Endogenous folate of normal fibroblasts using high-performance liquid chromatography and modified extraction procedure

✍ Scribed by Soudabeh A. Kashani; Bernard A. Cooper

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 695 KB
• Volume: 146
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90392-6

✦ Synopsis

The endogenous levels of the various folate monoglutamate compounds in cultured human fibroblasts were determined using high-performance liquid chromatography for the separation of folate monoglutamate. Endogenous folates were converted to monoglutamate forms using conjugase enzyme present in rat serum and incubation was carried out at pH 6.5. This minimized folate coenzyme interconversion during processing. Using methanol for precipitation of protein instead of heat minimized degradation of labile folates. Recovery of all folates except IO-formyltetrahydrofolic acid (IO-CHO HJ'teGlu) using this procedure was more than 90%. Disruption of cells by boiling appeared to cause less postextraction changes of cell folates than did freezing and thawing or sonication. When heat to release endogenous folate, conjugase treatment with rat serum at pH 6.5, and precipitation of protein with methanol were used, more than half of the intracellular folate of normal fibroblasts in confluent growth was 5methyltetrahydrofolic acid (5-CH, H&Glu).

and IO-CHO H&eGlu and tetrahydrofolic acid (H$teGlu) comprised 29 and 6%, respectively. o 1985 Academic PW. I~C.