Assay of 25-hydroxy vitamin D3-1 alpha-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry

Journal Article · Mon Dec 01 00:00:00 EST 1986 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(86)90348-9· OSTI ID:6576924

Holmberg, I; Saarem, K; Pedersen, J I; Bjoerkhem, I

An assay of 1 alpha-hydroxylation of 25-hydroxy vitamin D3 in pig kidney mitochondria, based on selected ion monitoring, has been developed. Trideuterium-labeled 1,25-dihydroxy vitamin D3 was synthesized and used as internal standard. This standard was added immediately after incubation of 25-hydroxy vitamin D3 with the mitochondrial fraction. The incubation extracts were purified by high-performance liquid chromatography. After formation of the trimethylsilyl derivative, the product was quantitated by mass fragmentography using the ion at m/z 452 and m/z 455. With the use of this assay it was found that formation of 1,25-dihydroxy vitamin D3 was linear with the amount of mitochondrial protein and time of incubation. Substrate saturation was obtained at about 20 microM of 25-hydroxy vitamin D3. The maximal rate of conversion obtained under the conditions employed was about 0.1 pmol/mg protein X minute.

• Research Organization: Huddinge Univ. Hospital, Sweden
• OSTI ID: 6576924
• Journal Information: Anal. Biochem.; (United States), Vol. 2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
HYDROXYLASES
BIOASSAY
VITAMIN D
HYDROXYLATION
BIOCHEMICAL REACTION KINETICS
DEUTERIUM
FRACTIONATION
ISOTOPE DILUTION
KIDNEYS
LIQUID COLUMN CHROMATOGRAPHY
MASS SPECTROSCOPY
MITOCHONDRIA
SWINE
ANIMALS
BODY
CELL CONSTITUENTS
CHROMATOGRAPHY
DOMESTIC ANIMALS
ENZYMES
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
NUCLEI
ODD-ODD NUCLEI
ORGANOIDS
ORGANS
OXIDOREDUCTASES
REACTION KINETICS
SEPARATION PROCESSES
SPECTROSCOPY
STABLE ISOTOPES
TRACER TECHNIQUES
VERTEBRATES
VITAMINS
550201* - Biochemistry- Tracer Techniques