A new fluorometric assay method for quinolinic acid

A new fluorometric assay method for quinolinic acid

ANALYTICAL 131, 194-197 (1983) BIOCHEMISTRY A New Fluorometric HIROSHI TAGUCHI, Department Assay Method for Quinolinic SHIRO KOYAMA, of Agricul...

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131, 194-197 (1983)


A New Fluorometric HIROSHI

TAGUCHI, Department

Assay Method for Quinolinic


of Agricultural Food Science





Chemistry, Me University, Tsu, Me 514, and *Department and Technology, Kyoto University, Kyoto 606, Japan



Received October 5, 1982 A new fluorometric assay method for quinolinic acid is introduced in this study. Quinolinic acid-hydrazine complex, a stable fluorescent compound, is formed after heating quinolinic acid with hydrazine at 215-220°C for 2 min. Fluorescence excitation and emission maxima of the complex are at 285 and 380 nm, respectively. This assay method is rapid and rather sensitive. It takes about 30 min to ascertain the amount of quinolinic acid as low as 50 ng. Specificity of this method is high among biological compounds. An ultrasensitive assaymethod for quinolinic acid (as low as 20 pg) with diphenylhydrazine instead of hydrazine is also found. After separating the quinolinic acid-diphenylhydrazine complex from residual diphenylhydrazine, this ultrasensitive assaymethod may be practically applicable.

Quinolinic acid (pyridine-2,3-dicarboxylic acid) is a key intermediate to the de novo biosynthetic pathways of NAD in animals (l), plants (2), and microorganisms (3,4). In animals, quinolinic acid is excreted in urine and its amount is one of the criteria of nicotinic acid deficiency (5-7). In plants the biosynthetic pathway through quinolinic acid is still unknown at present. Quinolinic acid is usually assayed microbiologically with Lactobacillus arabinosus 175 (ATCC 8014) as nicotinic acid after decarboxylation of quinolinic acid into nicotinic acid (8). Several chemical assay methods for nicotinic acid were also reported (9-l 1) and can be used for quinolinic acid assay after decarboxylation. In these methods not only quinolinic acid but also nicotinic acid and related compounds, including nicotinamide, nicotinic acid mononucleotide, nicotinamide mononucleotide, deamino-NAD, NAD(H), and NADP(H), are equally measurable. The microbiological assay method for nicotinic acid (12) is rather sensitive but time consuming. All methods mentioned above are indirect ones, and conversion of quinolinic acid into nicotinic acid before the assay is essential. A direct microbiological assay method 0003-2697/83 $3.00 Copyright 6 1983 by Academw Press. Inc. All rights of reproduction in any form rewrved.

for quinolinic acid with Pseudomonasjluorescence(ATCC 10838) was reported (13), but many compounds related to nicotinic acid interfere with the quinolinic acid assay. More sensitive, rapid, and direct assay method for quinolinic acid has been investigated and a new method meeting these requirements has just been discovered. This method is based on the formation of a fluorescent compound from quinolinic acid and hydrazine or related compounds under certain conditions. The proposed reaction scheme of quinolinic acid with hydrazine is shown in Fig. 1. MATERIALS


Chemicals. Diphenyhydrazine was purchased from Tokyo Chemical Industry Company, Ltd., Tokyo. Other chemicals were obtained from Nakarai Chemicals, Ltd., Kyoto. All chemicals used in this study were of the highest purity commercially available. Reaction of quinolinic acid with hydra&e.

A mixture of quinolinic acid (powder: less than 0.5 g, or solution: maximum 2 ml), 1 ml of 8% aqueous solution of hydrazine, and 2 ml of triethylene glycol in a 1.8 X 18-cm test tube





FIG. 1. Chemical drazine.



Quinolinic hydrazide

of quinolinic


acid with hy-

was clamped in a vertical position about 5 cm above a Bunsen burner. A thermometer and an aspirator connected to a suction pump were inserted in the test tube, and the mixture was boiled vigorously to distill the excess water at 1 lo- 130°C. Then the temperature was raised rapidly until it reached 215°C. After maintaining a temperature of 2 15-220°C for 2 min, the tube was removed and cooled to room temperature. If a dozen samples must be heated, heating blocks (test-tube heaters) instead of a Bunsen burner may be used. The reaction mixture obtained was used for fluorometric assay after diluting adequately with water. For the structure analysis of quinolinic acid-hydrazine complex, 0.5 g of quinolinic acid was used and 5 ml of water was added to the test tube after heating a mixture. Then crystals formed were collected by filtration. Other methods were the same as mentioned above. “he complex was recrystallized from ethyl alcohol solution. The recrystallized complex was dried completely and used for several spectrometric analyses to confirm the structure. Reactions of quinolinic acid with hydrazine analogs. For the comparison of relative intensity of fluorescence emitted by quinolinic acid-hydrazine analog complex, 0.1 g of quinolinic acid and each of 0.1 ml of methylhydrazine, 0.3 ml of phenylhydrazine, and 0.3 g of diphenylhydrazine were used. In the cases of phenylhydrazine and diphenylhydrazine, ethyl alcohol was added and the reaction mixture was diluted with ethyl alcohol after heating the mixtures. Fluorescence spectrophotometry. Fluorescence spectra and intensities were measured




with a Hitachi fluorescence spectrophotometer Model 650-60 equipped with a recorder. Band-pass slits of excitation and emission were set at 10 nm, each. Other spectrometries. To confirm the structure of quinolinic acid-hydrazine complex several spectrometries were carried out with recrystallized complex. Visible and ultraviolet spectra of the aqueous solution were measured with a Shimadzu double-beam/difference/dual-wavelength recording spectrophotometer Model UV-300. Mass spectra were measured with a Hitachi mass spectrometer Model RM-50. Infrared spectra were measured with a JASCO infrared spectrophotometer Model IR-G. NMR spectra were measured with a Hitachi high-resolution NMR spectrometer Model R-22. RESULTS

Fluorescence Spectra of Quinolinic Hydrazine Complex


Fluorescence excitation and emission spectra of recrystallized quinolinic acid-hydrazine complex are shown in Fig. 2. Excitation and emission maxima were at 285 and 380 nm, respectively. Very small peak found at 3 17 nm is Raman scattering of water (14). Even if the complex was not isolated from the reaction mixture, the spectra were essentially the same as those shown in Fig. 2. Hydrazine itself before and after the same treatment without quinolinic acid did not fluoresce at any wavelength.


O 200








I nm 1

FIG. 2. Excitation and emission spectra of recrystallized quinolinic acid-hydrazine complex. Excitation spectrum at 380 nm of emission (broken line) and emission spectrum at 285 nm of excitation (solid line).



Calibration Curve for Quinolinic Hydrazine Method


Results obtained by several spectrometries are summarized below: Visible and ultraviolet absorption spectra; x max, = 252 nm (t = 5080), Xmax2 = 300 nm (6 = 3370)~ Xmin 1 = 240 nm (E = 4240), and Xmin 2 = 276 nm (E = 27 10) were observed. No absorption was found in visible region. Mass spectrum: m/z (relative intensity) 163 (M+, loo), 147 (19), 105 (17) 78 (56) at 80 eV. Infrared spectrum; (KBr) 3150 (-NH-), 3050,2900,1690()C=O),


1390, 1350, 1210, 1095, 800,790,710 cm-‘. ‘H-NMR spectru m; 6MedSi (DMSO-de) 7.93 (lH, d of d, J = 8 Hz, J = 5 Hz), 8.56 (lH, d of d, J = 8 Hz, J = 2 Hz), 9.18 (lH, d of d, J = 5 Hz, J = 2 Hz). From these data the structure of quinolinic acid-hydrazine complex is confirmed as pyrido[2,3-dlpyridazine- 1,4(2H,3H)dione and the reaction scheme of quinolinic acid with hydrazine was proposed as shown in Fig. 1. Fluorescence Spectra of Quinolinic Hydrazine Analog Complex


Acid by

Calibration curve for quinolinic acid was prepared at very low concentrations of quinolinic acid (up to 1 pg/ml) under the optimum fluorometric conditions described above. Correlation coefficient between quinolinic acid concentration and fluorescence intensity of this curve was calculated at 0.999. The range of quinolinic acid concentration in which the assay is useful is 0.1-10 pg/ml. Other Spectrometries of Quinolinic Hydrazine Complex



Excitation and emission maxima of quinolinic acid-hydrazine analog complex and its relative fluorescence intensity are shown in Table 1. Fluorescence intensities of quinolinic acid-phenylhydrazine complex and of quinolinic acid-diphenylhydrazine complex were




Hydrazine Methylhydrazine Phenylhydrazine Diphenylhydrazine



Relative intensity at each X,,

285 265

380 400

100 (0)’ 15 (0)

240 281

335 395

180,000 (2,300)



n Numbers in parentheses are the blank values due to hydrazine analog without quinolinic acid at each X,, of the complex. The amount of hydrazine analog added to a test tube, the methods of heating, and the dilution rate of the reaction mixture were the same as those when quinolinic acid was also present in a test tube.

very high, compared with that of hydrazine complex. Unfortunately the blank values were also high. The X,,, of excitation and emission of phenylhydrazine after heating without quinolinic acid were 275 and 330 nm, respectively. Excitation and emission maxima of diphenylhydrazine after the same treatment as above were found at 240 and 340 nm, respectively. Specificity of the Method Various monocarboxylic acids, including nicotinic acid and dicarboxylic acids, were checked to see whether they react with hydrazine to produce any fluorescent compounds. As a result, most carboxylic acids were not converted to a fluorescent compound and no interference was observed for this quinolinic acid assay method except maleic acid (Ex = 313 nm, Em = 425 nm), cinchomeronic acid (Ex = 295 nm, Em = 410 nm), and phthalic acid (Ex = 305 nm, Em = 465 nm). Relative fluorescence intensities of these hydrazine complexes at the same concentration were 100 (quinolinic acid), 74 (maleic acid), 77 (cinchomeronic acid), and 355 (phthalic acid), respectively. Thin-Lawyer Chromatography R, values of quinolinic acid-diphenylhydrazine complex and diphenylhydrazine were


0.84 and 0.45, respectively, on a Merck silicagel plate (Type 5721) when the compounds were developed with benzene:petroleum ether (3:l) for 30 min at room temperature. DISCUSSION

A method for luminol synthesis by Fieser (15) is applied to synthesize quinolinic acidhydrazine complex and a new fluorometric assay method for quinolinic acid is introduced. The quinolinic acid-hydrazine complex is a yellow-colored stable fluorescent compound. This assay method for quinolinic acid is satisfactory: very low blank value (negligible), relatively high sensitivity, and high specificity. Sensitivity is as high as that of routine microbioassay of nicotinic acid with L. arubinosus. It takes about 24 h for the microbioassay but only 30 min is required for this new method to complete the assay. The recoveries of quinolinic acid from three independent samples of human urine were also satisfactory ( 10 1- 109%). From the data obtained above, which show several compounds reacting with hydrazine to form fluorescent compounds, two carboxyl groups attached in ortho position on benzene ring (or pyridine ring) are thought to be essential. In dicarboxylic acids, an en-dicarboxy1 group in cis form will be required in the molecule. A much more sensitive assay procedure for quinolinic acid was also found by condensing quinolinic acid with phenylhydrazine or diphenylhydrazine instead of hydrazine. Sensitivities of phenylhydrazine and diphenylhydrazine methods were respectively 700 and 1800 times higher than that of hydrazine method. However, the blank value of a mixture obtained from the diphenylhydrazine




method was high, as shown in Table 1. To lower the blank value, quinolinic acid-diphenylhydrazine complex was separated from diphenylhydrazine by thin-layer chromatography. The complex may be assayed directly on a thin-layer plate with a chromatogramscanning fluorometer, or extracted from the gel and then assayed with an ordinary fluorometer. These complexes are not water soluble, and ethanol or other organic solvents must be used to dissolve the complexes. Details for the application of this new assay method to various biological materials will be published later. REFERENCES Iwai, K., and Taguchi.

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