Acylcarnitine Analysis Research Articles

Chromatographic techniques in inborn errors of metabolism

Chromatography has played a pivotal role in the advances made during the last 30 years in our knowledge of inborn errors of metabolism. This review discusses the application of some of these techniques to the analysis of organic acids and acylcarnitines. The separation of organic acids needed a comprehensive approach that would permit all of the many organic acids present in urine or other complex mixtures to be extracted, analysed and identified in a single run. This required analytical methods of great resolving power, wide linear range and universal detectors such as gas chromatography (GC), or GC coupled with mass spectrometry. Sample preparation was another problem that has been tackled by a variety of approaches. Organic solvents have been employed widely for the extraction of organic acids from physiological fluids. Unfortunately, recoveries of the different organic acids by this method are sometimes less than quantitative and variable depending on the compound. Other methods, such as the use of DEAE-Sephadex columns, have the advantage of resulting in close to 100% recoveries, but are more tedious. Liquid partition chromatography on short silicic acid columns has also been recommended as a useful clean-up step prior to GC, permitting both the identification and quantitation of organic acids in urine, plasma or amniotic fluid. Although many derivatization procedure have been used to prepare organic acids for gas chromatography, the most common is trimethylsilylation. Oxo acids are usually reacted with one of several commonly used reagents to form oximes. GC analysis of organic acids was initially done using packed columns with methylsilicone-based, non-polar stationary phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of acylcarnitines in maternal urine for prenatal diagnosis of glutaric aciduria type 2

The urinary acylcarnitine profiles of two mothers whose first children were diagnosed to have glutaric aciduria type 2 (multiple acyl-CoA dehydrogenation deficiency, electron transfer flavoprotein (ETF) deficiency) were analysed in the second pregnancy. Large volumes of tigrylcarnitine and isovalerylcarnitine and a little glutarylcarnitine were detected. Each fetus was also diagnosed to be abnormal by enzyme activity and immunoassay of ETF protein. The acylcarnitines in the mothers' urine disappeared in 1 week after labour or artificial abortion. Acylcarnitines were never detected in the urine of controls.

Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism

ConclusionA method for analysis of acylcarnitines in blood at physiological concentrations has been developed. Preliminary results from umbilical cord blood and neonatal blood spotted onto Guthrie cards are encouraging. This method will detect up to at least eight inherited metabolic disorders which exhibit diagnostic acylcarnitine profiles, including medium‐chain acyl‐CoA dehydrogenase deficiency. The speed and simplicity of the method permit automation with existing technology, and could enable routine neonatal screening to be carried out in an efficient and cost‐effective manner.

Analysis of abnormal urinary metabolites in the newborn period in medium‐chain acyl‐CoA dehydrogenase deficiency

In order to determine which are useful early diagnostic markers for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we have analysed urine from an asymptomatic neonate. Profiling of urinary organic acids followed by peak confirmation by electron impact mass spectrometry revealed a high suberate/adipate ratio (greater than 1.0) and the presence of n-hexanoylglycine (HG). Acylcarnitine analysis by fast atom bombardment mass spectrometry (FAB-MS) was inconclusive, but FAB-MS/MS (tandem mass spectrometry) revealed diagnostic amounts of octanoylcarnitine and hexanoylcarnitine. Quantitative analysis of acylglycines by stable isotope dilution and chemical ionization mass spectrometry revealed a 30-fold increase in HG and increased suberylglycine, but no increase in 3-phenylpropionylglycine.

Application of fast atom bombardment with tandem mass spectrometry and liquid chromatography/ mass spectrometry to the analysis of acylcarnitines in human urine, blood, and tissue

Using a precursor-ion scan function on a triple quadrupole mass spectrometer, acylcarnitines were detected in the target matrices at or below concentrations of 1 nmol per gram by fast atom bombardment mass spectrometry. Acylcarnitine profiles from patients with known metabolic disorders were consistent with previously acquired data. Putative acylcarnitine signals were confirmed in one case by administration of stable isotope-labeled carnitine, which equilibrated rapidly with the endogenous pool. The addition of a continuous flow system enabled rapid sequential analysis without operator intervention, indicating the potential for automation of the analytical procedure. Incorporation of a micro-LC column enabled on-line liquid chromatographic/mass spectrometric analysis of selected patient samples. Large-scale screening and quantitative analysis of urine or blood for diagnostic acylcarnitines are now practicable.

Analysis of acylcarnitines in normal human urine with the radioisotopic exchange-high performance liquid chromatography (HPLC) method

The identification of camitine esters in the urine is important in the diagnosis of metabolic diseases which alter camitine metabolism [l]. Their quantitation may also give clues to the physiological role of carnitine in the metabolism of branched chain amino acids and fatty acids, the influence of nutrition on these processes and the possible function of camitine in the conjugation of potentially toxic endogenous or exogenous organic acids. The excretion of total, free and acylcamitine [2-41 and that of acetylcamitine [5] in healthy humans has been measured in several studies. However, the analysis of individual camitine esters in normal human urine has been incomplete due to a lack of methods sensitive and specific enough to detect low concentrations of these compounds. In the following, we report the quantitation of camitine esters in normal human urine using a modification of the radioisotopic exchange-HPLC method originally described by Kemer and Bieber for tissue extracts and colostrum WI.

Application of continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry to the analysis of diagnostic acylcarnitines in human urine.

Acylcarnitine profiles of diagnostic value were generated from the equivalent of 0.1 microL of raw urine using the continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry (LC/FAB-MS) interface for sample introduction. Further analysis was accomplished by gradient LC/MS using a commercially available packed fused-silica microbore column.

Application of high resolution fast atom bombardment and constant B/E ratio linked scanning to the identification and analysis of acylcarnitines in metabolic disease.

Acylcarnitines, a biologically important group of metabolites which have thus far eluded characterization by mass spectrometry, exhibit very intense fast atom bombardment mass spectra dominated by parent cations. The combination of high resolution selected ion detection and daughter ion analysis using the linked scan at constant B/E ratio has enabled the unequivocal identification of the acylcarnitines in the urine of children with propionic acidemia, methylmalonic aciduria and Reye's Syndrome. Quantitative analysis of acetylcarnitine and propionylcarnitine in selected samples was accomplished by isotope dilution, utilizing (2H3)acetyl- and (2H5)propionylcarnitines as internal standards. These techniques will allow assessment of the therapeutic use of L-carnitine in these disorders.