Transcript Urea cycle

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.
Urea
•The molecule has two amine (-NH2) residues joined by a
carbonyl (-CO-) functional group.
•Urea is derived from dietary protein and endogenous
protein catabolism.
The sources and fates of AAs
Sources of amino acids
Fates of amino acids
NH3
Dietary
proteins
Ketone bodies
Tissue degradation
Amino acid
proteins synthesis metabolic pool
Amino acids
synthesized
Urea
α-Keto acid
Oxidation
Glucose
conversion
Non- protein nitrogen
compounds
CO2
Amine
Synthesis of proteins
Deamination of AAs
Four types:
transamination
oxidative deamination
non-oxidative deamination
union deamination
• Ammonia is toxic , how to transport in blood ?
1. Alanine-glucose cycle
2. Transport by Gln
Pyruvate
Ala
Pyruvate
Alanine-glucose cycle
N H
C O O H
C H
2
C H
2
3
A T P
A D P+ P i
谷氨酰胺酶
C H N H
2
N H
C O O H
3
H
2
O
Glu
C O N H
C H
2
C H
2
C H N H
2
2
Brain\muscle
C O O H
Gln
？
CONH2
(CH2)2
HC
+
NH3
COO
glutamine
deamination
H2O
hydrolysis
NH3
COO
(CH 2)2
HC
+
NH 3
COO
Glutamic acid
liver
Urea cycle
CO2 + NH3 + H2O
2ATP
N-acetylglutamic acid
2ADP+Pi
Carbamoyl phosphate
ornithine
mitochondria
Pi
citrulline
citrulline
ATP
AMP + PPi
ornithine
Asp
α-ketoglutaric
acid
Amino
acids
Arginino succinate
urea
in cytosol
oxaloacetic
acid
Arg
Glutamic α-keto
Acid
acid
fumarate
malic acid
目录
BUN (Blood Urea Nitrogen)
• BUN teat is a measure of the amount of nitrogen in the blood
that comes from urea.
Non-protein nitrogen (or NPN):
which are not proteins but also contain nitrogen ,
mainly is the final product in the body , such as urea,
uric acid, creatine, creatinine and amino acids.
BUN
★ 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.
★ 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
deficiency, severe liver disease and water overload.
Tests 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
pCr,
pUrea
•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 %.
Normal
range->
0 mL/min
GFR 50%
(0%)
140 mL/min
(100%)
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.
( Urease method )
[ 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.
Urea
urease
ammonia + CO2
Ammonia + sodium hypochlorite
+ phenol
nitroso sodium
ferricyanide
blue colored
compound
Reagents:
1. urease:5000U/L
2. color reagentⅠ : sodium hypochlorite 10%
3. color reagent Ⅱ: Phenol 5%
4. Standard solution: 7.14mmol/L
Sample:
Serum or plasma are also used.
No hemolysis , no CM.
[PROCEDURE]
B
S
T
Working (ml)
0.3
0.3
0.3
Standard
solution(μl)
_
10
_
Sample(μl)
_
_
10
Mix, 37oc,10min
color reagentⅠ
(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
Set zero with B , 620nm, At , As
3.0
[ CALCULATION ]
At
C=
×Cs
As
( Standard solution: 7.14mmol/L )
[ Reference ]
2.8 mmol/L ~7.2 mmol/L
Next experiment
 Separation of Hemoglobin and Riboflavin by
Gel Filtration Chromatography（p50-52）