Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Alkaline Phosphatase - p-nitrophenol Method

     Introduction

Phosphatases are enzymes which catalyse the splitting of a phosphate from mono-phosphoric esters. Alkaline phosphatase (ALP), a mixture of isoenzymes from liver, bone, intestine and placenta, has maximum enzyme activity at about pH 10.5. Serum ALP measurements are of particular interest in the investigation of hepatobillary and bone diseases.

     Principle of the method

Paranitrophenyl phosphate, which is colourless, is hydrolysed by alkaline phosphatase at pH 10.5 & 37⁰C to form free paranitrophenol, which is coloured yellow. The addition of NaOH stops the enzyme activity and the final colour shows maximum absorbance at 410 nm.

     Specimen type, collection and storage

Serum or heparinized plasma can be used. Stable for 7 days at 2-8⁰C. The activity increases if samples are left at room temperature (25-35⁰C) for several hours.

     Reagents

All chemicals must be Analar grade.

     2-amino 2- methyl 1-propanol (AMP) buffer pH 10.5

Add 116 ml of AMP to 600 ml of distilled water. Mix and adjust the pH to 10.5 with 6 M HCI and then make up to 1 litre with distilled water. Stable for 6 months at 2-8⁰C.

     Magnesium chloride (1.5 mmol/1).

Dissolve 300 mg of magnesium chloride hexahydrate in distilled water and make up to 1 litre. Stable for 6 months at room temperature (25-35⁰ C)

     Substrate

Dissolve 83.5 mg of disodium paranitrophenyl phosphate in 1.0ml magnesium chloride solution. Stable for 24 hours at 2-8⁰C. This solution should be colourless; do not use it if the OD at 410nm > 0.800.

     Sodium hydroxide 0.25 M

Dissolve 10 g of NaOH in about 800 ml of distilled water and then make up to 1 litre with distilled water. Store in a polythene bottle at room temperature (25-35° C). Stable for 6 months.

     Stock paranitrophenol (PNP) 10.8 mmol/l.

Weigh out 150 mg of PNP and dissolve in about 80ml of NaOH (0.25M) and then make up to 100 ml with the same NaOH solution. Store ina brown glass bottle at room temperature (25-35⁰C). Stable for 3 months.

     Working PNP 54 m mol/l

Pipette 0.5 ml of the PNP stock solution into a 100ml volumetric flask and make up to the mark with NaOH solution (0.25 M). Prepare fresh before use.

     Equipment, glassware and other accessories

Refer to Section A (2), Introduction to SOP.

     Procedure

The protocol of the procedure is described below.

     Preparation of standards (SI -S6)

Pipette the following into appropriately labelled 18 x 150mm tubes

Set the spectrophotometer / filter photometer to zero absorbance at 410 nm / violet filter against 0.25M NaOH and measure the absorbance of the above standards.

     Enzyme measurement in test / QC

Pipette the following into another set of appropriately labelled 18 x 150 mm tubes.

(Note: NaOH should be added to each tube insequence maintaining timed intervals)

Mix and cool the tubes to room temperature (25-35⁰C). Measure the absorbance of test / QC at 410nm /violet filter, setting the spectrophotometer /filter photometer to zero with the blank.

     Calculation and calibration graph

The working PNP concentration is 54 m mol/L. Standard SI contains 0.5ml PNP

54
Concentration of PNP in SI = ------------- x 0.5 = 0.027m mol.
1000

ALP activity in U/L = Liberation of 1 m mol of PNP per minute at 37⁰C incubation per litre serum.

In the assay protocol, 0.05 ml serum is mixed with reagent and incubated for 15 minutes and the total volume is made up to 5.55ml. But the total volume in the case of each standard (SI to S6) is 5.0 ml.

PNP in umol/L or ALP activity in U/L in the test sample =

Test absorbance 0.027 5.55 1000
-------------------- x ------ x ------ x ------
Std absorbance 15 5.0 0.05

Test absorbance
= ----------------------- x 40
Std absorbance

i.e, ALP activity equivalent for S I = 40 U/L. Similarly the ALP activities represented by other standards are : S2 = 80, S3 = 160, S4 = 240, S5 = 320 and S6 = 400 U/L.

Construct a calibration graph by plotting the equivalent activity of ALP of the standards against their corresponding absorbance values. The measurable range with this graph is from 10 to 400 U/L.

Plot the absorbance values of test /QC on the calibration graph and read off the concentrations.

Once linearity is proved, it will be enough if a single standard is set up every time that patients' samples are analysed. Use standard S6 in the assay and calculate the results using the formula :

Test Absorbance
----------------------- x 400..... U/L.
Std Absorbance

     Analytical reliabilities

Refer to pages 7-9 of section 1 (General Introduction) on the use of internal QC and interpretation of daily QC data (for releasing patients' results).

Include one internal QC in every batch of samples analysed every day, irrespective of the number of samples in a batch. Since alkaline phosphatase isanalysed in a single batch in a day in an intermediate laboratory, it will not be possible to analyse several QC samples and calculate within-day precision. However, even if a single QC sample is analysed in a day, this value can be pooled with the preceding 20 values obtained in the previous days and between day precision can be calculated and expressed as %CV. Ensure that this is well within the acceptable limit, i.e. 10%.

Once a week it is good to analyse another QC serum from either a low QC or high QC pool.

"Assayed" QC sera with stated values (ranges) are available from several commercial sources, viz. Boehringer Mannheim, BioRad & Randox.

If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials.

     Hazardous materials

This procedure uses NaOH, which is caustic. Do not swallow, and avoid contact with the skin and mucous membranes. 4 nitro phenol is toxic. Do not pipette by mouth. Use a dispenser.

     Reference range and clinical interpretation

Serum alkaline phosphatase (adults) - 40-125 U/L. (Levels up to 3 times this may be normal in children) Liver, bone and placenta contain very high concentrations of ALP. Therefore, increase in ALP activity is usually related to hepatobiliary and bone disorders. Increased ALP levels are observed in liver diseases, osteomalacia, rickets and bone disorders. Moderate elevations are sometimes noted in congestive heart failure, intestinal disease and intra-abdominal bacterial infections.

     Limitations

Avoid exposure of the freshly dissolved substrate to strong sunlight, since the reagent is light sensitive. The change in absorbance will increase with an increase in temperature, since the pH of the reagent will be different at different temperatures. Haemolysed specimens are not suitable for analysis. Inform the requesting physician of the problem and ask for another specimen.

Any sample that gives a value > 400 U/L should be diluted either 1:2 or 1:3 with 0.9 g% sodium chloride solution and the correct value obtained by multiplying it by the appropriate dilution factor.

     Reference

1.      Bomers GN, McComb RB (1975). Clin.Chem. 21: 1988-1995.