ALT_riferimento
Reagents
1.Tris(hydroxymethyl)aminomethane (Tris)
(C4H11NO3), M r=121.1
2.L-Alanine, free acid (C3H7NO2), M r=89.09
3.2-Oxoglutaric acid, crystallized disodium salt, dihy-
drate (C5H4O5Na2· 2 H2O), M r=226.1
4.β-Nicotinamide adenine dinucleotide, reduced form
(NADH) (C21H27N7O14P2Na2), disodium salt, M r=709.4 Pyridoxal-5’-phosphoric acid, monohydrate
(C8H10O6NP · H2O), M r=265.2
https://www.wendangku.net/doc/3512069131.html,ctate dehydrogenase (LDH, EC 1.1.1.27), from pig
skeletal muscle, in glycerol
6.Sodium chloride (NaCl), M r=58.44
7.Sodium azide (NaN3), M r=65.01
8.Hydrochloric acid (HCl), M r=36.46, 1mol/l
9.Bovine serum albumin, Fraction V, M r=68000 Note: Ammonium sulphate suspension of the LDH reagent enzyme may not be used due to glutamate de-hydrogenase activity in the sample.
Note: The reagent enzyme preparation (LDH and bovine serum albumin) must be free from glutamate dehydrogenase and ALT. The absence of these contam-inants must be declared by the manufacturer or exper-imentally investigated in the reference laboratory. Note: Contamination of 2-oxoglutarate by pyruvate leads to a consumption of NADH and decreases the ini-tial absorbance of the final complete reaction mixture. Reagents of the highest purity must be used. If a chem-ical is suspected of containing impurities affecting the catalytic activity of the analyte, further investigations must be performed, https://www.wendangku.net/doc/3512069131.html,parisons with products from different manufacturers and different lots. It is recommended to use reagents which have already tested and approved in comparisons.
Charts for the Adjustment and the Control of
the pH Values (Procedure for the Adjustment of pH Values at Temperatures Diverging from 37°C)
Both the thermometer and the pH electrode are sus-pended in the mixed solution simultaneously. The stirred solution is then titrated to the pH value listed in the chart for the actually measured temperature. The speed of agitation should be the same during the cali-bration, the control and the adjustment of the pH value. The pH electrode should be positioned in the centre of the stirred solution.
The fact that the temperature can change during the titration must be taken into account. For this reason, the temperature in the proximity of the target value should be controlled again and the target pH value cor-rected according to Tables3 and 4, if necessary. The same applies to the adjustment of the temperature compensation of the pH meter.
Preparation of Solutions
The given mass of the compounds for the preparation of solutions refers to 100% content. If the content of the reagent chemical employed is less (e.g.yz %), the amount equivalent to the given mass is calculated by the use of a factor: F content =100/yz.
Highly purified water with a quality comparable to bi-distilled water (conductivity <2μS/cm, pH 6–7, sili-cate <0.1mg/l) shall be used for the preparation of the reagent solutions. The expanded (k=2) combined un-certainty (normally distributed) of each weighing pro-cedure (including the uncertainty of the purity of the substance) shall be ≤1.5%.Solution 1
1.47 g (121.2mmol/l) Tris
5.61 g (630.0mmol/l) L-Alanine, free acid 0.052 g (8.00mmol/l) Sodium azide
–Dissolve in about 80ml water.
–Adjust pH (37°C) 7.15 with 1mol/l hydrochloric acid.
–Transfer to a 100ml volumetric flask.
–Equilibrate the volumetric flask and water to 20°C.–Fill the water (20°C) up to the calibration mark of the volumetric flask.
Stability at 2°C – 8°C: 3 months Solution 2
1.47 g (121.2mmol/l) Tris
0.052 g (8.00mmol/l) Sodium azide –Dissolve in about 80ml water.
–Adjust pH (37°C) 7.15 with 1mol/l hydrochloric acid. –Transfer to a 100ml volumetric flask.
–Equilibrate the volumetric flask and water to 20°C.–Fill the water (20°C) up to the calibration mark of the volumetric flask.
Stability at 2°C – 8°C: 3 months
Solution 3 Array 16.7mg (6.300mmol/l) Pyridoxal-5’-phosphoric acid, monohydrate
–Dissolve in about 6ml Solution 2.
–Transfer to a 10ml volumetric flask.
–Equilibrate volumetric flask and Solution 2 to 20°C.
–Fill Solution 2 (20°C) up to the calibration mark of the volumetric flask.
–Store protected from light (e.g.in a brown bottle). Stability at 2°C– 8°C: 1 week
Solution 4
16.1mg (11.34mmol/l) NADH, disodium salt
–Dissolve in 2.00ml Solution 2.
–Store protected from light (e.g.in a brown bottle). Stability at 2°C– 8°C: 1 week
Diluent for reagent enzymes
1.20 g Bovine serum albumin
0.90 g (154mmol/l) NaCl
–Dissolve in about 80ml water.
–Transfer to a 100ml volumetric flask.
–Equilibrate the volumetric flask and water to 20°C.
–Fill the water (20°C) up to the calibration mark of the volumetric flask.
Stability at 2°C– 8°C: at least 1 month
Solution 5
Lactate dehydrogenase solution [3.57 mkat/l (214 kU/l) at 37°C]
–Dilute the LDH stock solution with diluent for reagent enzymes (see above) so that the dilution exhibits a catalytic LDH concentration of 3.57 mkat/l (214 kU/l) at 37°C.
Example: LDH stock: catalytic LDH concentration of the enzyme stock solution in mkat/l, see Appendix 1; V diluent: volume of diluent for reagent enzymes for the dilution of the LDH stock solution. V diluent=0.1 (LDH stock–3.57)/ 3.57. Add to 0.1ml enzyme stock solution the volume (ml) of V diluent .
Stability at 4°C: at least 2 days
Reaction solution
10.0ml Solution 1
0.200ml Solution 3
0.200ml Solution 4
0.100ml Solution 5
–Mix thoroughly and store light protected.
Stability at 2°C– 8°C: 1 day
Start reagent solution
0.407 g (180.0mmol/l) 2-Oxoglutaric acid, disodium salt, dihydrate
–Dissolve in about 6ml water.
–Transfer to a 10ml volumetric flask.
–Equilibrate the volumetric flask and water to 20°C.
Upper limit of the measurement range Array If the change of absorbance exceeds 0.0025 s–1 (0.15min–1) in the measurement interval, an analytical portion of the sample must be diluted with 9g/l (154mmol/l) sodium chloride solution and the mea-surement procedure must be repeated with the diluted specimen. The obtained value must then be multiplied by the corresponding factor of the dilution.
Sources of error
High pyruvate concentrations in the sample lead to high NADH consumption during the incubation period. This can reduce the upper limit of the measurement range and considerably lower the results of analyses.
Calculation
The temporal change of absorbance (s–1) is calculated with the analysis of regression (method of the least squares). After subtraction of the reagent blank rate the corrected change of absorbance is multiplied by the factor:
F=1905 (measurement at 339nm, ε339(NADH)= 630m2/mol)
The catalytic concentration of ALT is calculated in μkat/l.
?A/?t ALT: change of absorbance (in s–1) after correction of the reagent blank rate
b ALT: catalyti
c concentration of ALT
b ALT=1905 ?A/?t ALT
The catalytic concentration in μkat/l can be converted to U/l by multiplication by the factor f=60.
Preliminary Upper Reference Limits
The preliminary upper reference limits for adults (≥17 years) were investigated separately for men (n=422) and women (n=411).
Gender Upper reference limit* (and 90% confidence interval)
Women0.56 μkat/l (0.51 μkat/l– 0.60 μkat/l)
Men0.74 μkat/l (0.71 μkat/l– 0.77 μkat/l)
Gender Reference limit* (and 90% confidence inter-val)
Women34 U/l (31 U/l– 36 U/l)
Men45 U/l (42 U/l– 45 U/l)
*The upper reference limits are the 97.5th percentiles of the reference collectives. Inside brackets are the 90% confidence intervals of the 97.5th percentiles.
Appendix 1: Determination of the Catalytic Concentration of LDH in the Enzyme Stock Solution Additional reagents
Pyruvic acid, monosodium salt (C3H3O3Na), M r=110.0
LDH concentration in the final solution of Step 2. For calculation of the catalytic LDH concentration in the en-zyme stock solution (LDH stock) this result must be multi-plied by the dilution factor:
F dilution=40401
Calculation:
?A/?t LDH: change of absorbance (in s–1) in the reaction mixture after subtraction of the reagent blank rate LDH stock=1905 · 40401 ·
?A/?t LDH
The catalytic concentration in μkat/l can be conver-ted to kU/l by multiplication by the factor f=0.06. Appendix 2: Changes in the Reference Procedure for Measurements at 37°C Compared with the Reference Method for Measurements at 30°C as Described in
the Original IFCC Document
The primary reference procedure is deduced from the IFCC reference method (1) which provides optimised conditions for the measurement of catalytic activity concentrations of ALT. The measurement temperature of 37°C instead of 30°C requires only minimal changes of certain measurement parameters to retain the opti-mum measurement conditions. The modifications are listed and commented on in Table9. Furthermore, if in comparison to the 30°C reference method a more ac-curate specification has become necessary for improv-ing the high standardization of the measurements, it is also described here.
References
1.Bergmeyer HU, H?rder M, Rej R. International Federation of
Clinical Chemistry (IFCC). Approved recommendation (1985) on IFCC methods for the measurement of catalytic concentrations of enzymes. Part 3. IFCC method for alanine aminotransferase. J Clin Chem Clin Biochem 1986; 24:481–
95.