Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

Urea is the major end product of protein metabolism In humans.

Its formed in liver. Urea biosynthesis occurs in four stages: (1) transamination, (2) oxidative

deamination of glutamate, (3) ammonia

transport, (4) reactions of the

urea cycle

ammonia is produced in various ways by tissues are rapidly removed from circulation by the liver and converted to urea. This is essential, since ammonia is toxic to the central nervous system.

Urea is water soluble ,conversion of ammonia to urea is a kind of detoxification.

This is the importance of urea excretion and its estimation.

Any defect in any of the enzyme function of urea cycle can lead to various metabolic disorders.

More than 90% of urea is excreted through the kidneys(glomeruli), with losses through GIT and skin in minor fraction.

Determination of blood urea is important not only in many diseases of kidney but in a wide range of conds which r not primarily renal.

Causes of increase in serum urea

Causes r divided in pre-renal , renal and post-renal

PRE-RENAL :

dehydration pyloric n intestinal obstruction with vomiting sever n prolonged diarrhea chrn intestinal obstruction without vomiting ulcerative colitis with severe chloride loss diabetic coma

RENAL : acute glomerulonephritis malignant hypertension chronic pyelonephritis hydronephrosis congenital cystic kidney renal tuberculosis calcium deposition in kidney in hyperthyroidism n

hypervitaminosis D.

POST RENAL: enlargement of prostate

stones in urinary tract

stricture of the urethra

tumors of the bladder affecting the ureter

Cirrhosis of liver

Acute yellow atrophy of liver

Severe acidosis

Nephritis

Pregnancy

BLOOD UREA: Diacetyl monoxime method principle

when urea is heated with diacetyl(,containing two adjacent carbonyl group) under acidic condition yellow coloured compound is formed.the OD of the colour developed can be read at 420 nm.intensity of the color depends on conc of urea in serum.

additions BLANK STANDARD TESTProtein free filtrate

_ _ 1ml

Working standard _ 1ml _Distilled water 1ml _ _DAM reagent 0.4ml 0.4ml 0.4mlAcid mixture 1.6ml 1.6ml 1.6ml

OD of the test × conc of standard × 100 OD of the standard vol of the test

= T/S×100

BUN(blood urea nitrigen): = blood urea level× 28/60

Normal value - 5 -20 mg/dl

Principle: urease hydrolyses urea to ammonia and

CO2.the ammonia formed further reacts with a phenolic chromogen and hypo chloride to form a green colored complex. Intensity of colour is directly proportional to the amount of urea present in the sample. addition blank standard test

Working reagent

1.0 ml 1.0ml 1.0ml

Distilled water

0.01ml - -

Urea standard

- 0.01ml -

sample - - 0.01mlChorogen reagent

0.2ml 0.2ml 0.2ml

calculation: = T/S × 40

SERUM – 15 to 40mg/dl

The hypobromite method Principle: the vol of nitrogen liberated by the action of

hypobromite on the urea present in the urine is measured. the action is usually though not accurately, expressed

URINE - 15 to 30mg/day.

Urea clearance is less than GFR.Maximum urea clearance: =U × V/P U=mg of urea /ml of urine p=mg of urea/ml of plasma v=mg of urine excreted /minute

normal value - 75 ml /mint

Standard urea clearance: u × √v/p

normal value - 54ml/mint

Values below 75% is abnormal

Values fall progressively with increasing renal failure

Clearance values r the early indicator then plasma value

creatinine clearance is more preferred

Neither urea clearance nor blood urea r used as index of kidney function today

Creatine constitutes about 0.5% of total muscle weight. Its synthesized from 3 amino acids Glycine , Arginine &

Methionine. glycine arginine (occurs in mitochondia of kidney n

pancreas) Guanidoacetic acid ornithine SAM (in liver) SAH Creatine creatine phosphate (ck is pesent in muscle brain

n liver)

creatinine

CREATINE creatine kinase PHOSPHOCREATINE

SPONTANEOUS SPONTANEOUS

H₂O Pi

CREATININE

Stored creatine phosphate which is a high energy compound, stored in the muscle, serves as an immediate store of energy.

Inter conversion of phosphocreatine & creatine is an indicator of metabolic process of muscle contraction.

A particular proportion of muscle creatine is converted to creatinine spontaneously everyday.

So amount of creatinine produced in body is related to muscle mass .

Conc. Of creatinine in blood is mostly constant. Diet may influence the value with high meat intake. Its freely filtrated by glomerulus. Its an indicator of renal function.

Increased conc of creatine

Muscular disorders Myasthenia gravis Different myositis Starvation High fever Diabetes mellitus Hyperthyroidism

Causes of increase in serum Creatinine

Causes are divided in pre-renal , renal and post-renal

PRE-RENAL : Dehydration Pyloric & intestinal obstruction with vomiting Severe n prolonged diarrhea Chronic intestinal obstruction without vomiting Ulcerative colitis with severe chloride loss Diabetic coma

RENAL : Acute glomerulonephritis Malignant hypertension Chronic pyelonephritis Hydronephrosis Congenital cystic kidney Renal tuberculosis Calcium deposition in kidney in hyperthyroidism &

hyper-vitaminosis D .

POST RENAL: Enlargement of prostate

Stones in urinary tract

Stricture of the urethra

Tumors of the bladder effecting the ureter

Increased conc of creatinine

Conditions r same as increased urea conc. But in renal causes creatinine increases more

than the pre-renal causes.

Jaffe`s method (in urine) principle: creatinine in urine reacts with picric

acid in presence of NaOH to give orange colored compound.creatinine-picrate color read at 500 nm.

addition

BLANK STANDARD

TEST

Distilled water

3ml - -

Working standard

- 3ml -

Diluted urine

- - 3ml

Picric acid 1ml 1ml 1ml0.75 N NaOH

1ml 1ml 1ml

calculation: =gms of creatinine /lt =T/S×0.03/1000×1000/0.03 =T/S gm/lt

Normal value – 1 - 1.5gm/lt

Modified jaffe`s reaction: principle: cretinine reacts with alkaline

picrate to produce a reddish color. Absorbance of the color is directly proportional to creatinine conc in plasma n its measured in 500-520nm.

addition

blank standard

sample

Working reagent

- 1000ùl 1000ùl

Standard - 100ùl -serum - - 100ùl

Calculation: =T/S ×conc of standard(mg/lt)(2mg/dl)

normal value – 0.7 -1.4mg/dl (male) 0.6 – 1.2 mg/dl (female)

Uncorrected clearance =(u/p)× v

U=urinary creatinine conc. p=plasma creatinine conc v=urine flow in ml/min

Corrected clearance =u×v×1.73

p×A

Normal value: 85-125 ml/min (male) 80-115 ml/min (female)Clinical importance decreased creatinine clearance is an sensitive

indicator of GFR. UPTO 75% is nornal. Early detection of renal impairment Long term monitoring of renal patients Creatinine clearance is altered by body muscle mass

,drugs,age,sex