Determination of Urea (BUN) in serum

1. Determination of Urea (BUN) in serum ( Urease method )

2. 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.

3. 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.

4. N H U r e a 3 D i e t a r y a b s p r o t e i n s o n r o K e t o n e b o d i e s i p t a t i n i o m n a e d α - K e t o a c i d O x i d a t i o n d e g r a d a t i o n T i s s u e A m i n o a c i d p r o t e i n s m e t a b o l i c p o o l s y n t h e s i s d e G l u c o s e c a r b o x y l A m i n o a c i d s a c o n v e r s i o n t i o n s y n t h e s i z e d A m i n e N o n - p r o t e i n n i t r o g e n C O 2 c o m p o u n d s The sources and fates of AAs Sources of amino acids Fates of amino acids Synthesis of proteins

5. Deamination of AAs Four types: transamination oxidative deamination non-oxidative deamination union deamination

6. Ammonia is toxic , how to transport in blood ? 1. Alanine-glucose cycle 2. Transport by Gln

7. Pyruvate Ala Pyruvate Alanine-glucose cycle

8. N H C O N H C O O H 3 2 A T P + P i A D P C H C H 2 2 C H C H 2 2 谷氨酰胺酶 C H N H C H N H 2 2 H O N H 2 3 C O O H C O O H Glu Gln glutamine Brain\muscle ？ NH3 deamination liver H2O hydrolysis Glutamic acid

9. CO2 + NH3+ H2O 2ATP N-acetylglutamic acid Carbamoyl phosphate Pi citrulline ornithine citrulline α-ketoglutaric acid Amino acids ATP Asp AMP + PPi ornithine Arginino succinate Glutamic Acid oxaloacetic acid α-keto acid urea Arg fumarate malic acid Urea cycle 2ADP+Pi mitochondria in cytosol 目 录

10. 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.

11. 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.

12. 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.

13. glomerular filtration rate=GFR plasma creatinine= Pcr plasma urea-Purea urine volume= V urine urea- Uurea urine protein urine glucose hematuria Tests of renal function

14. 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.

15. Plasma Creatinine and Urea Concentration- hyperbolic correlation • 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 %. pCr, pUrea Normal range-> 140 mL/min (100%) 0mL/min (0%) GFR 50%

16. 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.

17. ( Urease method ) urease Urea ammonia + CO2 [ 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. nitroso sodium ferricyanide blue colored compound Ammonia + sodium hypochlorite + phenol

18. Reagents: • urease:5000U/L • color reagentⅠ : sodium hypochlorite 10% • color reagent Ⅱ: Phenol 5% • Standard solution: 7.14mmol/L Sample: Serum or plasma are also used. No hemolysis , no CM.

19. [PROCEDURE]

20. At C = ×Cs As [ CALCULATION ] ( Standard solution: 7.14mmol/L ) [ Reference ] 2.8 mmol/L ~7.2 mmol/L

21. Next experiment • Separation of Hemoglobin and Riboflavin by Gel Filtration Chromatography（p50-52）