For the determination of urea in serum, plasma and urine

(for in vitro diagnostic use only)

Summary

Urea is the end product of protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urease hydrolyzes urea to ammonia and CO2. The ammonia

formed further reacts with a phenolic chromogen and

hypochlorite to form a green colored complex. Intensity of

the color formed is directly proportional to the amount of

urea present in the sample.

 Urease

Urea + H2O Ammonia + CO2

Ammonia + Phenolic chromogen + Hypochlorite

 Green colored complex

Normal Reference Values

Serum/plasma : 14–40 mg/dL

Urine : Upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 assays 3 × 75 assays

L1: Buffer reagent 75 mL 3 × 75 mL

L2: Enzyme reagent 7.5 mL 3 × 7.5 mL

L3: Chromogen reagent 15 mL 45 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/Stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use for the given procedure.

Working enzyme reagent: For the flexibility and

convenience in performing large assay series, a working

enzyme reagent may be made by pouring 1 bottle of L2

(Enzyme reagent) into 1 bottle of L1 (Buffer reagent). For

smaller series combine 10 parts of L1 (Buffer reagent) and

1 part of L2 (Enzyme reagent). Use 1 mL of the working

reagent per assay instead of 1 mL of L1 and 0.1 mL of L2

as given in the procedure. The working enzyme reagent is

stable for at least 4 weeks when stored at 2–8°C.

Working chromogen reagent: For larger volume cuvettes,

dilute 1 part of L3 (Chromogen reagent) with 4 parts of

fresh ammonia free distilled/deionised water. Use 1 ml

of working chromogen instead of 0.2 mL in the assay. The

working chromogen reagent is stable for atleast 8 weeks

when stored at 2–8°C in a tightly stoppered plastic bottle.

Sample Material

Serum, plasma, urine. Dilute urine 1 + 49 with distilled

water before the assay (results × 50). Urea is reported to be

stable in the serum for 5 days when stored 2–8°C.

Procedure

Wavelength/filter : 570 nm (Hg 578 nm)/yellow

Temperature : 37°C/RT

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank (B),

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Buffer reagent (L1 ) 1.0 1.0 1.0

Enzyme reagent (L2) 0.1 0.1 0.1

Distilled water 0.01 — —

Urea standard (S) 0.01 —

Sample — — 0.01

Mix well and incubate for 5 minutes at 37°C or 10 minutes at RT

(25°C)

Chromogen reagent (L3) 0.2 0.2 0.2

470 Concise Book of Medical Laboratory Technology: Methods and Interpretations Mix well and incubate for 5 minutes at 37°C or

10 minutes at RT (25°C). Measure the absorbance of the

Standard (Abs S), and Test sample (Abs T) against the

Blank, within 60 minutes.

Calculations

 Abs T

Urea in mg/dL = ________ × 40 Abs S

Urea nitrogen in mg/dL = Urea in mg/dL × 0.467

Linearity

This procedure is linear upto 250 mg/dL. Using the working

chromogen reagent (1 mL) the linearity is increased to

400 mg/dL. If values exceed this limit, dilute the serum

with normal saline (NaCL 0.9%) and repeat the assay.

Calculate the value using the proper dilution factor.

Note

Any contamination by ammonia or ammonium salts lead

to erroneous results, hence plasma should not be collected

with fluoride or heparin ammonium salts. The working

enzyme reagent is not stable at elevated temperatures and

should be stored back at 2–8°C immediately after use. The

chromogen reagent contains chlorine. The bottle should

be opened only when required and closed tightly after use

to prevent the loss of active chlorine.

System Parameters

Reaction : End point No. of read :

Wavelength : 570 nm Interval :

Zero setting :  Reagent blank Sample

volume

: 0.01 mL

Incubation

temperature

: 37°C/RT Reagent

volume

: 1.30 mL

Incubated

time

:  5 min + 5 min

or

10 min +10 min

Standard

factor

React slope

: 40 mg/dL

: Increasing

Delay time : Linearity : 250 mg/dL

Read time : .... Units : mg/dL

Urea (GLDH Kinetic Method)

(Courtesy: Tulip Group of Companies)

For the determination of urea in serum or plasma (for

in vitro diagnostic use only).

Summary

Urea is the end product of protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urease hydrolyzes urea to ammonia and CO2. The ammonia

formed further combines with a ketoglutarate and NADH

to form glutamate and NAD. The rate of oxidation of NADH

to NAD is measured as a decrease in absorbance in a fixed

time which is proportional to the urea concentration in the

sample.

 Urease

Urea + H2O + 2 H+ 2 NH4 + CO2

 GLDH

2 NH4

+ + 2 α Ketoglutarate + ↓

 2 NADH 2 L-glutamate + 2

 NAD+ + 2 H2O

Normal Reference Values

Serum/plasma : 14–40 mg/dL

Urine : Upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 mL 2 ×75 mL

L1: Enzyme reagent 60 mL 2 × 60 mL

L2: Starter reagent 15 mL 2 × 15 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: For sample start assays a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Enzyme reagent).

This working reagent is stable for at least 10 days when

stored at 2–8°C. Alternatively for flexibility as much of

working reagent may be made as and when desired by

mixing together 4 parts of L1 (Enzyme reagent) and 1

part of L2 (Starter reagent). Alternatively 0.8 mL of L1 and

0.2 mL of L2 may also be used instead of 1 mL of the

working reagent directly during the assay.

Clinical Chemistry 471

Sample Material

Serum, plasma, urine. Dilute urine 1 + 49 with distilled

water before the assay (results × 50 ). Urea is reported to be

stable in the serum for 5 days when stored at 2–8°C.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Substrate Start Assay

Pipette into a clean dry test tube labeled standard (S) or

test (T):

Addition

Sequence

(S)/(T)

37°C/ 30°C/25°C

Enzyme reagent (L1) 0.8 mL