CN103499547A - Method for determination of L-carnitine by enzymatic spectrophotometry - Google Patents
Method for determination of L-carnitine by enzymatic spectrophotometry Download PDFInfo
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Abstract
The invention discloses a method for determination of L-carnitine by enzymatic spectrophotometry. The method comprises the following steps of 1, orderly adding NAD-containing buffer and L-carnitine standard solutions having different concentrations into a test tube, 2, adding L-carnitine dehydrogenase into the test tube to obtain an experiment tube, and adding distilled water which has the volume equal to the volume of the L-carnitine dehydrogenase and replaces the L-carnitine dehydrogenase into a test tube to obtain a control tube, 3, putting the experiment tube and the control tube in a water bath having a temperature of 37 DEG C for a water bath reaction, 4, measuring solution absorbance OD340 at the wave length of 340nm by a spectrophotometer to obtain experiment tube solution absorbance AOD340 and control tube solution absorbance BOD340, and drawing a standard curve by L-carnitine concentrations as horizontal coordinates and (AOD340-BOD340) as longitudinal coordinates, and 5, calculating L-carnitine content of a detected sample according to the standard curve. The method can simply, fast and accurately determine L-carnitine.
Description
Technical field
The present invention relates to the assay method of VBT, particularly, the present invention relates to the method for enzyme spectrophotometry VBT.
Background technology
VBT (L-carnitine), another name L-BETAIN, Cobastab t, levocarnitine etc., chemistry L-beta-hydroxy-gamma by name-trimethylamine butyric acid, be essential a kind of biostearin compound in human body.Its physiological function in human body has: participate in the beta-oxidation of long-chain fatty acid, at first long-chain fatty acid forms fatty acyl group CoA, then under the effect of carnitine transacetylase I, with VBT generation fatty acid carnitine, is transported in mitochondria and carries out beta-oxidation; Regulate the ratio between acyl-CoA-CoA in mitochondria, the stable of chondriosome acyl CoA-CoA ratio plays an important role to energetic supersession, because acyl-CoA-CoA ratio raises, pyruvic dehydrogenase had to inhibiting effect; Catch acyl group and generate fatty acyl carnitine, fatty acyl carnitine is transported cell and is entered the circulation system under the effect of fatty acyl carnitine invertase, by urine, excretes; What participate in membrane phospholipid in the film repair process goes acidylate-weight acidylate, is conducive to the timely reparation of film, plays the effect of secondary anti-oxidative defense barrier; Stimulate the metabolism of branched-chain amino acid, in time the metabolic product side chain keto acyl base of branched-chain amino acid is transported to outside film; Prevent that in body, the ammonia excess accumulation produces toxicity; Also the ketone process is given birth in participation gluconeogenesis and adjusting indirectly, can effectively reduce the concentration of lactic acid in the rear blood of motion, participates in the maturation of sperm etc.
VBT in mammalian body is mainly from synthetic two approach in diet and body, but the ratio of the carnitine obtained by these two approach depends on the synthetic required factor supply of the carnitines such as vitamin C, ferro element in age, eating habit and body.VBT from diet mainly is absorbed by the body at small intestine, and in body, the position of synthetic VBT is liver, and some mammiferous testis and kidney also have certain VBT synthesis capability.VBT is distributed in everywhere by the circulation system.In adult human body, approximately containing the 25g VBT, approximately 98% carnitine is distributed in skeletal muscle and cardiac muscle, the about 1:50 of the ratio of carnitine concentration in blood plasma and muscle.In male blood plasma, VBT concentration is 59 μ mol/L, and the women is 52 μ mol/L, and child is 35-45 μ mol/L.
The shortage of VBT can cause the disorder of a series of metabolic functions of human body, carntine deficiency is divided into primary carnitine deficiency disease and Secondary cases carntine deficiency, the clinical symptoms performance has skeletal muscle to carry out fatigue, myasthenia, flesh hypotonia, paralysis when serious, the visible obviously fat deposition of pathological analysis; The carrying out property such as DCM (dilated cardiomyopathy), amyocardia, arrhythmia cordis, heart failure cardiac damage; The different fatty liver of weight and dysfunction of liver are often arranged; Long-time hunger, fatigue, heating etc. can cause primary carnitine deficiency disease, the sudden death of fatty acid beta-oxidation obstacle infant; Under heating, the stress situation such as tired, hungry, ketotic hypoglycemia and metabolic acidosis etc. can appear hanging down.Primary VBT deficiency disease can be by the OCT N2 albumen in the VBT transporte to cells owing to lacking, and in patient's intestinal absorption food, the ability of VBT and the re-absorbed ability of kidney all descend.Secondary cases VBT deficiency disease can be caused by many reasons, comprises genetic fatty acid metabolism imbalance, branched-chain amino acid metabolism disorder, organic aciduria and valproic acid therapy.
VBT has been widely used in the industries such as food, feed, medicine at present, as added VBT etc. in infant food, diet food and sports drink.And VBT is also an important indicator clinically, in blood plasma, carnitine content can be used as the objective basis of carnitine supplementary therapy effect, and the people (2007) such as Ke Li think that the VBT content in refining can be used as an index of male sterility diagnosis.Therefore the mensuration of VBT is significant.
Current more for the method research of measuring VBT, have: bioanalysis, chemical method, tandem mass spectrometry, capillary electrophoresis etc.; But the method for successful Application enzyme spectrophotometry VBT but has no report.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of method of enzyme spectrophotometry VBT.This assay method adopts the enzyme spectrophotometric method, can measure easy, quickly and accurately VBT.
The present invention solves the problems of the technologies described above adopted technical scheme: the method for enzyme spectrophotometry VBT comprises the steps:
(1), in test tube, add successively the damping fluid that contains NAD, the VBT standard solution of variable concentrations;
(2) add again the VBT dehydrogenasa in the test tube of described step (1), as experiment tube; Add and the isopyknic distilled water of described VBT dehydrogenasa, to replace the VBT dehydrogenasa, manage in contrast;
(3) experiment tube and control tube all are placed in to 37 ℃ of water-baths;
(4) measure the absorbance OD340 of described step (3) gained solution at the 340nm place on spectrophotometer; Experiment tube is A
oD340, control tube is B
oD340; Take VBT concentration as horizontal ordinate, (A
oD340-B
oD340) be ordinate, the drawing standard curve;
(5), according to the typical curve of described step (4) gained, try to achieve the content of VBT in testing sample.
In described step (1), described damping fluid refers to the Tris-HCl damping fluid of pH8.9,0.1mol/L, and the concentration of the NAD wherein contained is 4.0～8.0mM; The interpolation volume is 1.5mL; In described step (1), the VBT standard solution scope of described variable concentrations is 0～0.30mmol/L.In described step (2), the described VBT dehydrogenasa that adds is that the work of interpolation enzyme is the VBT dehydrogenasa enzyme liquid 1.5mL of 7.49U/ml.It is the enzyme unit that lives that enzyme work is defined as enzyme amount that 37 ℃ of per minutes generate the NADH of 1 μ mol.
In described step (3), the water-bath time is 60 minutes.
The present invention compared with prior art, has following advantage:
(1) the VBT dehydrogenasa that the present invention uses can be used the method for VBT dehydrogenasa extraction and purification shown in embodiment 1 part to obtain, and the mode that also can use business to buy obtains.
(2) VBT in the enzyme spectrophotometry sample that the present invention adopts the VBT dehydrogenasa to participate in, detect and be limited to 1.90 μ mol/L, be limited to 100 μ mol/L on linearity, linearly dependent coefficient is 0.9957-0.9965, and the relative standard deviation is fine for the stability of 0.30-3.63%(proof the method).The recovery of the present invention, between 95.4-108.0%, has illustrated that accuracy of the present invention is higher.
(3) also to be to use instrument simple for advantage of the present invention, measures simple, convenient and rapidly, and high specificity, because the specificity of this enzyme can be distinguished D-carnitine and VBT.Although in prior art, capillary electrophoresis is rapider than enzyme spectrophotometry, be generally less than 30 minutes, the sensitivity of capillary electrophoresis and accuracy all are not so good as enzyme spectrophotometric method of the present invention.Method of the present invention is compared with other enzyme spectrophotometric method, does not need to add developer, and direct ultraviolet is measured the NADH that enzymatic reaction generates, and has avoided the inhibition problem of developer to enzymatic activity; But the linear upper limit of the present invention is lower, may be reversible due to enzymatic reaction, although react and mainly carry out to positive dirction, still there is a small amount of reversed reaction under the condition of pH8.9, this problem can suitably be diluted solution to sample when measuring.
The accompanying drawing explanation
The protein content typical curve that Fig. 1 is the Folin-phenol method in embodiment 1 " purifying of VBT dehydrogenasa " process.
Fig. 2 is that the enzyme of respectively managing in embodiment 1 " purifying of VBT dehydrogenasa " process is lived and protein content.
Fig. 3 is the VBT typical curve under different pH in embodiment 2.
Fig. 4 is the relation that enzyme dosage in embodiment 2, reaction time and absorbance change.
Embodiment
In order to understand better content of the present invention, below in conjunction with specific embodiment, be described further.Should be understood that these embodiment, only for the present invention is further described, limit the scope of the invention and be not used in.Should be understood that in addition after having read content of the present invention, the person skilled in art makes some nonessential change or adjustment to the present invention, still belongs to protection scope of the present invention.
Embodiment
Purification and enzyme activity determination that embodiment 1 VBT dehydrogenasa enzyme is lived
One, prepare VBT dehydrogenasa crude enzyme liquid
1. according to following one-tenth assignment system nutrient culture media, slant medium: 1.0%L-carnitine, 1.0% yeast extract, 1.0% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% epsom salt, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH7.0.
Fluid nutrient medium: the 0.5%L-carnitine, 0.1% yeast extract, 0.2% glucose, 0.2% 3 water dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate,, 0.05% epsom salt, 1.0% (v/v) liquid microelement, 1.5% agar powder, pH6.5.
2. bacterial strain activation: 30 ℃ of cultivation 20-30h of L-3 strains bacterial strain access slant medium that the strain that Ochrobactrum is belonged to can be produced the VBT dehydrogenasa (EC1.1.1.108) of advantageous property are activated;
3. one-level is cultivated: again the bacterial strain after activation is connect to a ring to fluid nutrient medium, condition of culture is: dress liquid 20mL in the 100mL conical flask, and 30 ℃, shaking table revolution 180rpm, cultivate 24 hours;
4. secondary is cultivated: then with 1% inoculum concentration, bacterium liquid is joined in fluid nutrient medium, with described one-level, cultivating under identical condition of culture and cultivating 24h;
5. three grades of cultivations, the solution culture fermentation 27h of dress 10L in the fermentation tank of 15L, the condition of fermentation: 30 ℃ of fermentation temperatures, stirring rate 180rpm, pH6.5;
6. the concentration of fermentation liquor: the centrifugal 30min of fermentation liquor 8000rpm after fermentation ends is collected to obtain to the 74.24g thalline, by 1:2(w/v) add 50mmol/L, the PB damping fluid of pH7.0, carry out broken wall with high pressure homogenizer, the centrifugal 30min of 8000rpm removes cell wall fragments afterwards, obtains crude enzyme liquid 670mL.Measure the crude enzyme liquid enzyme and live, and use the spectrophotometry protein content.
Two, the purifying of VBT dehydrogenasa
1. grade ammonium sulfate salting-out
Slowly add the ammonium sulfate powder in the crude enzyme liquid of embodiment 2 preparations, make saturation degree reach respectively 35%, 45%, 55%, 65%, constantly regulate pH makes it be stabilized in 7.0 simultaneously, saltout and spend the night, in each concentration by crude enzyme liquid with the centrifugal 30min of 8000rpm, ratio with 1:2 in precipitation adds 50mmol/L, surveys enzyme after the PB damping fluid of pH7.0 and lives and protein content.The results are shown in Table 1:
Table 1 grade ammonium sulfate salting-out result
As can be seen from Table 1, VBT dehydrogenasa sedimentation effect between ammonium sulfate saturation degree 35%-45% is best, and the specific activity that precipitation records after again dissolving is the highest.When being 35% and 55%, the ammonium sulfate saturation degree can precipitate a large amount of impurity albumen.In order to extract as much as possible the VBT dehydrogenasa, again carry out salt fractionation after 35% and 55% precipitation is dissolved again, but do not obtain obvious sediment 45% the time, therefore give up this part enzyme.45% precipitation and 65% precipitation are merged, measure enzyme and live and protein concentration, total enzyme is lived as 696.51U, and albumen is 3791.74mg, specific activity 0.184U/mg.By the enzyme liquid bag filter of packing into, use 0.01mol/L, dialysis under the Tris-HCl damping fluid of pH8.0 and 4 ℃, changed one time dislysate every 12 hours, change three times.The rear 8000rpm of dialysis end gets supernatant in centrifugal 30 minutes and carries out the DEAE-cellulose ion-exchange chromatography.
2. DEAE-cellulose ion-exchange chromatography
DEAE-cellulose chromatographic column (5.5 * 50cm) is first used 0.01mol/L, the Tris-HCl damping fluid balance of pH8.0, this ion exchange column on enzyme liquid after dialysing again, the 0.01mol/L that is 0 to 1mol/L by NaCl concentration, the Tris-HCl damping fluid of pH8.0 carries out gradient elution, flow velocity is 1mL/min, and 15min collects a pipe.Measure each tubulin content and measure and respectively manage enzyme work by Folin-phenol method.The protein content typical curve of Folin-phenol method is shown in Fig. 1.Each is managed, and enzyme is lived and protein content is shown in Fig. 2.
From No. 39 to No. 45, Guan Jun has enzyme work as can be seen from Figure 2, and their enzyme work, protein concentration are in Table 2.
No. 39-45 pipe enzyme of table 2 lived and the determination of protein concentration result
According to shown in table 2, by specific activity, 39,40,41 and No. 42 four higher pipes merge, the enzyme liquid enzyme obtained is lived as 7.49U/mL, protein concentration 2.63mg/mL, and specific activity 2.8U/mg, this enzyme liquid is for the mensuration of embodiment 2 enzyme spectrophotometric method.
Three, relevant experimental technique
1. the mensuration of thalli growth amount
The bacterial growth amount is determined in the absorbance at 600nm place by measuring fermentation liquor, adjusts the extension rate of bacterium liquid, makes absorbance be less than 1.000.
2.L-carnitin dehydrogenases enzyme activity determination
3.6mL after 37 ℃ of preheatings is containing the Tris-HCl(50mM of 10mM VBT and 1mM NAD; PH9.0) add the 0.4mL testing sample in damping fluid, measure at once the light absorption value at 340nm place after fully mixing, every a reading of 30 seconds records, adjust the extension rate of testing sample, the per minute absorbance is changed between 0.060-0.100.It is the enzyme unit that lives that enzyme work is defined as enzyme amount that 37 ℃ of per minutes generate the NADH of 1 μ mol.
3 determination of protein concentration
3.1 spectrophotometric method
By the absorption value at spectrophotometric determination 280nm place, and calculate according to formula 1OD280=1.67mg/mL.
3.2 Folin-phenol method
Folin-phenol method reagent: 1, (1) 4% sodium carbonate (2) 0.2M NaOH (3) 1% copper sulphate (4) 2% sodium potassium tartrate tetrahydrates.(1) and (2) equal-volume mix.(3) and (4) equal-volume mix.Then be mixed into to reagent first with 50:1 two nights.Used the same day.2,1M Folin-phenol reagent second.
Add 0,0.20,0.40,0.60,0.80 in test tube, 1.00mL bovine serum albumin(BSA) (500 μ g/mL), add the not enough 1mL of water, parallelly does two parts, respectively adds according to the order of sequence 5mL reagent first, shakes up, room temperature is placed 10 minutes, then adds successively 0.5mL reagent second, shakes up, and 30 ℃ are incubated 30 minutes.Then colorimetric estimation (OD on spectrophotometer
500).Horizontal ordinate is protein content, and ordinate is optical density value drawing standard curve.Testing sample as above reacts and measures, and the reference standard curve, draw protein content.
3.3 Lineweaver-Burk graphing method
The 1/s reciprocal of enzymatic reaction concentration of substrate of take is horizontal ordinate, and the 1/v reciprocal of enzyme ' s reaction speeding is that ordinate is made a rectilinear, and vertical axis intercept is-1/v
max, slope is Km/v
max, the transverse axis intercept is-1/Km.
Reaction system is as shown in table 3
Table 3 enzyme Spectrophotometric Determination VBT reaction system
The drafting of typical curve: the sample in system is changed into to the VBT standard solution of variable concentrations, horizontal ordinate is VBT concentration, and ordinate is A-B drawing standard curve.Testing sample is as above shown reaction and is measured, and the reference standard curve, draw VBT content.
The optimization of reaction system
The optimization of pH
With the Tris-HCl damping fluid of pH5.1,7.0,8.9 0.1mol/L, by table 4 reaction system, reacted respectively, the drawing standard curve, according to best pH of definite system such as the linearly dependent coefficient of typical curve, regression equation slopes.
The reaction system of the best pH choice experiment of table 4
Draw the VBT typical curve under 5.1,7.0,8.9 3 pH, result is as Fig. 3, and Fig. 3 is the VBT typical curve under different pH.
Fig. 3 shows when pH8.9, and VBT typical curve linearly dependent coefficient is greater than 99%, and slope is large, can reduce the impact that instrument error is brought, so this experiment adopts pH8.9, the Tris-HCl damping fluid of 0.1mol/L.
When the slope of VBT typical curve when pH8.9 is greater than pH7.0, reason may be the catalytic activity difference of VBT dehydrogenasa under these two kinds of pH, and variation has occurred the character of enzyme.In order to verify this possibility, measured again the Km of this enzyme when pH7.0.The Km of VBT while adopting Lineweaver-Burk graphing method (double-reciprocal plot method) to measure pH7.0.Reaction system is: the pH7.0 at 1.5mL containing 4.0mmol/L NAD, 0.1mmol/L add 37 ℃ of insulations of VBT standard solution of 1.5mL variable concentrations in the dPB damping fluid, add again 30 μ LL-carnitin dehydrogenases, mix absorbance under rear mensuration 340nm, every an absorbance of 30 seconds records, record 2 minutes.The Km that result records VBT is 1.77mmol/L, the 5.90mmol/L while being less than pH8.9.
The optimization of reaction time and enzyme dosage
The 100mmol/L containing the 0.5mmol/L VBT 37 ℃ of preheatings, after adding respectively 10,20,30,40,50 μ L enzyme liquid in the pH8.9Tris-HCl damping fluid, measure immediately 340nm place absorbance after mixing, then measured its absorbance at the 340nm place every 5 minutes, take the time as horizontal ordinate, take each absorbance constantly deducts initial absorbance as the ordinate mapping, determines reaction time and the enzyme dosage of system the best.The results are shown in Figure 4, Fig. 4 is the relation that enzyme dosage, reaction time and absorbance change.
As can be seen from Figure 4, add 30 μ L or more during multienzyme liquid absorbance with the curve of time, substantially overlap, and reach stable about 1 hour, so the reaction system enzyme dosage of drawing standard curve is 30 μ L, the reaction time is 1 hour.
Enzyme spectrophotometric method statistical analysis
The range of linearity of typical curve and detection limit
Reaction system is as shown in table 5, the VBT concentration of standard solution is from 0 to 0.30mmol/L, draw absorbance changing value-VBT concentration curve, by improving the VBT concentration of standard solution, till making absorbance and the appearance of standard carnitine solution concentration non-linear, with the range of linearity of the curve that settles the standard, the experiment triplicate.Detect the VBT concentration of 3 times of correspondences of the standard deviation that is limited to blank absorbance.The results are shown in Table 5.
The drafting of table 5 typical curve
Detection limit detects the VBT concentration of 3 times of correspondences of the standard deviation that is limited to blank absorbance, and result is 1.90 μ mol/L, illustrates that the method sensitivity is higher.
Be limited to 100 μ mol/L on the method linearity.
Repeated experiments
Get respectively the VBT solution of basic, normal, high three level concentration and measure by the reaction system shown in table 1, repeat 10 times.Result is respectively in Table 6,7,8.
Table 61 sample VBT concentration determination result
No. 2 sample VBT concentration determination results of table 7
No. 3 sample VBT concentration determination results of table 8
From above-mentioned three tables, can find out, the method stability when measuring the sample of varying level VBT content is all fine, the relative standard deviation all is less than 5%, when measuring 2, No. 3 samples, the relative standard deviation is less than 1%, and the relative standard deviation is more greatly because No. 1 sample VBT concentration is too low when measuring No. 1 sample.Repeatability and the good stability of presentation of results the method for repeated experiments.
The average recovery experiment
Get respectively the VBT solution of basic, normal, high three level concentration, first measure VBT concentration, then add respectively the standard VBT solution of isopyknic two kinds of concentration, mensuration adds the VBT after standard specimen, each sample replication three times, calculate recovery rate, result is as table 9.
Table 9 application of sample recovery experiment result
The recovery of this experiment, between 95.4-108.0%, illustrates that the method accuracy is better.
As mentioned above, just can realize preferably the present invention.
Claims (3)
1. the method for enzyme spectrophotometry VBT, is characterized in that, comprises the steps:
(1), in test tube, add successively the damping fluid that contains NAD, the VBT standard solution of variable concentrations;
(2) add again the VBT dehydrogenasa in the test tube of described step (1), as experiment tube; Add and the isopyknic distilled water of described VBT dehydrogenasa, to replace the VBT dehydrogenasa, manage in contrast;
(3) experiment tube and control tube all are placed in to 37 ℃ of water-baths;
(4) measure the absorbance OD340 of described step (3) gained solution at the 340nm place on spectrophotometer; Experiment tube is A
oD340, control tube is B
oD340; Take VBT concentration as horizontal ordinate, (A
oD340-B
oD340) be ordinate, the drawing standard curve;
(5), according to the typical curve of described step (4) gained, try to achieve the content of VBT in testing sample.
2. the method for enzyme spectrophotometry VBT according to claim 1, is characterized in that,
In described step (1), described damping fluid refers to the Tris-HCl damping fluid of pH8.9,0.1mol/L, and the concentration of the NAD wherein contained is 4.0～8.0mM; The interpolation volume is 1.5mL;
The VBT standard solution scope of described variable concentrations is 0～0.30mmol/L;
In described step (2), the described VBT dehydrogenasa that adds is that the work of interpolation enzyme is the VBT dehydrogenasa enzyme liquid 1.5mL of 7.49U/ml.
3. according to the method for the described enzyme spectrophotometry of right 4 VBT, it is characterized in that, in described step (3), the water-bath time is 60 minutes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106769943A (en) * | 2017-01-12 | 2017-05-31 | 南京欣迪生物药业工程有限责任公司 | A kind of refining carnitine detection kit and its application |
| CN110702623A (en) * | 2019-10-25 | 2020-01-17 | 徐詹程 | Photoelectric detection device and detection method thereof |
| CN115963169A (en) * | 2021-10-11 | 2023-04-14 | 生物岛实验室 | Detection method and detection kit for carnitine |
-
2013
- 2013-08-27 CN CN201310379341.XA patent/CN103499547A/en active Pending
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106769943A (en) * | 2017-01-12 | 2017-05-31 | 南京欣迪生物药业工程有限责任公司 | A kind of refining carnitine detection kit and its application |
| CN110702623A (en) * | 2019-10-25 | 2020-01-17 | 徐詹程 | Photoelectric detection device and detection method thereof |
| CN115963169A (en) * | 2021-10-11 | 2023-04-14 | 生物岛实验室 | Detection method and detection kit for carnitine |
| CN115963169B (en) * | 2021-10-11 | 2023-10-13 | 生物岛实验室 | Detection method of carnitine and detection kit |
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Application publication date: 20140108 |