Erich Bernt

UV-Assay with Pyruvate and NADH

Publisher Summary This chapter discusses about lactate dehydrogenase (LDH), which was first crystallized from rat muscle in 1943. LDHs from bacteria and yeast are not linked to the pyridine nucleotide coenzymes. The LDH activity in serum results from the presence of different enzyme proteins with the same action and substrate specificity, but of different origin. LDH is applied in biochemistry and clinical chemistry. The optimum conditions for the measurements of the enzyme in serum after myocardial infarction, in liver damage, blood diseases, and tumors have been more or less established. The sodium pyruvate content should be at least 99%. Deterioration of the buffer/substrate solution is usually because of bacterial contamination, which can be prevented by addition of a few drops of chloroform. Sudhof et al. have measured the loss of activity on storage of serum at various temperatures. With highly active serum a large proportion of the NADH can be oxidized between the addition of the serum and the first reading. LDH is a cytoplasmic enzyme; simple homogenization in a Potter–Elvehjem homogenizer is sufficient to completely extract the entire enzyme.

Glutamate-Oxaloacetate Transaminase

Publisher Summary This chapter elaborates the measurement and preparation of glutamate-oxaloacetate transaminase (GOT). GOT has been detected in micro-organisms and in all human and animal tissues. In humans the richest source is heart muscle, followed by brain, liver, gastric mucosa, adipose tissue, skeletal muscle, kidney, and finally serum with substantially smaller amounts. Several possibilities exist for the measurement of activity which includes paper chromatography of the substrates or reaction products after incubation and spectrophotometric measurement of the oxaloacetate formation at 280 mμ. The most widely used methods involve the measurement of the oxaloacetate formed from aspartate and α-oxoglutarate: one of them is by an enzymatic indicator reaction with malic dehydrogenase and reduced diphosphopyridine nucleotide. The pyridoxal phosphate required as a coenzyme is present in sufficient quantities in serum and all tissue samples. Animal organs, especially liver, kidney, and brain D are rich in glutamic dehydrogenase. It is necessary to determine the amount of interference due to this enzyme, as this occurs in spite of the use of ammonia-free reagents and the phosphate–aspartate buffer is substituted by one containing α-oxoglutarate and the GOT reaction is started with L -aspartate after measurements of the glutamic dehydrogenase activity.

Glutamate-Pyruvate Transaminase

Publisher Summary This chapter focuses on the enzymatic determination of glytamate–pyruvate transaminase (GPT). The GPT content of a human tissue decreases in the following order: liver, kidney, heart, skeletal muscle, pancreas, spleen, lung, and serum. This activity can be determined by several methods that include paper chromatography of the substrates or reaction products, manometric determination of the glutamic acid formed with glutamic acid decarboxylase and colorimetric determination of the pyruvate formed with salicylaldehyde. The most successful methods depend on the measurement of the pyruvate formed from alanine and α-oxoglutarate: one method is enzymatically with lactic dehydrogenase and reduced diphosphopyridine nucleotide (DPNH); and a second method is colorimetrically with 2,4-dinitrophenylhydrazine. This chapter also discusses the method for the determination of GPT with lactic dehydrogenase as an indicator enzyme. It is based on the principle that GPT catalyzes the reaction, as described in the chapter, and the activity of the transaminase is measured by the increase of pyruvate with time. The pyruvate is determined by the indicator reaction catalyzed by lactic dehydrogenase (LDH). The rate of oxidation of reduced diphosphopyridine nucleotide (DPNH) is proportional to the increase of pyruvate with time. The decrease of optical density at 340 or 366 mμ because of the oxidation of DPNH is then measured.

l-Glutamate UV-Assay with Gutamate Dehydrogenase and NAD

Publisher Summary Previous methods for the determination of glutamic acid are based on some reactions that are not specific and are often very complicated. Glutamic acid can be determined colorimetrically with ninhydrin after chromatographic separation or manometrically by means of glutamate decarboxylase or l -glutamate or d -glutamate oxidase. However, the necessary enzymes are not easily available. The determination of glutamate with glutamate dehydrogenase is easy to carry out, precise, and specific for the l -isomer. This method is applied in biochemistry, clinical chemistry, and food chemistry. Spectrophotometer or spectrum-line photometer is suitable for precise measurements at 340, 334, or 365 nm in the method. This chapter provides an overview of the entire procedure of the method, including the principle, reagents, and solutions used in it. It further describes a method of determining glutamate with diaphorase and tetrazolium salts. The method overcomes the unfavorable position of the equilibrium of the reaction because of the continuous reoxidation of NADH formed.

UV-Assay, Manual Method

Publisher Summary This chapter describes enzyme glutamate-oxaloacetate transaminase (GOT), which has been demonstrated in microorganisms and in all animal and human tissues so far investigated. It is most active in heart muscle, then follow in humans: brain, liver, gastric mucosa, adipose tissue, skeletal muscle, kidney, and so on, and finally serum with considerably smaller amounts. The UV-assay has proved most popular. The enzyme requires pyridoxal phosphate as coenzyme. This is usually present in sufficient amounts in serum and in all tissues. GOT is applied in biochemistry and clinical chemistry. The optimum pH and optimum substrate concentrations have been investigated by several authors. In the pH range 6.5–9.0 the activity is dependent on the aspartate concentration, but scarcely dependent on the 2-oxoglutarate concentration. With values around 15 U/l the standard deviation is 0.8 U/l, the coefficient of variation 5.5%. The mean upper limit of normal for GOT activity in serum for men is around 19 U/l and for women is 15 U/l. According to Bucher et al., GOT is located in both the intra- and extra-mitochondrial compartments of the cell.

Cholesterol and Esterified Cholesterol

Publisher Summary Cholesterol, an essential component of the human organism, is a building material for the cell membranes of all tissues and the parent substance of numerous hormones of the adrenals and sexual organs. Cholesterol occurs in a relatively high concentration in blood, where it is predominantly esterified with fatty acids. It is eliminated by liver, mainly in the form of bile acids. The determination of cholesterol is of great importance for clinical diagnosis, because a high serum cholesterol level is one of the important risk factors for arteriosclerosis and myocardial infarction. The formation of cholestenone, which is measured by the increase in extinction at 240 nm, is proportional to the quantity of cholesterol present. Esterified cholesterol is broken down into free cholesterol and fatty acid with ethanolic KOH or by enzymatic cleavage with cholesterol esterase. It is, therefore, possible to determine the free cholesterol, the esterified cholesterol, and the total cholesterol. This chapter describes the procedure in which the esterified cholesterol is hydrolyzed with ethanolic KOH. The pH optimum of the enzyme is pH 7–8. The enzymatic reaction proceeds fastest in 0.5 M phosphate buffer in the presence of a solubilizing agent.

Pyruvate Kinase Assay in Serum and Erythrocytes

Publisher Summary This chapter elaborates on pyruvate kinase (PK), which was discovered by Lohmann and Meyerhof and in 1942 first crystallized from rat muscle by Negelein. In the rat, the PKs from skeletal muscle, heart muscle, and brain differ from those from liver and kidney. In nonsphereocytic hemolytic anemia, a deficiency of PK in the erythrocytes was established. The disease is genetically determined and appears to be autosomal recessive. PK is applied in biochemistry and clinical chemistry. Lactate dehydrogenase (LDH) must not contain more than 0.01% PK; the Boehringer preparation conforms to this requirement. As the reaction is started with adenosine-5′-diphosphate (ADP) solution, all interfering compounds can react before the PK reaction and therefore, the PK assay is specific.

l-Glutamate: Determination with Glutamic Dehydrogenase

Publisher Summary This chapter presents an overview of a method for the enzymatic determination of L -glutamate with glutamic dehydrogenase. Glutamic dehydrogenase (G1DH) catalyzes the reaction described in the chapter. The equilibrium lies far to the left. However, by trapping the α-oxoglutarate with hydrazine, and with a large excess of DPN and alkaline medium (pH 9), the L -glutamate can be quantitatively oxidized to α-oxoglutarate. The increase of optical density at 340 or 366 mμ because of the formation of DPNH is a measure of the reaction. For purity, the G1DH should have a specific activity of at least 3 units /mg. The enzyme must be free from glutaminase. The DPN solution should be stored at 0–4°C and should be made fresh after ca. 2 weeks. The specific activity of the G1DH suspension should be checked each month. The suspension should be stored at 0–4°C. The buffer solution is stable at room temperature. Ammonium ions interfere with the determination; therefore they must be removed. Inaccurate timing of the addition of the DPN solution to the blank and experimental cuvettes leads to false results.

Colorimetric Assay with l-Lactate, NAD Phenazine Methosulphate and INT

This chapter focuses on colorimetric assay with l -lactate, NAD phenazine methosulfate (PMS), and 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride (INT). Optimum activity is obtained at pH 8.55 with 6 mM NAD and 45 mM l -lactate. All solutions should be prepared with fresh, doubly distilled water. All solutions should be stored, stoppered, in a refrigerator at ca. 4°C. Blood should be collected without venestasis. The calculations are best made on the basis of control sera of known activity. Not every batch of INT is suitable; a preliminary test should be made to check whether on reduction of the INT with ascorbic acid sufficient intensity of color is obtained. If diluted serum, urine or diluted tissue extract is used, the protein concentration is often not sufficient to hold the difficultly soluble formazan in solution. In this case 1 % ethoxy oleyl alcohol must be added to buffer solution.

Fructose-1, 6-diphosphate Aldolase UV Assay, Manuel Method

This chapter focuses on aldolase, which was first crystallized in 1943 by Warburg and Christian from rat muscle. The enzyme is widely distributed; it is found in all cells which catabolize carbohydrate via glycolysis. Fructose-1,6-diphosphate aldolase is applied in biochemistry and clinical chemistry. The pyruvate contained in the sample reacts with lactate dehydrogenase and NADH before the start of the assay. With collidine buffer the aldolase activity of serum, blood hemolysates, and muscle homogenates has a broad pH optimum between 7 and 8, while with veronal buffer it is between 8.5 and 9. In the presence of cyanide, crystalline aldolase from bovine liver has a pH optimum between 9.1 and 9.4 with fructose-1,6-diphosphate and between 8.1 and 8.4 with fructose-1-phosphate. All solutions should be prepared with fresh doubly distilled water. Store all solutions at ca 4°C. Aldolase is relatively thermostable in the presence of large amounts of other proteins. The crystalline enzyme obtained from ox liver catalyses the cleavage of fructose-1-phosphate and the synthesis of erythrulose phosphate from dihydroxyacetone phosphate to about the same extent, which suggest that existence of separate enzymes for these reactions is unlikely.