Influence of fulvic acid on the collagen secretion of bovine chondrocytes in vitro.

High concentrations of fulvic acid and selenium deficiency in drinking water are the main causative factors of Kashin-Beck disease, an endemic degenerative chronic osteoarticular disorder found in China. The influence of fulvic acid on collagen secretion was investigated in articular chondrocyte cultures from bovine interphalangeal joints. Collagen secretion in 7-day-old chondrocyte monolayers was determined by measuring [3H]-proline incorporation into collagen macromolecules after a 24-h application in cultured supernatants. Additionally, collagen secretion was measured with a collagen assay based on a dye-binding method of soluble collagens. Both methods showed a dose-dependent increase of collagen secretion after treatment with fulvic acid. The collagen was identified by immunoblotting and immunocytochemistry as type II collagen. Fulvic acid also induced H2O2 production in cartilage cells. After co-incubation with catalase and fulvic acid, the cells secreted the same amount of H2O2 or collagen as the non-treated controls, indicating an influence of H2O2 on collagen secretion. Chondrocytes were then treated directly with H2O2. This led to increased collagen secretion showing a positive correlation with the concentration of H2O2 up to 1 pM H2O2. Larger amounts of H2O2 decreased collagen secretion. Effects of reactive oxygen species, such as lipid peroxidation or lactate dehydrogenase (LDH) release from damaged cells, were not inducable by fulvic acid (<10 ppm). Our results demonstrate a fulvic-acid-induced stimulation of collagen secretion into the supernatant by articular chondrocytes via physiological amounts of H2O2.