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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)