Method: ultraviolet, kinetic

Principle:

• l-alanine +a-ketoglutarate +ALT = pyruvate +l-glutamate
• pyruvate + NADH+ LDH = l-lactate +NAD
• The rate of decrease in absorbance at 340 nm due to formation of NAD is directly proportional to ALT activity.
• Diagnostic significance: Increased levels of enzyme is associated with necrosis of hepatic cells of any etiology, severe shock, right heart failure, acute anoxia, extensive trauma, left heart failure, cirrhosis, mechanical jaundice, liver tumors, extensive myocardial infarction, myositis, myocarditis, muscular dystrophy. Decreased levels of enzyme is associated with pyridoxal phosphate deficiency

1. Bergmeyer HU, Scheide P, Wilhelm A, Wahlfeld Optimization of methods for AST and ALT Clin Chem 1978, v. 24, N 1, 58-73

2. Hafkenscheid JCM, Diggt CCM Determination of serum aminotransferases: activation by pyridoxal-5 phosphate in relation to substrate concentration Clin Chem 1979, v. 25, N 3, 353-362

Method: colorimetric, endpoint

Principle:

• l-alanine +a-ketoglutarate +ALT = pyruvate +l - glutamate
• The pyruvate then reacts with 2,4 dinitrophenylhydrazine producing highly colored complex with absorbance at 490-520 nm . Intensity of absorbance is proportional to ALT activity.
• Diagnostic significance: Increased levels of enzyme is associated with necrosis of hepatic cells of any etiology, severe shock, right heart failure, acute anoxia, extensive trauma, left heart failure, cirrhosis, mechanical jaundice, liver tumors, extensive myocardial infarction, myositis, myocarditis, muscular dystrophy. Decreased levels of enzyme is associated with pyridoxal phosphate deficiency

Method: colorimetric, endpoint

Principle:

• Bromcresol purple binds with HSA forming colored complex with absorbance at 600 nm. Intensity of absorbance is proportional to albumin concentration in serum.
• Diagnostic significance: True hyperalbuminemia does not happen. Any processes leading to a decrease in water content of the plasma lead also to an increase in the concentration of both albumin and all plasma proteins. Low levels associated with acute and chronic inflammation, rheumatic diseases, thermal burns, granulomotomous processes, bacterial and viral infections, leading to tissue destruction, acute and chronic liver disease, malignancy, hard heart failure, nephrotic syndrome.

Henry JB Clinical Chemistry 1967

Method: latex agglutination

Principle:

• Polystyrene latex beads coated with ds DNA in the presence of antibodies to it in human serum form visible agglutination. The rate of agglutination correlates with antibodies concentration in serum.
• Diagnostic significance: Antibodies to dsDNA are characteristics of the autoimmune diseases, SLE

Method: latex agglutination

Principle:

• Polystyrene latex beads coated with DNP in the presence of antibodies to it in human serum form visible agglutination. The rate of agglutination correlates with antibodies concentration in serum.
• Diagnostic significance: Antibodies to DNP are characteristics of the autoimmune diseases

1. Hall AP et al Latex agglutination and inhibition reactions: clinical experience in diagnosis of rheumatoid arthritis New Engl J. Med 1958, v. 258, 731

2. Dubois EL et al A latex nucleoprotein test for diagnosis of SLE: comparative evaluation JAMA 1961, v. 177, 141-143

Method: latex agglutination

Principle:

• Polystyrene latex beads coated with RNP in the presence of antibodies to it in human serum form visible agglutination. The rate of agglutination correlates with antibodies concentration in serum.
• Diagnostic significance: Antibodies to RNP are characteristics of the autoimmune diseases

Method: utraviolet, kinetic

Principle:

• Aspartate +a-ketoglutarate +AST = pyruvate +l- glutamate
• Pyruvate + NADH +MDH l-malate +NAD
• The rate of decrease in absorbance at 340 nm due to formation of NAD is directly proportional to AST activity.
• Diagnostic significance: Increased levels associated transient forms of hepatitis,especially of a viral origin necrosis of liver cells or their damage, chronic hepatitis, drug damage of liver cells, alcoholic hepatitis, infectious mononucleosis, necrosis or trauma of the heart or skeletal muscle, hypothyroidism, intestinal obstruction.

1. Bergmeyer HU, Scheide P, Wilhelm A, Wahlfeld Optimization of methods for AST and ALT Clin Chem 1978, v. 24, N 1, 58-73

2. Hafkenscheid JCM, Diggt CCM Determination of serum aminotransferases: activation by pyridoxal-5 phosphate in relation to substrate concentration Clin Chem 1979, v. 25, N 3, 353-362

Method: colorimetric, endpoint

Principle:

• aspartate +a-ketoglutarate +AST = pyruvate +l- glutamate
• The oxalacetate then reacts with 2,4 dinitrophenylhydrazine producing highly colored complex with absorbance at 490-520 nm Intensity of absorbance is proportional to AST activity.
• Diagnostic significance: Increased levels associated transient forms of hepatitis,especially of a viral origin necrosis of liver cells or their damage, chronic hepatitis, drug damage of liver cells, alcoholic hepatitis, infectious mononucleosis, necrosis or trauma of the heart or skeletal muscle, hypothyroidism, intestinal obstruction.

Method: screening test

Principle:

• paper strips are coated by reagents that become purple by action of nitrite. Many types of bacteria are producing nitrite and thus they can be detected by these strips

Method: colorimetric, endpoint (Walters Gerarde)

Principle:

• In the presence of dimethylsulfoxide both conjugated and nonconjugated bilirubin react with diazotized sulfanilic acid to yield azobilirubin with absorbance at 560 nm. Conjugated (direct) bilirubin reacts directly with diazotized sulfanilic acid to yield azobilirubin with absorbance at 560nm
• Diagnostic significance: Increased levels of direct bilirubin is associated with obturation of intrahepatic and extrahepatic bile ducts, damage of hepatic cells, especially in the late period of the disease, cholestasis. Increased levels of total bilirubin is associated with damage of hepatic cells (inflammatory, toxic, neoplastic) Obturation of intrahepatic and extrahepatic bile ducts hemolytic diseases, hypothyroidism,, salicilates

Walters M et al An ultramicromethod for the determination of conjugated and total bilirubin in serum and plasma Microchem J. 1970, v.15, 231-243

Method: colorimetric, endpoint

Principle:

• Serum ceruloplasmin oxidizes some substrates producing highly colored compounds in acid media. The intensity of color is proportional to ceruloplasmin activity.
• Diagnostic significance: Decreased levels associated with Wilson disease and Menkes syndrome.

Shrifrine M, Fisher G Ceruloplasmin levels in sera from human patients with osteosarcoma Cancer 1976, v. 39, 244-248

Method: latex agglutination

Principle:

• Polystyrene latex beads coated with antibodies to human C-Reactive protein in the presence of CRP in human serum form visible agglutination. The rate of agglutination correlates with CRP concentration in serum.
• Diagnostic significance: Increased levels of CRP is associated with inflammation, necrosis of tissues, trauma. In comparison to other acute phase proteins its concentration does not depend on the action of hormones and it is the best marker of inflammation

1. Wadsworth C et al Efficacy of latex agglutination and quantitation methods for determination of CRP in pediatric sera Clin Chim Acta 1984, v. 138, N 2, 309-319

2. Volanakis JE et al Human CRP and host defences UCLA Symp Mol Cell Biol New Ser 1990, v121 161-175

3.Griselli M et al CRP and complement are important mediators of tissue damage in acute myocardial infarction J.Expl Med 1999, v. 190, N 12, 1733-1741

Method: colorimetric endpoint (Jaffe, Slot modification)

Principle:

• Creatinine reacts with picric acid under alkaline conditions forming yellow-orange complex derived from creatinine as well as some nonspecific substances. Upon addition of acid the color contributed by creatinine is destroyed while that produced by non- specific substances remains. The difference in color intensity before and after acidification is proportional to the creatinine concentration
• Diagnostic significance: Increased concentration of creatinine is associated Impaired renal function (acute and chronic) of any etiology, active acromegaly and gigantism, hyperthyroidism, meat food. Decreased concentration is associated with weakness caused by age or by decreasing in muscle mass, pregnancy especially in the first and second trimester

Method: colorimetric kinetic

Principle:

• Creatinine reacts with an alkaline picrate, forming a red complex. A short time interval of reaction excludes the influence of other components of the serum. The change in the optical density of the complex is proportional to the concentration of creatinine in the sample
• Diagnostic significance:Increased concentration of creatinine is associated Impaired renal function (acute and chronic) of any etiology, active acromegaly and gigantism, hyperthyroidism, meat food. Decreased concentration is associated with weakness caused by age or by decreasing in muscle mass, pregnancy especially in the first and second trimester

Method: colorimetric, endpoint

Principle:

• fructosamine (glycated serum protein) reduces nitroblue tetrazolium under appropriate conditions forming a purple colored formazan. Intensity of absorbance is proportional to fructosamine concentration.
• Diagnostic significance: Fructosamine is the index of the average glycemic state during previous 2-3 weeks. The test is useful short term diabetic control

1. Baker J et al Clinical usefulness of estimation of serum fructosamine concentration as a screening test for diabetes melititus Br.Med J. 1983, v. 287, N 6396 863-867

2. Johnson RN et alFructosamine: a new approach to the estimation of serum glycoprotein. An index of diabetic control Clin Chim Acta 1983, v. 127, 87-95

3. Johnson RH et al The alkaline reducing activity of glycated serum proteins and its relevance to diabetic mellitus Clin Chem 1986, v. 32, 368-370

Method: colorimetric, endpoint

Principle:

• Heating of samples under appropriate conditions with specific reagents brings to selective coloring of fructose. Intensity of the color is proportional to fructose concentration
• Diagnostic significance: Decreased levels of fructose in sperm may be the cause of infertility.

Davis J, Gander J A reevaluation of the Roe procedure for the determination of fructose Anal Biochem 1967, v. 19, 72

Method: colorimetric, endpoint

Principle:

• Hemoglobin reacts with acetoncyanhydrine in appropriate medium. All forms of hemoglobin in blood are converted to cyanmethemoglobin. The absorbance at 530-550 nm is proportional to the hemoglobin concentration in blood.
• Diagnostic significance: Increased levels are associated with polycythemia, high physical load, stay at high altitudes, dehydratation, persistent vomiting, burns, intestinal obstruction.Decreased levels associated with anemia.

Rice E Rapid determination of total Hb as hemoglobin cyanide in blood containing carboxy hemoglobin Clin Chim Acta 1967, v. 18, N 1, 89-91

Method: ultraviolet, kinetic

Principle: Pyruvate + NADH+LDH=Lactate+NAD

• The rate of decrease in absorbance at 340 nm due to formation of NAD is directly proportional to LDH activity.
• Diagnostic significance: Increased levels of enzyme takes place after 8-12 hours after myocardial infarction. Moderate increases also observed in cases of liver diseases.

Vassault A. Lactate dehydrogenase. UV method with pyruvate and NADH in Bergmeyer HU Ed Methods of Enzymatic Analysis 1983, v3

Method: latex agglutination

Principle:

• Polystyrene latex beads coated with antibodies to human myoglobin in the presence of myoglobin in human serum form visible agglutination. The rate of agglutination correlates with myoglobin concentration in serum.
• Diagnostic significance: Increased levels associated infarction, uremia, trauma, inflammatory damage of muscles, physical load, burns. Decreased levels are associated presence of circulated antibodies against myoglobin, rheumatoid arthritis.

1.Mair J Myoglobin Dev Cardiovasc Med 1998, v. 205 (myocardial Damage) 53-59

2.Ohman EM et al Early detection of acute myocardial infarction . Additional diagnostic information from serum concentration of myoglobin in patients without ST elevation Br.Heart J 1990, v. 63, N 6, 335-338

3. Chapell JP et al Semi-quantitative estimation of serum myoglobin by a rapid latex agglutination method: an emergency screening test for acute myocardial infarction Clin Chim Acta 1985, v. 145, 143-150

Method: colorimetric, endpoint (biuret)

Principle:

• The copper ions in alkaline biuret reagent react with peptide bonds of serum proteins to form a purple color. The intensity of the color is proportional to the total protein concentration
• Diagnostic significance: Increased levels is observed in cases of polycolonal or monoclonal hyperimmunoglobulinemia and dehydration. Decreased levels are associated with protein loss as a result of gastroenteropathy, nephrotic syndrome, burns

Tietz N (ed) Fundamentals of Clinical Chemistry 3rd ed Philadelphia 1987

Method: colorimetric endpoint.

Principle:

• brilliant blue G reacts with protein in acid alcohol medium to form a blue colored complex. The intensity of color is proportional to protein concentration.
• Diagnostic significance: Increased level of protein in urine is observed during impaired kidney function

Macart M, Gerbaut L Evaluation of an improved Coomassie dye binding method for urinary protein assay Clin Chim Acta 1984, v. 141, N 1, 77-85

Principle:

• Albumin in urine reacts with dye on strip bring to color change. Color intensity is correlated with amount of protein. The strips are applied for semi quantitative determination of protein in urine

Method: Latex Agglutination

Principle:

• Polystyrene latex beads coated with denaturated human IgG in the presence of rhematoid factors in human serum form visible agglutination. The rate of agglutination correlates with rheumatoid factor concentration in serum.
• Diagnostic significance: Rheumatoid factor has been associated with some bacterial and viral infections, some chronic infections. Its determination is applied for differentiating rheumatoid arthritis from other inflammatory diseases

1.Singer JM et al Latex fixation test I. Application to the serologic diagnosis of rheumatoid arthritis Am. J. Med 1956, v. 21, 888

2. Rheins ML et al Effect of animal sera and serum albumin on latex fixation test for rheumatoid arthritis Proc Soc Expl Biol Med 1957, v. 96, 67

Method : colorimetric, endpoint

Principle:

• Urea + H20+urease= NH3+ C02 nitroprusside
• NH3 + salicilate-hypochlorite = colored complex
• Diagnostic significance: Increased levels associated with decreased renal perfusion, congestive heart failure, depletion of salt reserves, abundant hydrosis, Decreased levels are associated with diet with low content of protein , hard liver diseases

Tobacco A, Meiattini F, Moda E, tarli P Simplified enzymic/colorimetric serum urea nitrogen determination Clin Chem 1979, v. 25, N 2, 336-337

Method: ultraviolet, endpoint

Principle:

• Uric acid + H2O +O2 +uricase= allantoin + CO2 +H2O2
• Absorbance at 293 nm is measured before and after reaction. Differences in absorbances show concentration of uric acid.
• Diagnostic significance: Increased levels associated with gout, leukemia, severe renal impairment.

Method: colorimetric, endpoint

Principle:

• Uric acid + H2O +O2 +uricase = allantoin + CO2 +
• H2O2 + peroxidase + phenol +4-aminoantipyrine= dyed complex
• Diagnostic significance: Increased levels associated with gout, renal failure, leukemia, myeloma, polycythemia , lymphoma, psoriasis toxicosis. Decreased levels are associated with disease of Wilson- Konovalov, Fanconi syndrome, Hodgkin's disease, xantinuria, pathological secretion of antidiuretic hormone.

1.Domagk GF, Schlicke HH A colorimetric method using uricase and peroxidase for the determination of uric acid Anal Biochem 1968, v. 22, 219-224

2. Sanders GTB, Pasma AJ,Hock FJ Determination of uric acid with uricase and peroxidase Clin Chim Acta 1980, v. 100, N 2-3, 299-305