Linked Life Data

8660623

Source:http://linkedlifedata.com/resource/pubmed/id/8660623

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1996-10-17
pubmed:abstractText
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)+.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
239
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
41-6
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Luminometric measurement of subnanomole amounts of key metabolites in extracts from isolated heart muscle cells.
pubmed:affiliation
Institute of Physiology, Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't