Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Sample mechanical transport means in or for automated...
Reexamination Certificate
1998-03-09
2001-08-07
Alexander, Lyle A. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Sample mechanical transport means in or for automated...
C436S063000, C436S808000, C436S813000, C436S094000, C422S051000
Reexamination Certificate
active
06270725
ABSTRACT:
BACKGROUND OF THE INVENTION
1 . Field of the Invention
This invention relates to a diagnostic kit and a method for determining the concentration of sucrose in a sample of a physiological fluid. The kit and method are useful in connection with a method for the non-invasive detection of gastric damage.
2. Related Background Art
Stomach ulcers can pose a serious health threat as, in many instances, ulcers are asymptomatic. Since stomach ulcers can develop and be present without any symptoms, the damage brought about by ulcers to the stomach and the bleeding associated with such ulcers can be a serious, and possibly fatal health risk.
Traditional methods for detecting gastric ulcers include endoscopy, barium meal followed by x-rays, and radiolabeled detecting agents. Endoscopy causes patient discomfort, requires anesthesia, and must generally be performed in a clinic or a hospital. X-rays and radiolabeled detecting agents have the common disadvantage of exposing the patient to radiation. In addition, all of these procedures require a skilled evaluation of the results in order to properly diagnose the patient's condition.
A method for detection of gastric epithelial damage, particularly ulcers and lesions in the stomach, using non-invasive, non-radioactive and non-x-ray techniques or procedures is disclosed in U.S. Pat. No. 5,620,899. The method of this reference employs a disaccharide which can be orally administered to a patient, which does not transport across cell membranes, which is metabolized within the small intestine to its monosaccharide components, and which is not broken down elsewhere in the body. Damage to the gastric epithelium will allow the disaccharide to enter the blood without being metabolized. Hence, the disaccharide will appear in the blood or urine to an extent that can be correlated with the extent of gastric epithelial damage. Typically, the disaccharide is administered to a patient, followed by collection of blood or urine, which is assayed for the disaccharide. The use of sucrose in particular as a diagnostic marker in detection of gastric epithelial damage is described in U.S. patent application Ser. No. 08/456,203.
The methodology utilized to evaluate the level of sucrose in the blood or urine in conjunction with the performance of the above-described gastric assay must be useful for analyzing physiological fluids. A hexokinase/glucose-6-phosphate method has been suggested for analysis of glucose in serum, plasma, or urine. United States Department of Health, Education and Welfare, Food and Drug Administration. In Vitro Diagnostic Products for Human Use, Proposed Establishment of Product Class Standard for Detection or Measurement of Glucose, Fed. Regist. Vol. 39, No. 126, 24146 (1974). There is, however, no suggestion of measuring sucrose in physiological fluids.
A method is described in S. Sekin, Tobacco International, Vol. 181, 27-27 (1979) for determination of sucrose in tobacco. This method converts glucose in an aliquot of a tobacco sample to 6-phosphogluconate, with concomitant production of NADH, by addition of ATP, hexokinase, glucose-6-phosphate dehydrogenase and NAD. An absorbance measurement at 340 nm is used to quantify the NADH, and hence the endogenous glucose. Another aliquot is treated with invertase, ATP, hexokinase, glucose-6-phosphate dehydrogenase and NAD. The invertase cleaves the sucrose to glucose and fructose. The glucose produced from cleavage, along with the glucose initially present in the sample, is converted to 6-phosphogluconate, producing NADH. An absorbance measurement at 340 nm quantifies NADH, and hence total glucose. The difference between total glucose and endogenous glucose gives the molar amount of sucrose present. However, there is no suggestion that this method would be compatible with analyzing sucrose in complex physiological fluids. In addition, the reagents used for the analysis are non-stabilized solutions which cannot be stored or transported for long periods of time.
A method suitable for determination of sucrose in physiological fluids would be highly desirable, as would a diagnostic kit containing the necessary reagents preformulated for use in such a method.
SUMMARY OF THE INVENTION
This invention is directed to a kit for use in a diagnostic method for detecting sucrose in physiological fluids. The kit comprises: (a) a first container of a solid first reagent mixture comprising ATP, NAD, hexokinase, G-6-PDH, and a buffer; which, after reconstitution with a specified amount of water, results in a first reagent solution comprising (i) ATP in a concentration in the range from about 0.5 mM to about 5.0 mM, (ii) NAD in a concentration in the range from about 0.5 mM to about 3.0 mM, (iii) hexokinase in a concentration in the range from about 200 U/L to about 2,000 U/L, and (iv) G-6-PDH in a concentration in the range from about 500 U/L to about 2,500 U/L, the first reagent solution having a pH in the range from about 7 to about 8; and (b) a second container of a solid second reagent mixture comprising ATP, NAD, hexokinase, G-6-PDH, invertase, and a buffer; which, after reconstitution with a specified amount of water, results in a second reagent solution comprising (i) ATP in a concentration in the range from 0.0 mM to about 5.0 mM, (ii) NAD in a concentration in the range from 0.0 mM to about 3.0 mM, (iii) hexokinase in a concentration in the range from 0 U/L to about 2,000 U/L, (iv) G-6-PDH in a concentration in the range from 0 U/L to about 2,500 U/L, and (v) invertase in a concentration in the range from about 50 kU/L to about 500 kU/L, the second reagent solution having a pH in the range from about 7 to about 8. The kit may also contain a sucrose solution as a standard, and at least one lyophilized urine control sample.
The invention is also directed to a diagnostic method for detecting sucrose in physiological fluids that may be performed with the above-described kit. The method comprises the steps of (a) treating a sample of physiological fluid with ATP, NAD, hexokinase, and G-6-PDH in amounts effective to substantially convert any glucose present in the sample to 6-phosphogluconate; (b) determining an absorbance of a solution formed in step (a) at about 340 nm; (c) treating the solution formed in step (a) with invertase, ATP, NAD, hexokinase, and G-6-PDH in amounts effective to substantially convert any glucose formed from sucrose present in the sample to 6-phosphogluconate; (d) determining an absorbance of a solution formed in step (c) at about 340 nm; and (e) calculating a sucrose concentration from the difference between the absorbance measured in step (b) and the absorbance measured in step (d).
A variation of the method comprises the steps of: (a) treating a first sample of the fluid with ATP, NAD, hexokinase, and G-6-PDH in amounts effective to substantially convert any glucose present in the sample to 6-phosphogluconate; (b) determining an absorbance of a solution formed in step (a) at about 340 nm; (c) treating a second sample of the fluid with invertase, ATP, NAD, hexokinase, and G-6-PDH in amounts effective to substantially convert any glucose present in the sample and glucose formed from sucrose present in the sample to 6-phosphogluconate; (d) determining an absorbance of a solution formed in step (c) at about 340 nm; and (e) calculating a sucrose concentration from the difference between the absorbance measured in step (b) and the absorbance measured in step (d).
DETAILED DESCRIPTION OF THE INVENTION
The following terms are a s defined herein . The term “NAD” indicates the compound nicotinamide adenine dinucleotide. The term “NADH” indicates reduced nicotinamide adenine dinucleotide. The term “ATP” indicates the compound adenosine triphosphate. The term “ADP” indicates the compound adenosine diphosphate. The term “G-6-PDH” indicates the enzyme glucose-6-phosphate dehydrogenase. The term “U” indicates an amount of enzyme measured in units and “U/L” is a measure of enzyme concentration in units per liter. The term “PIPES” indicates the compound 1,4-piperazinebis(ethanesulfonic acid). T
Alexander Lyle A.
Fitzpatrick ,Cella, Harper & Scinto
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