In vivo regulation of phenylalanine hydroxylation to tyrosine, studied using enrichment in apoB-100.
Phenylalanine hydroxylation is necessary for the conversion of phenylalanine to tyrosine and disposal of excess phenylalanine. Studies of in vivo regulation of phenylalanine hydroxylation suffer from the lack of a method to determine intrahepatocyte enrichment of phenylalanine and tyrosine. apoB-100, a hepatic export protein, is synthesized from intrahepatocyte amino acids. We designed an in vivo multi-isotope study, [(15)N]phenylalanine and [2H2]tyrosine to determine rates of phenylalanine hydroxylation from plasma enrichments in free amino acids and apoB-100. For independent verification of apoB-100 as a reflection of enrichment in the intrahepatocyte pool, [1-(13)C]lysine was used as an indicator amino acid (IAA) to measure in vivo changes in protein synthesis in response to tyrosine supplementation. Adult men (n = 6) were fed an amino acid-based diet with low phenylalanine (9 mg.kg(-1).day(-1), 4.54 mumol.kg(-1).,h(-1)) and seven graded intakes of tyrosine from 2.5 (deficient) to 12.5 (excess) mg.kg(-1).day(-1). Gas chromatography-quadrupole mass spectrometry did not detect any tracer in apoB-100 tyrosine. A new and more sensitive method to measure label enrichment in proteins using isotope ratio mass spectrometry demonstrated that phenylalanine hydroxylation measured in apoB-100 decreased linearly in response to increasing tyrosine intake and reached a break point at 6.8 mg.kg(-1).day(-1). IAA oxidation decreased with increased tyrosine intake and reached a break point at 6.0 mg.kg(-1).day(-1). We conclude: apoB-100 is an accurate and useful measure of changes in phenylalanine hydroxylation; the synthesis of tyrosine via phenylalanine hydroxylation is regulated to meet the needs for protein synthesis; and that plasma phenylalanine does not reflect changes in protein synthesis.
Rafii
Mahroukh
M
Division of Gastroentrology and Nutrition, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada.
McKenzie
Jane M
JM
Roberts
Susan A
SA
Steiner
George
G
Ball
Ronald O
RO
Pencharz
Paul B
PB
D000328:Adult; D053299:Apolipoprotein B-100 / Q000378:metabolism*; D004032:Diet; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D053299:Apolipoprotein B-100; D014443:Tyrosine; D010649:Phenylalanine; D008239:Lysine