Luminometric measurement of subnanomole amounts of key metabolites in extracts from isolated heart muscle cells.
In principle, luminometry allows very sensitive metabolite measurements as shown with standards in aqueous solutions (e.g., buffers). However, components of complex biological samples may largely interfere with luminometric reactions. We now describe a procedure by which subnanomole amounts of intermediary metabolites (malate, glucose 6-phosphate) can be measured by luminometry in extracts from isolated mammalian cells, namely rat heart muscle cells. Basically, measurements occur in two steps: (i) Enzymatically catalyzed reactions involving the metabolite to be measured lead to the stoichiometric production of NAD(P)H; (ii) the oxidation of this NAD(P)H in a luciferase/reductase system results in light production which is proportional to the original concentration of the metabolite. The reaction scheme is thus as follows: (1) Metabolite (malate, glucose 6-phosphate) + NAD(P)+ --> X + NAD(P)H + H+; (2) NAD(P)H + O2 + RCOH --> NAD(P)+ + RCOOH + H2O + hnu. The cardiomyocytes used are previously subjected to an ethanolic extraction in which the cellular NAD(P)H is destroyed by acidification. Subsequent evaporation of the extracts allows to neutralize and to concentrate the samples. This contributes, along with other experimental maneuvers, to increasing the sensitivity of the method. With this procedure, we were able to detect amounts of approximately 70 pmol of malate and approximately 90 pmol of glucose 6-phosphate in cardiomyocyte samples. In addition, the calculated cellular concentrations of malate and glucose 6-phosphate (101.1 +/- 4.5, and 202.8 +/- 26.1 microM, respectively, in the absence of exogenous substrate) correspond to values previously reported for heart tissue. In principle, the procedure described could be applied to the measurement of any ethanol-extractable metabolite that can be converted in reactions involving NAD(P)+.
Jüngling
E
E
Institute of Physiology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany.
Timmerman
M
M
Ionescu
A A
AA
Mertens
M
M
Löken
C
C
Kammermeier
H
H
Fischer
Y
Y
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002951:Citrates / Q000032:analysis; D019343:Citric Acid; D005260:Female; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000032:analysis; D007656:Ketoglutaric Acids / Q000032:analysis; D008163:Luminescent Measurements; D008293:Malates / Q000032:analysis; D008832:Microchemistry; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D009243:NAD / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
D002951:Citrates; D005958:Glucosephosphates; D007656:Ketoglutaric Acids; D008293:Malates; D009243:NAD; D019343:Citric Acid; D019298:Glucose-6-Phosphate; C030298:malic acid