Preventive Care Profile Plus

Alanine Aminotransferase (ALT)

The method developed for use on the VetScan VS2 Chemistry Analyzer is a modification of the Wróblewski and LaDue procedure recommended by the International Federation of Clinical Chemistry (IFCC).1,2 In this reaction, ALT catalyzes the transfer of an amino group from L-alanine to α-ketoglutarate to form L-glutamate and pyruvate. Lactate dehydrogenase catalyzes the conversion of pyruvate to lactate. Concomitantly, NADH is oxidized to NAD+ , as illustrated in the following reaction scheme.

L-Alanine + α-Ketoglutarate → L-Glutamate + Pyruvate

Pyruvate + NADH + H⁺ → Lactate + NAD⁺

The rate of change of the absorbance difference between 340 nm and 405 nm is due to the conversion of NADH to NAD⁺ and is directly proportional to the amount of ALT present in the sample.

Albumin (ALB)

Dye binding techniques are the most frequently used methods for measuring albumin. Bromcresol green (BCG) is the most commonly used of the dye binding methods.

BCG + Albumin → BCG-Albumin Complex

Bound albumin is proportional to the concentration of albumin in the sample. This is an endpoint reaction that is measured bichromatically at 630 nm and 405 nm.

Alkaline Phosphatase (ALP)

The VetScan procedure is modified from the AACC and IFCC methods. Alkaline phosphatase hydrolyzes p-NPP in a metalion buffer and forms p-nitrophenol and phosphate. The use of p-nitrophenyl phosphate (p-NPP) increases the speed of the reaction. The reliability of this technique is greatly increased by the use of a metal-ion buffer to maintain the concentration of magnesium and zinc ions in the reaction. The American Association for Clinical Chemistry (AACC) reference method uses pNPP as a substrate and a metal-ion buffer.

p-Nitrophenyl Phosphate + H2O → p-Nitrophenol + Phosphate

The amount of ALP in the sample is proportional to the rate of increase in absorbance difference between 405 nm and 500 nm.

Aspartate Aminotransferase (AST)

The Abaxis AST method is a modification of the IFCC reference method. This method catalyzes the reaction of L-aspartate and α-ketoglutarate into oxaloacetate and L-glutamate. Oxaloacetate is converted to malate and NADH is oxidized to NAD⁺ by the enzyme malate dehydrogenase (MDH).

L-aspartate + α-Ketoglutarate → Oxaloacetate + L-glutamate

Oxaloacetate + NADH + H⁺ → Malate +NAD⁺

The rate of absorbance change caused by the conversion of NADH to NAD⁺ is determined bichromatically at 340 nm and 405 nm. This rate is directly proportional to amout of AST present in the sample.

Blood Urea Nitrogen (BUN)

Urea can be measured both directly and indirectly. The diacetyl monoxime reaction, the only direct method to measure urea, is commonly used but employs dangerous reagents. Indirect methods measure ammonia created from the urea; the use of the enzyme urease has increased the specificity of these tests. The ammonia is quantitated by a variety of methods, including nesslerization (acid titration), the Berthelot technique and coupled enzymatic reactions. Catalyzed Berthelot procedures, however, are erratic when measuring ammonia. Coupled-enzyme reactions are rapid, have a high specificity for ammonia, and are commonly used. One such reaction has been proposed as a candidate reference method.

In the coupled-enzyme reaction, urease hydrolyzes urea into ammonia and carbon dioxide. Upon combining ammonia with α-ketoglutarate and reduced nicotinamide adenine dinucleotide (NADH), the enzyme glutamate dehydrogenase (GLDH) oxidizes NADH to NAD⁺ .

Urea + H2O → 2NH3 + CO2

NH3 + α-Ketoglutarate + NADH + H⁺ → L-Glutamate + H2O + NAD⁺

The rate of change of the absorbance difference between 340 nm and 405 nm is caused by the conversion of NADH to NAD⁺ and is directly proportional to the amount of urea present in the sample.

Calcium (CA)

The reference method for total calcium is atomic absorption spectroscopy; however, this method is not suited for routine use. Spectrophotometric methods using either o-cresolphthalein complexone (CPC) or arsenazo III metallochromic indicators are most commonly used. Arsenazo III has a high affinity for calcium and is not as temperature dependent as CPC.

Calcium in the patient sample binds with arsenazo III to form a calcium-dye complex.

Ca²⁺ + Arsenazo III → Ca²⁺ – Arsenazo III Complex

The endpoint reaction is monitored at 405 nm, 467 nm and 600 nm. The amount of calcium in the sample is proportional to the absorbance.

Chloride (Cl^– )

The method is based on the determination of chloride-dependent activation of α-amylase activity. Deactivated α-amylase is reactivated by addition of the chloride ion, allowing the calcium to re-associate with the enzyme. The reactivation of α-amylase activity is proportional to the concentration of chloride ions in the sample. The reactivated α-amylase converts the substrate, 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNPG3) to 2-chloro-p-nitrophenol (CNP) producing color and α-maltotriose (G3). The reaction is measured bichromatically and the increase in absorbance is directly proportional to the reactivated α-amylase activity and the concentration of chloride ion in the sample.

CNPG3 → CNP + G3

Creatinine (CRE)

The Jaffe method, first introduced in 1886, is still a commonly used method of determining creatinine levels in blood. The current reference method combines the use of Fuller’s earth (floridin) with the Jaffe technique to increase the specificity of the reaction. 24,25 Enzymatic methods have been developed that are more specific for creatinine than the various modifications of the Jaffe technique. Methods using the enzyme creatinine amidohydrolase eliminate the problem of ammonium ion interference found in techniques using creatinine iminohydrolase.

Creatinine + H2O → Creatine

Creatine + H2O → Sarcosine + Urea

Sarcosine + H2O + O2 → Glycine + Formaldehyde + H2O2

H2O2 + TBHBA + 4-AAP → Red Quinoneimine Dye + H2O

Two cuvettes are used to determine the concentration of creatinine in the sample. Endogenous creatine is measured in the blank cuvette, which is subtracted from the combined endogenous creatine and the creatine formed from the enzyme reactions in the test cuvette. Once the endogenous creatine is eliminated from the calculations, the concentration of creatinine is proportional to the intensity of the red color produced. The endpoint reaction is measured as the difference in absorbance between 550 nm and 600 nm.

Glucose (GLU)

Measurements of glucose concentration were first performed using copper-reduction methods (such as Folin-Wu and SomogyiNelson). The lack of specificity in copper-reduction techniques led to the development of quantitative procedures using the enzymes hexokinase and glucose oxidase. The Abaxis glucose is a modified version of the hexokinase method, which has been proposed as the basis of the glucose reference method. The reaction of glucose with adenosine triphosphate (ATP), catalyzed by hexokinase (HK), produces glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G-6-PDH) catalyzes the reaction of G-6-P into 6-phosphogluconate and the reduction of nicotinamide adenine dinucleotide (NAD⁺ ) to NADH.

Glucose + ATP → G-6-P + ADP

G-6-PDH G-6-P + NAD⁺ → 6-Phosphogluconate + NADH + H⁺

Potassium (K⁺ )

Spectrophotometric methods have been developed that allow the measurement of potassium concentration on standard clinical chemistry instrumentation. The Abaxis enzymatic method is based on the activation of pyruvate kinase (PK) with potassium and shows excellent linearity and negligible susceptibility to endogenous substances. Interference from sodium and ammonium ions are minimized with the addition of Kryptofix and glutamine synthetase respectively.

In the coupled-enzyme reaction, PK dephosphorylates phosphoenolpyruvate (PEP) to form pyruvate. Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate. Concomitantly, NADH is oxidized to NAD + . The rate of change in absorbance between 340 nm and 405 nm is due to the conversion of NADH to NAD⁺ and is directly proportional to the amount of potassium in the sample.

ADP + PEP → Pyruvate + ATP

Pyruvate + NADH + H⁺ → Lactate + NAD⁺

Sodium (NA⁺)

Colorimetric and enzymatic methods have been developed that allow the measurement of sodium concentration on standard clinical chemistry instrumentation. In the Abaxis enzymatic reaction, β-galactosidase is activated by the sodium in the sample. The activated enzyme catalyzes the reaction of ο-nitrophenyl-β-D-galactopyranoside (ONPG) to ο-nitrophenol and galactose. The reaction rate between 405 nm and 500 nm is proportional to sodium concentration.

ONPG + H2O → ο-Nitrophenol + Galactose

Total Bilirubin (TBIL)

Total bilirubin levels have been typically measured by tests that employ diazotized sulfanilic acid. A newer, more specific method has been developed using the enzyme bilirubin oxidase. In addition to using the more specific total bilirubin test method, photodegradation of the analyte is minimized on the analyzer because the sample can be tested immediately after collection.

In the enzymatic procedure, bilirubin is oxidized by bilirubin oxidase into biliverdin. Bilirubin is quantitated as the difference in absorbance between 467 nm and 550 nm. The initial absorbance of this endpoint reaction is determined from the bilirubin blank cuvette and the final absorbance is obtained from the bilirubin test cuvette. The amount of bilirubin in the sample is proportional to the difference between the initial and final absorbance measurements.

Bilirubin + O2 → Biliverdin + H2O

Total Carbon Dioxide (tCO2)

Total carbon dioxide in serum or plasma exists as dissolved carbon dioxide, carbamino derivatives of proteins, bicarbonate and carbonate ions and carbonic acid. Total carbon dioxide can be measured by pH indicator, CO2 electrode and spectrophotometric enzymatic methods, which all produce accurate and precise results. The enzymatic method is well suited for use on a routine blood chemistry analyzer without adding complexity.

In the enzymatic method, the specimen is first made alkaline to convert all forms of carbon dioxide (CO2) toward bicarbonate (HCO3 – ). Phosphoenolpyruvate (PEP) and HCO3 – then react to form oxaloacetate and phosphate in the presence of phosphoenolpyruvate carboxylase (PEPC). Malate dehydrogenase (MDH) catalyzes the reaction of oxaloacetate and reduced nicotinamide adenine dinucleotide (NADH) to NAD⁺ and malate. The rate of change in absorbance due to the conversion of NADH to NAD⁺ is directly proportional to the amount of tCO2 in the sample.

PEP + HCO3^– → Oxaloacetate + Phosphate

Oxaloacetate + NADH + H^– → NAD^– + Malate

Total Protein (TP)

The total protein method is a modification of the biuret reaction, noted for its precision, accuracy, and specificity. It was originally developed by Riegler and modified by Weichselbaum, Doumas, et al. The biuret reaction is a candidate total protein reference method.

In the biuret reaction, the protein solution is treated with cupric [Cu(II)] ions in a strongly alkaline medium. Sodium potassium tartrate and potassium iodide are added to prevent the precipitation of copper hydroxide and the auto-reduction of copper, respectively. The Cu(II) ions react with peptide bonds between the carbonyl oxygen and amide nitrogen atoms to form a colored Cu-Protein complex.

Total Protein + Cu(II) → Cu-Protein Complex

The amount of total protein present in the sample is directly proportional to the absorbance of the Cu-protein complex. The total protein test is an endpoint reaction and the absorbance is measured as the difference in absorbance between 550 nm and 850 nm.