dr sidhu.pptx

phd biochemistry

1. Urea is the main end product of protein catabolism. Deamination of amino acids takes place in the liver. The site of urea cycle is liver , whereby ammonia released is converted into urea and is excreted through urine. Introduction

2. It represents about 40-50% of nonprotein nitrogen of blood and about 80-90% of total urinary nitrogen excretion. Urea nitrogen varies directly with protein intake and inversely with rate of excretion of urea.

3. Determination of Blood Urea Blood urea can be estimated either by: Enzymatic method employing urease 2. Non-Enzymatic method

4. Enzymatic Method Nessler’s Method Berthelot Reaction

5. Nessler’s Method Urea is converted to ammonia by the enzyme urease. Ammonia reacts with Nessler’s reagent giving rise to a brown coloured compound which is read at 450 nm. The enzyme acts optimally at 55 , Ph 7.0-8.0 and is inhibited by ammonia and flouride . Nessler’s reagent is Potassium mercuric iodide.  

6. Disadvantages of Nessler’s Method Colour instability. Turbidity. Nonlinear calibration besides suceptibility to contamination with NH3 from the laboratory and endogenous ammonia in the sample.

7. Berthelot Reaction Ammonia released by the action of urease reacts with phenol in the presence of hypochlorite to form an indophenol which gives a blue coloured compound in alkali. Nitroprusside acts as a catalyst increasing the rate of reaction.

8. Berthelot Reaction Advantages The colour form is soluble. It obeys Beer’s and Lambert’s law at high concentration. It is more sensitive technique. Disadvantages Contamination of NH3 can occur endogenously and exogenously. This effects the result . So it is replaced by the glutamate dehydrogenase method.

9. Glutamate Dehydrogenase Method (GLDH Kinetic Method) In this method glutamate production from ammonia and 2-oxaloglutarate is measured by the absorbance change at 340 nm with conversion of NADH to NAD+. Each molecule of urea hydrolysed causes the production of two molecules of NAD+.

10. Non-Enzymatic Method ( Diacetyl Monoxime Method) In this method urea may be measured without conversion to ammonia by making use of ability to react with diacetyl monoxime . Principle: Under acidic conditions when urea is heated with compounds such as diacetyl monoxime coloured products are formed. On heating, it decomposes to give diacetyl and hydroxylamine, which then condenses with urea to give diazine .

11. Reagents of DAM Method Reagent A - Fecl3 + Orthophosphoric acid Reagent B - DAM + Thiosemicarbazide H2SO4 Standard (40 mg%)

12. Procedure Reagents Test Standard Blank Reagent A 0.3 ml 0.3 ml 0.3 ml Reagent B 0.3 ml 0.3 ml 0.3 ml Reagent C 0.4 ml 0.4 ml 0.4 ml Sample 0.01 ml - - Std. (40 mg%) - 0.01 ml - D.w Mix well and boil test - tubes for 15mintues. - Cool them and take reading at 520 nm. 0.01 ml

13. Calculations : Blood urea x Conc. Of Std. (mg%) Normal Value of blood urea is 20-40 mg% Blood Urea Nitrogen(mg%) = Blood Urea x 0.467 Linearity : The procedure is linear upto 100 mg%.  

14. Interpretations: Higher in men than in women. There is a slow rise with age. Pre-Renal Uremia – Important cause is reduction of plasma. This diminishes the blood pressure and GFR. This leads to urea retention. Renal Uremia – Renal diseases which diminishes the GFR lead to urea retention. Post Renal Uremia – Obstruction to the flow of urine after it leaves the kidneys leads back pressure on the renal pelvis and diminishes GFR.

15. Urinary Urea Urinary urea can be estimated as blood urea but urine must be diluted 1:10 or 1:20 with D.w . Total daily excretion of urinary urea is 30g and avg. of 20g/L on ordinary diet. The excretion varies with dietary intake of protein. Normal value is 10-30 gm /L.

16. Urea Clearance It is the amount of plasma in ml being cleared of urea by the both the kidneys in every minute. Urea Clearance = UV/P U is Urine conc. , V is Urine Volume and P is Plasma conc.

17. Interpretations : In acute renal impairment urea excretion falls and serum conc. rises rapidly, so urine excretion over successive 24hours period is helpful in the management of cases of acute renal failure.

18. Urea Clearance Calculations : If the excretion of urine is at the rate of 2ml or more per min, the clearance is designed as Maximum urea clearance and calculated as: If the excretion of urine is at a less than 2ml/min the clearance is designed as Std. Urea Clearance and calculated as : Normal Value : is 54ml/min  

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