MATERIAL IV DETERMINATION OF ALBUMIN LEVELS (QUANTITATIVE) Basic Theory Albumin is the most abundant plasma protein in the human body, namely around 55-60% and the total normal serum protein level is 3.8-5.0 g/dl. Albumin consists of a single chain of polypeptides with a molecular weight of 66.4 kDa and consists of 58 amino acids. In the albumin molecule there are 17 disulfide bonds that connect amino acids that contain sulfur. The albumin molecule is elliptical so that this molecular shape will not increase plasma viscosity and is completely soluble. Serum albumin levels are determined as a function of synthesis rate, degradation rate, and distribution between intravascular and extravascular compartments. Total albumin reserves are 3.5-5.0 g/kg BW or 250-300 g in healthy adults weighing 70 kg, of this amount 42% is in the plasma compartment and the rest is in the extravascular compartment (Evans, 2002). Human albumin is made from ,human plasma precipitated with alcohol. Albumin is widely used for volume replacement and treating hypoalbuminemia In detail, the functions and roles of albumin in the body are as explained below: a) Albumin as a binder and transporter. Albumin will bind weakly and reversibly to negatively and positively charged particles, and function as a carrier and transporter of metabolite and drug molecules. Although there are many theories about the importance of albumin as a transporter and binding protein, there is still little about the changes that occur in patients with hypoalbuminemia. b) Anticoagulant effect of albumin Albumin has an effect on blood clotting. It works like heparin, because it has a similar molecular structure. Heparin has a negative charge on the sulfate group that binds to the positively charged antithrombin III, which causes an anticoagulant effect. Serum albumin is also negatively charged. c) Albumin as a buffer Albumin acts as a buffer due to the presence of residual charges and albumin molecules and is relatively abundant in plasma. At normal pH conditions, albumin is negatively charged and plays a role in the formation of anion groups which can affect acid-base status. A decrease in albumin levels will cause metabolic alkalosis, because a decrease in albumin of 1 g/dl will increase bicarbonate levels by 3.4 mmol/L and base production >3.7 mmol/L as well as a decrease in anions by 3 mmol/L. d) Antioxidant effect of albumin Albumin in serum acts to block a state of neurotoxic oxidant stress induced by hydrogen peroxide or copper, ascorbic acid which, when oxidized, will produce free radicals. e) Apart from what is mentioned above, albumin also plays a role in maintaining microvascular integrity, thus preventing the entry of intestinal germs into the blood vessels, thereby avoiding bacterial peritonitis. Albumin synthesis only occurs in the liver. In healthy people the rate of albumin synthesis is 194 mg/kg/day (12-25 grams/day). Under normal circumstances only 20-30% of hepatocytes produce albumin Albumin in clinical aspects is used in several ways, namely: a) Hypovolemia Hypovolemia is characterized by intravascular volume deficiency due to lack of external fluid or redistribution of internal and extracellular fluid. If hypovolemia occurs and is accompanied by hypoalbuminemia with inadequate hydration or edema, it is better to use 25% albumin rather than 5% albumin. If hydration is excessive, 5% albumin or 25% albumin dissolved in crystalloid should be used. Although crystalloids or colloids can be used for emergency treatment of hypovolemic shock, human albumin has a long intravascular halflife. b) Hypoalbuminemia The association between hypoalbuminemia and poor outcomes has motivated clinicians to administer exogenous albumin to patients with hypoalbuminemia. Human albumin has been indicated for the treatment of hypoalbuminemia in the United States and other countries. But there is still controversy, even though hypoalbuminemia directly causes poor treatment outcomes. Hypoalbuminemia is not an indication for administering albumin because hypoalbuminemia is not directly related to plasma and other fluid volumes, but is caused by excess and deficit of intravascular fluid caused by dilution, disease and distribution factors. c) Albumin burns are given at 24 hours post-trauma to help withdraw fluid from extravascular to intravascular. d)Adult Respiratory Distress Syndrome(ARDS) The characteristic of ARDS is a state of hypoproteinemia caused by pulmonary edema. If pulmonary overload occurs accompanied by hypoalbuminemia, a 25% albumin solution will provide a therapeutic effect if combined with a diuretic. e) Nephrosis Albumin may be useful to help treat edema in nephrosis patients receiving steroids and/or diuretics. f) Cardiopulmonary By Pass Operation g) To bind and remove toxic bilirubin in neonates with hemolytic disease. Method Principles Bromocresol green at a slightly acidic pH in serum albumin can produce a change in indicator color from green-yellow to green-blue Procedure Wavelength : Hg 546 nm (540-600 nm) Optical path : 1 cm Incubation temperature : 20 – 25°C or 37° Material : Blood Sample 10-15 ml Reactor 1. Citrate buffer, pH 4.2 : 30 mmol/L 2. Bromocresol green : 0.26 mmol/L 3. Standard : 5 g/dL Specimen (biological sample) Serum, plasma heparin or plasma EDTA Stability: • 10 weeks at a temperature of 20-250C, • 5 months at temperature 4-80C and • 3 months months at a temperature of -200C Tool : 1. Reaction tube 2. EDTA 3. Centrifuge 4. Kits 5. Spectrophotometer 6. Myropipette Implementation Pipette in cuvette Blank Standard Sample Reagent 1000 µl 1000 µl 1000 µl Sample - 10 µl 10 µl aquabides 10 µl - - Mix, incubate for 10 minutes at temperature 37oC. Measure the absorbance of the standard and sample against the reagent blank within 60 minutes. (∆A)