Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism.

Abstract

OBJECTIVES:

To set up a novel simple, sensitive, and reliable ion-pairing HPLC method for the synchronous separation of several purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis and screening of inborn errors of metabolism (IEM).

DESIGN AND METHODS:

The separation was set up using a Hypersil C-18, 5-microm particle size, 250 x 4.6 mm column, and a step gradient using two buffers and tetrabutylammonium hydroxide as the pairing reagent. A highly sensitive diode array UV detector was set up at a wavelength between 200 and 300 nm that revealed purines and pyrimidines at 260 nm and other compounds at 206 nm.

RESULTS:

Compounds were determined in the plasma of 15 healthy adults, in the urine of 50 healthy subjects (1-3 years, 4-6 years, 8-10 years, 12-18 years, 25-35 years), and in 10 non-pathological amniotic fluid samples. To assess the validity of the chemical diagnosis of IEM, plasma and urine samples were analyzed in patients affected by Canavan disease (n = 10; mean age 4.6 +/- 2.3). Low plasma levels of N-acetylaspartate (16.96 +/- 19.57 micromol/L plasma; not detectable in healthy adults) and dramatically high urinary N-acetylaspartate concentrations (1872.03 +/- 631.86 micromol/mmol creatinine; 450 times higher than that which was observed in age-matched controls) were recorded. Neither N-acetylglutamate nor N-acetylaspartylglutamate could be detected in the plasma or urine of controls or patients with Canavan disease.

CONCLUSIONS:

The results demonstrate the suitability of the present ion-pairing HPLC separation with UV detection of cytosine, cytidine, creatinine, uracil, uridine, beta-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, N-acetylaspartate, N-acetylglutamate, N-acetylaspartylglutamate, malonic acid, methylmalonic acid, GSH, and GSSG as a reliable method for the prenatal and neonatal chemical diagnosis and screening of IEM using biological fluids.

• Authors: Barbara Tavazzi, Giuseppe Lazzarino, Paola Leone, Angela Maria Amorini, Francesco Bellia, Christopher G Janson, Valentina Di Pietro, Lia Ceccarelli, Sonia Donzelli, Jeremy S Francis, Bruno Giardina
• Journal: Clinical biochemistry (Clin Biochem) Vol. 38 Issue 11 Pg. 997-1008 (Nov 2005) ISSN: 0009-9120 [Print] United States
• PMID: 16139832 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Amino Acids
• Dicarboxylic Acids
• Purines
• Pyrimidines
• Aspartic Acid
• N-acetylaspartate

Topics

• Adolescent
• Adult
• Amino Acids (isolation & purification)
• Amniotic Fluid (chemistry)
• Aspartic Acid (analogs & derivatives, blood, urine)
• Canavan Disease (diagnosis)
• Child
• Child, Preschool
• Chromatography, High Pressure Liquid (methods)
• Dicarboxylic Acids (isolation & purification)
• Humans
• Infant
• Mass Screening (methods)
• Metabolism, Inborn Errors (diagnosis)
• Middle Aged
• Prenatal Diagnosis (methods)
• Purines (isolation & purification)
• Pyrimidines (isolation & purification)
• Reproducibility of Results
• Sensitivity and Specificity
• Spectrophotometry, Ultraviolet