Determination of Urea (BUN) in serum

( Urease method )

DESCRIPTION

Urea is a waste product which produced in the liver, dissolved in blood (in a concentration of 2.5 - 7.5 mM), and secreted by the kidney.

Urea also plays a very important role in protein catabolism, removal of toxic ammonia from the body.

Urea determination is very useful for the medical clinician to assess kidney and other organs function of patients.

•The molecule has two amine (-NH2) residues joined by a

carbonyl (-CO-) functional group.

•Urea is derived from dietary protein and endogenous

protein catabolism.

Urea

Synthesis of proteins

Fates of amino acidsSources of amino acids

The sources and fates of AAs

Amino acidmetabolic pool

deamination

decarboxylation

NH3

α-Keto acid

Ketone bodies

Oxidation

Glucose

Urea

AmineCO2

conversion

Non- protein nitrogen compounds

absorption

degradation

synthesis

Dietary proteins

Tissue proteins

Amino acids synthesized

Four types:

transamination

oxidative deamination

non-oxidative deamination

union deamination

Deamination of AAs

• Ammonia is toxic , how to transport in blood ?

1. Alanine-glucose cycle

2. Transport by Gln

Ala

Pyruvate

Pyruvate

Alanine-glucose cycle

H2O

NHNH33COO

(CH2)2

CH NH3

COO

+

(CH2)2

CH NH3

COO

CONH2

+

glutamine Glutamic acid

hydrolysis

deaminationliver

GlnGlu

谷氨酰胺酶

H 2 ON H 3

+P iA D PA T PN H3 C O N H 2

C H 2

C H 2

C H N H 2

C OO H

C OO H

C H 2

C H 2

C H N H 2

C OO H Brain\muscle

？

2ADP+Pi

CO2 + NH3 + H2O

Carbamoyl phosphate

2ATP N-acetylglutamic acid

Pi

ornithine citrulline

Amino acids

α-ketoglutaric acid

Glutamic Acid

α-keto acid

citrulline

Arginino succinate

AspATP

AMP + PPi

Arg

ornithine

urea

mitochondria

in cytosol目 录

fumarate

malic acid

oxaloacetic acid

Urea cycle

• BUN teat is a measure of the amount of nitrogen in the blood that comes from urea.

BUN (Blood Urea Nitrogen)

Non-protein nitrogen (or NPN):

which are not proteins but also contain nitrogen , mai

nly is the final product in the body , such as urea, uric

acid, creatine, creatinine and amino acids.

★ BUN is used to assess the state of protein metabolism. Increased production occurs on high protein diets or after gastrointestinal hemorrhage and when there is increased tissue breakdown as in starvation, trauma and inflammation.

BUN

★ It is used as a marker of renal function. A plasma urea concentration above 15 mmol/l almost certainly indicates renal impairment.

• The plasma urea is the most useful test of 'renal excretory function', as it correlates well with the clinical consequences of retained metabolic products (uremia) in renal insufficiency.

Increased Urea

A marked and prolonged increase in urea level is indicative

of damaged renal function. Depending on the duration of the

damage, the term acute or chronic renal failure is used.

Disease causing obstruction of urine outflow may also lead to

kidney failure, e.g. urethral strictures, prostatic enlargement and

cancer of the bladder.

Decreased Urea Low level of urea may be found in pregnancy, protein d

eficiency, severe liver disease and water overload.

Tests of renal functionTests of renal function

glomerular filtration rate=GFR

plasma creatinine= Pcr

plasma urea-Purea

urine volume= V urine urea- Uurea

urine protein urine glucose hematuria

•Glomerular filtration rate (GFR): describes the flow rate of filtered fluid through the kidney.•The capacity of the normal kidney to excrete urea is high and in the presence of normal renal function urea levels rarely rise above normal despite increased production.

Plasma Creatinine and Urea Concentration- hyperbolic correlation

GFRGFR 50% 50%

pCr,pCr,pUreapUrea

140 mL/min140 mL/min(100%)(100%)

00 mL/minmL/min(0%)(0%)

•Plasma urea concentration increases with decreased GFR.•In the absence of increased urea production, plasma urea does not usually rise above normal until GFR has fallen by at least 50 %.

NormalNormal rangerange->->

As a kidney function test, urea is inferior to serum creatinine because:

High protein diet increases urea formation.

Any condition of proteins catabolism urea formation.

Urea test is a useful test but must be

interpreted with great care.

Most useful when considered along with

creatinine.

[ PRINCIPLE ]• Urea is catalized by urease to form ammonia and carbon dioxide. • Ammonia combine with sodium hypochlorite in presenst of nitroso sodium ferricyanide , to form blue colored compound. • The absorption is proportional to the concentration of ammonia.

Ureaurease

ammonia + CO2

Ammonia + sodium hypochlorite + phenol

nitroso sodium ferricyanide blue colored

compound

( Urease method )

Reagents:

1.urease:5000U/L

2.color reagent : sodium hypochlorite 10%Ⅰ

3.color reagent : Phenol 5%Ⅱ

4.Standard solution: 7.14mmol/L

Sample:

Serum or plasma are also used.

No hemolysis , no CM.

B S T

Working (ml) 0.3 0.3 0.3

Standard solution(μl)

_ 10 _

Sample(μl) _ _ 10

Mix, 37oc,10min

color reagent Ⅰ(ml)

0.2 0.2 0.2

color reagent Ⅱ(ml)

0.2 0.2 0.2

Mix, 37oc, 5 min

ddH2O(ml) 3.0 3.0 3.0

Set zero with B , 620nm, At , As

[PROCEDURE]

[ CALCULATION ]

C = At

As×Cs

[ Reference ]

2.8 mmol/L ~7.2 mmol/L

( Standard solution: 7.14mmol/L )

Next experiment

Separation of Hemoglobin and Riboflavin by

Gel Filtration Chromatography（ p50-52）