CN1987473A - Method for detecting lithium concentration and lithium diagnostic kit - Google Patents
Method for detecting lithium concentration and lithium diagnostic kit Download PDFInfo
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- CN1987473A CN1987473A CN 200610161231 CN200610161231A CN1987473A CN 1987473 A CN1987473 A CN 1987473A CN 200610161231 CN200610161231 CN 200610161231 CN 200610161231 A CN200610161231 A CN 200610161231A CN 1987473 A CN1987473 A CN 1987473A
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- lithium
- reagent
- glyceraldehyde
- inositol
- diagnostic kit
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Abstract
Enzymatic reaction continuous monitoring method of using lithium ion needed sensitized cyclohexanhexol -1(4) - phosphatase (CP) united to glyceraldehyde -3- phosphate dehydrogenase (GPD) is adopted in the invention. CP carries out reaction of enzymolysis for cyclohexanhexol 1- phosphoric acid to generate phosphate radical. Using action of united GPD deoxidizes coenzyme to reduced coenzyme so as to be able to measure upward velocity of absorbency of the reduced coenzyme at 340nm. Concentration of lithium can be calculated from the said upward speed of absorbency at 340nm. Features are: high specificity, and accurate test result. Using a pair of agent or Triple agent for kit, the invention can reduce intercross influence from each component. Through ultraviolet / visible light analytical apparatus, or semi-automatic/full automatic biochemical analysis apparatus the invention can obtain measured result quickly so that it is in low cost and is convenient to be popularized further.
Description
Technical field
The present invention relates in the fields such as medicine, food, environment the detection of lithium concentration, relate in particular to and utilize enzymic colorimetric and enzyme (even) united method to measure the method for lithium concentration, and the lithium diagnostic kit that makes thus, the lithium concentration technical field of analysis and detection belonged to.
Background technology
Lithium is a trace element, is monovalent cation.Occurring in nature does not have free lithium, therefore actual lithium ion or the lithium salts of referring to.The forties in 20th century, Cade is first with the success of lithium salts treatment mania.
Lithium ion does not combine with blood plasma and histone, is distributed to whole body with body fluid, and each tissue concentration differs, and thyroid gland and kidney concentration are the highest.Cerebrospinal fluidconcentration be about blood concentration half.Lithium enters cell through ion channel, and sodium in the displacement cell causes the cell excitement reduction.In addition, many chemical property of lithium are similar with magnesium ion to calcium, perhaps can replace some physiological function of calcium and magnesium, as the mediator that influences calcium ion regulatory discharges and influence ability CAMP generation that magnesium participates in etc.
When the blood lithium concentration meets or exceeds 1.5mmol/L, toxicity symptom in various degree can appear, and early stage poisoning manifestations is increasing the weight of of bad reaction, as vomiting that takes place frequently and diarrhoea, and unable, indifferent, limb tremor becomes thick, exagger by tiny.Serious poisoning can appear more than the blood lithium concentration 2.0mmol/L, performance is fuzzy consciously, incoordination, pronounce indistinctly, epileptic attack and even stupor, shock, kidney function damage.But the above threat to life of blood lithium concentration 3.0mmol/L.Because the lithium salts safe range is little, answer the concentration of periodic monitoring serum lithium.Blood drawing detects and should use lithium after 12 hours at last.Toxic reaction appears or suspect blood concentration low excessively (＜0.6mmol/L) time, should tightly observe, and check the blood lithium concentration at any time.
At present, generally use electrochemical electrode sensor lithium content.
Summary of the invention
The objective of the invention is: a kind of method of measuring lithium concentration is provided, and uses the formulated lithium diagnostic kit of this method.
For realizing purpose of the present invention, a kind of enzymic colorimetric and enzyme (even) united method are measured the method for lithium concentration, adopt following steps to carry out:
At first,, make it to take place following reaction with sample of measuring and the reagent mixing that contains inositol 1-phosphoric acid, inositol-1 (4)-phosphatase, glyceraldehyde-3-phosphate, coenzyme and glyceraldehyde-3-phosphate dehydrogenase,
Then, the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance rises, calculate the concentration of lithium.
Above-mentioned enzymic colorimetric and enzyme (even) united method are measured in the method for lithium concentration, and described coenzyme is NADP
+, NAD
+Or thio-NAD
+In a kind of.
Realize that lithium diagnostic kit of the present invention can be single agent, is grouped into by following one-tenth:
Damping fluid 20～500mmol/L,
Stabilizing agent 5～100g/ml,
DPN diphosphopyridine nucleotide～6mmol/L,
Inositol-1 (4)-phosphatase 10 00～80000U/L,
Glyceraldehyde-3-phosphate dehydrogenase 1000～80000U/L,
Inositol 1-phosphatase 11～12mmol/L,
Glyceraldehyde-3-phosphate 1～12mmol/L.
Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also the various compositions in above single agent can be carried out formulated in combination and become two agent, such as:
The prescription of two agent is not limited only in the above-mentioned table listed, wherein the composition of reagent I: coenzyme, inositol 1-phosphoric acid, glyceraldehyde-3-phosphate can be placed on reagent II; Inositol among the reagent II-1 (4)-phosphatase, glyceraldehyde-3-phosphate dehydrogenase also can be put into reagent I, so can form multiple formulations, enumerate no longer one by one.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Reagent can also be made into following three reagent:
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the coenzyme among the reagent I can be placed among reagent II or the reagent III, inositol 1-phosphoric acid among the reagent II, glyceraldehyde-3-phosphate can be placed among reagent I or the reagent III, inositol among the reagent III-1 (4)-phosphatase, glyceraldehyde-3-phosphate dehydrogenase also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent II/ reagent III, concentration is at 0.1～3mol/L, or 5～100g/ml, or within 5～50%v/v scope.
Material with used as stabilizers can be: at least a in ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), the ethylene glycol (Ethylene glycol).
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent, two agent or three doses, and the diagnosis/detection kit of following formula components relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 100mmol/L,
Stabilizing agent 2mol/L,
Coenzyme 3mmol/L,
Inositol-1 (4)-phosphatase 10 00～80000U/L,,
Glyceraldehyde-3-phosphate dehydrogenase 1000～80000U/L,
Inositol 1-phosphatase 11～12mmol/L,
Glyceraldehyde-3-phosphate 1～12mmol/L.
The present invention utilizes enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is measured the method for lithium concentration in the variation of 340nm wavelength place absorbance.Simultaneously, the present invention gives in order to realize the lithium diagnosis/determination kit of this method, adopt this kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out lithium concentration determination, and finding speed is fast, accuracy is high, thereby can obtain practical applying.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes enzymic colorimetric and enzyme (even) united method to measure lithium concentration fully, and test result is accurate;
(2) composition of participation reaction all adds, and is not subjected to the pollution of inside and outside source material, test process degree of accuracy height;
(3) this method is easy, easy to operate, can obtain testing result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) but this method just fast detecting on general ultraviolet/visible light analysis instrument or semi-automatic/automatic clinical chemistry analyzer does not need special or additional instruments, testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that assay method provided by the invention can be made various ways such as liquid reagent, powdered reagent, be used for measuring the size of various sample lithium concentrations;
(6) liquid lithium diagnosis/detection kit provided by the invention, good stability has guaranteed the application testing effect well.Be made into after two agent or three doses, can further reduce the cross influence between the various compositions, testing result is more credible, and reagent is more stable, can store for a long time.
Embodiment
The assay method of a kind of lithium concentration of the present invention and lithium diagnostic kit utilize inositol-1 (4)-phosphatase (inositol-1 (or4)-monophosphatase that needs lithium ion to activate; EC3.1.3.25) enzyme (idol) connection glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphatedehydrogenase; EC1.2.1.59) enzymatic reaction continuous monitoring method.Inositol-1 (4)-phosphatase enzymolysis inositol 1-phosphatase reaction produces phosphate radical, the effect of uniting glyceraldehyde-3-phosphate dehydrogenase again by (idol), coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured the speed that reduced coenzyme rises in 340nm place absorbance, by measuring the speed that 340nm place absorbance rises, can calculate the concentration of lithium.
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare lithium diagnostic kit by following composition and consumption:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 80g/ml,
NADP
+ 3mmol/L，
Inositol-1 (4)-phosphatase 6000U/L,
Glyceraldehyde-3-phosphate dehydrogenase 12000U/L,
Inositol 1-phosphoric acid 6mmol/L,
Glyceraldehyde-3-phosphate 12mmol/L.
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, tested lithium sample is 1: 25 with the body ratio of reagent, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of lithium.
Embodiment two (two agent)
Prepare lithium diagnostic kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 2mol/L,
NAD
+ 3mmol/L，
Inositol 1-phosphoric acid 8mmol/L,
Glyceraldehyde-3-phosphate 16mmol/L;
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Propylene glycol 50%v/v,
Inositol-1 (4)-phosphatase 8000U/L,
Glyceraldehyde-3-phosphate dehydrogenase 10000U/L.
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested lithium sample and reagent I, reagent II is 2: 20: 5, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of lithium.
Embodiment three (three doses)
Prepare lithium diagnostic kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ethylene glycol 2mol/L,
thio-NAD
+ 3mmol/L；
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 80g/ml,
Inositol 1-phosphoric acid 8mmol/L,
Glyceraldehyde-3-phosphate 16mmol/L;
Reagent III---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 50%v/v,
Inositol-1 (4)-phosphatase 9000U/L,
Glyceraldehyde-3-phosphate dehydrogenase 16000U/L.
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring lithium concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested lithium sample and reagent I, reagent II, reagent III is 4: 40: 5: 5, the Direction of Reaction is positive reaction (reaction of rising), and about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of lithium.
Through experimental verification, adopt other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.
Claims (6)
1. the assay method of lithium concentration may further comprise the steps:
1. with sample of measuring and the reagent mixing that contains inositol 1-phosphoric acid, inositol-1 (4)-phosphatase, glyceraldehyde-3-phosphate, coenzyme and glyceraldehyde-3-phosphate dehydrogenase, make it to take place following reaction,
2. the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance rises, calculate the concentration of lithium.
2. lithium diagnostic kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 20～500mmol/L,
Stabilizing agent 0.1～3mol/L, or
5～100g/ml, or
5～50％v/v，
DPN diphosphopyridine nucleotide～6mmol/L,
Inositol-1 (4)-phosphatase 10 00～80000U/L,
Glyceraldehyde-3-phosphate dehydrogenase 1000～80000U/L,
Inositol 1-phosphatase 11～12mmol/L,
Glyceraldehyde-3-phosphate 1～12mmol/L.
3. lithium diagnostic kit according to claim 2 is characterized in that: described coenzyme is NADP
+, NAD
+Or thio-NAD
+In a kind of.
4. lithium diagnostic kit according to claim 2 is characterized in that: described stabilizing agent is at least a in ammonium sulfate, glycerine, propylene glycol, the ethylene glycol.
5. any one lithium diagnostic kit in the claim 2～4 is characterized in that: described reagent is made into single agent, two agent or three doses.
6. any one lithium diagnostic kit in the claim 2～4, it is characterized in that: described kit is powdered reagent box or liquid reagent box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610161231 CN1987473A (en) | 2006-12-15 | 2006-12-15 | Method for detecting lithium concentration and lithium diagnostic kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610161231 CN1987473A (en) | 2006-12-15 | 2006-12-15 | Method for detecting lithium concentration and lithium diagnostic kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1987473A true CN1987473A (en) | 2007-06-27 |
Family
ID=38184351
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610161231 Pending CN1987473A (en) | 2006-12-15 | 2006-12-15 | Method for detecting lithium concentration and lithium diagnostic kit |
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| Country | Link |
|---|---|
| CN (1) | CN1987473A (en) |
-
2006
- 2006-12-15 CN CN 200610161231 patent/CN1987473A/en active Pending
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Open date: 20070627 |