Inborn Errors of Metabolism


Inborn errors of metabolism range in frequency from very common, e.g., 1 in 20 for glucose-6-phosphate dehydrogenase deficiency, the most common disease-producing enzyme defect in the world (affected individuals are at risk of hemolytic anemia when exposed to certain drugs or foods), to extremely rare, with only a few reported cases in the world. Although most individual metabolic gene deficiencies are sufficiently rare to be considered 'orphan diseases', their biomedical impact is better appreciated when they are grouped by metabolic pathway. For example, Oxidative Phosphorylation (OXPHOS) disorders affect at least 1 in 5,000, fatty acid oxidation (FAO) deficits 1 in 10,000, urea cycle disorders 1 in 10,000, and peroxisomal disorders 1 in 50,000.

MitoSciences has developed a wide range of mAbs and mAb-based kits that can help characterize patients with defects in the major pathways of intermediary metabolism. These are available as individual mAbs and cocktails for Western blotting, and for single cell analysis by immunocytochemistry and immunohistochemistry. In addition, the protein level and in some cases activity, of many of these enzymes can be measured accurately and sensitively with mAb-based immunocapture kits. These include quantitative ELISA-based tests for high-throughput analysis and lateral flow "dipstick" assays for high sensitivity, rapid measurement in clinically-relevant samples such as fingerprick (unfractionated) blood, cheek swabs, urinary sediment and cultured cells, e.g. fibroblasts and lyphoblasts from biopsies. These products can be found categorized both by protein and assay type using the appropriate metabolic pathway link on the intermediary metabolism overview chart.

OXPHOS Deficiencies

Products for the study of OXPHOS deficiencies.

Inherited OXPHOS disorders comprise a wide range of defects of either mtDNA encoded or nuclear DNA encoded proteins and are the most common set of inherited mitochondrial diseases with an incidence of at least 1 in 5,000. Moreover, it was recently established that at least 1 in 200 apparently unaffected individuals carry known pathogenic mutations mtDNA. These individuals (and many more who are carriers for nDNA-encoded mutations) may have clinically sub-threshold OXPHOS deficits that put them at increased risk to environmental toxins or therapeutic drugs known to have adverse effects on mitochondria. Such predispositions could account for the idiosyncratic mitotoxicities of many therapeutic drugs and so are of great interest to pharmaceutical drug developers (see our MitoTox™ page for more information on this subject and on mitotoxicity drug screening tools).

It is noteworthy that deficits of OXPHOS enzyme complexes I and IV are involved in the majority of inherited OXPHOS disorders, either as isolated enzyme defects due to mutations in complex-specific assembly factors or structural subunits of the affected complex, or as part of a constellation of multiple OXPHOS enzyme complex deficits due to mtDNA or nDNA-encoded mutations affecting mitoribosome translation. Therefore, an opportunity exists to screen and identify the majority of inherited OXPHOS disorders with just a few simple assays that measure the protein level and/or enzyme activities of complexes I and IV. MitoSciences ELISA assays for complex I and complex IV activities (MS141 and MS441) offer high throughput options for such screening while MitoSciences lateral flow "dipstick" assays (MS130, MS131, MS430 and MS431) provide a convenient and rapid method to assess levels of complex I and IV protein and/or activities in non-invasive samples such as cheek swabs. For example, using fibroblasts from patients with isolated deficiencies of complex I or IV, we recently reported that the activity dipsticks correctly identified all complex IV patients tested and almost all complex I patients tested (Willis et al., 2009 Biochim Biophys Acta. 2009 May;1787(5):533-8).

MitoSciences also has a wide range of mAbs and assays that can be used to study the expression levels, subunit composition, function and dysfunction of all 5 OXPHOS enzyme complexes. For convenience and efficiency, cocktails of mAbs are available that allow rapid and efficient screening for alterations of all 5 complexes simultaneously, either by western blot (MS601 and MS604) or immunocytochemistry (MS602 and MS602a). In each case, the cocktail contains a mixture of mono-specific mAbs that allow precise identification of the levels of single representative subunits from each of the 5 complexes.

The protein levels of all 5 complexes can also be measured in parallel on a single ELISA plate by using our new FlexPlex™ assay modules (MSFX-1, MSFX-2, MSFX-3, MSFX-4 and MSFX-5). These modules can also be combined with any of another 25 modules specific for additional mitochondrial, cytoplasmic and peroxisomal proteins. In this way the expression of key proteins in a wide range of pathways can be assessed quickly and accurately.

Once alterations are identified, it is possible to use additional tests, such as immunopurification of individual OXPHOS complexes or other enzymes, to drill down further and analyze the structure and function of altered enzymes, e.g., to investigate for evidence of post-translational modifications or oxidative damage to specific subunits.

Mitochondrial Fatty Acid Oxidation Disorders

Products for the study of mitochondrial fatty acid oxidation disorders.

MitoSciences has a unique set of mAbs and kits to analyze 6 of the 13 enzymes involved in FAOX (click on the FAOX pathway sidebar figure for links to each enzyme and its set of analytic tools). The MCAD toolkit is especially noteworthy as deficiencies of MCAD account for almost 70% of all diagnosed FAOX disorders and a single mutation (985A > G) accounts for approximately 80% of symptomatic MCAD disorders. This mutation affects assembly and stability of the enzyme and therefore is expressed as a deficiency of the steady-state levels of the MCAD enzyme, both by protein quantity and activity. Although reliable metabolite and genetic screening methods exist to identify MCAD deficiencies, questions remain about the geneotype:phenotype relationship, in particular an incomplete correlation of metabolite levels and symptoms, and so protein based tests and analytic tools are needed to fully investigate and understand the disorder. MitoSciences has mAbs and kits to immunocapture and purify MCAD from complex mixtures for detailed structural analysis (MS726), to measure the steady state levels of the protein in either high-throughput ELISA (MSFX-9) or rapid dipstick (MSX32) assays or western blot (MS726), and to assess the levels of MCAD protein expression at the single cell level by immunocytochemistry or immunohistochemistry (MS726).

Peroxisomal Disorders

Products for the study of peroxisomal disorders.

Recent studies have established a clear functional link between peroxisomes and mitochondria. Although less complex than mitochondria which mitochondria contain their own DNA, replication and protein translation systems and are involved in many more metabolic pathways) peroxisomes are considered to be important processing centers to convert complex fatty acids such as branched chain and omega fatty acids into forms that can be further metabolized by the mitochondrion. An important difference between fatty acid oxidation in mitochondria and peroxisomes is that electrons released in the former are captured as NADH for use in energy production, while those released within the peroxisome are removed by catalase to produce hydrogen peroxide.

An inherited inability of peroxisomes to modify complex fatty acid species leads to a number of relatively common diseases including Zellwegers syndrome (through altered organelle biogenesis), adreno-leukodystrophy (mutations in ABCD1), Refsum disease (PHYH), non-specific X linked mental retardation (mutations in FACL4) and others.

Good antibodies to peroxisomal proteins have been scarce until the introduction of the set provided recently by MitoSciences. Antibodies to a total of 6 different enzymes involved in peroxisomal fatty acid oxidation are available for use in immunocytochemistry and In-Cell ELISA, and all but one of these are also available for the FlexPlex™ ELISA system.

Pyruvate Dehydrogenase (PDH) Deficiency

Products for the study of pyruvate dehydrogenase deficiency.

Pyruvate dehydrogenase regulates the balance of glucose utilization between aerobic (OXPHOS) and anaerobic (glycolysis) pathways and thus sits at one of the most important and central control points in intermediary metabolism. The importance of this control has been affirmed by the re-discovery and understanding of the "Warburg" effect (the tendency of proliferating cancer cells to use glycolysis rather than OXPHOS even under aerobic conditions) and the potential to take advantage of this bias for new anti-cancer therapies.

PDH in turn is regulated by a complex, dynamic interplay of PDH activity modifiers that are driven by the cell's immediate energy balance and the competing needs of catabolic and anabolic pathways for Acetyl-CoA. Phosphorylation status of PDH is one of the most important such factor and is controlled by a specific set of PDH kinases (PDK1-4) and phosphatases (PDP1 and PDP2). MitoSciences has a complete set of mAbs and mAb-based capture kits to measure PDH activity and protein level in cell and tissue samples and to assess the functional consequences of physiologic or pathogenic alterations on activity. We also offer all 4 PDKs and both PDPs as in the form of enzymatically active recombinant human proteins. The use of the PDH toolbox is described in our PDH playbook and in a set of detailed protocols for measurement and manipulation of PDH phosphorylation.

Almost all inherited PDH deficiencies are caused by mutations in the nuclear DNA encoded gene for the E1-α subunit, resulting in low expression of the E1-α protein and deficiencies in PDH activity. Consequences are severe, and PDH deficiency, along with OXPHOS deficiencies, is a cause of Leigh's disease a devastating neurologic disorder. PDH deficiencies can therefore be identified either by PDH activity measurements, e.g., with activity dipsticks or microplate assays, or by dual-color immunocytochemistry to measure the ratio of PDH-E2/PDH-E1α protein expression in individual cells. Because the gene for E1-α is X-linked while E2 is not, dual-color (PDH-E2/PDH-E1α) immunocytochemistry has also been used to identify female carriers of E1-α deficiencies who are cellular mosaics of normal and E1-α deficient cells due to random X chromosome inactivation.

Products for the study of Urea Cycle Disorders

Urea cycle pathway diagram

The urea cycle is a set of coordinated reactions occurring in the mitochondrial matrix and cytosol that convert ammonia, produced during amino acid degradation, to urea in the liver. An important enzyme in this process is mitochondrial carbamoyl phosphate synthetase 1 (CPS1). CPS1 deficieny is an inherited autosomal recessive disorder that causes hyperammonemia, and can result in developmental delay and mental retardation. Infants with defects in CPS1 also have increased risk of transitional pulmonary hypertension in infancy which has an incidence of 1.9 per 1000, and has an 11% mortality rate. MitoSciences CPS1 kits and mAbs can be used to quantitate levels of the protein in tissue samples by 2-site immunocapure ELISA or in cell populations exposed in vitro to test conditions by high-throughput In-Cell-ELISA, and to purify the protein by immunoaffinity for detailed molecular analysis.

Products for the study of Friedreich's Ataxia

Products for the study of Friedreich's Ataxia

Iron metabolism requires a complex interplay of cell-surface receptors, cytoplasmic storage proteins, and mitochondrial and cytoplasmic enzymes. Heme biosynthesis begins and ends in the mitochondrion, with mid-process steps taking place in the cytoplasm. Iron-sulfur clusters (ISCs), which are necessary prosthetic groups in re-dox enzymes of both the mitochondrion and cytoplasm, are also formed through a multistep process spanning both the mitochondrion and cytoplasm. Mitochondrial frataxin plays a key role in both processes and frataxin deficits result in lowered levels of both ISC and heme-containing proteins, and increased cellular oxidative stress.

Inherited frataxin deficiency causes Friedreich's Ataxia (FA), a progressive neurodegenerative disease that is the most common inherited ataxia. Recent progress in understanding the molecular pathophysiology of FA has generated considerable excitement as it suggests that FA is a solvable problem and will likely be the first mitochondrial disease cured by rational application of small drug therapies. This optimism is derived from the discovery that frataxin deficiency in FA is caused by an expanded triplet nucleotide repeat in a the first intron of the frataxin gene, that the coding sequence for the protein itself is normal, and that FA patients had only slightly lower levels of frataxin (5-30% of normal) than carriers (40-50% of normal) who are essentially unaffected. Therefore, it seems likely that for many patients only a modest increase in frataxin levels is needed to raise levels above the threshold needed for normal function. Recent advances of new therapeutic drugs tested in cell culture and animal model studies have shown extremely promising results and a number of drugs designed to boost frataxin production or to address downstream effects of reduced frataxin levels are now entering the clinic.

MitoSciences has a broad array of mAb and mAb-based immunocapture kits that can be used to measaure frataxin levels in cell and tissue samples, including tissues suitable for repetitive, non-invasive sampling such as cheek swabs and blood. These tests, in particular the rapid lateral flow "dipstick" assay and high-throughput ELISA assay, therefore offer the means to both investigate new therapeutic approaches in vitro and also assess efficacy of these potential treatments in vivo.

We also offer a wide range of products to measure downstream effects of frataxin deficiency, including quantitative assays for activity and protein level of ISC and heme containing enzymes such as aconitase and OXPHOS complexes I, II, III and IV. A complete listing and a set of examples of the use of these products are described in our Frataxin brochure.

Browse Products By:
Product Search:

Related Pathways:

Intermediary Metabolism

Oxidative Phosphorylation

Mitochondrial Fatty Acid Oxidation Pathway

Fatty Acid Oxidation (Peroxisomal)

Urea Cycle

Subscribe to MitoNews
MitoNews is a free periodic annotated review of important publications in mitochondria and metabolic research.

MitoNews Latest Issue
Volume 08 Issue 05 (May 2012)

MitoNews Archives

Sales & Customer Support:

[email protected]

© 2004-2012 MitoSciences Inc, an Abcam company. All rights reserved.