Dr. Urender S. GhanghasMBBS, MD IIyr

For BMLT, DMLTBIOCHEMISTRY

SGT MEDICAL COLLEGEGURGOAN

Principle Sample Reagents Procedures Wavelength Reading Calculation Normal values Interpretation

1. Jaffe method(Manual method) Disadvantage: non creatinine

chromagen interfere with the reaction e.g. protein , glucose, Ketones, Pyruvate, Bilirubin…….

2. Kinetic analyses modes(Automated method)

PFF + Saturated picrate acid

(alkaline media) NaOH

Creatinine picrate orange red complex (absorbed at 510 nm)

Serum or Plasma Protein free filtrate: to avoid non

creatinine chromagen interfering with the reaction

Saturated picrate acid Creatinine standard -1 mg% NaOH-5% Sodium tungstate solution – 10% H2SO4

In a centrifuge tube take;1. 1 ml of DW 2. 1 ml serum3. 1 ml of sodium tungstate4. 1 ml of H2SO4

Mix and centrifuge for 5 min. to obtain clear supernatant PFF

BlankStand.Test

Sample :PFF------2 ml

DW4 ml3 ml2 ml

Standard (0.01mg/ml)--1 ml--

NaOH1 ml1 ml1 ml

Sat. Picric acid1 ml1 ml1ml

Mix

Incubate at Room Temp. for 10 min.

Read the OD test & Stand. against the blank at 510 nm

510 nm

Read the sample against its blank at 510 nm

O.D. =…………..

According to Beer Lambert law C T = ODT X CSTD

ODSTD

Serum Creatinine (mg%) = O.D Test X Amount of standard X 100 O.D Std. Volume of sample

Conc. = …………. mg/dl

KINECTIC METHOD :- they were developed in a quest both for specificity and for faster and automate analysis .Rate is measured:

Faster More specific Most widely used Implemented on various automated instruments. JAFFE’S KINETIC ASSAY ENZYMATIC KINETIC ASSAY

In this method also Jaffe’s reaction principle is used but defined experimental condition, by using special type of spectrophotometer or autoanalyser, equipped with a thermocuvette. Jaffe’s reaction is carried out at 30 degree centigrade as follows:

First reading is recorded at 20 sec. Most of the interfering non-creatinine chromogens react with alkaline picrate reagent fast and are recorded after 20 seconds.

Creatinine and alkaline picrate react relatively slowly after 20 seconds,upto 80 seconds.Hence second reading is noted after 80 seconds.

Same procedure is used for a standard.

The result are calculated as follows-

Mg/dl, Creatinine= O.D.T[80 sec]-O.D.T[20sec] X conc. of std. O.D std[80sec]-O.D std[20sec]

Dilute urine 1:100 with distilled water so that the creatinine values come within the range of Lambert-Beer’ s law and the interfering substances get diluted. Urine creatinine is then estimated by using Jaffe’s method.

BSTDilute Urine------3 ml

Distilled water3 ml------

Standard(0.01 mg/ml)

---3 ml---

NaOH 5%1 ml1 ml1 ml

Sat.Picric acid1 ml1 ml1 ml

Mix for 15 min. at room temp.Take OD at 520 nm. Calculate the result.

Increased values = (Azotemia)

1.Prerenal: systemic infection e.g. babesiosis2.Renal: e.g. Amyloidosis, Glomerulosclerosis

and Polycystic disease 3.Post renal: e.g. Ureteral obstruction

Decreased creatinine blood level

Muscle diseaseCachexiaPortosystemic shunts