Abstract
113 Objective: To measure in standard sections of needle biopsies all amino acids (AA) physiologically occuring in proteins, especially AA that indicate amount/function of specific proteins, e.g. hydroxyproline (Hyp) in collagens. Methods: Total tissue volume is calculated from the known thickness and the surface of a section, the latter determined by digital planimetry in a graphics facility (Nikon Microphot-SA microscope; Kontron ProgRes 3012 imaging system; Apple 8600/200 computer; Adobe Photoshop 4.0/NIH Image software). AA are measured in an automated HPLC facility using a Waters 474 scanning fluorescence detector. Each standard section of a needle biopsy, obtained for diagnostic purposes from patients with serologically confirmed hepatitis C and staged by a liver pathologist, was scanned, its total tissue volume calculated in µm3, hydrolyzed, and pre-column derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) [Cohen et al., Anal. Biochem. 211:279-87; 1993]. Separations were generated on a C18 column. This strategy, applicable to any histological specimen, generates results per unit volume, independent of tissue content of water or DNA. Results: 16 AA in each section were quantified in amole/µm3 (a, atto = 10-18). AA variability in repeat runs of the same section averaged 1%, in sequential sections of the same tissue 6%. With increasing disease activity, volume content of all AA decreased, but Hyp gradually increased 9-fold. Representative results for each major AA class are shown in the table.TABLEConclusions: In hepatitis C, volume content of only Hyp correlates with distinct patterns of fibrosis. The technique's minimal tissue demand will enhance localization studies that use microdissection or RT-PCR methods.
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