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Technical Briefs |
Academic Medical Centre, Departments of Clinical Chemistry and Pediatrics, Emma Children’s Hospital, University of Amsterdam, Amsterdam; The Netherlands
aaddress correspondence to this author at: Academic Medical Centre, Laboratory Genetic Metabolic Diseases (F0-224), Department of Pediatrics, Meibergdreef 9, Amsterdam, 1105 AZ The Netherlands; fax 31-20-696-2596, e-mail R.J.Wanders{at}amc.uva.nl
3-Methylglutaconic aciduria (3MGA) type I (McKusick 250950) is biochemically characterized by increased excretion of 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid in urine. Affected individuals display a range of clinical manifestations varying from mildly delayed speech development to severe neurologic involvement (1)(2). 3MGA type I is an autosomal recessive disorder caused by a deficiency of 3-methylglutaconyl-CoA hydratase (3MGH; EC 4.2.1.18). Three additional forms of 3MGA have been recognized—type II (Barth syndrome, McKusick 302060); type III (Costeff syndrome, McKusick 258501); and type IV ("unspecified", McKusick 250951)—all characterized by normal hydratase activities (3). Recently, the gene encoding 3MGH was identified by two independent groups (4)(5). As shown in Fig. 1A
, this mitochondrial enzyme catalyzes the penultimate step in leucine catabolism, which is the reversible conversion of 3-methylglutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA).
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Eleven patients have been described with isolated 3MGH deficiency (1)(2)(5)(6)(7)(8)(9)(10)(11). The hydratase deficiency in these patients was identified by use of a radioactive enzyme assay measuring three consecutive steps of leucine degradation, from 3-methylcrotonyl-CoA to acetoacetic acid (12). However, this procedure lacks specificity and is labor-intensive because of the need to purify the coupling enzyme, 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4), from bovine liver. Furthermore, the assay is not very practical for use in clinical laboratories because it involves the use of radiochemicals.
The need to differentiate patients with 3MGA type I from patients with other forms of 3MGA requires the availability of a sensitive and specific enzyme assay. From our knowledge that, in general, hydratase reactions are readily reversible and that 3-methylglutaconyl-CoA is not commercially available, we studied the 3MGH activity in the reverse direction, using HMG-CoA as a substrate. We quantified the formation of 3-methylglutaconyl-CoA by reversed-phase HPLC with ultraviolet detection. The described assay allows the precise measurement of residual 3MGH activity in cultured human skin fibroblasts.
We obtained KH2PO4, H3PO4, Tris, EDTA, HCl, KOH, MES, and acetonitrile (chromatography grade) from Merck. Bovine serum albumin (essentially fatty acid free), HMG-CoA, and bicinchoninic acid were purchased from Sigma.
We obtained skin fibroblasts from 2 individuals with confirmed 3MGA type I (1)(6), 5 individuals with confirmed Barth syndrome (13), 2 individuals with 3MGA type IV (14), and 13 control individuals with no evidence of an inborn error of branched chain amino acid oxidation or mitochondrial fatty acid oxidation. All cells were initially obtained during the process of diagnosing inborn errors of metabolism.
Fibroblasts were grown and harvested as described elsewhere (15). For use in determining the intraassay (within-day) and interassay (between-day) variation, we harvested six cell cultures from three control individuals, pooled them, divided them into 20 pellets, and stored them frozen at –80 °C.
Cell pellets were suspended in 200 µL of phosphate-buffered saline by repeated pipetting and were sonicated three times on ice for 15 s at 8 W at 45-s intervals. We determined the protein concentration by the bicinchoninic acid assay (Sigma), using bovine serum albumin as the calibrator. The assay mixture contained, in a final volume of 100 µL, 100 mmol/L Tris (pH 8.0), 10 mmol/L EDTA, 1 g/L bovine serum albumin, 100 µmol/L HMG-CoA, and 10–50 mg/L fibroblast protein. After incubation at 37 °C for 60 min, the reaction was terminated by addition of 10 µL of 2 mol/L HCl. The samples were homogenized, and the assay tubes were placed on ice. After 5 min, the homogenates were brought to pH 6 with 2 mol/L KOH–1 mol/L MES (pH 6) and centrifuged at 21 000g for 10 min at 4 °C. Finally, 100 µL of the supernatant was transferred to a HPLC vial.
The system for HPLC separation consisted of a Perkin-Elmer pump (PE series 200), a Gilson 234 autosampling injector, a C-402X frit (Upchurch scientific), a 20 x 4.6 (i.d.) mm SUPELCOSIL LC-18-DB (5 µm bead size) guard column (Supelco), and a 250 x 4.6 (i.d.) mm column filled with the same packing material. Separation was at ambient temperature, and compounds were detected by use of an ultraviolet detector (SPD-10A VP UV-VIS; Shimadzu) monitoring the absorbance at 260 nm.
For gradient elution, we used a binary solvent system. Solvent A was 100 mmol/L KH2PO4 adjusted to pH 4 with 100 mmol/L H3PO4, filtered before use through a 0.45 µm nitrocellulose membrane under reduced pressure. Solvent B was 200 mL/L acetonitrile–800 mL/L solvent A. Both solvents were degassed for 15 min in an ultrasonic bath (Branson 3510). We injected 50 µL of sample, and the acyl-CoA esters were eluted at a flow rate of 1 mL/min by a 15-min linear gradient of 20% B to 100% B. Peak areas of interest were integrated by use of Chromeleon software package (Dionex).
We first developed a HPLC-based method to allow baseline separation between HMG-CoA and 3-methylglutaconyl-CoA. Separation of acyl-CoA esters by reversed-phase HPLC is dependent on the salt concentration and the pH of the eluent (16). We found that a linear gradient of acetonitrile in potassium phosphate buffer (100 mmol/L, pH 4.0) allowed baseline separation between substrate and product.
Typical chromatograms for incubations with fibroblast extracts from a control individual and a patient with 3MGA type I are shown in Fig. 1B
. Using acyl-CoA calibrators, we were able to identify free CoA (9.0 min) and HMG-CoA and acetyl-CoA, which coeluted at 11.8 min. Comparison of the chromatograms of a patient with 3MGA type I and a control individual suggested that the extra peak at 16.5 min was 3-methylglutaconyl-CoA. Indeed, after hydrolysis and derivatization of the fraction that eluted at 16.5 min, we verified the presence of 3-methylglutaconate on the basis of its retention time and confirmed these results by mass spectrometry (results not shown) as described by Röschinger et al. (17). Thus, the acyl-CoA that eluted at 16.5 min was the product 3-methylglutaconyl-CoA.
HMG-CoA is converted into 3-methylglutaconyl-CoA by the action of 3MGH, and its activity can be quantified by determining the amount of 3-methylglutaconyl-CoA formed. However, the acyl-CoA esters present in the reaction mixture are susceptible to enzymatic and chemical hydrolysis. We measured the rate of hydrolysis for HMG-CoA and 3-methylglutaconyl-CoA by incubating homogenates of 3MGH-deficient fibroblasts with these acyl-CoA esters. Under the assay conditions used, both the substrate and the product were equally hydrolyzed to the extent of 
2.5%. We therefore corrected for hydrolysis by calculating the 3MGH activity as the ratio of product to the sum of product and substrate. However, because HMG-CoA is also a substrate for other enzymes, calculating the 3MGH activity in such manner would give an overestimation of the 3MGH activity.
HMG-CoA could first be converted into mevalonic acid by the action of HMG-CoA reductase (EC1.1.1.34) with NADPH as a coenzyme. However, because NADPH was not added, the action of the reductase on HMG-CoA under the assay conditions was negligible. In addition, preliminary studies revealed that, under the incubation conditions described above, <5% of HMG-CoA was converted into acetyl-CoA by the action of HMG-CoA lyase (results not shown). For correct calculation of the 3MGH activity, the contribution of acetyl-CoA has to be added to the total amount of the CoA esters in the incubation. To summarize, the 3MGH activity was calculated according to the following equation, which expresses the 3MGH activity as nmol · min–1 · (mg protein)–1:
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1 and 2 are 22.6 cm2/µmol and 16.0 cm2/µmol (18), respectively; the input is 10 nmoles; time is 60 min; and protein is expressed in mg. The activity of 3MGH in human fibroblast extracts showed little variation as a function of pH in the range 7.0–9.0 (data not shown) as reported by others using the coupled enzyme assay (19). For the standard assay, we selected a pH of 8.0.
The assay was linear with an incubation time up to at least 60 min with 50 mg/L fibroblast protein per assay (data not shown). For the standard assay, an incubation time of 60 min and a protein content of 10–50 mg/L fibroblast protein per assay were chosen.
Using optimized assay conditions, we reinvestigated fibroblasts from two patients with established 3MGA type I (1)(6) and detected no formation of 3-methylglutaconyl-CoA in either case. Inclusion of 10 mL/L control homogenate in a homogenate from a patient with 3MGA type I, corresponding to a 3MGH activity of 20 pmol · min–1 · (mg protein)–1, could readily be detected. Hence, the 3MGH activity in both patients was <20 pmol · min–1 · (mg protein)–1.
The intraassay variation, estimated by assaying 10 of the pooled control pellets in a single experiment, was 4.0% [mean (SD) = 2.26 (0.09) nmol · min–1 · (mg protein)–1]. The interassay variation was 4.8% [mean (SD) = 2.2 (0.1) nmol · min–1 · (mg protein)–1; n = 10 days].
Measurement of 3MGH activity in 13 control fibroblast homogenates revealed a mean (SD) 3MGH activity of 2.1 (0.7) nmol · min–1 · (mg protein)–1 [range, 1.0–3.6 · min–1 · (mg protein)–1]. The mean (SD) 3MGH activity in fibroblasts from five Barth syndrome patients was 2.8 (0.8) nmol · min–1 · (mg protein)–1 [range, 2.2–4.2 · min–1 · (mg protein)–1]. The two patients with confirmed 3MGA type IV had 3MGH activities of 1.59 and 1.02 nmol · min–1 · (mg protein)–1, respectively. These values for 3MGH activity were within the range for controls. Hence, the 3MGH activity in cultured fibroblast homogenates from patients with Barth syndrome or 3MGA type IV appears to be within reference values. This corresponds with the finding that 3MGH activity was normal in patients with Barth syndrome and 3MGA type IV by the coupled enzyme assay (3).
In conclusion, we present a sensitive and specific enzymatic assay for 3MGH that enables the rapid enzymatic diagnosis of 3MGA type I in cultured human skin fibroblasts without the need for radiochemicals. Baseline separation between the substrate HMG-CoA and the product 3-methylglutaconyl-CoA was achieved. Our novel procedure allows detection of 1% residual 3MGH activity in patients with 3MGA type I. To date, all of our patients with 3MGA type I demonstrated a 3MGH activity below this limit. Our method could be useful for studies of genotype/phenotype correlation as well as for studies of the enzymatic characterization of mutated proteins produced in Escherichia coli.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Overbeek, T. Begley, R. M. Butler, J. V. Choudhuri, H.-Y. Chuang, M. Cohoon, V. de Crecy-Lagard, N. Diaz, T. Disz, R. Edwards, et al. The Subsystems Approach to Genome Annotation and its Use in the Project to Annotate 1000 Genomes Nucleic Acids Res., October 7, 2005; 33(17): 5691 - 5702. [Abstract] [Full Text] [PDF]
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