MEASUREMENT OF METABOLIC EVENTS IN THE AVIAN EPIPHYSEAL GROWTH CARTILAGE USING A BIOLUMINESCENCE TECHNIQUE

We developed a technique to map the distribution of selected metabolit es in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidor eductase. Chick tibial epiphyses were freeze-trapped, sectioned, and f reeze-dried. For evaluating lactate, luciferase was suspended in a buf fer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydro genase (LDH). The buffer was frozen into a layer 800 mum thick and pla ced in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was fo cused through a photographic lens and collected on film. We found that lactate was synthesized by cells in all regions of the growth plate. The highest concentration of the metabolite was observed in the calcif ied hypertrophic region. Substantial levels of lactate were also prese nt in articular cartilage. By modifying the composition of the buffer solution, we were able to map the distribution of glucose and glucose- 6-phosphate and the activity of LDH. Maximal levels of each of the thr ee components were present in hypertrophic cartilage. Chemical analysi s of the tissue section confirmed the luminographic studies and provid ed further evidence that there was reliance on glycolytic metabolism i n terminally differentiated chondrocytes. Use of enzyme couples simila r to those described above should permit the technique to be used to s tudy most, if not all, of the major metabolic components of cartilage.