CN109883972A - Dissociate Methods For The Determination of Iron in a kind of red blood cell - Google Patents
Dissociate Methods For The Determination of Iron in a kind of red blood cell Download PDFInfo
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 122
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 29
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 25
- 239000000523 sample Substances 0.000 claims abstract description 26
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- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 13
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- 206010018910 Haemolysis Diseases 0.000 claims description 10
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- 238000005259 measurement Methods 0.000 claims description 10
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- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical class [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
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- 229910021641 deionized water Inorganic materials 0.000 claims description 6
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- XGGLLRJQCZROSE-UHFFFAOYSA-K ammonium iron(iii) sulfate Chemical compound [NH4+].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XGGLLRJQCZROSE-UHFFFAOYSA-K 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
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- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 102000001554 Hemoglobins Human genes 0.000 description 7
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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Abstract
The present invention provides the Methods For The Determination of Iron that dissociates in a kind of red blood cell, comprising: standard solution is prepared；The preparation of sample test solution；The preparation of blank sample；Under selected running parameter, standard solution is measured using flame atomic absorption spectrophotometer, draws working curve；Sample test solution is measured, the absorbance of sample is obtained after deducting blank, the content of free iron in sample can be calculated according to working curve.The invention has the advantages that: sample handling processes are simple, and high sensitivity, the range of linearity is wide, and specificity is strong, meet the demand in scientific experiment.
Description
Technical field
The present invention relates to blood metal element content determination techniques field, in particular to dissociate iron content in a kind of red blood cell
Measuring method.
Background technique
Ferro element is the highest microelement of body burden, is almost distributed in each organ of whole body.The intracorporal iron of people is big
Part is present in blood rbc in the form of hemoglobin, and remainder exists in the form of myoglobins, ferritin etc..Iron
Existence form in blood includes that the heme iron in conjunction with hemoglobin, serum levels of iron in conjunction with transferrin etc. combine iron
And free iron.The content of iron is of great significance for medical diagnosis on disease, grasp body health situation etc. in detection blood.For example,
The ferro concentration in serum of chronic hemolytic anemia patient is higher than normal level；Body is there are malignant tumour, cirrhosis, obstructive Huangs
When the diseases such as subcutaneous ulcer, ferro concentration in serum is then lower than normal level.
Iron is the composition indispensable element of hemoglobin, the content and the direct phase of its oxygen carrying capacity of iron in erythrocyte
It closes.When human body is by external irritants such as environmental contaminants stress, body may generate series reaction, cause in red blood cell
The ferro element of hemoglobin is released, and is transformed into free state from reference state, so as to cause the decline of red blood cell oxygen carrying capacity, danger
Evil human health.Therefore, the same important in inhibiting of free iron content in blood rbc is further detected.
Currently, being rarely reported about the dissociate detection technique of iron content of erythrocyte, has iron content in detection blood
Method is mainly for whole blood or serum: (1) spectrophotometry.This method be added in blood sample reagent and iron appropriate from
Son complexing is added color developing agent and is inhaled using ultraviolet-uisible spectrophotometer measurement after separating the iron ion of complex state with blood sample
Luminosity, then the content by standard curve calculating tapping.This method is although more common, but the sensitivity and linear measurement range detected
It is lower.(2) atomic absorption spectrophotometry.This method directly clears up blood sample with acid or direct calcination dry ash
Change, be configured to the solution of certain volume, absorbance is measured by atomic absorption spectrophotometer and iron content is calculated.The party
Although method sensitivity is higher, the free iron in blood cannot be isolated well.In addition, Bao layers of Se Pu Fa ﹑ coulometric titration and
Redox Di Dings the methods of Fa ﹑ atomic emission spectrometry and is also used for the detection of blood iron content, but also has detection Xian ﹑ spirit
The disadvantages of sensitivity is poor, resists other metallic element interference performances weak, and specificity is not strong.
Summary of the invention
The present invention in view of the drawbacks of the prior art, provides the Methods For The Determination of Iron that dissociates in a kind of red blood cell, can be effective
Ground solves the above-mentioned problems of the prior art.
In order to realize the above goal of the invention, the technical solution adopted by the present invention is as follows:
Dissociate Methods For The Determination of Iron in a kind of red blood cell
Step 1: standard solution is prepared
The configuration of iron standard reserving solution: 0.8631g ammonium ferric sulfate is accurately weighed, is dissolved in water, 1.00mL sulfuric acid solution (1+ is added
3) 100mL volumetric flask, is moved into, water is added to be settled to scale, is mixed.This ferrous solution mass concentration is 1000mg/L.
The configuration of iron standard intermediate fluid: the accurate iron standard reserving solution 10mL that draws adds nitric acid solution (5 in 100mL volumetric flask
+ 95) it is settled to scale, is mixed.This ferrous solution mass concentration is 100mg/L.
Iron standard serial solution: respectively it is accurate draw iron standard intermediate fluid (100mg/L) 0mL, 0.100mL, 0.200mL,
0.300mL, 0.400mL, 0.500mL add nitric acid solution (5+95) to be settled to scale in 100mL volumetric flask, mix.This iron mark
In quasi- serial solution iron speciation be respectively 0mg/L, 0.100mg/L, 0.200mg/L, 0.300mg/L, 0.400mg/L,
0.500mg/L。
Step 2: the preparation of sample test solution
It extracts a certain amount of blood sample to be put into centrifuge tube anticoagulant in advance, 3mL physiological saline is added, after gently shaking
400 × g of centrifugal force is centrifuged five minutes, is abandoned supernatant, is repeated 3 times；Deionized water is added into centrifuge tube to 5mL, obtains haemolysis
Liquid.Hemolysate is centrifuged 20 minutes, Aspirate supernatant 3mL by 2000 × g of relative centrifugal force, is centrifuged 20 minutes, is taken under the same terms
Into super filter tube, 3500 × g of relative centrifugal force is centrifuged 20 minutes supernatant 2.4mL.1.6mL filtrate is collected, 1.0mL nitric acid is added
With 0.25mL hydrogen peroxide, it is placed in 95 DEG C of resolution 3h of water-bath, other disturbing factors such as removal organic matter.Ultra-filtration centrifuge tube used
Filter material is styrol copolymer/butadiene, and filter membrane is Ultracel low adhesion regenerated cellulose film, and filtering out liquid pipe is
Polypropylene, filter liquor lid and lining are polyethylene.
Step 3: the preparation of blank sample
1.6mL water is added in colorimetric cylinder, 1.0mL nitric acid and 0.25mL hydrogen peroxide is added, is placed in 95 DEG C of water-bath and disappears
Solve 3h.
Step 4: measurement
Under selected running parameter, standard solution is measured using flame atomic absorption spectrophotometer, draws out working curve；
The absorbance for measuring blank sample and sample test solution deducts the absorbance of blank sample to eliminate reagent interference, according to work song
Line can calculate the content of free iron in sample.
Preferably, the red blood cell best hemolysis time is 12h in step 2.
Preferably, super filter tube described in step 2 is the super filter tube of aperture 30KD.
Preferably, the running parameter that step 4 is selected are as follows: wavelength 248.3nm, slit 0.2nm, lamp current 10mA, burning
Grease head highness 3mm, air mass flow 9L/min, acetylene flow 2L/min.
Preferably, the measuring method replication 3 times or more are averaged to improve accuracy.
Compared with prior art the present invention has the advantages that providing a kind of separation of free iron in blood rbc sample
Detection method, sample handling processes are simple, and high sensitivity, the range of linearity is wide, and specificity is strong, meet the need in scientific experiment
It asks.
This method can also be used for serum, in whole blood free iron measurement.For serum, after serum is isolated in sampling, directly
Carry out separating step；For whole blood, sampling is added after hemolysate is made in deionized water and carries out separating step again.
Detailed description of the invention
Fig. 1 is the comparison of 1 sample of embodiment of the present invention iron content after the filtering of the super filter tube of different pore size；
Fig. 2 is SD rat erythrocyte free iron under the conditions of 1DBP of the embodiment of the present invention (dibutyl phthalate) contamination
The measurement result of content.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention more comprehensible, it develops simultaneously embodiment below in conjunction with attached drawing, it is right
The present invention is described in further details.
Dissociate Methods For The Determination of Iron in a kind of red blood cell, comprising the following steps:
Step 1: standard solution is prepared
The configuration of iron standard reserving solution: 0.8631g ammonium ferric sulfate is accurately weighed, is dissolved in water, 1.00mL sulfuric acid solution (1+ is added
3) 100mL volumetric flask, is moved into, water is added to be settled to scale, is mixed.This ferrous solution mass concentration is 1000mg/L.
The configuration of iron standard intermediate fluid: accurate iron standard reserving solution (1000mg/L) 10mL that draws adds in 100mL volumetric flask
Nitric acid solution (5+95) is settled to scale, mixes.This ferrous solution mass concentration is 100mg/L.
Iron standard serial solution: respectively it is accurate draw iron standard intermediate fluid (100mg/L) 0mL, 0.100mL, 0.200mL,
0.300mL, 0.400mL, 0.500mL add nitric acid solution (5+95) to be settled to scale in 100mL volumetric flask, mix.This iron mark
In quasi- serial solution iron speciation be respectively 0mg/L, 0.100mg/L, 0.200mg/L, 0.300mg/L, 0.400mg/L,
0.500mg/L。
Step 2: the preparation of sample test solution
It extracts a certain amount of blood sample to be put into centrifuge tube anticoagulant in advance, 3mL physiological saline is added, after gently shaking
It is centrifuged five minutes with 400 × g of relative centrifugal force, abandoning supernatant, repetitive operation 3 times；Deionized water is added extremely into centrifuge tube
5mL obtains hemolysate.With 2000 × g of relative centrifugal force, hemolysate is centrifuged 20 minutes, Aspirate supernatant 3mL, is continued in phase
It is centrifuged 20 minutes under the conditions of, then takes supernatant 2.4mL, the supernatant being centrifuged twice moved in super filter tube, with opposite
3500 × g of centrifugal force is centrifuged 20 minutes.1.6mL filtrate is collected, 1.0mL nitric acid and 0.25mL hydrogen peroxide is added, is placed in water-bath
95 DEG C of resolution 3h of pot.
Step 3: the preparation of blank sample
1.6mL water is added in colorimetric cylinder, 1.0mL nitric acid and 0.25mL hydrogen peroxide is added, is placed in 95 DEG C of water-bath and disappears
Solve 3h.
Step 4: measurement
(1 is shown in Table) under selected running parameter, standard solution is measured using flame atomic absorption spectrophotometer, drafting is gone to work
Make curve；The absorbance for measuring blank sample and sample test solution, deducts the absorbance of blank sample to eliminate reagent interference, according to
Working curve can calculate the content of free iron in sample.
The running parameter of 1 flame atomic absorption spectrophotometer of table
It is to reduce the loss of iron in ultrafiltration centrifugal process to the greatest extent the present invention relates to the key of operating procedure, is closed to measure
Manage effective result.Therefore, it is necessary to optimize to operating condition, guarantee ultrafiltration effect.
The determination of best hemolysis time
Deionized water is added in deposition red blood cell, red blood cell water suction, which is risen brokenly, obtains hemolysate, after being centrifuged with centrifuge, obtains
To the supernatant of red, transparent and minimal amount of sediment (broken cell membrane).The different hemolysis times may generate different
Hemolyzing effect to influence the clarification degree of centrifuged supernatant, and then influences the effect of ultrafiltration centrifugation.In order to avoid ultra filtration
Iron loss in journey influences measurement result, determines the best hemolysis time by observation solution Zhuan ﹑ centrifugal effect and ultrafiltration effect.
It the results are shown in Table 2.
The centrifugal treating effect of hemolysate after the different hemolysis times of table 2
As shown in Table 2, as the hemolysis time extends, the precipitating object amount after centrifugation is increasing, to energy when alleviating ultrafiltration
By supernatant liquid measure influence, ultrafiltration effect becomes better and better.But as the hemolysis time continues growing, hemolysate gradually becomes black
Brown, and supernatant becomes viscous, and can not separate it with sediment by centrifugation, influence ultrafiltration effect.It is final true by analyzing
Determining the red blood cell best hemolysis time is 12h.
The determination in super filter tube aperture
The present invention recycles filtered fluid using hemoglobin and other high molecular weight proteins in ultra-filtration centrifuge tube retention hemolysate
For measuring the content of free iron.Usual super filter tube answers molecular cut off to should not exceed the 1/3 of destination protein molecular weight, it is contemplated that
The molecular weight of hemoglobin is 64KD, we choose four kinds of common super filter tubes, and (molecular cut off is respectively 3KD, 10KD, 30KD
And 50KD), its ultrafiltration effect is compared.Sample is handled under the same conditions, after different super filter tube ultrafiltration, uses flame
Atomic Absorption Spectrometer measures absorbance, and the concentration of iron ion in sample solution is calculated according to standard curve, as a result such as Fig. 1
It is shown.
In four kinds of super filter tubes, aperture is that the super filter tube effect of 50KD is best, but 50KD and haemoglobin molecule amount 64KD connects
Closely, and filtrate is with faint yellow, therefore does not select this pipe.Aperture is that the super filter tube strainability of 3KD is minimum, and is easy to block.
Aperture is that the super filter tube filter effect of 10KD and 30KD is not much different, but in view of ferro element is after hemoglobin release, having can
It can be combined with some small molecular proteins, therefore final choice aperture is the super filter tube of 30KD.
The determination of relative centrifugal force and centrifugation
(1) it directly uses ultra-filtration centrifuge tube: hemolysate is added directly into ultra-filtration centrifuge tube, and attempt respectively with 6000
The relative centrifugal forces such as × g, 8000 × g ten minutes, discovery super filter tube was blocked, ineffective.
(2) by first using common centrifuge tube centrifugation reuse ultra-filtration centrifuge tube be centrifuged, reduce relative centrifugal force, increase from
The stimulation optimizations centrifugation step such as heart time can effectively avoid super filter tube from being blocked when discovery is using following below scheme, realize preferable
Centrifugal effect: it first uses common 2000 × g of centrifuge tube relative centrifugal force 20 minutes, takes supernatant；Continue using commonly from
The heart 2000 × g of pipe relative centrifugal force is centrifuged 20 minutes, takes supernatant；It supernatant samples will be added in ultra-filtration centrifuge tube twice, with
3500 × g of relative centrifugal force is centrifuged 20 minutes.
Embodiment 1
Studies have shown that DBP (dibutyl phthalate) can cause the iron of erythrocyte to discharge.The method of the present invention is used for
In the experiment that measurement DBP causes SD rat blood red blood cell iron to discharge:
Male SD rat is taken, after adaptable fed 7 days, is divided into 2 groups, including solvent control group (jade by random digits table
Rice bran oil), DBP group (2.4g/kg), every group 3, in experimentation rat can free water feed.Stomach-filling is carried out to each group rat
Contamination, duration 3h.
After contamination, SD rat is fixed, 0.5mL whole blood is obtained into centrifuge tube anticoagulant in advance from tail vein, adds
Enter 3mL physiological saline, with 400 × g of relative centrifugal force centrifugation 5 minutes after concussion, sucks supernatant, repetitive operation is three times；It is added
Deionized water is settled to 5mL and obtains hemolysate.In order to guarantee hemolyzing effect, it is further processed again after standing 12h.
By hemolysate with 2000 × g of relative centrifugal force centrifugation 20 minutes, then Aspirate supernatant 3mL, continue with it is opposite from
2000 × g of mental and physical efforts is centrifuged 20 minutes, then takes supernatant 2.4mL, and the supernatant being centrifuged twice is moved in ultra-filtration centrifuge tube,
With 3500 × g of relative centrifugal force centrifugation 20 minutes.1.6mL filtrate is collected, 1.0mL nitric acid and 0.25mL hydrogen peroxide is added, sets
In 95 DEG C of resolution 3h of water-bath.
Resolution sample is settled to 6mL, is measured with flame atomic absorption spectrophotometer, according to standard song after deduction blank
Line computation obtains the concentration of iron ion in sample, as a result as shown in Figure 2.Compared with the control group, DBP contamination group has measured very bright
Aobvious iron release (the free iron content of red blood cell obviously increases), shows the feasibility and advance of this method.
Those of ordinary skill in the art will understand that the embodiments described herein, which is to help reader, understands this hair
Bright implementation method, it should be understood that protection scope of the present invention is not limited to such specific embodiments and embodiments.Ability
The those of ordinary skill in domain disclosed the technical disclosures can make its various for not departing from essence of the invention according to the present invention
Its various specific variations and combinations, these variations and combinations are still within the scope of the present invention.
Claims (5)
1. dissociate Methods For The Determination of Iron in a kind of red blood cell, which comprises the following steps:
Step 1: standard solution is prepared
The configuration of iron standard reserving solution: accurately weighing 0.8631g ammonium ferric sulfate, be dissolved in water, add 1.00mL sulfuric acid solution (1+3),
100mL volumetric flask is moved into, water is added to be settled to scale, is mixed；This ferrous solution mass concentration is 1000mg/L；
The configuration of iron standard intermediate fluid: the accurate iron standard reserving solution 10mL that draws adds nitric acid solution (5+95) in 100mL volumetric flask
It is settled to scale, is mixed；This ferrous solution mass concentration is 100mg/L；
Iron standard serial solution: respectively it is accurate draw iron standard intermediate fluid (100mg/L) 0mL, 0.100mL, 0.200mL,
0.300mL, 0.400mL, 0.500mL add nitric acid solution (5+95) to be settled to scale in 100mL volumetric flask, mix；This iron mark
In quasi- serial solution iron speciation be respectively 0mg/L, 0.100mg/L, 0.200mg/L, 0.300mg/L, 0.400mg/L,
0.500mg/L；
Step 2: the preparation of sample test solution
It extracts a certain amount of blood sample to be put into centrifuge tube anticoagulant in advance, 3mL physiological saline is added, with phase after gently shaking
400 × g of centrifugal force is centrifuged five minutes, abandoning supernatant, repetitive operation 3 times；Deionized water is added into centrifuge tube to 5mL, obtains
To hemolysate；Hemolysate is centrifuged 20 minutes, Aspirate supernatant 3mL with 2000 × g of relative centrifugal force, is continued under the same conditions
Centrifugation 20 minutes, then supernatant 2.4mL is taken, the supernatant being centrifuged twice is moved in super filter tube, with relative centrifugal force
3500 × g is centrifuged 20 minutes；1.6mL filtrate is collected, 1.0mL nitric acid and 0.25mL hydrogen peroxide is added, is placed in 95 DEG C of water-bath
Clear up 3h, other disturbing factors such as removal organic matter；Ultra-filtration centrifuge tube filter material used is styrol copolymer/fourth two
Alkene, filter membrane are Ultracel low adhesion regenerated cellulose film, and filtering out liquid pipe is polypropylene, and filter liquor lid and lining are poly- second
Alkene；
Step 3: the preparation of blank sample
1.6mL water is added in colorimetric cylinder, 1.0mL nitric acid and 0.25mL hydrogen peroxide is added, is placed in 95 DEG C of resolution 3h of water-bath；
Step 4: measurement
Under selected running parameter, standard solution is measured using flame atomic absorption spectrophotometer, draws out working curve；Measurement
The absorbance of blank sample and sample test solution, deduct the absorbance of blank sample with eliminate reagent interference, can according to working curve
Calculate the content of free iron in sample.
2. according to the method described in claim 1, it is characterized by: the red blood cell best hemolysis time is 12h in step 2.
3. according to the method described in claim 2, it is characterized by: super filter tube described in step 2 is the super filter tube of aperture 30KD.
4. according to the method described in claim 1, it is characterized by: the selected running parameter of step 4 are as follows: wavelength 248.3nm, it is narrow
0.2nm, lamp current 10mA are stitched, burn grease head highness 3mm, air mass flow 9L/min, acetylene flow 2L/min.
5. according to the method described in claim 1, it is characterized by: the Methods For The Determination of Iron that dissociates in the red blood cell repeats to survey
Determine 3 times or more to be averaged to improve accuracy.
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| CN201910287323.6A CN109883972A (en) | 2019-04-11 | 2019-04-11 | Dissociate Methods For The Determination of Iron in a kind of red blood cell |
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| CN114609070A (en) * | 2022-03-16 | 2022-06-10 | 云锦华彰(北京)生物科技有限公司 | Free Fe in hemoglobin oxygen carrier3+Content detection method |
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