Novel biomarkers for diagnosing and understanding pathophysiology of metabolic diseases.

BackgroundTandem mass spectrometric analysis of simultaneously measuredblood spot acylcarnitines and amino acids is a widely accepted andapplied method for the early detection of a number of disorders offatty acid, organic acid and amino acid metabolism. Adaptation of thisapproach has led to vastly improved clinical outcomes for a number ofdisorders including medium chain acyl-CoA dehydrogenase (MCAD)deﬁciency [1], very long-chain acyl-CoA dehydrogenase (VLCAD)deﬁciency, glutaric acidemia type 1 and maple syrup urine disease.Outcome data for other conditions are awaited but, intuitively, mostinvestigators believe that these conditions should also result in betteroutcomes if diagnosed presymptomatically [2]. The sensitivity andspeciﬁcity of the current process are very high for disorders such asMCAD deﬁciency but there are numerous examples where sensitivityis not sufﬁciently high such that positive cases may be missed. Thereis a signiﬁcant literature documenting missed cases of subsequentlyfatal VLCAD deﬁciency that was not diagnosed in the newborn perioddespite screening for the disorder [3]. We are lookingfor analternativescreening method to reduce this false negative rate.MethodAcylcarnitinemeasurementinbody ﬂuidsrepresentsbiomarkeraccu-mulation that is several steps away from the primary accumulating me-tabolites. For fatty acid oxidation and many organic acid defects theprimary accumulating metabolite is an intra-mitochondrial coenzyme Aspecies. This species is then converted to an acylcarnitine which exitsthe mitochondria, may be further metabolized elsewhere in the cytosol(peroxisomes, microsomes) and eventually reaches the circulationthrough unknown mechanisms. For VLCAD deﬁciency for instance, theprimaryaccumulatingmetabolitesshouldbeC16:0,C18:1andC18:2spe-cies but the diagnostic measurands ar e unsaturated C14 species suggest-ing extensive modiﬁcation of the primary species. Presently, mostpublished acyl-CoA assays require a chromatographic separation stepwhich is generally regarded as too time consuming for screening pur-poses. To address this issue we have developed a ﬂow injection tandemmass spectrometric protocol which can analyze all species of acyl-CoA'sin a Multiple Reaction Mode (MRM) [4]. Acyl-CoA's are intracellular bio-markers and would not be expected to accumulate outside of a cellulardomain so the method was developed initially for tissue analysis for val-idation purposes.ResultsWe analyzed normal mouse tissues and those from both the short-chain acyl-CoA dehydrogenase (SCAD) mouse and the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) knockout mouse. A normalproﬁle from the wild-type mouse liver demonstrated the expectedspecies including C16, C18:1 and C18:1 in addition to acetyl-CoA andfree CoA. There was little intermediate species accumulation. TheSCAD knockout mouse liver demonstrated the expected accumulationof butyryl-CoA whilst the SCHAD knockout mouse demonstrated accu-mulation of medium-chain species consistent with the known chain-length speciﬁcity of the enzyme which includes medium chain species.Future directionsWearenowworkingonincreasingthesensitivityoftheassayinorderto be able to analyze acyl-CoA's in the cellular component of a wholeblood sample, a requisite for newborn screening. Initial work withwhitebloodcellssuggeststhatthisisfeasibleandmaybecomeapplicableas mass spectrometers continue to evolve in terms of sensitivity.WehavebeenabletoutilizetheassaytostudyfattyacidoxidationinthegastrointestinaltractandhaverecentlydeterminedthattheGItractcan generate energy from long-chain fatty acids derived fromthedietandshortchainfattyacidsthataregeneratedbythemicrobiomeinapathwaythatappearstobeindependentofmalonyl-CoAregulation.References