Sodium azide reacts with lead and copper plumbing, to

form potentially explosive azides. When disposing of such

reagents flush with large volumes of water to prevent azide

build up. Exposed metal surfaces should be cleaned with

10% sodium hydroxide.

The reagents must be used only for the purpose

intended by suitably qualified laboratory personnel, under

appropriate laboratory conditions.

Stability and Preparation of Reagents

Buffer

Contents ready for use. Stable until expiry date when

stored at +2 to +8°C.

Substrate

Reconstitute the contents of one vial of substrate 2 with the

appropriate volume of buffer 1:

2.5 mL for the 20 × 2.5 mL kit

30 mL for the 4 × 30 mL kit

Stable for 2 weeks at +2 to +8°C for 5 days at +15 to

+25°C.

Standard

Dissolve the contents of one vial of standard 3 in 3.0 mL

of redistilled water, swirling gently for 30 mins before use.

Stable for 5 days at +2 to +8°C.

Materials Provided

¾ Buffer

¾ Substrate

¾ Standard.

Materials Required but not Provided

Randox assayed multisera level 2 and level 3.

Procedure Notes

In rare cases, a patient’s serum may give an increase in

absorbance rather than a decrease. The lipase activity

of these samples usually falls within the normal range.

Extremely high lipase activities can lead to considerable

substrate consumption, with A1 being less than 0.500.

In such cases, dilute the sample 1+9 with 0.9% NaCl

solution and repeat the assay. Multiply the result by 10.

It is preferable to use disposable cuvettes. Glass cuvettes

should be cleaned thoroughly especially after being used

for triglyceride or cholesterol assays.

Manual—Lipase

Procedure

Wavelength: 340 nm (Hg 365 nm or Hg 334 nm)

Cuvette: 1 cm light path

Temperature: 37°C

Measurement: Against air or distilled/deionized water

Pipette into cuvette:

Standard Sample

Macro Semi Macro Semi

Micro Micro

Contd...

522 Concise Book of Medical Laboratory Technology: Methods and Interpretations Reagent 2.5 mL 1.0 mL 2.5 mL 1.0 mL

Sample — — 0.1 mL 0.04 mL

Standard 0.1 mL 0.04 mL — —

Mix. Avoid the formation of foam. Read absorbance A1 of standard

and sample after 4 minutes. After a further 5 minutes read

absorbance A2 of standard and sample.

∆A of sample of standard = A1 – A2

Calculation

Calculate the assay factor as follows:

Activity standard Factor = ∆A standard

Sample lipase activity = Factor × ∆Asample

Quality Control

Randox Assayed Multi-sera, level 2 and level 3 are

recommended for daily quality control. Two levels of

controls should be assayed at least once a day. Values

obtained should fall within a specified range. If these values

fall outside the range and repetition exclude errors, the

following steps should be taken:

1. Check instrument settings and light source.

2. Check cleanliness of all equipment in use.

3. Check water, contaminants, i.e. bacterial growth may

contribute to inaccurate results.

4. Check reaction temperature.

5. Check expiry date of kit and contents.

Interference

Hemolysis interferes with the assay.

Normal Values

Serum: Up to 190 U/l (37°C).

It is recommended that each laboratory establish

its own reference range to reflect the age, sex, diet and

geographical location of the population.

Linearity

If the lipase activity exceeds 500 U/l, dilute the sample 1 +

1 with 0.9% NaCl solution and reassay. Multiply result by 2.

Normal Value

< 200 U/L with triolein; < 160 U/L with olive oil.

SI Units

Adults 13–141 U/L 0.22–2.40 µKat/L

Age 20–60 31–186 U/L 0.53–3.16 µKat/L

Over age 60 < 302 U/L < 5.13 µKat/L

Over age 90 26–267 U/L 0.44–4.54 µKat/L

Children 20–136 IU/L

Infants 9–105 IU/L

Clinical Relevance

Increased

Cholecystitis, cirrhosis, duodenal ulcers, fat embolism,

gallstone colic, pain (abdominal), pancreatic carcinoma,

pancreatic cholera, pancreatic trauma, pancreatitis,

peritonitis trauma, pancreatitis, peritonitis, renal

disease with impaired output, and strangulated bowel.

Drugs include bethanechol, heparin, and narcotic

analgesics.

Description

Lipase is a pancreatic enzyme that changes fats and

triglycerides into fatty acids and glycerol. The pancreas is

the only body organ that demonstrates significant lipase

activity. In acute pancreatitis, serum lipase begins to

increase in 2–6 hours, peaks at 12–30 hours, and remains

elevated, but slowly decreases for 2–4 days. Lipase rises

and falls in tandem with amylase in acute pancreatitis, but

is a more specific marker for this condition.

PHOSPHATASES

Phosphatases belong to the class of enzymes called

hydrolases, and they are characterized, by their ability to

hydrolyze a large variety of organic phosphate esters with

the formation of an alcohol and a phosphate ion.

Phosphatases of diagnostic significance are of two

kinds: alkaline phosphatase and acid phosphatase. These

are differentiated by their reaction in alkaline and acidic

medium. The pH for measuring the alkaline phosphatase

activity is 10, and for acid phosphatase, it is 5.0. Various

substrates have been used from time to time, but the

current use of p-nitrophenyl phosphate (PNPP) appears to

be universal. The PNNP is not a natural substrate for the

phosphatases. It is used because it gives a reasonably rapid

rate of reaction and because it is analytically convenient

to measure the product formed (p-nitrophenol). The

liberated phenol is yellow in color in alkaline medium

and is colorless in acid medium. Continuous assay can be

done for alkaline phosphatase by measuring the rate of

formation of p-nitrophenol at pH 10. For this one will need

a recording spectrophotometer. The manual two-point

method is described here. Contd...

Contd... Contd...

Enzymology 523

Units for Reporting Phosphatase Activity

Several units are used in expressing phosphatase activity—

Bodansky unit, King-Armstrong unit, Bessey-Lowry-Brock

unit and U/L (International unit). As the current trend is

to express all enzyme activity by the International unit,

one U/L signifies 1.0 mmole of chromogen from the

substrate used per minute (and thus releases 1.0 mmole

of chromogen from the substrate per minute) under the

conditions of the assay.