Abstract 667
Fibrosis of parenchymal and hollow organs represents a major, often outcome-determining complication of chronic diseases in adults and children. The assessment of tissue fibrosis rests on the qualitative and subjective interpretation of stained histological sections, which in only a few tissues is graded according to organized staging systems. Surrogate antigenic markers in serum, derived from the extracellular matrix, are often compromised by their implicit lack of organ specificity and their susceptibility to altered turnover kinetics. Biochemical determinations of tissue fibrosis are usually referenced to wet weight or DNA content, both of which are subject to disease-related changes, such as presence of edema or inflammatory cells. To overcome these inadequacies, we developed a fluorescence-based chromatographic method that qualifies all protein amino acids in a histological section, including the collagen-specific hydroxyproline residues, and relates the results to the true tissue volume in the histological section analyzed. Since this technique measures protein amino acids relevant for fibrosis at the microscopic level, we coined the term LAAF, an acronym for localization of amino acids of fibrosis. Applying LAAF to routine sections from biopsies of adult patients with hepatitis C, we correlated in a blinded manner the histological staging according to Knodell, with the numeric results for protein amino acid content per unit volume of true tissue. Relative to the values obtained for normal liver, only the amount of tissue hydroxyproline increased as the histological degree of tissue fibrosis advanced. From normal to stage 1, the increase in tissue hydroxyproline was three-fold; from normal to stage 3, four-fold; and from normal to stage 4, nine-fold. Sections of fine needle biopsies from two neonates with α1-antitrypsin deficiency, both subsequently requiring liver transplantation, revealed that the tissue content of protein amino acids was globally higher in adults. However, the ratio of amino acids not prominent in collagen, e.g. [alanine/serine], was maintained irrespective of age and degree of fibrosis. By contrast, all ratios involving peptide-bound hydroxyproline were markedly changed. The ratio [hydroxyproline/(hydroxyproline+proline)] was elevated by one order of magnitude in the pediatric samples and achieved values that corresponded to end-stage liver disease in adults. LAAF, apparently useful for objective measurements of fibrosis in parenchymal organs, was also used to analyze tissue from a representative hollow organ. Superficial esophageal biopsies were obtained endoscopically from 5 pediatric patients with symptoms of gastroesophageal reflux. Results did not correlate with tissue reaction, however. Thus, although LAAF objectively measures fibrosis at the microscopic level, the method requires adequate sampling of the interstitial connective tissue.
