Quantitative Acylcarnitine Profiling in Peripheral Blood Mononuclear Cells Using In Vitro Loading With Palmitic and 2-Oxoadipic Acids: Biochemical Confirmation of Fatty Acid Oxidation and Organic Acid Disorders

Abstract

Organic acid (OAD) and fatty acid oxidation disorders (FAOD) are inborn errors of metabolism often presenting with life-threatening metabolic decompensation followed by (irreversible) organ failure, and even death during catabolic state. Most of these diseases are considered as treatable, and metabolic decompensations can be avoided by early diagnosis and start of therapy. Confirmation of suspected diagnosis currently relies on enzymatic and mutation analyses and in vitro loading of palmitic acid in human skin fibroblast cultures. Furthermore, in some cases potentially life-threatening in vivo loading or fasting tests are still performed. In this study, we established a standardized in vitro loading test in peripheral blood mononuclear cells (PBMC) that allows reliable biochemical confirmation of a suspected diagnosis within 1 week. Patients with confirmed diagnosis of short-, medium-, very-long-chain, and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies, methylmalonic, propionic, isovaleric acidurias, and glutaric aciduria type I were included in the study. PBMC, isolated from heparinized venous blood samples of these individuals were incubated for 5 days with palmitic acid or 2-oxoadipic acid (glutaric aciduria type I), respectively, and quantitative acylcarnitine profiling was subsequently performed in supernatants using electrospray ionization tandem mass spectrometry. All patients were clearly identified, including those with mild biochemical phenotypes who, in particular, are at risk to be missed under balanced metabolic conditions. In glutaric aciduria type I, the same results were also obtained using lymphoblasts. In conclusion, our assay allows biochemical confirmation of a number of FAOD and OAD and could easily be implemented into the confirmatory diagnostic work-up. Organic acids and fatty acids comprise a group of physiologically occurring intermediates in a variety of intracellular metabolic pathways, such as catabolism of amino acids, mitochondrial ␤-oxidation of fatty acids, tricarboxylic acid cycle, and cholesterol biosynthesis. Organic acid (OAD) and fatty acid oxidation disorders (FAOD) are caused by autosomal recessive inherited deficiencies of single enzymes (or in the metabolism of required coenzymes) in the outlined metabolic

Metabonomic analysis of HIV-infected biofluids

“…Metabolic stress (induced by HIV) is also associated with a decrease in glucose levels. 67 Elevated levels of adipic acid could be an indication of the development of a sugar disorder in infected patients. Oxidative phosphorylation is a process which primarily takes place in mitochondria.…”

Ethylmalonic Encephalopathy: Clinical and Biochemical Observations

“…Evaluation of the OXPHOS enzyme activities is possible by comparing the band intensities from the patient and the tissue specifi c controls. Acylcarnitine profi ling in peripheral blood mononuclear cells (PBMC) using in vitro loading with palmitic acid Quantitative acylcarnitine profi ling was performed as previously described [15] . Briefl y, PBMC were isolated from 3 mL heparinized venous blood samples using Ficoll density gradient centrifugation [21] .…”

Enzymatic evaluation of glutaric acidemia type 1 by an in vitro probe assay of acylcarnitine profiling using fibroblasts and electrospray ionization/tandem mass spectrometry (MS/MS)

“…Since 2-oxoadipic acid used by Schulze-Bergkamen et al [13] is no longer commercially available, we determined whether the other molecules involved in GCDH represent alternative substrates. Lys and 2AA were used as substrates as they locate upstream of glutaryl-CoA synthesis pathway (Fig.…”

“…Schulze-Bergkamen et al reported enzymatic evaluation for GA1 on peripheral blood mononuclear cells (PBMC) using an in vitro probe assay and MS/MS, in which 2-oxoadipic acid was used as a substrate [13]. However, 2-oxoadipic acid is no longer available on a commercial basis, which led us to determine the alternative substrate.…”