Reactivity of creatinine with alkaline 3,5-dinitrobenzoate: a new fluorescent kidney function test.

OBJECTIVES: To develop a new quantitative fluorometric assay for creatinine with enhanced specificity and inherent fluorescence sensitivity. DESIGN AND METHODS: Creatinine is reacted with 3,5-dinitrobenzoate under alkaline reaction conditions to produce a fluorophore. Alternatively, 3,5-dinitrobenzoyl chloride or methyl-3,5-dinitrobenzoate may be employed as reagents. Fluorophore development was evaluated for Group 1A bases under aqueous and mixed solvent conditions. RESULTS: The chemical reaction between creatinine and 3,5-dinitrobenzoate performed under highly alkaline conditions produced a fluorescent product with excitation and emission maxima near 410 and 475 nm, respectively. The reaction proceeds rapidly in the presence of group IA bases under both aqueous and mixed solvent conditions, in particular with water/dimethylsulfoxide and water/1,4-butanediol solvent systems containing 1.0 M LiOH. A linear calibration curve was obtained when the fluorescence intensity measured in microamperes was plotted versus creatinine concentration in the range of 5-50 mumol/L. The detection limit for creatinine was well below 1 mumol/L. CONCLUSIONS: A simple, sensitive and highly specific fluorometric method for the determination creatinine has been developed.

Effect of glucose upon alkaline picrate: a Jaffé interference.

The reactivity of glucose in aqueous alkaline picrate was investigated by spectrophotometry and polarography at 25 degrees C in 0.51 mol/l sodium hydroxide. Thin-layer chromatography and infrared spectroscopy studies have conclusively identified the presence of picramic acid in 5:1 and 10:1 glucose picrate test solutions incubated at 25 degrees C. The polarographic data of an alkaline picrate blank with a concentration of 0.284 mmol/l, show three well-defined nitro group reduction waves with approximate half-wave potentials of -0.62 V, -0.78 V, and -0.93 V and a fourth broad wave appearing near -1.31 V versus a saturated calomel electrode. The addition of glucose to alkaline picrate resulted in a decreased diffusion current for reduction waves 1-3, with little change in reduction wave 4. The reactivity of test solutions containing glucose:picrate in 1:1, 2:1, 5:1 and 10:1 molar ratios was investigated at varied time intervals between 10 and 180 minutes. The absorption spectra of a 10:1 glucose:picrate solution shifted from 356 nm to 375 nm and a broad tailing shoulder absorbance formed in the 450-600 nm region. An orange coloured minor product, separated by thin-layer chromatography, was observed to fluoresce. The maximum excitation and emission wavelengths were 318 nm and 545 nm, respectively. A major, red-coloured product was isolated and identified as picramic acid by infrared spectroscopy. For 10:1 glucose:picrate test solutions incubated at 25 degrees C, picramic acid formed within 10 minutes. Within the first minute, the colour was observed to change from yellow to orange and then to red.

Reactivity of acetoacetate with alkaline picrate: an interference of the Jaffé reaction.

Spectrophotometric, kinetic, and polarographic studies of the interaction of acetoacetate with alkaline picrate have been undertaken in the presence of aqueous NaOH concentrations ranging between 0.50 mol/L and 2.50 mol/L. Spectrophotometric data has substantiated formation of the following acetoacetate-picrate complexes: 1:1 red, 490 nm; 2:1 orange, 390 nm; and 3:1 colorless, 265 nm. Depending upon the time of measurement, the composition of alkaline picrate, and the acetoacetate level in the test samples, acetoacetate may be either a positive or negative interference in kinetic Jaffé methods for the determination of creatinine. Polarograms of alkaline picrate in 0.50 mol/L NaOH showed three well-defined nitro group reduction waves and a more diffuse fourth reduction wave with approximate half-wave potentials of -0.62 V, -0.79 V, -0.94 V, and -1.32 V, respectively. Increasing the concentration of hydroxide and/or acetoacetate resulted in the disappearance of reduction waves 1 to 3 with only reduction wave 4 remaining. Based upon the polarographic results, a trinitro anion structure has been assigned for the 2:1 acetoacetate-picrate complex.

Jaffe' reaction products.

Polarographic and spectrophotometric studies of the interaction of creatinine with alkaline picrate have been undertaken in sodium hydroxide concentrations ranging between 0.95 and 4.5 mol/l. Red colored 1:1 and orange colored 2:1 creatinine-picrate complexes readily formed along with orange colored 1:1:1 creatinine-picrate-hydroxide complexes. The red and orange colored complexes were easily identified by their corresponding absorption maxima near 490 nanometers and 390 nanometers, respectively. Alkaline picrate polarograms showed three well-defined nitro group reduction waves with approximate half-wave potentials of -0.60 volts, -0.77 volts, and -0.91 volts. Increased concentrations of hydroxide and/or creatinine resulted in a decreased diffusion current for reduction waves 1-3 and the appearance of a fourth reduction wave, with an approximate half-wave potential of -1.24 volts. Further increases in base and/or creatinine concentration resulted in the disappearance of reduction waves 1-3, with only reduction wave 4 remaining. Based upon the experimental data, a tri-nitro anion structure has been assigned for the 2:1 complex.

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 mum). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.

Miniature two-dimensional thin-layer chromatographic separation of lecithin and sphingomyelin.

A miniature two-dimensional thin-layer chromatographic procedure employing silica gel impregnated glass-microfiber chromatography sheets (commercial product, ITLC-type SG sheets) has been developed for the separation of lecithin (L) and sphingomyelin (S) from a standard lipid mixture containing L, S, lysolecithin, phosphatidyl inositol (PI), phosphatidyl serine (PS), phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The newly developed procedure eliminates possible interference from PI and PS. Complete separation of L and S was easily achieved with chromatographic solvent migration times of approximately 3 and 2 min for the first and second dimensions, respectively. The lipids were visualized by charring and fluorescent staining techniques. The procedure has been adapted for the separation of L and S from amniotic fluid samples.

A rapid, sensitive method for accurate determination of the lecithin/sphingomyelin ratio by thin-layer chromatography and reflectance spectrofluorometry.

A highly reproducible thin-layer chromatographic procedure has been developed for accurate determination of the lecithin/sphingomyelin ratio. Two interfering compounds, phosphatidyl inositol and phosphatidyl serine, have been investigated and eliminated by adsorption onto DEAE-cellulose. A uniform fluorescence staining procedure employing 2',7'-dichlorofluorescein has been developed. Accurate quantitation was performed by direct measurement of the reflected fluorescence intensity of the lecithin and sphingomyelin fluorophore spots with a spectrofluorometer equipped with a thin-layer scanning attachment. Stability and reproducibility studies are reported.