Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving glucose or galactose
Patent
1991-10-16
1994-01-11
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving glucose or galactose
435 4, 435 10, 435 11, 435 26, 435 95, 422 57, 422102, C12Q 100, C12Q 154, C12Q 162, C12P 1922
Patent
active
052780473
DESCRIPTION:
BRIEF SUMMARY
The invention relates to glucose determination and more specifically quantitative total glucose determination in undiluted whole blood. The invention is directed to a method of analysis, a reagent composition therefor and the use of this reagent composition.
An important routine analysis in clinical chemistry is blood glucose determination. This type of analysis is used for diagnosis and therapy of diabetes mellitus and for diagnosing a number of other metabolic disorders. Several methods for quantitative glucose determination in whole blood are known.
One of these known methods is based on enzymatic glucose determination with glucose dehydrogenase, and a reagent composition for this method is described in U.S. Pat. No. 4,120,755. This composition comprises a buffer, a pyridine coenzyme and glucose dehydrogenase. The buffer has a pH between 7.9 and 9.0. Another known composition for glucose determination is described in U.S. Pat. No. 3,964,974. This composition primarily consists of a glucose dehydrogenase, specified in respect of purity and type, a pyridine coenzyme such as NAD, a buffer system for maintaining the pH of an aqueous solution of the composition between 6.5 and 8.5, and mutarotase. Neither of these two patents discloses a method or a composition suited for direct quantitative determination of total glucose in undiluted whole blood, that is without a preceding dilution or precipitation step. The only information on how glucose in whole blood can be analysed is given in Example 7 in U.S. Pat. No. 3,964,974, where wet-chemical glucose determination is effected on a sample of whole blood. The proteins are however removed from the whole blood sample before the glucose measurement. By removing the proteins, the red blood cells will also be removed. Thus, the glucose measurement is not effected on whole blood. Reference is here made to p 9, lines 19-24 of the U.S. publication where the reader is explicitly directed away from the idea of retaining the red blood cells in whole blood measurements. After the undiluted blood has been deproteinised in a pre-dilution step, it is mixed with an enzyme/buffer solution. The reaction is then started by adding a coenzyme solution (NAD), whereupon the glucose content is determined. It is stated that the glucose content can be measured, inter alia, by a NADH.sub.2 -indicating dye or color reaction, e.g. by hydrogenating a tetrazolium salt into a corresponding formazan under the influence of the enzyme diaphorase. The publication does however not suggest using this NADH.sub.2 -indicating technique for total glucose measurements in a whole blood hemolysate, but only for glucose measurements in blood plasma. Furthermore, in the blood glucose measurements in U.S. Pat. No. 3,964,974, e.g. according to Example 7 thereof, measurements are performed throughout on diluted blood, as opposed to the present invention relying on measurements directly on undiluted blood samples.
It is evident that a simple and quick test for quantitative determination of total glucose in undiluted whole blood would be an important aid in clinical laboratories.
One object of the present invention therefore is to provide a method for quantitative determination of total glucose in undiluted whole blood by transmission spectrophotometry.
Another object is to provide a dry reagent composition for such determination.
The invention relates in particular to total glucose determination in small volumes of undiluted whole blood. In this context, "small volumes" means volumes between 0.1 and 0.001 ml, preferably between 0.02 and 0.001 ml.
According to the invention, the blood preferably is not only undiluted but also untreated.
According to a first aspect of the invention, a method is provided for quantitative determination of total glucose in undiluted whole blood, which method is characterised by contacting a sample, preferably less than 20 microlitre, of undiluted whole blood with a reagent in dry form comprising: methosulphate, phenazine ethosulphate, phenazine phenosulphate and Meldola blue; thio-NAD
REFERENCES:
patent: 3964974 (1976-06-01), Banauch et al.
patent: 3977944 (1976-08-01), Muller-Matthesius et al.
patent: 4120755 (1978-10-01), Pierre et al.
patent: 4898813 (1990-02-01), Albarella et al.
patent: 4975367 (1990-12-01), Albarella et al.
patent: 5059394 (1991-10-01), Phillips et al.
Lilja Jan E.
Nilsson Sven-Erik L.
Leary Louise N.
Wityshyn Michael G.
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