Albumin is the most abundant protein in human serum. A dye-binding method is commonly used in clinical laboratories for its estimation using different types of dyes. However, all these dye methods were interfered by a variety of compounds. Here we present a method for the detection of albumin in human serum and other biological fluids. The principle is based on the fact that lactate dehydrogenase isoenzyme-5 (LDH-5) binds specifically to Dextran-Blue (DB). Albumin inhibits the binding of LDH-5 with DB. Absence of LDH activity in DB fraction after gel filtration indicates the presence of albumin in sample and vice versa

Javed, M U

Waqar, S N

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eCommons@AKUDepartment of Biological & Biomedical Sciences Medical College, PakistanJune 2001An enzymatic method for the detection of humanserum albuminM U. JavedAga Khan UniversityS N. WaqarAga Khan UniversityFollow this and additional works at: https://ecommons.aku.edu/pakistan_fhs_mc_bbsPart of the Biochemistry CommonsRecommended CitationJaved, M. U., Waqar, S. N. (2001). An enzymatic method for the detection of human serum albumin. Experimental and MolecularMedicine, 33(2), 103-105.Available at: https://ecommons.aku.edu/pakistan_fhs_mc_bbs/386EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 33, No. 2, 103-105, June 2001An enzymatic method for the detection of human serum albuminMasood-ul-Hassan Javed1,2 and Saima N. Waqar11 Department of Biochemistry, The Aga Khan University MedicalCollege, Stadium Road, Karachi 74800, Pakistan2 Corresponding author: Tel, +00-92-51-4446801-Ext: 3364; Fax, +00-92-51-4435046; E-mail, masoodjaved@hotmail.comAccepted 4 June 2001Abbreviations: DB, dextran blue; HSA, human serum albumin; LDH,lactate dehydrogenase; CSF, cerebrospinal fluidAbstractAlbumin is the most abundant protein in humanserum. A dye-binding method is commonly used inclinical laboratories for its estimation using differenttypes of dyes. However, all these dye methods wereinterfered by a variety of compounds. Here wepresent a method for the detection of albumin inhuman serum and other biological fluids. The prin-ciple is based on the fact that lactate dehydrogenaseisoenzyme-5 (LDH-5) binds specifically to Dextran-Blue (DB). Albumin inhibits the binding of LDH-5with DB. Absence of LDH activity in DB fraction aftergel filtration indicates the presence of albumin insample and vice versa.Keywords: albumin, human, LDH-5, serumIntroductionHuman serum albumin (HSA) is synthesized in liver andis the most abundant protein in human serum, repre-senting 40-60% of the total protein (Lindsey, 1996). Theearliest method for its determination involved the salting-out of the globulins with sodium sulfate, leaving thealbumin in solution (Silverman and Christenson, 1994).The albumin was then determined by the Kjeldaholmethod and, later, by the Biuret color development(Gomes et al., 1998). The method commonly used todayis one involving dye binding and shift in color when adye is bound by albumin (Silverman and Christenson,1994; Lindsey, 1996; Gomes et al., 1998). A variety ofdyes have been used, including methyl orange, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), bromocresolgreen (BCG), and bromocresol purple (BCP) (Thakkaret al., 1997; Kessler et al., 1997a). Methyl orange isnon-specific for albumin; β-lipoproteins and some α1-and α2-globulins also bind to albumin and have a lowsensitivity (Kessler et al., 1997a). In addition, severalcompounds such as salicylates, penicillin, conjugatedbilirubin, and sulfonamides, interfere with the binding ofalbumin to the dye. Interfering substances such asbilirubin and salicylates do not affect BCG; however,hemoglobin can inhibit the binding of albumin to dye(Kessler et al., 1997b). Even though BCG is the dyemost commonly used today, there are repeated reportsthat the measurement of albumin by BCG overestimatesalbumin levels (Karon et al., 1997). Several formats ofelectrophoresis, radioimmunoassays (RIA) and immuno-assays are available for albumin determination (Karonet al., 1997; Kessler et al., 1997b), which are moreappropriate for body fluids in which albumin concent-ration is normally low (i.e., urine and CSF). The RIA andimmunoassays are relatively expensive and unlikely tobe applied except for special diagnostic purposes.Recently, the albumin-blue (AB) series of fluorescentalbumin dyes have been described (Kessler et al.,1997a). The limited solubility of the reagent solution sofar prevented its application in routine analysis. A newderivative of AB, AB580, with improved solubility hasbecome available recently and is used in autoanalyserfor albumin determination in serum and urine (Karon etal., 1997). Except for blood serum, albumin concent-rations in body fluids such as urine are usually low and,therefore, require sensitive and selective method fortheir detection and estimation. Enzymes have beenextensively used nowadays in the analytical laboratoriesdue to their high specificity and sensitivity (Whitaker,1991; Rej, 1998). In this study we present a simplemethod for the detection of human albumin in biologicalsample using LDH-5 and DB.Materials and MethodsChemicalsNADH, sodium pyruvate, Dextran Blue (DB), humanserum albumin (HSA), bovine serum albumin (BSA) andpotassium phosphate were from Sigma, St. Louis, U.S.A.Sepharose-CL-6B gel was from Pharmacia-LKB Bio-technology, Uppsala, Sweden. The LDH-5 was purifiedfrom rat liver as described elsewhere (Javed et al.,1995). All biological fluids were obtained from theclinical laboratory of the Aga Khan University Hospital104 Exp. Mol. Med. Vol. 33(2), 103-105, 2001(AKUH) and assayed after centrifugation within 3 days.This study followed the protocol of the Human SubjectProtection Committee of AKUH and all volunteers wereinformed prior to participation.Principle of the assayThis assay is based on high affinity binding of DB withLDH-5 (Leatherbarrow and Dean, 1980; Javed andNaru, 1998; Javed et al., 1999). This binding of LDH-5with DB was specifically inhibited by HSA (Javed andNaru, 1998; Javed et al., 1999). The presence of theLDH activity in DB fraction after gel filtration indicatesthe absence of albumin while the absence of LDHactivity indicates the presence of albumin.ProcedureThe Sepharose-CL-6B gel was packed in column (17cm × 0.5 cm) and equilibrated with 10 mM potassiumphosphate buffer containing 0.02% sodium azide, pH7.5. One mg DB was added to 1 ml sample (± HSA) andapplied on top of the column. The microfractionator(Gilson, Wisconsin, USA) collected one ml fractions andthe flow rate was controlled to about 40 ml/h byMicroperpex peristaltic pump (LKB, Ujppsala, Sweden).To measure LDH activity, we added 465 µl of DB fractionto 1ml cuvette. Twenty five µl substrate buffer (12 mMsodium pyruvate and 1 M potassium phosphate pH 7.5)were mixed and kept at 30oC for about 10 min. Thereaction was then started by adding 10 µl of 0.9 mMNADH and the rate was monitored at 340 nm inspectrophotometer (Beckman DU70, USA) for 2-3 min(Javed et al., 1995; Javed and Naru 1998).ResultsIn the presence of DB in the samples, LDH-5 co-elutedwith DB in void volume of sepharose column (Figure 1).However, in the presence of HSA in samples along withDB, LDH-5 activity was absent from DB fractions andeluted in later fractions of column chromatography(Figure 1). All human body fluids that contained albumin(serum, albuminuric urine, plural fluid, ascites, peritonialfluids, etc) showed no LDH-5 activity in DB fractionsafter gel filtration and LDH-5 activity was observed in thelater fractions. The minimum volume of normal humanserum needed to completely block the LDH-5 bindingwith DB was found to be 2 µl, that equals to be about0.08 mg of albumin. We tried to estimate the albumin bycalibrating a standard curve but we could not reproducethe results. By adding serum to DB-LDH samples fromanimals (i.e., animal albumin) like sheep, rabbit, mouse,rat, hamster and pigeon, the DB-LDH complex appear-ed in void volume. There was no effect on binding of DBwith LDH-5 even up to 40 mg/ml of animal albumin.Similarly there was no effect of BSA on the binding ofDB with LDH-5. DiscussionVarious methods have been used for the detection ofalbumin in different biological samples (Karon et al.,1997; Kessler et al., 1997a; Kessler et al., 1997b; Thakkaret al., 1997). Each method has some merits and demeritsand the major drawback is the interference by otherproteins and metabolites (Kessler et al., 1997a). Here,we described a new method for the detection of HSA.We believe that the gel filtration method is more specificthan the dye binding methods (Karon et al., 1997;Kessler et al., 1997a; Kessler et al., 1997b; Thakkar etal., 1997) because the DB specifically binds to LDH-5and it does not have any affinity even for LDH-1.Similarly, the HSA specifically compete with the bindingof LDH-5 with DB and albumin from other animals andother serum proteins from human serum do not competewith binding of DB with LDH-5 (Leatherbarrow andDean, 1980). We have also observed that the binding ofDB with LDH-5 is inhibited only by HSA, as the serumfrom animals, like sheep, rabbit, mouse, rat, hamsterand pigeon (which contained animal albumin and nothuman albumin) were unable to block the binding ofLDH-5 with DB and LDH activity appeared in the voidvolume in the presence of even up to 40 mg/ml ofanimal albumin (speculating that these animal serumalso have about 4 gm/dl of albumin with total serumprotein as about 6-8 gm/dl). This method is more simpleand sensitive to detect micro quantity of albumin. Thesame gel has been used for many months and evencan be used for many years in the presence of 0.02%sodium azide. Furthermore, LDH-5 can easily be puri-fied in large quantities from rat liver (Javed et al., 1995)Figure 1. Gel filtration of DB-LDH-complex in the presence (: -: ) andabsence (;-;) of human serum albumin. The method is described in thetext. DB peak is shown as (5-5) which is eluted in the void volume. Thepattern of the Fig was like (;-;) when we added the serum in the samplesfrom various animal species or BSA as mentioned in the results.Human serum albumin 105and was found to be stable for years at 4oC.In conclusion, the detection of HSA in biological fluidsby this method is more specific relative to dye bindingmethods and can be applied in many pathologicalconditions. The dye binding and electrophoresis arestandard but need special equipments, which may notbe available in ordinary clinical laboratories, particularlyin developing countries. Furthermore this new assay forHSA detection is a useful addition to the analyticalsciences.AcknowledgementsWe gratefully thank to Miss Farzana A. Yousuf for hertechnical help. ReferencesGomes MB, Dimetz T, Luchetti MR, Goncalves MF, GazzolaH, Matos H. Albumin concentration is underestimated infrozen urine. Ann Clin Biochem 1998;35:434-35Javed MH, Yousuf FA, Hussain AN, Ishaq M, Waqar MA.Purification and properties of LDH from liver of Uromastixhardwickii. Comp Biochem Physiol 1995;111B:27-34Javed MH, Naru TY. A simple method for the detection of LDHisoenzyme M:H ratio by gel filtration. Ann Clin Biochem 1998;35:439-41Javed MH, Jafri AH, Yousuf FA. Detection of LDH isoenzyme-M in body fluids containing albumin. Ann Clin Biochem 1999;36:250-51Karon BS, Kampe KC, Scott MG. Heparin interference withsodium and albumin assays. Clin Chem 1997;43:697-98Kessler MA, Meinitzer A, Petak W, Wolfbeis OS. Micro-albuminuria and borderline-increased albumin excretion deter-mined with a centrifugal analyzer and the albumin blue 580fluorescence assay. Clin Chem 1997a;443:996-1002Kessler MA, Meinitzer A, Wolfbeis OS. (1997b) Albumin blue580 fluorescence assay for albumin. Anal Biochem 1997b;248:180-82Leatherbarrow RJ., Dean DG. Studies on the mechanism ofbinding of serum albumins to immobilized cibacron blueF3GA. Biochem J 1980;189:27-34Lindsey BJ. Amino acids and proteins. In Clin. chem.:principles, procedures, correlation (ed M.L. Bishop, J.L.Duben-Engelkirk & E.P. Fody, EP), 1996, pp. 169-214.Philadelphia: J. B. Lippincott Co.Rej R. They use enzymes for every things! Clin Chem1998;44:1149-53 Silverman LM. Christenson RH. Amino acids and proteins InTietz text book of clinical chemistry (ed C. A. Burtis & E. R.Ashwood), 1994, pp. 191-205. Philadelphia: W. B. SaundersCo.Thakkar H, Newman DJ, Holownia P, Davey CL, Wang CC,Lloyd J, Graig AL, Price CP. Development and validation of aparticle-enhanced turbidimetric inhibition assay for urinealbumin on the ACA analyzer. Clin Chem 1997;43:109-13Whitaker JR. Enzymes in analytical chemistry, In Food enzy-mology (ed P. F. Fox), 1991, pp. 287-308. London: ElsevierApplied Science.

An enzymatic method for the detection of human serum albumin

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An enzymatic method for the detection of human serum albumin

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