Organic acids analysis

G. D. Guerrand and co-workers, "Organic Acid Analysis," / Clin. Microbiol 16, 355 (1982). 

Many laboratories use quantitative urinary organic acid analysis as an alternative to a qualitative approach and may not use stable isotope dilution as a more rigorous means of quantitation. The results from EQA schemes in the area reflect this variability of practice and the lack of internationally agreed standardisation. 

Some important assays commonly used in biochemical genetics laboratories do not provide quantitative data (e.g. MPS-EP, qualitative urinary organic acid analysis, AA-TLC). In addition, all successful investigations depend heavily upon selection of the correct analytes to measure and the appropriate interpretation of the quantitative or qualitative results in their clinical context. These challenges suggest a requirement for external quality assessment or proficiency testing schemes that can inform participants about their performance in these areas when compared with other centres. 

Similarly, there is an increasing degree of consistency of methodological approach between laboratories. For instance, a recent survey of those undertaking qualitative urinary organic acid analysis revealed that more than 90% (83 of 91) of laboratories indicated that they extracted the samples with ethylacetate or ethylacetate/ether, formed trimethylsilyl derivatives and employed gas chromatography-mass spectrometry as a means of analysis. 

Downing M, Bonham JR, Allen JC, Heap SJ, Manning NJ, Olpin SE, Pollitt RJ (1999) Is quality assurance for quality urinary organic acid analysis improving performance J Inherit Metab Dis 22 148... 

In the absence of tandem mass spectrometry equipment, almost equally reliable estimations of the PA concentrations can be made using gas chromatography-mass spectrometry (GC-MS). A standard quadrupole instrument, such as the one used for organic acid analysis, will be sufficient. Depending on the derivative, a choice between positive and negative ionization will have to be made. In general, a more extensive prepurification of the biological samples, will have to be realized. 

Table 3.1.3 Pathologic acylglycine species detected by organic acid analysis. CoA coenzyme A, FAO fatty acid oxidation, ILE isoleucine, LEU Leucine, MCAD medium-chain acyl-CoA dehydrogenase, MET methionine,...

Organic acid analysis could be performed in other biological specimens [14, 22] when targeting specific analyte(s) in previously diagnosed patients, or fetuses at risk for a specific condition. Plasma/serum profiling is not helpful in the laboratory evaluation of patients without a specific diagnosis, and should be avoided [2]. 

Organic acid analysis is one of the most challenging tests apphed to the diagnosis of inborn errors of metabolism. Several hundreds of compounds are excreted in the urine of individuals free of apparent disease, and the excretion of informative markers could be marginal depending on clinical status. Therefore, pattern recognition and descriptive interpretation are essential for proper utilization of this test. 

Organic acid analysis is probably the most complex test performed in a biochemical genetics laboratory [2]. Errors and oversights may happen at all stages of the testing process and can be categorized as shown in Table 3.1.11. 

Table 3.1.11 Common pitfalls in organic acid analysis...

Hoffman GP. Organic acid analysis. In Blau N, Duran M, Blaskovics ME (eds) Physicians Guide to the Laboratory Diagnosis of Metabolic Diseases. Chapman Hall, London, pp 31-49... 

Sewell AC, Bohles HJ (1991) 4-Hydroxycyclohexanecarboxylic acid a rare compound in urinary organic acid analysis. Clin Chem 37 1301-1302... 

Sweetman L (1991) Organic acid analysis. In Hommes FA (ed) Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York, pp 143-176... 

Acylcarnitine analysis was first performed in urine specimens in the evaluation of patients with organic acidemias. However, because it was found that acylcarnitine analysis of plasma is more informative for the diagnosis of FAO disorders than analysis of urine specimens, plasma has become the preferred specimen [17]. It is only recently that it was shown that urine acylcarnitine analysis still has a role in the diagnostic evaluation of patients with organic acidurias but uninformative or borderline abnormal results of plasma acylcarnitine and urine organic acid analysis [18-21]. In our laboratory, sample preparation and analysis is identical to that of plasma once a urine aliquot has been prepared that is based on the creatinine concentration. 

Standard GC-MS used for organic acid analysis (see Chap. 3.1). Data regarding calibration and quality control in this procedure can be found in Chap. 3.1 of this book. 

In the past decade, eight inherited disorders have been linked to specific enzyme defects in the isoprenoid/cholesterol biosynthetic pathway after the finding of abnormally increased levels of intermediate metabolites in tissues and/or body fluids of patients (Table 5.1.1) [7, 9, 10]. Two of these disorders are due to a defect of the enzyme mevalonate kinase, and in principle affect the synthesis of all isoprenoids (Fig. 5.1.1) [5]. The hallmark of these two disorders is the accumulation of mevalonic acid in body fluids and tissues, which can be detected by organic acid analysis, or preferably, by stable-isotope dilution gas chromatography (GC)-mass spectrometry (GC-MS) [2]. Confirmative diagnostic possibilities include direct measurement of mevalonate kinase activities in white blood cells or primary skin fibroblasts [3] from patients, and/or molecular analysis of the MVK gene [8]. 

With regard to AADC, vanillactic acid (a metabolite of 3-MD) appears in the urine and can be detected by organic acid analysis. As predicted, an increase in urinary vanillactic acid has also been reported in PNPO deficiency. The PLP concentration in CSF, which can be determined by HPLC, is also reported to be decreased in PNPO deficiency. Mutation analysis can also now confirm or refute a suggestion of PNPO deficiency. 

Urinary organic acid analysis is useful for differentiating isolated carboxylase deficiencies from the biotin-responsive multiple carboxylase deficiencies. P-Hydroxyisovalerate is the most common urinary metabolite observed in isolated P-methylcrotonyl-CoA carboxylase deficiency, biotinidase deficiency, biotin holo-carboxylase synthetase deficiency, and acquired biotin deficiency. In addition to P-hydroxy-isovalerate, elevated concentrations of urinary lactate, methylcitrate, and P-hydroxypropionate are indicative of multiple carboxylase deficiency. 

The organic acid analysis in the urine of this child was consistent with biotin deficiency or multiple carboxylase deficiency. Biotin deficiency usually can be excluded unless there is a history of dietary indiscretion, such as consuming a diet containing raw eggs or few biotin-containing foods, or there is a history of prolonged parenteral hyperalimentation without biotin supplementation. Low serum biotin concentrations can be useful in differentiating... 

Organic Acids, Analysis by Thin Layer Chromatography... 

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