Measurement of metabolic events in the avian epiphyseal growth cartilage using a bioluminescence technique.
We developed a technique to map the distribution of selected metabolites in the growth cartilage in situ using luciferase-NAD(P)H:FMN oxidoreductase. Chick tibial epiphyses were freeze-trapped, sectioned, and freeze-dried. For evaluating lactate, luciferase was suspended in a buffer containing polyvinylalcohol, gelatin, NAD, FMN, and lactic dehydrogenase (LDH). The buffer was frozen into a layer 800 microns thick and placed in contact with the tissue section. The temperature of the frozen reagent mixture was then allowed to increase; the emitted light was focused 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 calcified hypertrophic region. Substantial levels of lactate were also present 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 three components were present in hypertrophic cartilage. Chemical analysis of the tissue section confirmed the luminographic studies and provided further evidence that there was reliance on glycolytic metabolism in terminally differentiated chondrocytes. Use of enzyme couples similar to those described above should permit the technique to be used to study most, if not all, of the major metabolic components of cartilage.
Kim
J K
JK
Department of Orthodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003.
Haselgrove
J C
JC
Shapiro
I M
IM
D000818:Animals; D002356:Cartilage / Q000378:metabolism*; D002645:Chickens; D005486:Flavin Mononucleotide / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism*; D006132:Growth Plate / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism*; D019344:Lactic Acid; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D009249:NADP / Q000378:metabolism
D005958:Glucosephosphates; D007773:Lactates; D019344:Lactic Acid; D009249:NADP; D019298:Glucose-6-Phosphate; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D008156:Luciferases; D005947:Glucose