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GTR Home > Tests > very long chain fatty acids (cultured cells)

Methodology

Methodology

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Biochemical Genetics
AAnalyte
Metabolite levels
  • Agilent 6890 capillary gas chromatograph
EEnzyme assay
Enzyme activity
  • Beckman scintillation counter

Test comments

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C26:0 and C22:0 fatty acid measurements and beta oxidation C16:0 and C24:0 enzyme assays are performed using cultured cells: skin fibroblasts, amniocytes, or chorionic villi.

Test development

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Test developed by laboratory (no manufacturer test name)

Test procedure

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Method 1: Harvest 1 T-25 confluent flask with trysin, wash the cells twice with PBS, and homogenize the pellet in 100ul distilled water. Take a 5ul aliquot for protein analysis. Extract the remaining cultured cell homogenate with 2:1 chloroform adding 1ug of C19:0 and C27:0 fatty acids as internal standards. Dry the lipid extract and add to the dried lipid extract 1.5ml 1N methanolic HCL and heat at 75 deg C. for 3 hours. Cool and extract the fatty acid methyl esters using 3 ml hexane. Dry the hexane extract and add 50ul hexane, transfer to injection vials and inject 1ul on 50 meter DB-1 capillary column, identify the fatty acids, C19:0, C27:0, C22:0 and C26:0 by retention time found by calibrating the column with known fatty acid methyl ester standards and measure the fatty acids by flame ionization against the internal standards C19:0 and C27:0. Method 2: Harvest 1 T-25 confluent flask with trysin, wash the cells twice with PBS, and homogenize the pellet in PBS. Take a 5ul aliquot for protein analysis. Take 25ul of cell homogenate and add to the separate tubes containing either 1-C14 labled C16:0 fatty acid, or 1-C14 labled C24:0 fatty acid. Incubate for 1 hour, add 10 volumes 2:1chloroform:methanol to form two phases and take an aliquot of the water soluble products in the upper phase and transfer to scintillation vial, dry, add scintillation cocktail and measure the radioactivity in the Beckman scintillation counter. C16:0 fatty acid is oxidized mainly in the mitochonria, whereas C24:0 fatty acid is oxidized primarily in the peroxisome. Results are reported as a ratio of radioactivity of C24:0/C16:0.

Citations
  • Peroxisomal disorders: complementation analysis using beta-oxidation of very long chain fatty acids. - PubMed ID: 2222480
  • Method 1: Moser HW, and Moser AB, 1991. Measurement of saturated very long chain fatty acids in plasma. In Techniques in Diagnostic Human Biochemical Genetics. Hommes FA (Ed). New York: Wiley-Liss. Chapter 12, pp. 177-191.

Confirmation of results

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Duplicate analysis using the same method.

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Practice guidelines

  • ACMG ACT, 2023
    American College of Medical Genetics and Genomics, Newborn Screening ACT Sheet, Elevated lysophosphatidylcholines, X-Linked Adrenoleukodystrophy (X-ALD), 2023
  • ACMG Algorithm, 2023
    ACMG Algorithm, X-ALD: Elevated lysophosphatidylcholines C24:0, C26:0, 2023
  • AAP, 2021
    Leukodystrophies in Children: Diagnosis, Care, and Treatment, Pediatrics (2021) 148 (3): e2021053126.
  • EuroGentest, 2011
    Clinical utility gene card for: adrenoleukodystrophy.

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