Determination of blood ammonia concentration in study_3105

By Matthew James,2014-10-30 14:56
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Determination of blood ammonia concentration in study_3105

Determination of blood ammonia concentration in study

     Abstract Objective: To establish UV spectrophotometry in the blood ammonia concentration method for hepatic coma, hepatic encephalopathy, severe liver disease, uremia and other

    metabolic disorders diagnosis of reference. Methods: Blood from the body immediately after the accession of quantitative excessive sodium tungstate and sulfuric acid solution, so that protein precipitation at the same time, blood ammonia and sulfuric acid to form ammonium sulfate and to survive in the blood filtrate, and then with phenol - hypochlorite

    significantly color wavelength of 630 nm was measured after the optical density OD values (a blank correction). Results: The blood ammonia in the 20 ~ 60 μmol / L within the

    concentration showed good linearity between OD. Conclusion: This method is fast, simple and suitable for clinical detection of blood ammonia concentration.

     Key words ammonia; methodology; optical density

     Normal in vivo of free ammonia (blood ammonia, BA) were

    very low (normal 20 ~ 60 μmol / L) [1], its main protein

    comes from in vivo metabolism of amino acids generated in the process, as well as decomposition by Deamination to endogenous ammonia, ammonia can form such a normal amide and synthesis of

    other nitrogen-containing compounds continuously being

    transformed; another source of protein foods in the intestine of bacterial decomposition of exogenous made of ammonia, a normal cases, this ammonia through the portal vein into the

    liver were synthesized as urea, excreted through the kidneys. However, in the event of metabolic disorders, such as hepatic coma, hepatic encephalopathy, severe hepatitis, uremia, etc.

    can not be due to the normal metabolism of ammonia excreted caused BA increased. Extreme failure of liver function or blood flow through the liver can not be properly converted and other serious liver disease, ammonia can not be removed from the circulation in a timely manner so that ammonia can be increased [1]. High-BA had neurological toxicity, BA

    determination for portal hypertension, hepatic coma, Reve's syndrome and a number of pediatric patients with congenital metabolic disorders such as diagnosis, observation and prognosis of great significance [2]. BA determination methods

    of trace diffusion method, colorimetry, ion exchange, ammonia electrode method, enzymatic and so on. The author of this laboratory in the context of existing conditions, refer to the literature at home and abroad to establish rapid and accurate

    detection of BA method for the clinical type of disease diagnosis and treatment of reference.

     An instrument and reagent

     1.1 Instrument

     Electronic Balance COBS 120 type (Korea COBS Company),

    UV Spectrophotometer HP8453 (Hewlett-Packard), centrifugal

    precipitators 800 (Shanghai Surgical Instrument Factory), YKH

     I liquid fast mixer (Jiangxi instrumentarija) , DHW 420

    3 water tanks with electric thermostat (Beijing Scientific Instrument Factory Dong Ha).

     1.2 reagent

     Sodium tungstate (Tianjin Chemical Reagent Factory big-

    mao, batch number 030,906), ammonium sulfate (Beibei, Chongqing Chemical Reagent Factory, batch number 050,329),

    sodium nitroprusside (Shanghai 3 Ace Products Corporation, batch number 20,020,204) , phenol (Beibei, Chongqing Chemical Reagent Factory, batch number: 20,000,924), sodium hydroxide (Chongqing Chemical Reagent Factory Valleys and Lakes, batch

    number 20,000,312), sodium hypochlorite (Chongqing, eastern Sichuan Chemical Group Chemical Reagent Factory, batch number 20,040,904), ammonium sulfate (Beibei, Chongqing Chemical

    Reagent Factory, batch number 050,329), sulfuric acid (Chongqing Chuanjiang Chemical Reagent Factory, batch number 20,000,105), test drugs used were of analytical grade, water, ammonia-free distilled water for the new system.

     2 Methods and Results

     2.1 Stock Solution Preparation [2]

     Preparation of ammonia-free: reference literature [3] to take re-distilled water 1 000 mL, add 1 mL dilute sulfuric acid and potassium permanganate test solution 1 mL, distilled or get back.

     Phenol reagent: refined to take sodium nitroprusside 25.00 mg, after being mixed with the amount of phenol-free

    ammonia to 100 mL, filter derived.

     Alkaline sodium hypochlorite solution: fine to take 250 mg of sodium hypochlorite, sodium hydroxide, 2 500 mg, dissolved in 100 mL non-ammonia derived.

     0.5 mol / L sulfuric acid solution, 5 mmol / L ammonium sulfate solution, refer to literature method [3] preparation.

     2.2 Preparation of Standard Solution

     Were refined take "2.1" item ammonium sulfate stock solution 20,200,500,1 000,1 500,2 000,4 000 μL placed in 50

    mL flask and immediately add to the scale that no ammonia was 10,20, 50,100,150,200,400 μmol / L of ammonium sulfate series of standard solution.

     2.3 Preparation of standard curve

     Take "2.2" series of standard solution of ammonium sulfate item each 1 mL, respectively, a 10% sodium tungstate solution, 0.5 mol / L sulfuric acid solution of 0.5 mL, vortex turbulence 1 min, 4 000 r / min centrifugation 5 min, Sub-

    supernatant 1 mL, followed by adding phenol reagent and

    alkaline sodium hypochlorite solution of 1 mL, vortex 1 min water bath at 37 ? after 20 min, in order to correct the

    blank tube was measured after the OD value of 630 nm ; will be measured by the absorbance OD values of the concentration of

    linear regression handled regression equation: OD = 0.0017C 0.23, correlation coefficient r = 0.9964, according to (NH4) 2SO4 and the free ammonia and a quantitative relationship, that was the concentration of free ammonia In the 5 ~ 200μmol

    / L of the quantitative linear range (see Figure 1).

     2.4 Determination of clinical samples

     Reference to the literature at home and abroad, through the optimized method for the determination of BA: In vitro quantification of blood immediately joined the excess of 10%

    sodium tungstate, and 0.5 mol / L sulfuric acid solution, so that protein precipitation at the same time, blood ammonia and sulfuric acid to form sulfuric acid Ammonium and to survive in the blood filtrate, and then to sodium hypochlorite phenol

    reagent color wavelength of 630 nm was measured after the OD value, and can be quantitatively determined BA. Reposted elsewhere in the paper for free download http://

     2.5 Recovery Test Method

     Click "2.2" method of entry were the preparation of high, medium and low (400,200,40 μmol / L) of ammonium sulfate

    standard solution appropriate, depicting 1mL was added 1 mL normal plasma ammonia-free, after the precise amount of mixing to take a mL by 2.4, respectively, under the method precision

    by adding 0.5 mL 10% sodium tungstate, and 0.5 mol / L sulfuric acid solution of 0.5 mL, following by a corresponding

    under quasi-curve approach, and OD values were determined, the measured OD into the standard curve calculated by substituting

    the measured volume and the recovery rate, the results in Table 1. Table 1 ammonia recovery test (abbreviated)

     2.6 Precision tests

     The preparation of low, medium, high (25,50,75 μmol / L)

    3 containing different concentrations of ammonia-free solution

    to normal plasma, all three copies of ammonium sulfate, days 4 ? cold preservation, for every interval of 1 h by 2.3 under the standard curve method Determination of a second, continuous measurement 5 times. Another take the same samples

     18 ? cryopreservation, daily determination of a second, continuous measurement on the 5th. OD obtained by substituting into the standard curve equation calculated free ammonia concentration, the results in Table 2. Table 2 ammonia precision test (abbreviated)

     2.7 Stability Test

     Take adequate preparation of ammonium sulfate solution 75 μmol / L and normal plasma containing ammonia-free ammonium

    sulfate solution, respectively, placed in 4 ? ice 18 ?

    water bath and stored under the conditions of storage, each depicting a balanced 5 min after the 1 mL sample, according to "2.6" with precision of determination under, 4 ? specimens

    stored under the 1 d interval of 2 h each measured a times, each time the parallel determination of five copies, up to 14

    h were measured eight times; 18 ? under the conditions of

    preserved specimens from time to time determine a time each day, each parallel determination five copies of continuous determination of 7 d, the measured OD value of the standard

    curve equation by the actual amount of free ammonia, the results shown in Table 3. From the table we can see new blood in the free ammonia is very unstable and at room temperature and 4 ? cold preservation are unstable and should, within 2 h in the determination of the 18 ? is also useful to next

    stored within 24 h measured. Table 3 ammonia stability test (omitted)

     2.6 Clinical Application

     I choose my hospital inpatients the past two years, except for liver, gallbladder, kidney disease. 28 patients

    with cerebral infarction, of which 16 males and 12 females, mean age (55.8 ? 10.5) years of age; stroke patients with 25 cases, including 15 males and 10 females, mean age (60.4 ?

    15.4) years; encephalitis patients 10 cases, of which 6 were

    male and 4 female, mean age (39.8 ? 13.5) years of age. All

    patients were characterized by clinical and CT and (or) MRI examination. Control group, 25 cases, after checking all the healthy and normal, of which 13 males and 12 females, mean age

    (57.2 ? 14.3) years of age. All patients were in a coma period and the recovery of all to take the elbow vein 1 ~ 2 mL, heparin anticoagulation immediately determined using the above method of ammonia in the control group also measured blood ammonia.

     Results ammonia content of the control group (56.53 ?

    29.15) μmol / L, unconscious patients with cerebral infarction period of ammonia content (179.00 ? 108.59) μmol

    / L, recovery of (66.89 ? 26.67) μmol / L, coma period with

    the control The χ2 test differences between groups was

    statistically significant (χ2 = 12.68, P

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