Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving urea or urease
Patent
1986-11-13
1990-09-11
Kepplinger, Esther M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving urea or urease
435 14, 435 15, 435 26, 435 28, C12Q 158, C12Q 154, C12Q 132, C12Q 128
Patent
active
049562762
DESCRIPTION:
BRIEF SUMMARY
The invention relates to biological analysis, more particularly to the determination of co-enzymes in which the molecule contains nicotinamide, inter alia NADH, NADPH and APADH.
As is known, these abbreviations denote the following substances: also called co-enzyme I or DPN (diphosphopyridine nucleotide) also called co-enzyme II or TPN (triphosphopyridine nucleotide)
The structure of NAD is made up in succession of a 3-amidopyridinium group fixed in the 1-position of a ribose unit connected by its -5- position to a diphosphate group fixed at -5'- to a second ribose group comprising an adenine group at -1'-.
The reversible conversion of NAD to NADH is diagrammatically represented as follows (where R denotes the ribose-diphosphate-ribose-adenine chain): ##STR1##
Nicotinamide co-enzymes play a part in a large number of biochemical enzymatic reactions used as clinical tests. These include oxidation of alphahydroxyacids to the corresponding ketonic acids in the presence of a suitable dehydrogenase. One example is the oxidation of lactic acid or lactates to pyruvic acid ##STR2## Similarly glucose-6-phosphate is oxidised in the presence of NAD and glucose-6-phosphate dehydrogenase (G6PDH) to glucono-.delta.-lactone-6-phosphate and NADPH. The determination of NADP or NADPH in this reaction is very important since it can be indirectly, used to determine glucose in biological fluids after it has been converted to glucose-6-phosphate in the presence of ATP (adenosine triphosphate) and hexokinase.
NADH also acts as a co-enzymatic factor in the conversion by ammonium salts of 2-oxoglutarate to L-glutamate in the presence of GLDH (glutamade dehydrogenase), so that the ammonium in the reaction medium can be determined by determining the remaining NADH (or the NAD.sup.+ formed). This reaction is of use for determining urea in biological fluids which, in the presence of urease, supply the NH.sub.3 occurring as the NH.sub.4 ion in the aforementioned conversion.
Other applications associated with the determination of the NAD.sup.+, NADH and APADH factors are described in the following documents: EP-A-29 104 (MILES), FR-A-2 299 644 (AKZO).
In view of the great importance of determining the aforementioned co-enzymes in one or the other of their states of oxido-reduction, numerous techniques have been proposed for this purpose.
For example, since NAD and NADH have different absorptions in UV, one form can be determined in the presence of the other by spectrophotometry. The sensitivity of spectrophotometric determination can also be increased by combined use of coloured redox compounds, e.g. tetrazolium compounds which, in the presence of NADH or NADPH and an electron acceptor such as phenazine methosulphate, give intensely coloured formazan salts. (See e.g. document EP-A-114 267). Use may also be made of fluorimetric techniques as described e.g. in document FR-A-2 266 644.
An electrochemical method may also be used as described in document J 56 035 050, where NADH or NADPH is oxidized by Meldola blue, after which the reduced form of the dye is oxidized electrochemically and the oxidation current is measured.
It has recently been recommended (EP-A-29 104) to use the following reaction: ##STR3##
The resulting released hydrogen peroxide is then determined by conventional methods, e.g. by its action, catalyzed by peroxidase, on a redox indicator, the oxidised form whereof is determined by colorimetry.
Document EP-A-124 909 describes a process which is similar but simplified since peroxidase is the only enzyme involved. The method consists in reacting the co-enzyme with peroxidase in the presence of metal ions such as Mn.sup.+2 or Co.sup.+2, resulting in quantitative formation of hydrogen peroxide which is then determined as before by conventional methods, e.g. by colorimetry, inter alia the system comprising 4-aminoantipyrine as coupler and a phenolic compound or an aromatic amine as the chromophore. In the absence of metal ions (see page 5, paragraph 1 of this document) the formation of H.sub.2 O.sub.2 is not quantitative and cons
REFERENCES:
patent: 4353983 (1982-10-01), Siddiqi
patent: 4551427 (1985-11-01), Draeger et al.
patent: 4608335 (1986-08-01), Fossati
Siddiqi, Clin. Chem., vol. 28/9, 1982, p. 1962-1967.
Brochot Jean
Siddiqi Igbal
Battelle (Memorial Institute)
Hoffer Florina B.
Kepplinger Esther M.
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