Hans Ulrich Bergmeyer

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.

Adenosine-5′-diphosphate and Adenosine-5′-monophosphate

Publisher Summary Adenosine-5-diphosphate (ADP) and adenosine-5-monophosphate (AMP) are used in biochemistry and clinical chemistry. The enzymatic determination with myokinase (MK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) estimates ADP and AMP in a single assay system. This method is preferable to the existing chromatographic methods because of its simplicity and speed. The principle is that the decrease of NADH measured by the change in extinction at 340 nm, 334nm, and 365 nm is proportional to the amount of AMP and ADP present. There can be interference in the assay technique because the NADH preparation may contain AMP and that can be corrected by blank. In the assay, spectrophotometer or spectrum-line photometer is suitable for precise measurements at 340 nm, 334 nm, or 365 nm. The chapter describes the preparation of sample and procedure for the assay. Drugs and other therapeutic measures can affect the result of the assay.

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.

Ein neues Prinzip enzymatischer Analyse

Enzymes fixed to a water-insoluble carrier can be applied for analysis in the same way as inorganic catalysators. They can be used continuously and with a single defined activity more than 10000 determinations can be carried out instead of only one. For the example of a glucose determination a simple apparatus is described for analysing non-deproteinised samples. The buffer solution is pumped continuously and the samples (ca. 60/h) are fed into the circuit accurately dosed by a novel applicator. An O2-sensitive electrode is used as detector. The result is obtained within < 1 min.ZusammenfassungAn wasserunlösliche Träger fixierte Enzyme lassen sich wie anorganische Katalysatoren zur Analyse verwenden. Sie können laufend wieder verwendet werden und es können mit einer definierten Aktivität statt einer mehr als 10000 Bestimmungen ausgeführt werden. Am Beispiel der Glucose-Bestimmung wird eine einfache Meßanordnung beschrieben, mit der nicht enteiweißte Proben analysiert werden können. Die Pufferlösung wird ständig umgepumpt. Über einen neuartigen Probenapplikator werden die Probelösungen (ca. 60/h) exakt dosiert in den Strom eingespeist. Meßdetektor ist eine O2-empfindliche Elektrode. Das Meßergebnis liegt in < 1 min vor.

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.

Analytical Differentiation of Purine and Pyrimidine Nucleotides Determination of ADP, ATP, and Sum of GTP + ITP in Biological Material

Publisher Summary This chapter presents the determination of adenosine-5′-diphosphate (ADP), adenosine-5′-triphosphate (ATP), and the sum of guanosine-5′-triphosphate (GTP) and ionosine-5′-triphosphate (ITP) in biological material. The specific enzymatic determinations of ATP, for example by using luciferase, have been known for some time; however, in the past, ATP has been determined mainly by two non-specific methods: (1) with hexokinase (HK) and glucose-6-phosphate dehydrogenase and (2) with phosphoglycerate kinase (PGK) and glyceraldehyde phosphate dehydrogenase (GAPDH). HK and PGK act on the other nucleoside triphosphates as well as on ATP. Only myokinase (MK) is specific for ADP and ATP. When nucleoside triphosphates are converted non-specifically into the diphosphates by any kinase, only ADP, out of all the diphosphates, can be specifically converted by myokinase into ATP and AMP. This chapter describes a method in which the creatine kinase (CK) reaction is used for the specific determination of the ADP originally present in the sample. ATP inhibits the F-6-PK reaction, but this inhibition can be eliminated by the addition of A-5-MP.

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.

UV-Assay after Separation on DEAE-Sephadex

Publisher Summary This chapter describes the isoenzymes of lactate dehydrogenase (LDH). The individual types of LDH proteins can be separated from each other by electrophoresis or chromatography. LDH isoenzymes can be differentiated by heat denaturation or by inactivation in the presence of high urea concentrations. Under defined conditions, DEAE-Sephadex A-50 adsorbs the heart muscle-type LDH protein, while the liver-type LDH remains in solution. The optimum concentration for LDH1, LDH3, and LDH5 is 0.15 mM. NADH must be free from inhibitors. If the adsorption is taken into account, the coefficient of variation is ca. ±6%. The adsorption values for sera with normal LDH activity are between 30% and 60%.

Alkaline Phosphatase in Serum Determination with Automatic Analysers

Publisher Summary This chapter focuses on phosphatase activities from 60–300 U/l that can be determined with optimum conditions for measurements. Alkaline phosphatase in serum is applied in routine clinical chemistry. p-Nitrophenylphosphate is hydrolyzed by phosphatases to phosphate and p-nitrophenol. All solutions should be prepared with fresh, doubly distilled water and also the dilutions should be prepared very accurately. Only fresh serum free from hemolysis should be used. Tygon tubing should be used for connections. The assays are carried out with cam set at 40–1/2, that is, at the rate of 40 assays per hour and a ratio of sample to washing fluid of 1:2. The sample collector tube is then immersed for 30 seconds in the sample and 60 seconds in the washing fluid. Read off the enzyme activity in the samples (serum) in U/l from the standard curve. The variance coefficient for the automated assay of alkaline phosphatase is 1.2% at ca 80 U/l.