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Method for measurement of peroxisomal very-long-chain fatty acid ([beta]-oxidation in human skin fibroblasts using stable-isotope-labeled tetracosanoic acid.

Peroxisomes are present in virtually every eukaryotic cell type except the mature erythrocyte. In higher eukaryotes, one of the main functions of peroxisomes is the [beta]-oxidation of very-long-chain fatty acids (VLCFA; > 22 carbon atoms) (1). The importance of peroxisomal [beta]-oxidation is emphasized by the existence of a variety of different diseases in which peroxisomal [beta]-oxidation is impaired and VLCFA concentrations are increased (1-4). Peroxisomal disorders can be categorized as (a) single peroxisomal enzyme deficiencies, including X-linked adrenoleukodystrophy (X-ALD) and disorders attributable to defects in one of the peroxisomal (3-oxidation enzymes, such as acyl-CoA oxidase (AOX) deficiency and bifunctional protein (DBP) deficiency; and (b) disorders attributable to defects in peroxisome biogenesis. The peroxisome biogenesis disorders (PBDs) represent a continuum of clinical features ranging from the most severe form, Zellweger syndrome, through neonatal adrenoleukodystrophy to the least severe form, infantile Refsum disease.

Currently, measurement of the peroxisomal fatty acid [beta]-oxidation activity is performed with 1-[[sup.14]C]-radio-labeled VLCFA substrates and one of two available methods: either in intact human skin fibroblasts cultured in monolayer (5); or in isolated fibroblasts permeabilized with digitonin (6). We investigated the feasibility of using deuterium-labeled tetracosanoic acid ([D.sub.3]-C24:0) as an alternative substrate to radiolabeled 1-[[sup.14]C]-labeled C24:0 for the measurement of peroxisomal ([beta]-oxidation activity in cultured primary human skin fibroblasts.

Before use, the purity of 24,24,24-[D.sub.3]-C24:0 (Larodan Fine Chemicals AB) was determined. The [D.sub.3]-C24:0 substrate contained ~6% deuterium-labeled octadecanoic acid ([D.sub.3] C18:0). Acetone was used to purify [D.sub.3]-C24:0 according to the following procedure: 4 mL of acetone was added to 20 mg of [D.sub.3]-C24:0. The sample was vortex-mixed vigorously, left at room temperature for 30 min, and centrifuged at 1600g for 10 min; approximately 80% of the acetone was then removed, and 3 mL of fresh acetone was added. This procedure was repeated two more times. After three washing steps with acetone, ~80% of the acetone was removed, and the remaining acetone was evaporated at room temperature under a constant stream of nitrogen. The residue was weighed, and a stock solution of 10 mmol/L [D.sub.3]-C24:0 in absolute ethanol was prepared. After purification, the purity of [D.sub.3]-C24:0 was analyzed, and the contribution of the [D.sub.3]-C18:0 contaminant was determined to be <0.2%.

Fibroblasts from healthy controls and patients with X-ALD were cultured in the absence or presence of 20 [micro]mol/L [D.sub.3]-C24:0 in HAM-F10 tissue culture medium supplemented with 100 mL/L fetal calf serum, penicillin (100 IU/mL), streptomycin (100 IU/mL), and glutamine (2 mmol/L). Before usage, the [D.sub.3]-C24:0 stock solution was put in a water bath for 5 min, vortex-mixed, and diluted in HAM-F10 tissue culture medium to a final concentration of 20 [micro]mol/L. Cells were used between passage numbers 6 and 18. For fatty acid analysis, cells were harvested with trypsin, washed twice with phosphate-buffered saline (PBS) and once with 9 g/L NaCl, dissolved in 200 [micro]L of deionized water, and sonicated, and the protein concentration was determined. The peroxisomal ([beta]-oxidation activity was calculated by measurement of the amount of intracellular deterium-labeled hexadecanoic acid ([D.sub.3]-C16:0) present in nmol/mg of protein. In our method we chose [D.sub.3]-C16:0 as a marker for peroxisomal [beta]-oxidation because of the availability of a [D.sub.3]-C16:0 internal standard, which enabled accurate calculation of the amount of [D.sub.3]-C16:0 present in the cells.

Fatty acids were analyzed by electrospray ionization mass spectrometry using a recently described isotopedilution method (7). For calculation of the amount of [D.sub.3]-C16:0, we constructed a five-point calibration curve. Of a calibration mixture containing [D.sub.3]-C16:0 (40 [micro]mol/L), we added 0, 25, 50, 100, and 200 [micro]L to 100 [micro]L of internal standard containing deuterium-labeled behenic acid ([D.sub.4]-C22:0; 50.0 [micro]mol/L), [D.sub.4]-C24:0 (50.0 [micro]mol/L), and deuterium-labeled hexacosanoic acid ([D.sub.4]-C26:0; 1.0 [micro]mol/L). Samples were extracted and analyzed as described previously (7). The input concentration of [D.sub.3]-C16:0 (in nmol) was plotted against the ratio of the peak height of [D.sub.3]-C16:0 to the peak height of the [D.sub.4]-C22:0 internal standard. The trend line and the intercept were used to calculate the [D.sub.3]-C16:0 concentration in the samples.

The effect of incubation time on the production of [D.sub.3]-C16:0 from [D.sub.3]-C24:0 in fibroblasts from healthy individuals and patients with X-ALD is shown in Fig. 1. At all time points investigated, the amount of [D.sub.3] C16:0 in the X-ALD cell lines was markedly lower than that in the control cell lines. Because [D.sub.3] C16:0 is an intermediate of peroxisomal [beta]-oxidation and not an end product, the amount of [D.sub.3] C16:0 measured in the cells at the different time points reflects the flux through the [beta]-oxidation pathway and hence is an indicator of the overall activity of the pathway. After 2-3 days, the amount of [D.sub.3] C16:0 present in control and X-ALD cell lines plateaued, indicating that a steady state was reached. To exclude depletion of the substrate in the medium, we measured the amount of [D.sub.3] C24:0 present in the medium after 72 h. The medium of the control cells still contained >80% of the initial [D.sub.3] C24:0 concentration. On the basis of the data presented in Fig. 1, we selected a 3-day incubation period with 20 [micro]mol/L [D.sub.3] C24:0 for subsequent studies.

The intraassay CV was determined by the following procedure: the cells were divided into five separate tissue culture flasks, treated with 20 [micro]mol/L [D.sub.3] C24:0, and after 3 days, the amount of [D.sub.3]-C16:0 present in the cells was measured. The intraassay CV obtained was 5.8%. The interassay CV, determined by assaying control cell lines during 5 separate weeks, was 8.8%.

The [D.sub.3]-C16:0 concentrations present after 3 days of incubation with 20 [micro]mol/L [D.sub.3]-C24:0 of fibroblasts from controls and patients with different peroxisomal ([beta]-oxidation disorders, including X-ALD, AOX deficiency, and DBP deficiency, are summarized in Table 1. In addition, cells from different PBD patients were analyzed as well.

[FIGURE 1 OMITTED]

In none of the six AOX--or DBP-deficient cell lines could [D.sub.3]-C16:0 be detected. The cells had taken up the [D.sub.3]-C24:0 substrate, as we concluded from measurement of intracellular [D.sub.3]-C24:0 concentrations. These data indicate that [beta]-oxidation of C24:0 to C16:0 takes place exclusively in peroxisomes and not in mitochondria.

In fibroblasts derived from PBD patients, the amount of [D.sub.3]-C16:0 was only 5% of the amount in control fibroblasts (Table 1). Among the different PBD patient cell lines analyzed, however, we observed variation in residual peroxisomal [beta]-oxidation activity, as indicated by the amount of intracellular [D.sub.3]-C16:0 present. No [D.sub.3]-C16:0 was detectable in eight cell lines derived from patients with the severe Zellweger phenotype, whereas [D.sub.3]-C16:0 was detectable in four cell lines derived from patients with the milder neonatal adrenoleukodystrophy or infantile Refsum disease phenotypes. These observations are in agreement with a previous study that reported the predictive value of dihydroxyacetonephosphate acyltransferase (DHAPAT) activity and residual peroxisomal VL-CFA [beta]-oxidation activity, measured with 1-[[sup.14]C]-C24:0 as substrate, for the life expectancy of PBD patients (8).

Fibroblasts derived from patients with X-ALD had the highest (15%) relative amount of [D.sub.3]C16:0 formed (Table 1). The mean (SD) amount of [D.sub.3]C16:0 in X-ALD fibroblasts was 0.60 (0.42) nmol/mg of protein. Within the group of 12 X-ALD patients included in the analysis, no correlation was observed between the peroxisomal [beta]-oxidation activity and the phenotype of the patient.

In conclusion, we have developed an easy, sensitive, nonradioactive method for analysis of peroxisomal [beta]-oxidation activity in fibroblasts.

We thank Herman ten Brink and Rob Ofman for helpful technical suggestions and discussion. This work was supported by grants from the Netherlands Organization for Scientific Research (NWO-MW: No. 903-42-077), the European Leukodystrophy Association, and European Union Project LSHM-CT-2004-502987.

References

(1.) Singh I, Moser AE, Goldfischer S, Moser HW. Lignoceric acid is oxidized in the peroxisome: implications for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy. Proc Natl Acad Sci U S A 1984;81:4203-7.

(2.) Singh I, Moser AE, Moser HW, Kishimoto Y. Adrenoleukodystrophy: impaired oxidation of very long chain fatty acids in white blood cells, cultured skin fibroblasts, and amniocytes. Pediatr Res 1984;18:286-90.

(3.) Lazo O, Contreras M, Hashmi M, Stanley W, Irazu C, Singh I. Peroxisomal lignoceroyl-CoA ligase deficiency in childhood adrenoleukodystrophy and adrenomyeloneuropathy. Proc Natl Acad Sci U S A 1988;85:7647-51.

(4.) Wanders RJ, van Roermund CW, van Wijland MJ, Schutgens RB, van den BH, Schram AW, et al. Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids. Biochem Biophys Res Commun 1988;153:618-24.

(5.) Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund CW, Jacobs BS. Measurement of peroxisomal fatty acid [beta]-oxidation in cultured human skin fibroblasts. J Inherit Metab Dis 1995;18 Suppl 1:113-24.

(6.) Watkins PA, Ferrell EV Jr, Pedersen JI, Hoefler G. Peroxisomal fatty acid [beta]-oxidation in HepG2 cells. Arch Biochem Biophys 1991;289:329-36.

(7.) Valianpour F, Selhorst JJ, van Lint LE, van Gennip AH, Wanders RJ, Kemp S. Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet Metab 2003;79:189-96.

(8.) Gootjes J, Mooijer PA, Dekker C, Barth PG, Poll-The BT, Waterham HR, et al. Biochemical markers predicting survival in peroxisome biogenesis disorders. Adv Exp Med Biol 2003;544:67-8.

DOI : 10.1373/clinchem.2004.038539

Stephan Kemp, * Fredoen Valianpour, Petra A.W. Mooyer, Willem Kulik, and Ronald J.A. Wanders (University of Amsterdam, Academic Medical Center, Departments of Pediatrics/Emma Children's Hospital, and Clinical Chemistry, Laboratory for Genetic Metabolic Diseases, Amsterdam, The Netherlands; * address correspondence to this author at: Laboratory for Genetic Metabolic Diseases, Room F0-224, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands; fax 31-20-6962596, e-mail s.kemp@amc.uva.nl)
Table 1. [D.sub.3]-C16:0 concentrations in skin fibroblasts
from controls and patients after 3 days of incubation
with [D.sub.3]-C24:0.

 [D.sub.3]-C16:0, (a)
 nmol/mg
 n of protein

Controls
 Mean (SD) 10 3.93 (1.07)
 Range 2.33-5.50
Patients
 X-ALD
 Mean (SD) 12 0.58 (0.40)
 Range 0.21-1.54
 PBD
 Mean (SD) 12 0.19 (0.26)
 Range 0.0-0.64
 AOX/DBP deficiency
 Mean (SD) 6 0.0 (0.0)
 Range 0.0-0.0

 Relative
 amount, (b) % P (c)

Controls
 Mean (SD) 100
 Range
Patients
 X-ALD
 Mean (SD) 15 <0.0001
 Range
 PBD
 Mean (SD) 5 <0.0001
 Range
 AOX/DBP deficiency
 Mean (SD) 0 <0.0001
 Range

(a) [D.sub.3]-C16:0 concentrations in cultured skin fibroblasts
after 3 days of incubation with 20 [micro]mol/L [D.sub.3]-C24:0.

(b) [D.sub.3]-C16:0 concentrations in control fibroblasts were
used to calculate the relative amounts in patient cell lines.

(c) P values were calculated by use of the two-tailed Student
t-test.
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Title Annotation:Technical Briefs
Author:Kemp, Stephan; Valianpour, Fredoen; Mooyer, Petra A.W.; Kulik, Willem; Wanders, Ronald J.A.
Publication:Clinical Chemistry
Date:Oct 1, 2004
Words:2008
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