II. Determination of albumin and globulin fraction with Biuret Method Reagent : 1.

Standard protein solution (5 mg albumin/ml and 5 mg globulin/ml), freshly made. 2. Biuret Reagent (contains CuSO4, Na-K Tartrate, NaOH and KI) 3. Sample solution. Procedure : 1. Add Biuret Reagent into 2 tubes (Tube I and II), 3 ml in each tube. 2. Into Tube I add 2 ml of standard protein solution and into Tube II add 2 ml of sample solution. 3. Incubate at 37oC for 10 minutes 4. Cool down, read with spectrophotometer at wavelength of 540 nm. III. Determination of albumin and globulin fraction with Folin-Lowry Method Principle Protein reacts with Folin-Ciocalteau reagent to yield a compound with complex color. The color is formed by the reaction between Cu-alkali and protein as in Biuret reaction and phosphomolibdate reduction by tyrosine and tryptophan present in protein. Reagent 1. Sodium carbonate base solution (2% of Na2CO3 in 0,1 N of NaOH) 2. CuSO4NaK tartrate solution (0.5% of CuSO4 in 1% of NaK-tartrate), freshly made. 3. Alkali solution. This solution is made just before used by mix 50 ml of solution no.1 and 1 ml of solution no.2. 4. Diluted Folin-Ciocalteau Reagent (contains Na-tungstate dan Na-molibdate in phosphoric acid and chloride acid). Dilute the reagent with water by 1:1 volume. 5. Standard protein solution 6. Sample solution Procedure 1. In a reaction tube, add 5 ml of solution no. 3 (alkali solution) with 1 ml of sample solution. 2. Mix thoroughly; incubate at room temperature for approximately 10 minutes. 3. Add 0.5 ml of diluted Folin-Ciocalteau reagent; shake strongly for 3-5 minutes. 4. Incubate at room temperature for 30 minutes. 5. Read the absorbance at wavelength of 750 nm. 6. Calculate protein level of sample solution after the curve of standard solution has been determined. Note It is not easy to obtain urine from patient with albuminuria. As for the sample solution, urine + several drops of plasma or serum can be utilized. References Bray, WE. 1957. Clinical Laboratory Methods 5th Ed. The CV Mosby Co. Davidson, I., Henry, JB., 1974. Todd Stanford Clinical Diagnosis by Laboratory Methods 15th Ed.. WB Saunders Co., Philadelphia Harrison, GA., 1957. Chemical Methods in clinical Medicines 4th Ed.. J & A Churchill Ltd.,

Laboratory Manual BIOCHEMISTRY DETERMINATION OF ALBUMIN URINE WITH SALTING OUT PENENTUAN KREATININ URIN DENGAN SPEKTROFOTOMETER DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITAS GADJAH MADA DETERMINATION OF ALBUMIN URINE WITH SALTING OUT Principle Normal urine contains a trace of protein material, but the amount present is generally less than 250 mg per 24 hours and is so slight as to escape detection by any of the simple tests in general use. Globulin is not a constituent of normal urine and frequently is found in pathological conditions. Proteoses have been found in the urine in cases of pneumonia, diphtheria, intestinal ulcer, carcinoma, dermatitis, osteomalacia, atrophy of the kidneys, and in condition of partially digested pus. Manifestation of diabetic kidney disease is the presence of small amount of albumin in the urine, called microalbuminuria. Protein excretion in the urine normally does not exceed 100 to 200 mg per 24 hours. Much of this protein comes from the tubules, but a percentage is filtered through the glomeruli. Microalbuminuria is the presence in levels exceed 300 mg per 24 hours, this condition is called proteinuria. Some causes of transient increases in albuminuria include exercise, dietary intake, pregnancy, drugs, and other factor that can alter both protein delivery and glomerular hemodynamics. Common Reagents 1. Reagent to determine protein with Biuret/ Folin-Lowry method. 2. Sample solution 3. Concentrated ammonium sulfate solution (dissolve 767 g of ammonium sulfate with aquadest up to 1 L) 4. Standard protein solution (bovine albumin dan gamma-globulin) Procedures I. Separation of albumin and globulin fraction 1. Add 100 ml of aquadest into sample solution which contains protein. Stir well; globulin will precipitate whereas albumin still dissolves in the suspension. 2. Centrifuge the suspension at 3000 g for 10 minutes. 3. Move supernatant which contains albumin into a new tube. 4. Dissolve pellet which contains globulin with diluted-salt solution as little as possible. 5. Keep both fractions before used in the next experiment.

London. Henry, R.J., Cannon, D.C., Wikleman, J.W. 1974. Clinical Chemistry Principles and Techniques

2nd Ed.. Harper & Row Publishers, Hagertown. Todd, J.C., and Sanford, A.H. 1975. Clinical Diagnosis by Laboratory Methods 11th Ed..WB Saunders Co., Philadelphia PENENTUAN KREATININ URIN DENGAN SPEKTROFOTOMETER Cara Kerja Kedalam 3 labu pengukur 100 ml beri tanda masing-masing U (untuk sampel urin ) , S ( untuk larutan standar) dan B ( untuk blanko ) Larutan standar : 1 mg kreatinin / ml (dibuat dengan melarutkan kreatinin zinc chloride dalam HCl encer ) REAGEN SAMPEL (U) STANDAR (S) BLANKO (B) URIN 1 ml STANDAR 1 ml AKUADES 1 ml ASAM PIKRAT JENUH 20 ml 20 ml 20 ml NaOH 10% 1,5 ml 1,5 ml 1,5 ml - Campur dan biarkan selama 10 menit agar warna timbul - Tambahkan pada masing-masing labu akuades hingga 100 ml, campur dan baca absorbansinya denga spektrofotometer pada panjang gelombang 520 nm Perhitungan A. sampel A blanko Kadar Kreatinin = ------------------------------ X 100 mg% A standar A blanko Kepustakaan HAWK, P.B., Oser, B.L., and Summerson, W.H., 1954. Practical Physiologycsl Chemistry. 13rd ed, Mc Graw Hill Book Company, Inc. New York. 900-902