ml of an aqueous solution cooled in an ice-water bath; 5 ml of conof DL-glyceraldehyde . dimer and dihydroxycentrated sulfuric acid was added slowly acetonemdimer (Tokyo Kasei Co.), 5-50 with cooling, and the tube was then shaken. nmoYm1 of an aqueous solution of acetaldeIn the case of the trioses or lactic acid, each hyde (3), 5.5- 112.5 nmoYm1 of an aqueous tube was placed in a water bath kept at 80°C solution of lithium lactate (3). OPP, 1.8% for 5 min; the tube was then cooled in an (w/v), was used in 0.5% sodium hydroxide ice-water bath. Next, 10 ,ul of 0.15% sodium solution and sodium nitrite was used in nitrite solution was added into tube, and the 0.15% (w/v) aqueous solution, freshly pretube was gently shaken. Subsequently, 10 ~1 pared before use. All of the reagents and of 1.8% OPP solution was added. After mixsamples were stored in a refrigerator. Methylglyoxal was prepared according to the ing the contents, the tube was placed in a 20°C water bath for 15 min; the fluorescence literature (10) and acetaldehyde-semicarbaintensity was measured with excitation at zone was prepared by the usual method. All 405 nm and emission at 465 nm. For blank other organic and inorganic chemicals used test, a water instead of the sample solution were analytical grade. was treated in the same way as described Measurements. The fluorescence spectra above. and their intensities were measured by a For the assay of each component in the Hitachi fluorescence spectrophotometer mixed samples, 2 ml of the sample solution Model MPF-2A in the same way as reported containing the trioses (within ca. 400 nmol), earlier (3). A Shibata microdiffusion unit acetaldehyde (within ca. 400 nmol), and was used for the Conway’s microdiffusion analysis of acetaldehyde, and the pH was lactic acid (within ca. 500 nmol) was poured measured with a Model HM-18B digital pH on the top of the anion-exchange resin column. During eluting, the initial 5 ml eluted meter (Toa Denpa Kogyo Co.). by 0.005 M phosphate buffer (pH 6.0) was Thin layer chromatography. A tic plate collected for analysis of the trioses and acetcoated with Merck silica gel HF,,, (thickness, aldehyde. Then, the following 5-ml fraction 0.25 mm) was activated at 110°C for 40 min eluted by 0.1 M phosphate buffer (pH 6.0) before use. After developed with a chlorowas also collected for analysis of lactic acid. form-ethanol (10: l), each spot was detected For the determination of trioses and acetalwith a color reaction of phosphomolybdic dehyde, a 200~,ul sample solution containing acid. 210 nmol of trioses and 1- 10 nmol of acetPreparation of anion-exchange resin colaldehyde was placed in two test tubes. One umn. Three hundred milligrams of Amberwas treated with the method for acetaldelite CG 400 (Rohm & Haas Co.; Type I, lOOhyde and the other with the method for the 200 mesh, Cl-), a strongly basic anion-extrioses. The fluorescence intensity due to change resin equilibrated with 0.005 M the trioses was given by the difference in phosphate buffer (pH 6.0), was packed in a fluorescence intensity between those obglass column (5 X 100 mm). Standard method.3 A 200~~1 sample con- tained for the former and the latter. 3 The amounts of the reagents added in the assay of acetaldehyde were modified to those used for lactic acid because it seemed favorable to unify the reaction conditions with regard to the assay of both compounds, and it was confirmed that acetaldehyde could be determined under the conditions of the assay for lactic acid; see Ref. (3).
RESULTS AND DISCUSSION Fluorescence Reaction of Trioses with OPP
The fluorescence spectra of the reaction products obtained by the standard method for methylglyoxal and the trioses were iden-
FLUOROMETRIC
DETERMINATION
tical with that of acetaldehyde. It has been reported that glyceraldehyde and dihydroxyacetone produce easily methylglyoxal when heated with sulfuric acid (1 l), and it is quite likely that methylglyoxal liberates acetaldehyde. A thin layer chromatogram for the reaction products obtained in a microdiffusion test [absorbent, semicarbazide hydrochloride solution, (12)] of the decomposition products of the above compounds when heated with sulfuric acid is shown in Fig. 1; the Rf value of every semicarbazone produced in the absorbent was equal to that of acetaldehyde-semicarbazone. Therefore, it was confirmed that the fluorescence was due to the reaction product between OPP and acetaldehyde, which was liberated from these compounds by sulfuric acid. Reaction Conditions for Determination of Trioses The relationship between fluorescence intensity and heating time in the decomposition of the trioses into acetaldehyde with sulfuric acid was studied at a temperature range 40-100°C. As is shown in Fig. 2, a constant intensity for glyceraldehyde as well as dihydroxyacetone was obtained when heating was done at 60°C for 10 min or 80°C for 5 min; at the higher temperature, the intensity decreased slightly with increasing heating time. In the proposed method, the reaction was carried out at 80°C for 5 min because it was advantageous to use the same condition for the assay of trioses and lactic acid. From the relationship obtained between fluorescence intensity and the amounts of the reagents added, it was revealed that the same amounts of OPP and sodium nitrite as those used in the assay for lactic acid can also be used in this case. Furthermore, concentrated sulfuric acid was found to give the best result for the decomposition of trioses. Every interference with the assay for acetaldehyde and lactic acid reported earlier was found to affect on the assay of trioses because of the same reaction employed.
OF TRIOSES
1.0 -
LI
II
III
ABCDE FIG. 1. Thin layer chromatogram of semicarbazones of acetaldehyde and reaction products obtained by decomposition of methylglyoxal and trioses. (A) Acetaldehyde-semicarbazone; (B) semicarbazone obtained from methylglyoxal, (C) semicarbazone obtained from glyceraldehyde; (D) semicarbazone obtained from dihydroxyacetone; (E) semicarbazide hydrochloride. Developer, chloroform-ethanol (10: 1). Detecting reagent, 10% phosphomolybdic acid-50% ethanol.
Calibration
Curves
Calibration curves for the standard solution of glyceraldehyde and dihydroxyacetone showed the straight lines passing through the origin with the same slope for 1.8-72 nmol in a 200-~1 sample solution. Therefore, it was concluded that glyceraldehyde , dihydroxyacetone , and their mixtures, so called glycerose, could be determined with the calibration curve for either of the compounds. The coefficients of variation for reproducibility of the measurements were 4.8 1 and 2.40% at 9 and 72 nmol of glyceraldehyde (in 10 experiments, n = lo), respectively; 4.10 and 3.60% at the same concentration of dihydroxyacetone (n = 10). Determinations of Trioses, Acetaldehyde, and Lactic Acid in the Mixtures After examining the conditions for separation of lactic acid from the other two com-
4
ASABE ET AL.
20 Time,
30
40
50
min.
FIG. 2. Effect of temperature on decomposition time. Glyceraldehyde, 40 nmoV200 ~1; (A) boiling water; (0) 80°C; (0) 60°C; (A) 40°C.
ponents, 0.005 M phosphate buffer (pH 6.0) was chosen for the solvent of lactic acid from which it adsorbs on the anion-exchange resin. Under this condition, the trioses and acetaldehyde could be immediately eluted without adsorption. Then, 0.1 M phosphate buffer (pH 6.0) was used for elution of the lactic acid adsorbed on the resin. Although the trioses and acetaldehyde in mixture can not be separated from each other by the column treatment, they differ in the reaction conditions for fluorescence to appear. Hence, the influence of the trioses on the fluorescence reaction of acetaldehyde was studied. As the result, the fiuorescence intensity was found to decrease when more than twofold the amounts of the trioses were added to 10 nmol of acetaldehyde in 200 ~1, while no effect of the trioses added was observed at additions below 10 nmol, and the calibration curve of acetaldehyde containing 10 nmol of the trioses was identical with that of the standard acetaldehyde solution. Next, the effect of acetaldehyde on the determination of the trioses was studied. When the fluorescence intensity due to 10 nmol of acetaldehyde was subtracted from those of the trioses containing 10 nmol of acetaldehyde obtained by the assay for the trioses, the resulting curve coincided with
the calibration curve of the standard triose solution. Therefore, the fluorescence intensity due to the trioses was evaluated in the way described above. As is shown in Fig. 3, a good separation was obtained by the present method using 2 ml of the prepared sample solution containing 250 nmol each of glyceraldehyde and acetaldehyde, and 500 nmol of lactic acid. Recoveries (n = 10) were 98.6 + 1.7% for glyceraldehyde, 99.7 -+ 1.1% for acetaldehyde, and 100.4 +- 1.4% for lactic acid, respectively. Recovery Examination with the Chemistry Control Serum
In order to minimize the amount of the serum to be taken in practical assay, the standard procedure was modified as follows: A 200~~1 sample of the serum containing the trioses, acetaldehyde, and lactic acid was poured onto the top of the resin column and then eluted with 0.005 M phosphate buffer (pH 6.0). The first 0.5 ml eluted was discarded and the following 1.O ml of eluate was collected for assay of the trioses and acetaldehyde, and then, 10 ml of the eluate by 0.1 M phosphate buffer (pH 6.0) was col+
(I)
-+e--
(2)
IOOC
-+A
LA
Effluent,
ml
FIG. 3. Elution curve of acetaldehyde (AA), glyceraldehyde (GLA), and lactic acid (LA). Sample taken, 2 ml of the mixture (containing AA 250 nmol, GLA 250 nmol, and LA 500 nmol). Eluting solution: (1) 0.005 MKH,PO,-0.005 M N%HPOa, pH 6.0; (2) 0.1 M KH,PO,-0.1 M N%HPOb, pH 6.0.
FLUOROMETRIC
DETERMINATION
OF TRIOSES
5
lected for assay of lactic acid. The eluate 100.1 2 3.0% (100 nmol added) and 98.0 containing the trioses and acetaldehyde was 2 2.9% (1 pmol added) for lactic acid. pretreated with 200 ~1 of 2.5% uranylacetate ACKNOWLEDGMENT solution (centrifuged at 2500 rpm for 5 min) before the fluorescence reaction. This work was supported in part by a Grant-in-Aid It was necessary to remove proteins con- for Scientific Research from the Ministry of Education tained in the serum which may interfere with of Japan. the fluorescence reaction because they could REFERENCES not be separated from the smaple solution Asabe, Y., Someya, K., Suzuki, M., and Takitani, by the column treatment and hence they S. (1977) Bunseki Kagaku 26, 43. contaminated the eluate containing the tri2. Kojima, S., Asabe, Y., Suzuki, M., and Takitani, oses and acetaldehyde. The uranylacetate S. (1975) Bunseki Kagaku 24, 167. method gave the best result for deproteini3. Asabe, Y., Kojima, S., Suzuki, M., and Takitani, zation. After examining the effect of uranylS. (1977) Anal. Biochem. 19, 73. acetate on the fluorescence reaction, an 4. Tsai, M. U., and Schwartz, E. L. (1961) Anni. Biochem. 2, 107. amount of 200 ~1 of 2.5% concentration was 5. Ardenne, M. v., and Tuemmler, R. (1964) Deur. chosen. The treatment by the reagent was Gesundheitsw. 19, 49, 709. found to give the best result when it was 6. Pinter, J. K., Hayashi, J. A., and Watson, J. A. made for the eluate before the fluorescence (1967) Arch. Biochem. Biophys. 121, 404. 7. Charlton, J. M., and van Heyningen, R. (1969) reaction. Anal. Biochem. 30, 313. Finally, recoveries of glyceraldehyde, ac8. Nakai, T., Koyama, M., and Demura, H. (1970) etaldehyde, and lactic acid added to the J. Chromatogr. 50, 338. chemistry control serum (Hyland Division 9. Momose, T. (1959) Talanta 3, 155. Travenol Lab., Inc.) were surveyed, The 10. Riley, H. L., Morely, J. F., and Friend, N. A. C. (1932) J. Chem. Sot. 1875. mean recoveries (n =lO) in 200 ,ul serum 11. Neuberg, C., Farber, E., Levite, A., and Schwenk, were 99.1 + 5.9% (25 nmol added) and 98.3 E. (1917) Biochem. Z. 83, 244. -+ 3.9% (50 nmol added) for glyceraldehyde; 12. Ishizaka, 0. (1969) in Experimental Methods of 98.9 -+ 3.2% (25 nmol added) and 99.7 Microdiffusion Analysis, p. 105, Nankodo, -+ 1.1% (50 nmol added) for acetaldehyde; Tokyo (in Japanese).
BIOCHEMISTRY
Fluorometric
94, l-5 (1979)
Determination
YOSHIHIRO Faculty
ASABE,
of Trioses
with o-Phenylphenoll
SHIGEYOSHI MOMOSE, AND SHOJI TAKITANI
of Pharmaceutical Ichigaya-funagawara-machi,
Sciences.
Science Shinjuku.
MASAO
SUZUKI,
University of Tokyo, Tokyo 162, Japan
12,
Received December 14, 1977 It was found that trioses, such as glyceraldehyde and dihydroxyacetone, could be determined by using the assay for acetaldehyde reported earlier, on the basis of the fluorescence reaction between acetaldehyde and o-phenylphenol in sulfuric acid. The calibration curves for the standard solutions of glyceraldehyde and dihydroxyacetone showed straight lines passing through the origin with the same slope for 1.8-72 nmoU200 ~1. Then, the standard method for the assay of trioses, acetaldehyde, and lactic acid in mixed sample solutions was established by a combination of separation of lactic acid from the other two components with an anion-exchange resin treatment and simultaneous determination of trioses and acetaldehyde based upon the differences in the reaction condition between the both compounds. Finally, almost 100% recovery was obtained for each of the components in the examination of the recovery of these three compounds added to the chemistry control serum.
In previous papers (2,3), the authors have reported the microdeterminations of lactic acid and muramic acid based upon a fluorescence reaction of acetaldehyde with o-phenylphenol (OPP)* in sulfuric acid. Because of its simplicity, rapidity and high sensitivity, this method seemed applicable to rapid analysis of other samples which liberate acetaldehyde. In fact, it was found that methylglyoxal and trioses (glyceraldehyde and dihydroxyacetone) liberate acetaldehyde when heated with sulfuric acid, which then gives blue fluorescence by the reaction with OPP. The present study aimed at establishing a fluorometric determination of trioses on the basis of this reaction. Previously published methods for the assay of trioses include the spectrophotometric method (4), the polarographic method (5), and also the enzymatic methods (6,7). On the other hand, their fluorescence reactions with chromo-
tropic acid (8) and Shydroxy-1-tetralone (9) are reported but these reactions are interfered with formaldehyde and common sugars, and hence no application of these reactions to quantitative determination of trioses is reported. Our method should be useful for the assay of biological samples if it allows simultaneous determination of trioses, acetaldehyde, and lactic acid in a mixture because acetaldehyde, and lactic acid naturally occur in usual biological fluids. Therefore, the standard procedure for the assay of the three components in the mixture was established by a combination of separation of lactic acid from the other two components with an anion-exchange resin treatment and simultaneous determination of trioses and acetaldehyde based upon the differences in reaction condition in the fluorescence reactions of both compounds. Finally, recovery of the added trioses, acetaldehyde, and lactic acid to the chemistry control serum was examined, as a preliminary attempt, in order to apply the proposed method to biological samples.
’ This paper constitutes Part V of a series entitled “Color Reactions of Carbonyl Compounds with Substituted-phenols”; Part IV in the series is Ref. (1). * Abbreviations used: OPP, o-phenylphenol. 1
0003-2697/79/050001-05$02.00/O Copynght 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
2
ASABE ET AL.
MATERIALS
AND METHODS
taining 1J-72 nmol of trioses (or 1- 10 nmol of acetaldehyde or 1.1-22.5 nmol of lactic Materials. The standard samples used acid) in a glass-stoppered test tube was were 9-360 nmol/ml of an aqueous solution cooled in an ice-water bath; 5 ml of conof DL-glyceraldehyde . dimer and dihydroxycentrated sulfuric acid was added slowly acetonemdimer (Tokyo Kasei Co.), 5-50 with cooling, and the tube was then shaken. nmoYm1 of an aqueous solution of acetaldeIn the case of the trioses or lactic acid, each hyde (3), 5.5- 112.5 nmoYm1 of an aqueous tube was placed in a water bath kept at 80°C solution of lithium lactate (3). OPP, 1.8% for 5 min; the tube was then cooled in an (w/v), was used in 0.5% sodium hydroxide ice-water bath. Next, 10 ,ul of 0.15% sodium solution and sodium nitrite was used in nitrite solution was added into tube, and the 0.15% (w/v) aqueous solution, freshly pretube was gently shaken. Subsequently, 10 ~1 pared before use. All of the reagents and of 1.8% OPP solution was added. After mixsamples were stored in a refrigerator. Methylglyoxal was prepared according to the ing the contents, the tube was placed in a 20°C water bath for 15 min; the fluorescence literature (10) and acetaldehyde-semicarbaintensity was measured with excitation at zone was prepared by the usual method. All 405 nm and emission at 465 nm. For blank other organic and inorganic chemicals used test, a water instead of the sample solution were analytical grade. was treated in the same way as described Measurements. The fluorescence spectra above. and their intensities were measured by a For the assay of each component in the Hitachi fluorescence spectrophotometer mixed samples, 2 ml of the sample solution Model MPF-2A in the same way as reported containing the trioses (within ca. 400 nmol), earlier (3). A Shibata microdiffusion unit acetaldehyde (within ca. 400 nmol), and was used for the Conway’s microdiffusion analysis of acetaldehyde, and the pH was lactic acid (within ca. 500 nmol) was poured measured with a Model HM-18B digital pH on the top of the anion-exchange resin column. During eluting, the initial 5 ml eluted meter (Toa Denpa Kogyo Co.). by 0.005 M phosphate buffer (pH 6.0) was Thin layer chromatography. A tic plate collected for analysis of the trioses and acetcoated with Merck silica gel HF,,, (thickness, aldehyde. Then, the following 5-ml fraction 0.25 mm) was activated at 110°C for 40 min eluted by 0.1 M phosphate buffer (pH 6.0) before use. After developed with a chlorowas also collected for analysis of lactic acid. form-ethanol (10: l), each spot was detected For the determination of trioses and acetalwith a color reaction of phosphomolybdic dehyde, a 200~,ul sample solution containing acid. 210 nmol of trioses and 1- 10 nmol of acetPreparation of anion-exchange resin colaldehyde was placed in two test tubes. One umn. Three hundred milligrams of Amberwas treated with the method for acetaldelite CG 400 (Rohm & Haas Co.; Type I, lOOhyde and the other with the method for the 200 mesh, Cl-), a strongly basic anion-extrioses. The fluorescence intensity due to change resin equilibrated with 0.005 M the trioses was given by the difference in phosphate buffer (pH 6.0), was packed in a fluorescence intensity between those obglass column (5 X 100 mm). Standard method.3 A 200~~1 sample con- tained for the former and the latter. 3 The amounts of the reagents added in the assay of acetaldehyde were modified to those used for lactic acid because it seemed favorable to unify the reaction conditions with regard to the assay of both compounds, and it was confirmed that acetaldehyde could be determined under the conditions of the assay for lactic acid; see Ref. (3).
RESULTS AND DISCUSSION Fluorescence Reaction of Trioses with OPP
The fluorescence spectra of the reaction products obtained by the standard method for methylglyoxal and the trioses were iden-
FLUOROMETRIC
DETERMINATION
tical with that of acetaldehyde. It has been reported that glyceraldehyde and dihydroxyacetone produce easily methylglyoxal when heated with sulfuric acid (1 l), and it is quite likely that methylglyoxal liberates acetaldehyde. A thin layer chromatogram for the reaction products obtained in a microdiffusion test [absorbent, semicarbazide hydrochloride solution, (12)] of the decomposition products of the above compounds when heated with sulfuric acid is shown in Fig. 1; the Rf value of every semicarbazone produced in the absorbent was equal to that of acetaldehyde-semicarbazone. Therefore, it was confirmed that the fluorescence was due to the reaction product between OPP and acetaldehyde, which was liberated from these compounds by sulfuric acid. Reaction Conditions for Determination of Trioses The relationship between fluorescence intensity and heating time in the decomposition of the trioses into acetaldehyde with sulfuric acid was studied at a temperature range 40-100°C. As is shown in Fig. 2, a constant intensity for glyceraldehyde as well as dihydroxyacetone was obtained when heating was done at 60°C for 10 min or 80°C for 5 min; at the higher temperature, the intensity decreased slightly with increasing heating time. In the proposed method, the reaction was carried out at 80°C for 5 min because it was advantageous to use the same condition for the assay of trioses and lactic acid. From the relationship obtained between fluorescence intensity and the amounts of the reagents added, it was revealed that the same amounts of OPP and sodium nitrite as those used in the assay for lactic acid can also be used in this case. Furthermore, concentrated sulfuric acid was found to give the best result for the decomposition of trioses. Every interference with the assay for acetaldehyde and lactic acid reported earlier was found to affect on the assay of trioses because of the same reaction employed.
OF TRIOSES
1.0 -
LI
II
III
ABCDE FIG. 1. Thin layer chromatogram of semicarbazones of acetaldehyde and reaction products obtained by decomposition of methylglyoxal and trioses. (A) Acetaldehyde-semicarbazone; (B) semicarbazone obtained from methylglyoxal, (C) semicarbazone obtained from glyceraldehyde; (D) semicarbazone obtained from dihydroxyacetone; (E) semicarbazide hydrochloride. Developer, chloroform-ethanol (10: 1). Detecting reagent, 10% phosphomolybdic acid-50% ethanol.
Calibration
Curves
Calibration curves for the standard solution of glyceraldehyde and dihydroxyacetone showed the straight lines passing through the origin with the same slope for 1.8-72 nmol in a 200-~1 sample solution. Therefore, it was concluded that glyceraldehyde , dihydroxyacetone , and their mixtures, so called glycerose, could be determined with the calibration curve for either of the compounds. The coefficients of variation for reproducibility of the measurements were 4.8 1 and 2.40% at 9 and 72 nmol of glyceraldehyde (in 10 experiments, n = lo), respectively; 4.10 and 3.60% at the same concentration of dihydroxyacetone (n = 10). Determinations of Trioses, Acetaldehyde, and Lactic Acid in the Mixtures After examining the conditions for separation of lactic acid from the other two com-
4
ASABE ET AL.
20 Time,
30
40
50
min.
FIG. 2. Effect of temperature on decomposition time. Glyceraldehyde, 40 nmoV200 ~1; (A) boiling water; (0) 80°C; (0) 60°C; (A) 40°C.
ponents, 0.005 M phosphate buffer (pH 6.0) was chosen for the solvent of lactic acid from which it adsorbs on the anion-exchange resin. Under this condition, the trioses and acetaldehyde could be immediately eluted without adsorption. Then, 0.1 M phosphate buffer (pH 6.0) was used for elution of the lactic acid adsorbed on the resin. Although the trioses and acetaldehyde in mixture can not be separated from each other by the column treatment, they differ in the reaction conditions for fluorescence to appear. Hence, the influence of the trioses on the fluorescence reaction of acetaldehyde was studied. As the result, the fiuorescence intensity was found to decrease when more than twofold the amounts of the trioses were added to 10 nmol of acetaldehyde in 200 ~1, while no effect of the trioses added was observed at additions below 10 nmol, and the calibration curve of acetaldehyde containing 10 nmol of the trioses was identical with that of the standard acetaldehyde solution. Next, the effect of acetaldehyde on the determination of the trioses was studied. When the fluorescence intensity due to 10 nmol of acetaldehyde was subtracted from those of the trioses containing 10 nmol of acetaldehyde obtained by the assay for the trioses, the resulting curve coincided with
the calibration curve of the standard triose solution. Therefore, the fluorescence intensity due to the trioses was evaluated in the way described above. As is shown in Fig. 3, a good separation was obtained by the present method using 2 ml of the prepared sample solution containing 250 nmol each of glyceraldehyde and acetaldehyde, and 500 nmol of lactic acid. Recoveries (n = 10) were 98.6 + 1.7% for glyceraldehyde, 99.7 -+ 1.1% for acetaldehyde, and 100.4 +- 1.4% for lactic acid, respectively. Recovery Examination with the Chemistry Control Serum
In order to minimize the amount of the serum to be taken in practical assay, the standard procedure was modified as follows: A 200~~1 sample of the serum containing the trioses, acetaldehyde, and lactic acid was poured onto the top of the resin column and then eluted with 0.005 M phosphate buffer (pH 6.0). The first 0.5 ml eluted was discarded and the following 1.O ml of eluate was collected for assay of the trioses and acetaldehyde, and then, 10 ml of the eluate by 0.1 M phosphate buffer (pH 6.0) was col+
(I)
-+e--
(2)
IOOC
-+A
LA
Effluent,
ml
FIG. 3. Elution curve of acetaldehyde (AA), glyceraldehyde (GLA), and lactic acid (LA). Sample taken, 2 ml of the mixture (containing AA 250 nmol, GLA 250 nmol, and LA 500 nmol). Eluting solution: (1) 0.005 MKH,PO,-0.005 M N%HPOa, pH 6.0; (2) 0.1 M KH,PO,-0.1 M N%HPOb, pH 6.0.
FLUOROMETRIC
DETERMINATION
OF TRIOSES
5
lected for assay of lactic acid. The eluate 100.1 2 3.0% (100 nmol added) and 98.0 containing the trioses and acetaldehyde was 2 2.9% (1 pmol added) for lactic acid. pretreated with 200 ~1 of 2.5% uranylacetate ACKNOWLEDGMENT solution (centrifuged at 2500 rpm for 5 min) before the fluorescence reaction. This work was supported in part by a Grant-in-Aid It was necessary to remove proteins con- for Scientific Research from the Ministry of Education tained in the serum which may interfere with of Japan. the fluorescence reaction because they could REFERENCES not be separated from the smaple solution Asabe, Y., Someya, K., Suzuki, M., and Takitani, by the column treatment and hence they S. (1977) Bunseki Kagaku 26, 43. contaminated the eluate containing the tri2. Kojima, S., Asabe, Y., Suzuki, M., and Takitani, oses and acetaldehyde. The uranylacetate S. (1975) Bunseki Kagaku 24, 167. method gave the best result for deproteini3. Asabe, Y., Kojima, S., Suzuki, M., and Takitani, zation. After examining the effect of uranylS. (1977) Anal. Biochem. 19, 73. acetate on the fluorescence reaction, an 4. Tsai, M. U., and Schwartz, E. L. (1961) Anni. Biochem. 2, 107. amount of 200 ~1 of 2.5% concentration was 5. Ardenne, M. v., and Tuemmler, R. (1964) Deur. chosen. The treatment by the reagent was Gesundheitsw. 19, 49, 709. found to give the best result when it was 6. Pinter, J. K., Hayashi, J. A., and Watson, J. A. made for the eluate before the fluorescence (1967) Arch. Biochem. Biophys. 121, 404. 7. Charlton, J. M., and van Heyningen, R. (1969) reaction. Anal. Biochem. 30, 313. Finally, recoveries of glyceraldehyde, ac8. Nakai, T., Koyama, M., and Demura, H. (1970) etaldehyde, and lactic acid added to the J. Chromatogr. 50, 338. chemistry control serum (Hyland Division 9. Momose, T. (1959) Talanta 3, 155. Travenol Lab., Inc.) were surveyed, The 10. Riley, H. L., Morely, J. F., and Friend, N. A. C. (1932) J. Chem. Sot. 1875. mean recoveries (n =lO) in 200 ,ul serum 11. Neuberg, C., Farber, E., Levite, A., and Schwenk, were 99.1 + 5.9% (25 nmol added) and 98.3 E. (1917) Biochem. Z. 83, 244. -+ 3.9% (50 nmol added) for glyceraldehyde; 12. Ishizaka, 0. (1969) in Experimental Methods of 98.9 -+ 3.2% (25 nmol added) and 99.7 Microdiffusion Analysis, p. 105, Nankodo, -+ 1.1% (50 nmol added) for acetaldehyde; Tokyo (in Japanese).
