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)