Hyaluronic acid N-deacetylase assay in whole skin.
Hyaluronic acid (HA) N-deacetylase(s) was quantified in whole skin, using a novel method that involved reaction of skin with exogenous HA as substrate. Acetyl (CH(3)CO-) moieties generated were converted chemically to MeOAc and quantified using gas chromatography/mass spectrometry. HA (1.7 mg) and skin (1.0 g) yielded 3.32 and 769.00 microg of MeOAc from the 69.0- and 76.5-year-old-patient samples, respectively. Without added HA, 194.00 microg of product was obtained from the 76.5-year-old-patient samples. With chondroitin as substrate, the yields were 2.89 and 818.04 microg of MeOAc from the 69.0- and 76.5-year-old-patient samples, respectively. The K5 (capsular, Escherichia coli polysaccharide) substrate yielded no detectable product, except for 170.02 microg from the 76.5-year-old-patient samples. This highly sensitive method was used to demonstrate that human-skin-HA N-deacetylase(s) was first detectable at 69 years of age, highly active at 76.5 years of age, and specific for N-acetyl moieties of d-GlcNAc and d-GalNAc where C(1) is beta-linked as in HA and CH.
Longas
María O
MO
Department of Chemistry and Physics, Purdue University Calumet, 2200 169th Street, Hammond, Indiana 46323, USA. mol@calumet.purdue.edu
Burden
James D
JD
Lesniak
John
J
Booth
Ryan M
RM
McPencow
James A
JA
Park
Jung I
JI
D000328:Adult; D000368:Aged; D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D001940:Breast; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007700:Kinetics; D008875:Middle Aged; D012867:Skin / Q000201:enzymology*; D015595:Skin Aging / Q000502:physiology*; D013379:Substrate Specificity
D000581:Amidohydrolases; C476607:hyaluronic acid N-deacetylase