Enzymes of the Arterial Wall

By John Esben Kirk

Enzymes of the Arterial Wall is a comprehensive up-to-date monograph, and is the first publication dealing specifically with quantitative determinations of enzyme activities in human and animal vascular tissue. All available information concerning this subject is included. This summary of all current knowledge will be very useful to scientists who lack extensive library facilities and knowledge of foreign languages necessary for a thorough and time-consuming personal search of the original literature. A systematic description is made of 98 different enzymes; nearly all enzymes in the carbohydrate metabolic pathways are included. Brevity of discussion has made it possible to incorporate all available data. The results represent 27,200 quantitative biochemical assays performed with reliable analytical techniques on both normal and arteriosclerotic tissue; 70 enzymic procedures are described. The framework for the arrangement of facts throughout the book was designed to make information easily accessible. Each enzyme is described separately, using the sequence of The Commission on Enzymes of The International Union of Biochemistry, and is followed by literature references with full titles.

• Language: English
• Publisher: Academic Press
• Release date: Jun 28, 2014
• ISBN: 9781483261461

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ENZYMES OF THE ARTERIAL WALL

JOHN ESBEN KIRK

Washington University, Division of Gerontology, St. Louis, Missouri

St. Louis, Missouri

Table of Contents

Cover image

Title page

Copyright

Dedication

Preface

Introduction

Chapter 1: Oxidoreductases

α-Glycerophosphate Dehydrogenase (L-Glycerol-3-phosphate : NAD Oxidoreductase; 1.1.1.8)

Sorbitol Dehydrogenase (l-Iditol : NAD Oxidoreductase; 1.1.1.14)

Aldose Reductase (Alditol : NADP Oxidoreductase; 1.1.1.21)

Lactic Dehydrogenase (l-Lactate : NAD Oxidoreductase; 1.1.1.27)

α-Hydroxybutyric Dehydrogenase

NAD-Linked Adrenaline Dehydrogenase

β-Hydroxyacyl-Coenzyme A Dehydrogenase (l-3-Hydroxyacyl-CoA : NAD Oxidoreductase; 1.1.1.35)

Malic Dehydrogenase (L-Malate : NAD Oxidoreductase; 1.1.1.37)

NADP-Malic Enzyme (l-Malate : NADP Oxidoreductase [Decarboxylating]; 1.1.1.40)

Isocitric Dehydrogenase (NADP-specific) (threo-Ds-Isocitrate : NADP Oxidoreductase [Decarboxylating]; 1.1.1.42)

6-Phosphogluconate Dehydrogenase (6-Phospho-d-gluconate : NADP Oxidoreductase [Decarboxylating]; 1.1.1.44)

Glucose-6-phosphate Dehydrogenase (d-GIucose-6-phosphate: NADP Oxidoreductase; 1.1.1.49)

GlyceraIdehyde-3-phosphate Dehydrogenase (D-Glyceraldehyde-3-phosphate: NAD Oxidoreductase [Phosphorylating]; 1.2.1.12)

Aldehyde Oxidase

Succinic Dehydrogenase and Succinic Oxidase

Glutamic Dehydrogenase (l-Glutamate: NAD Oxidoreductase [Deaminating]; 1.4.1.2)

Monoamine Oxidase (Monoamine: Oxygen Oxidoreductase [Deaminating]; 1.4.3.4)

Benzylamine Oxidase

Glutathione Reductase (Reduced-NAD(P): Oxidized Glutathione Oxidoreductase; 1.6.4.2)

Diaphorase (Reduced-NAD: Lipoamide Oxidoreductase; 1.6.4.3)

Cytochrome c Reductase (NADH-linked) (Reduced-NAD: [Acceptor] Oxidoreductase; 1.6.99.3)

Cytochrome c Oxidase (Ferrocytochrome c: Oxygen Oxidoreductase; 1.9.3.1)

Catalase (Hydrogen Peroxide: Hydrogen Peroxide Oxidoreductase; 1.11.1.6)

Peroxidase (Donor: Hydrogen Peroxide Oxidoreductase; 1.11.1.7)

Chapter 2: Transferases

Catechol-O-methyltransferase (S-Adenosylmethionine: Catechol O-methyltransferase; 2.1.1.6)

Transketolase (D-Sedoheptulose-7-phosphate: D-Glyceraldehyde-3-phosphate Glycolaldehyde-Transferase; 2.2.1.1)

Transaldolase (Sedoheptulose-7-phosphate: D-Glyceraldehyde-3-phosphate Dihydroxyacetonetransferase; 2.2.1.2)

Glycogen Phosphorylase (α-1,4-Glucan: Orthophosphate Glucosyltransferase; 2.4.1.1)

Purine Nucleoside Phosphorylase (Purine Nucleoside: Orthophosphate Ribosyltransferase; 2.4.2.1)

Glutamic-Oxalacetic Transaminase (L-Aspartate: 2-Oxoglutarate Aminotransferase; 2.6.1.1) and Glutamic-Pyruvic Transaminase (L-Alanine: 2-Oxoglutarate Aminotransferase; 2.6.1.2)

Hexosamine-Synthesizing Enzyme (L-GIutamine: D-Fructose-6-phosphate Aminotransferase; 2.6.1.16)

Hexokinase (ATP: D-Hexose 6-Phosphotransferase; 2.7.1.1)

Phosphofructokinase (ATP: D-Fructose-6-phosphate 1-Phosphotransferase; 2.7.1.11)

Pyruvate Kinase (ATP: Pyruvate Phosphotransferase; 2.7.1.40)

Phosphoglyceric Kinase (ATP: 3-Phospho-D-glycerate 1-Phosphotransferase; 2.7.2.3)

Creatine Phosphokinase (ATP: Creatine Phosphotransferase; 2.7.3.2)

Myokinase (ATP: AMP Phosphotransferase; 2.7.4.3)

Phosphoglucomutase (α-D-Glucose-1,6-diphosphate: α-D-Glucose-1-phosphate Phosphotransferase; 2.7.5.1)

Phosphoglyceric Acid Mutase (2,3-Diphospho-D-glycerate: 2-Phospho-D-glycerate Phosphotransferase; 2.7.5.3)

Uridine Diphosphate Glucose Pyrophosphorylase (UTP: α-D-Glucose-1-phosphate Uridyltransferase; 2.7.7.9)

Ribonuclease (Ribonucleate Pyrimidine-nucleotido-2′-transferase [Cyclizing]; 2.7.7.16)

Rhodanese (Thiosulfate: Cyanide Sulfurtransferase; 2.8.1.1)

Chapter 3: Hydrolases

Carboxylic Esterases (Carboxylic Ester Hydrolases; 3.1.1)

Lipase (Glycerol Ester Hydrolase; 3.1.1.3)

LIPOPROTEIN LIPASE

Phospholipase A (Phosphatide Acyl-hydrolase; 3.1.1.4)

Phospholipase B (Lysolecithin Acyl-hydrolase; 3.1.1.5)

Sphingomyelin Cholinephosphohydrolase

Acetylcholinesterase (Acetylcholine Hydrolase; 3.1.1.7) and Cholinesterase (Acylcholine Acyl-hydrolase; 3.1.1.8)

Cholesterol Esterase (Sterol Ester Hydrolase; 3.1.1.13)

Alkaline Phosphatase (Orthophosphoric Monoester Phosphohydrolase; 3.1.3.1) and Acid Phosphatase (Orthophosphoric Monoester Phosphohydrolase; 3.1.3.2)

5′-NucIeotidase (5′-Ribonucleotide Phosphohydrolase; 3.1.3.5)

Fructose-1,6-diphosphatase (d-Fructose-1,6-diphosphate 1-Phosphohydrolase; 3.1.3.11)

Arylsulfatase A + B and Arylsulfatase C (Aryl-sulfate Sulfohydrolase; 3.1.6.1)

Chondroitin-4-sulfatase

α-Glucosidase (α-d-Glucoside Glucohydrolase; 3.2.1.20) and β-Glucosidase (β-d-Glucoside Glucohydrolase; 3.2.1.21)

β-Galactosidase (β-d-Galactoside Galactohydrolase; 3.2.1.23)

α-Mannosidase (α-D-Mannoside Mannohydrolase; 3.2.1.24)

α-N-Acetylglucosaminidase

β-N-Acetylglucosaminidase (β-2-Acetamide-2-deoxy-d-glucoside Acetamidodeoxyglucohydrolase; 3.2.1.30)

β-Glucuronidase (β-d-Glucuronide Glucuronohydrolase; 3.2.1.31)

Hyaluronidase (Hyaluronate Glycanohydrolase; 3.2.1.35)

β-Xylosidase (β-d-Xyloside Xylohydrolase; 3.2.1.37)

NAD Nucleosidase (NAD Glycohydrolase; 3.2.2.5)

Leucine Aminopeptidase (L-Leucyl-peptide Hydrolase; 3.4.1.1)

Tripeptidase (Amino-acyl-dipeptide Hydrolase; 3.4.1.3)

Carboxypeptidase (A peptidyl-amino-acid Hydrolase; 3.4.2 Subgroup)

Glycyl-glycine Dipeptidase (Glycyl-glycine Hydrolase; 3.4.3.1) and Leucyl-leucine Dipeptidase

Elastase

Thromboplastin

Vasculokinase

Plasmin (3.4.4.14) and Associated Factors

Cathepsin (3.4.4.23), Total Proteolysis and Autolysis

Adenosine Deaminase (Adenosine Aminohydrolase; 3.5.4.4) and Adenylic Acid Deaminase (AMP Aminohydrolase; 3.5.4.6)

Inorganic Pyrophosphatase (Pyrophosphate Phosphohydrolase; 3.6.1.1)

Adenosinetriphosphatases (ATP Phosphohydrolase; 3.6.1.3, and ATP Pyrophosphohydrolase; 3.6.1.8)

Acylphosphatase (Acylphosphate Phosphohydrolase; 3.6.1.7)

FAD-Hydrolyzing Enzyme

Chapter 4: Lyases

Aldolase (Fructose-16-diphosphate D-glyceraldehyde-3-phosphate-lyase; 4.1.2.13)

Citrate Condensing Enzyme (Citrate Oxaloacetate-lyase CoA-Acetylating; 4.1.3.7)

Carbonic Anhydrase (Carbonate Hydro-lyase, 4.2.1.1)

Fumarase (L-Malate Hydro-lyase; 4.2.1.2)

Aconitase (Citrate [Isocitrate] Hydro-lyase; 4.2.1.3)

Enolase (2-Phospho-D-glycerate Hydro-lyase; 4.2.1.11)

Glyoxalase I (S-Lactoyl-glutathione Methylglyoxal-lyase [Isomerizing]; 4.4.1.5)

Chapter 5: Isomerases

Triosephosphate Isomerase (D-Glyceraldehyde-3-phosphate Ketol-isomerase; 5.3.1.1)

Ribose-5-phosphate Isomerase (D-Ribose-5-phosphate Ketol-isomerase; 5.3.1.6)

Phosphomannose Isomerase (D-Mannose-6-phosphate Ketol-isomerase; 5.3.1.8)

Phosphoglucoisomerase (D-GIucose-6-phosphate Ketol-isomerase; 5.3.1.9)

Chapter 6: Comparison of Enzyme Activities in Vascular Samples from Male and Female Subjects

Publisher Summary

Chapter 7: Enzyme Activities of Arterial Grafts

Publisher Summary

Chapter 8: Concluding Remarks

Publisher Summary

Author Index

Subject Index

Copyright

Copyright © 1969, by Academic Press, Inc.

all rights reserved

no part of this book may be reproduced in any form, by photostat, microfilm, retrieval system, or any other means, without written permission from the publishers.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by

ACADEMIC PRESS, INC. (LONDON) LTD.

Berkeley Square House, London W.1

Library of Congress Catalog Card Number: 69-13477

printed in the united states of america

Dedication

Dedicated to My Wife and Dr. Donald D. Van Slyke

Preface

Vascular tissue enzymology, the field to which this book is devoted, has expanded greatly during the last two decades. This has led to the accumulation of a great deal of quantitative data that, if properly collated, would enable one to recognize the most promising trends in this discipline. In this book I have attempted to achieve this goal.

This is a comprehensive up-to-date monograph, and is the first publication dealing specifically with quantitative determinations of enzyme activities in human and animal vascular tissue. All available information concerning this subject is included. Research contributions from 28 different countries are presented. This summary of all current knowledge will therefore be very useful to scientists who lack extensive library facilities and knowledge of foreign languages necessary for a thorough and time-consuming personal search of the original literature.

A systematic description is made of 98 different enzymes; nearly all enzymes in the carbohydrate metabolic pathways are included. Brevity of discussion has made it possible to incorporate all available data. The results represent 27,200 quantitative biochemical assays performed with reliable analytical techniques on both normal and arteriosclerotic tissue; 70 enzymic procedures are described.

The framework for the arrangement of facts throughout the book was designed to make information easily accessible. Each enzyme is described separately, using the sequence of The Commission on Enzymes of The International Union of Biochemistry, and is followed by literature references with full titles. The enzymic values and statistical calculations are presented in 278 tables. The latter show differences in activities exhibited by various types of blood vessels obtained from the same individuals, both male and female, and by arteriosclerotic and normal tissue portions from the same arterial specimens. In addition, coefficients of correlation between age and enzyme activities are methodically listed.

These facts, which emphasize the importance of quantitative vascular enzymology in biology, gerontology, and pathology, provide a guide of prime importance for the study of the mechanisms by which the process of atherogenesis occurs. The extensive and clearly presented information makes it a standard book for all interested in this basic subject.

I am very grateful to Dr. Nathan W. Shock and The Gerontological Society for allowing me to reproduce segments of previous publications which appeared in the Journal of Gerontology; and to the St. Louis City Morgue for fresh human tissue samples provided for over a twenty-year period. My special thanks are due to my wife for the care and attention she has given to all details in the preparation of the manuscript.

Sincere thanks are also due to the following present and former members of our research group who participated in the experimental elucidation of this field: Drs. N. Brandstrup, C. Bruni, Y. O. Chang, S. P. Chiang, M. Chieffi, M. Dyrbye, P. G. Effersøe, P. F. Hansen, F. Haruki, S. Hosoda, K. Iversen, S. G. Johnsen, T. Kheim, T. E. Kirk, T. J. S. Laursen, J. R. Matzke, E. Praetorius, E. Ritz, R. Sanwald, R. Schaus, L. B. Sørensen, and I. C. Wang.

Our research has been supported by the Washington University Ina Champ Urbauer Fund and by grants from Public Health Service (HE-00891), Life Insurance Medical Research Fund (G-56-54), and the St. Louis Heart Association.

Finally, I wish to record my gratitude to the publisher, Academic Press, and its staff for exceedingly helpful assistance.

March, 1969

JOHN ESBEN KIRK

Introduction

Biochemical research on enzyme activities of normal and arteriosclerotic tissue has become a subject of considerable attention in recent years, and it is now widely agreed that such investigation constitutes an important approach for future evaluation of the pathogenesis of arteriosclerosis. Both systemic and local factors are undoubtedly involved in atherogenesis; since enzymes are essential to the biological functioning of tissues, comprehensive studies on the metabolic aspects of arterial walls and alterations in enzyme activities associated with aging and pathological vascular changes are of fundamental significance.

On the basis of quantitative biochemical research performed in the author’s laboratory, the main metabolic pattern of human arteries has been established. These investigations have shown that the aortic wall distinguishes itself by having a low respiratory rate, a rather high rate of glycolysis, and a low Pasteur effect (Kirk et al., 1954). These results have subsequently been confirmed by Fontaine et al. (1960). In the blood vessel wall systematic studies on vascular tissue enzyme activities have demonstrated the functioning of the glycolytic and glycogen pathways, the tricarboxylic acid cycle, the hexose monophosphate shunt, oxidative chain, malate shunt, and sorbitol pathway. Additional analyses on other special groups of enzymes and on tissue cofactor concentrations have also been made. Reviews of the general metabolism of the human arterial wall have been presented (Kirk, 1963, 1968).

The specific area of enzyme activities in human and animal vascular tissue is presented in this book. It covers quantitative values for more than 90 different enzymes and describes the assay procedures. Several important observations are mentioned in the text, and in general, only brief comments are made. Because the book is an annotated bibliography the enzymes are purposely arranged in the sequence given by the Commission on Enzymes of The International Union of Biochemistry (1965); in agreement with this commission, trivial names are used in the text. To avoid arbitrary units, most of the activities have been expressed as millimoles of substrate metabolized per gram wet tissue and per gram tissue nitrogen per hour. Since values recorded for arteriosclerotic samples may be partly influenced by a replacement of the arterial tissue with inert non-nitrogenous material, the inclusion of activity values expressed per gram tissue nitrogen is appropriate.

The majority of assays of human vascular tissue have been performed by the author and his associates. The enzymic values are listed as (1) mean activity observed for each decade of subjects with calculated standard deviation of distribution (s.d.distr.). (2) In addition, tables are presented in which comparisons have been made of enzyme activities of thoracic descending aorta, pulmonary artery, normal coronary artery, and vena cava inferior samples from the same persons. Because of the low susceptibility of the pulmonary artery and the vena cava to pathological changes, these data may be of great significance. (3) Variations in enzyme activities with age have been determined systematically; since the values recorded for children frequently are quite different from those observed for adults (Kirk, 1963, 1966), coefficients of correlation have been calculated for 0- to 89-year and 20- to 89-year age groups. (4) For most of the enzymes, a table is also provided in which the mean enzyme values for arteriosclerotic tissue portions are tabulated in percentages of the activities exhibited by normal segments of the same blood vessels. These latter tables make it possible to compare enzymic changes associated with aging and with the development of arteriosclerosis.

A special chapter is included in which comparisons are made of enzyme activities of vascular samples derived from sexually mature men and women (18- to 54-year-old subjects). This review covers assays of 58 different enzymes. When sufficient samples were available, statistical calculations were made on aortic, pulmonary artery, coronary artery, and vena cava inferior specimens; a total of 7454 enzyme determinations from the 18- to 54-year age group were used. These quantitative data were selected because it has been well established that premenopausal women are less susceptible to atherosclerosis than men. Finally, a brief review is presented about enzyme activities in vascular grafts.

The enzyme activity measurements by the author’s research group have been made on homogenates of vascular tissue obtained fresh at autopsy shortly after death. Most of the assays were conducted on intima-media samples of the thoracic descending aorta, pulmonary artery, coronary artery, and inferior vena cava; for tissue homogenization a Kontes Duall grinder attached to a controlled electric stirrer was employed. A large number of samples was generally included in each investigation to permit evaluation of the relation of enzyme activity to age. The aortic and coronary artery analyses were performed separately on normal and arteriosclerotic tissue portions. The enzymic assays were made at optimal pH, in the presence of required cofactors, and usually at a substrate concentration permitting zero order reaction; techniques were selected which eliminate interference by other enzymes. Nitrogen determinations were performed by the Kjeldahl method on portions of the same tissue specimens from which the homogenates were prepared.

It is generally agreed that quantitative assays of enzyme activities under such optimal conditions afford a reliable measurement of the enzyme content present in the tissue. But because the substrate and coenzyme concentrations undoubtedly are considerably higher than those present in the tissue, it is not certain to what extent the recorded values correspond to activities exhibited by the vascular wall in vivo. However, it is reasonable to assume that the presence of a compound with high specific biological activity in a tissue is a strong indication of its physiological significance in relation to the functioning of the tissue.

A great disparity exists in the anatomical structure of large, elastic and of medium-sized, muscular-walled arteries. Even though such arteries show several metabolic similarities, the differences in tissue structure are to some extent reflected in the enzyme activities of the blood vessels. For this reason, observations made on 1 type of arteries are not necessarily applicable to other arteries; biochemical studies on separate types of blood vessels are therefore desirable. The salient difference in susceptibility to arteriosclerosis observed for various arteries may actually provide a special opportunity for identification of local metabolic factors involved in the pathogenesis of arteriosclerosis.

In spite of the great differences between structure of human and animal arteries, biochemical measurements of enzyme activities of normal animal vascular tissues and of arterial samples with experimentally induced arteriosclerosis may similarly supply significant information. The enzymic pattern of animal arterial tissue has been studied by several investigators, and the correlation between enzyme activity rates in normal aortic tissue of some animal species and their susceptibility to atherosclerosis has been outlined by Zemplényi et al. (1963, 1965). Research on experimental arteriosclerosis may also prove to be of great value since it gives the opportunity to study enzymic changes occurring in aortic tissue during the initial stage of atheromatosis. However, such findings in animal tissues with newly induced arteriosclerosis cannot be directly compared with human samples showing advanced arteriosclerosis and old morphological lesions.

REFERENCES

Commission on Enzymes of The International Union of BiochemistryEnzyme Nomenclature. Amsterdam: Elsevier, 1965.

Fontaine, R., Mandel, P., Pantesco, V., Kempf, E. Le métabolisme de la paroi artérielle et ses variations au cours du vieillissement. Strasbourg Med.. 1960; 11:605–615.

Kirk, J. E. Intermediary metabolism of human arterial tissue and its changes with age and atherosclerosis. In: Sandler M., Bourne G.H., eds. Atherosclerosis and Its Origin. New York: Academic Press; 1963:67–117.

Kirk, J. E., Aging in enzyme activities of human arterial tissueShock N.W., ed. Perspectives in Experimental Gerontology. Thomas: Springfield, Illinois, 1966:182–192

Kirk, J. E., Arteriosclerosis and arterial metabolismBittar, E.E., Bittar, N., eds. The Biological Basis of Medicine; I. Academic Press, New York, 1968:493–519.

Kirk, J. E., Effersøe, P. G., Chiang, S. P. The rate of respiration and glycolysis by human and dog aortic tissue. J. Gerontol.. 1954; 9:10–35.

Zemplényi, T., Lojda, Z., Mrhová, O. Enzymes of the vascular wall in experimental atherosclerosis in the rabbit. In: Sandler M., Bourne G.H., eds. Atherosclerosis and Its Origin. New York: Academic Press; 1963:459–513.

Zemplényi, T., Mrhová, O., and Grafnetter, D. (1965). The lipolytic activity and the activity of some other enzymes of the arterial wall in different species. Bull. Soc. Roy. Zool. Anvers No. 37, 55–73.

Oxidoreductases

Publisher Summary

This chapter focuses on oxidoreductases, which are the enzymes concerned with biological oxidation and reduction and participate in respiration and fermentation processes. In the oxidation of biological substances, three factors are involved—the hydrogen donator, the hydrogen acceptor, and the catalyst. The oxidoreductase section of enzymes has 14 subgroups. Some of the tissue oxidases catalyze the reduction of molecular oxygen to water, whereas the functioning of monoamine oxidase leads to the formation of hydrogen peroxide. Because an appreciable catalase activity has been demonstrated in human aortic tissue, it may be assumed that hydrogen peroxide produced by monoamine oxidase is quickly converted to oxygen and water.

Oxidoreductases are the enzymes concerned with biological oxidation and reduction and therefore participate in respiration and fermentation processes. In the oxidation of biological substances 3 factors are involved : the hydrogen donator, the hydrogen acceptor, and the catalyst. As pointed out by Baldwin (1963), the NAD- and NADP-linked dehydrogenases have outstanding properties of specificity toward their substrate and toward their hydrogen acceptor; they are apparently all capable of acting reversibly.

The oxidoreductase section of enzymes has 14 subgroups. In the present chapter activities of 24 arterial enzymes of the following subgroups are reported : 1.1 (No. = 12); 1.2 (No. = 2); 1.3 (No. = 1); 1.4 (No. = 3); 1.6 (No. = 3); 1.9 (No. = 1); and 1.11 (No. = 2).

The 1.1 and 1.2 enzymes act, respectively, on the CH-OH group and on aldehyde or keto groups as donors with NAD or NADP as acceptors; for the 1.3 enzymes the CH-CH group of the substrate is the hydrogen donor. The 1.4 subsection consists of enzymes which bring about oxidative deamination. The 1.6 subgroup distinguishes itself by the fact that the reduced forms of the coenzymes are named as donors.

Subgroup 1.9 contains important aerobic enzymes (e.g., cytochrome c oxidase, 1.9.3.1) which function as the terminal steps of the hydrogen transport chains; they act on heme groups of donors with oxygen as acceptor. The 1.11 enzymes (catalase, 1.11.1.6; peroxidase, 1.11.1.7) use hydrogen peroxide as oxidant.

For biological reasons it should be mentioned that some of the tissue oxidases (e.g., cytochrome c oxidase) catalyze the reduction of molecular oxygen to water, whereas the functioning of monoamine oxidase (1.4.3.4) leads to the formation of hydrogen peroxide. Because an appreciable catalase activity has been demonstrated in human aortic tissue (see pp. 109–111), it may be assumed that hydrogen peroxide produced by monoamine oxidase is quickly converted to oxygen and water.

REFERENCE

Baldwin, E. Dynamic Aspects of Biochemistry,, 4th ed. London and New York: Cambridge Univ. Press, 1963.

α-Glycerophosphate Dehydrogenase (L-Glycerol-3-phosphate : NAD Oxidoreductase; 1.1.1.8)

Publisher Summary

This chapter focuses on α-glycerophosphate dehydrogenase, which is a NAD-dependent enzyme located in the cellular cytoplasm that catalyzes the interconversion of α-glycerophosphate and dihydroxyacetone phosphate. The chapter presents research on the α-glycerophosphate dehydrogenase activity in human vascular tissue. The final millimolar concentrations employed in the test were dihydroxyacetone phosphate, 0.35; NADH, 0.155; and triethanolamine-EDTA buffer, pH 7.5, 50.0. The average α-glycerophosphate dehydrogenase activities recorded for various types of blood vessels show conspicuously higher values for the pulmonary artery, coronary artery, and inferior vena cava than for the thoracic descending aorta. Both the aorta, pulmonary artery, and vena cava samples from children displayed notably lower activities than samples from adults. Assays performed on normal and arteriosclerotic aortic tissue portions showed significantly reduced activities for the pathological specimens.

This dehydrogenase is a NAD-dependent enzyme located in the cellular cytoplasm which catalyzes the interconversion of α-glycerophosphate and dihydroxyacetone phosphate. Several biological functions of this enzyme have been considered. Because of its linkage with NAD, it has been suggested that under anaerobic conditions it exerts an influence on the ratio of lactate/pyruvate contents in the tissue. Evidence has been presented that the α-glycerophosphate compound derived from dihydroxyacetone phosphate is a major fatty acid acceptor in arterial tissue. This biochemical process in which synthesized fatty acids are converted to neutral fat has been studied in detail by Stein and co-workers (1962, 1963); the fatty acids penetrating into the arterial wall may similarly be converted to neutral fat. It should also be mentioned that the equilibrium constant of the α-glycerophosphate dehydrogenase reaction greatly favors the reduction of dihydroxyacetone phosphate and thus the formation of α-glycerophosphate.

HUMAN VASCULAR TISSUE

Analytical Procedure

Research on the α-glycerophosphate dehydrogenase activity in human vascular tissue has been done by Kirk and Ritz (1967). The enzymic assays were performed by spectrophotometric measurement of oxidation of NADH by dihydroxyacetone phosphate. Aqueous 2% homogenates were prepared at 0°C; the homogenates were subsequently centrifuged, and the supernatants immediately used for enzyme determination.

The final millimolar concentrations employed in the test (total volume, 3.0 ml) were: dihydroxyacetone phosphate, 0.35; NADH, 0.155; and triethanolamine-EDTA buffer, pH 7.5, 50.0. The reaction was conducted at 37°C using a Beckman DU spectrophotometer provided with thermospacer equipment. The buffer solution, NADH reagent, and 0.1–0.5 ml homogenate supernatant were first placed in a silica cuvette and the volume adjusted to 2.9 ml. After 20 minutes’ preincubation, 0.1 ml dihydroxyacetone phosphate solution was added to the sample and optical density readings at 340 mµ were then made at 5- to 10-minute intervals against a tissue blank over a 30-minute period. Under these conditions, zero order kinetics were usually obtained for 20 minutes; the enzyme activity was calculated on the basis of the linear part of the curve. A close relationship was observed between quantity of tissue utilized and recorded values. A reagent blank was run with each test; no notable changes in optical density were found for the blank.

Results

The average α-glycerophosphate dehydrogenase activities recorded for various types of blood vessels show conspicuously higher values for the pulmonary artery, coronary artery, and inferior vena cava than for the thoracic descending aorta (Tables I-1 and I-2). In view of the fact that the α-glycerophosphate compound produced in the reaction catalyzed by this enzyme may be an important factor in the formation of triglycerides in arterial tissue, the very high α-glycerophosphate dehydrogenase level in human coronary artery tissue deserves attention pertaining to the pathogenesis of atherosclerosis.

TABLE I-1

MEAN α-GLYCEROPHOSPHATE DEHYDROGENASE ACTIVITIES OF HUMAN VASCULAR TISSUEa

aValues expressed as millimoles of substrate metabolized per gram wet tissue and per gram tissue nitrogen per hour.

bThoracic descending aorta.

cFrom Kirk and Ritz, 1967.

dFrom Kirk and Ritz, supplementary. (Throughout this volume, supplementary refers to supplementary studies performed after the original publication.)

TABLE I-2

MEAN αGLYCEROPHOSPHATE DEHYDROGENASE ACTIVITIES OF PULMONARY ARTERY, NORMAL CORONARY ARTERY, AND VENA CAVA INFERIOR SAMPLES EXPRESSED IN PERCENT OF ACTIVITIES OF NORMAL AORTIC TISSUE FROM THE SAME SUBJECTSa

aFrom Kirk and Ritz, 1967.

bt of diff. = statistical significance of difference.

Both the aorta, pulmonary artery, and vena cava samples from children displayed notably lower activities than samples from adults (Table I-1). For the 20- to 87-year age group, no changes with age were found for the pulmonary artery or coronary artery, whereas the activities of normal and lipid-arteriosclerotic aortic tissue declined (Table I-3); the decrease was most pronounced in the arteriosclerotic specimens. In contrast to this, a tendency to increase in α-glycerophosphate dehydrogenase with age was noted for the inferior vena cava.

TABLE I-3

COEFFICIENTS OF CORRELATION BETWEEN AGE AND α-GLYCEROPHOSPHATE DEHYDROGENASE ACTIVITYa

aFrom Kirk and Ritz, 1967.

br = coefficient of correlation; t = statistical significance.

cThoracic descending aorta.

Assays performed on normal and arteriosclerotic aortic tissue portions showed significantly reduced activities for the pathological specimens (Table I-4).

TABLE I-4

MEAN α-GLYCEROPHOSPHATE DEHYDROGENASE ACTIVITIES OF HUMAN ARTERIOSCLEROTIC TISSUE EXPRESSED IN PERCENT OF ACTIVITIES OF NORMAL TISSUE PORTIONS FROM THE SAME ARTERIAL SAMPLEa

aFrom Kirk and Ritz, 1967.

bThoracic descending aorta.

REFERENCES

Kirk, J. E., Ritz, E. The glyceraldehyde-3-phosphate and α-glycerophosphate dehydrogenase activities of arterial tissue in individuals of various ages. J. Gerontol.. 1967; 22:427–432.

Stein, Y., Stein, O. Incorporation of fatty acids into lipids of aortic slices of rabbits, dogs, rats, and baboons. J. Atherosclerosis Res.. 1962; 2:400–412.

Stein, Y., Stein, O., Shapiro, B. Enzymic pathways of glyceride and phospholipid synthesis in aortic homogenates. Biochim. Biophys. Acta. 1963; 70:33–42.

Sorbitol Dehydrogenase (l-Iditol : NAD Oxidoreductase; 1.1.1.14)

Publisher Summary

The second enzyme of the sorbitol pathway, sorbitol dehydrogenase, oxidizes sorbitol to fructose with NAD as the cofactor. In a study described in this chapter, vascular samples obtained freshly at autopsy for sorbitol dehydrogenase determination were limited to those derived from individuals whose serum activity did not exceed 1 IU/ml. The sorbitol dehydrogenase activities are rather low, but the demonstrated existence of this enzyme in the vascular wall is of definite interest. Essentially similar sorbitol dehydrogenase values were found for the aorta, pulmonary artery, and coronary artery, whereas conspicuously lower activity was exhibited by the inferior vena cava. A comparison of enzymic values of lipid-arteriosclerotic and normal aortic tissue specimens showed no great differences, whereas statistically lower activities were observed for fibrous-arteriosclerotic tissue.

The second enzyme of the sorbitol pathway, sorbitol dehydrogenase, oxidizes sorbitol to fructose with NAD as the cofactor. Through the combined action of aldose reductase and sorbitol dehydrogenase, D-glucose can thus be converted to D-fructose via sorbitol. The demonstration of both aldose reductase (Kirk, 1967) and sorbitol dehydrogenase (Ritz and Kirk, 1967) in human vascular tissue indicates the complete functioning of the sorbitol metabolic pathway in the vascular wall.

HUMAN VASCULAR TISSUE

Analytical Procedure

The vascular samples obtained fresh at autopsy for sorbitol dehydrogenase determination (Ritz and Kirk, 1967) were limited to those derived from individuals whose serum activity did not exceed 1 IU/ml. Aqueous 5% homogenates were prepared at 0°C; after centrifugation, aliquots of the supernatants were immediately used for enzyme activity assays.

The method employed for sorbitol dehydrogenase measurement was based on that described by Gerlach (1957). In this procedure, tissue extracts are incubated with D-fructose and NADH and the conversion of reduced NAD to NAD is measured spectrophotometrically. The final millimolar concentrations in the test (total volume, 3.0 ml) were: D-fructose, 222.0; NADH, 0.16; and triethanolamine buffer, pH 7.4, 120.0. All reagents were obtained from the Boehringer-Mannheim Co., New York. The reaction was conducted at 37°C using a Beckman spectrophotometer. The buffer solution, NADH reagent, and 1.0 ml 5% homogenate supernatant were first pipetted into a silica cuvette. After 15 minutes’ preincubation the fructose substrate was added, and optical density readings at 340 mμ were then made at 15-minute intervals against a tissue blank over a 1-hour period. Under these conditions zero order kinetics were obtained, and a close relationship was observed between quantity of tissue utilized and measured enzymic values. A reagent blank was run with each test.

Results

The sorbitol dehydrogenase activities listed in Table I-5 are rather low, but the demonstrated existence of this enzyme in the vascular wall is of definite interest. The mean activity displayed by human aortic samples is only about one-tenth of that reported by Kmoniček (1961) for human muscular tissue; it is also consistently lower than his data for the human lens.

TABLE I-5

MEAN SORBITOL DEHYDROGENASE ACTIVITIES OF HUMAN VASCULAR TISSUEa,b

aValues expressed as micromoles of substrate metabolized per gram wet tissue and per gram tissue nitrogen per hour.

bFrom Ritz and Kirk, 1967.

cThoracic descending aorta.

As seen from Tables I-5 and I-6, essentially similar sorbitol dehydrogenase values were found for the aorta, pulmonary artery, and coronary artery, whereas conspicuously lower activity was exhibited by the inferior vena cava. Studies of variation in sorbitol dehydrogenase activity with age displayed markedly lower values for samples from children than from adults, this difference being most apparent for the 0- to 1-year infants (Table I-5). For the 20- to 87-year age group, no statistically significant variations with age in enzyme activity were recorded, but a tendency was noted for the sorbitol dehydrogenase level of the vena cava to decrease in aging individuals (Table I-7).

TABLE I-6

MEAN SORBITOL DEHYDROGENASE ACTIVITIES OF PULMONARY ARTERY, NORMAL CORONARY ARTERY, AND VENA CAVA INFERIOR SAMPLES EXPRESSED IN PERCENT OF ACTIVITIES OF NORMAL AORTIC TISSUE FROM THE SAME SUBJECTSa

aFrom Ritz and Kirk, 1967.

TABLE I-7

COEFFICIENTS OF CORRELATION BETWEEN AGE AND SORBITOL DEHYDROGENASE ACTIVITYa

aFrom Ritz and Kirk, 1967.

bThoracic descending aorta.

Comparison of enzymic values of lipid-arteriosclerotic and normal aortic tissue specimens showed no great differences (Table I-8), whereas statistically lower activities were observed for fibrous-arteriosclerotic tissue. Assays of coronary artery samples revealed distinctly lower sorbitol dehydrogenase activities of the lipid-arteriosclerotic than of the normal tissue specimens of that artery (Table I-8).

TABLE I-8

MEAN SORBITOL DEHYDROGENASE ACTIVITIES OF HUMAN ARTERIOSCLEROTIC TISSUE EXPRESSED IN PERCENT OF ACTIVITIES OF NORMAL TISSUE PORTIONS FROM THE SAME ARTERIAL SAMPLEa

aFrom Ritz and Kirk, 1967.

bThoracic descending aorta.

REFERENCES

Gerlach, U. Pathologischer Übertritt von Sorbitdehydrogenase ins Blut bei Lebererkrankungen. Klin. Wochschr.. 1957; 35:1144–1145.

Kirk, J. E. Transaldolase and aldose reductase activities of human vascular tissue. J. Lab. Clin. Med.. 1967; 70:889–890. [(abstract)].

Kmoniček, J. Enzyme activity of the human lens. Cesk. Ofth.. 1961; 17:102–106. [(in Czechoslovakian)].

Ritz, E., Kirk, J. E. The phosphofructokinase and sorbitol dehydrogenase activities of arterial tissue in individuals of various ages. J. Gerontol.. 1967; 22:433–438.

Aldose Reductase (Alditol : NADP Oxidoreductase; 1.1.1.21)

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This chapter focuses on aldose reductase, which is the first enzyme of the sorbitol pathway. It catalyzes the conversion of glucose to its reduced alcohol form, sorbitol. The occurrence of sorbitol and other polyols in body fluids and organs of mammals is explainable by the action of aldose reductase. The chapter presents a few aldose reductase investigations in which supernatant aliquots of centrifuged aqueous homogenates were incubated at 37°C with d-glyceraldehyde and NADPH, and the rate of oxidation of NADPH was measured spectrophotometrically at 340 m/μ. The mean aldose reductase values observed for various types of human blood vessels showed an average activity for arterial samples of about 0.007 mmole of substrate metabolized/gm wet tissue/hour. Significantly lower aldose reductase values were recorded for the vena cava inferior than for the aorta, whereas moderately higher mean activities were found in the pulmonary and coronary arteries.

Aldose reductase is the first enzyme of the sorbitol pathway; it catalyzes the conversion of glucose to its reduced alcohol form, sorbitol. NADPH is the specific hydrogen donor for this initial reaction. Besides glucose, a number of other aldoses with free aldehyde groups or free glycosidic hydroxyl groups are reduced by aldose reductase in the presence of NADPH. Since in every case the aldehyde group is reduced, the name aldose reductase was proposed by Hers (1956) for the responsible enzyme. One of the most suitable substrates for assay of aldose reductase is purified D-glyceraldehyde, the Km for this compound being much lower than that of glucose.

The occurrence of sorbitol and other polyols in body fluids and organs of mammals is explainable by the action of aldose reductase. The accumulation of polyols in rat lens with cataract has received much attention, but studies on aldose reductase activities in animal vascular tissue have not yet been reported. However, it has been demonstrated by Ritz in the author’s laboratory (Ritz and Kirk, 1967) that when rabbit aortic homogenates are incubated with ¹⁴C-1-labeled glucose and NADPH in tris buffer, pH 7.4, a formed sorbitol compound can be identified by paper chromatography. Determinations of aldose reductase activity in human vascular tissue have been made by Kirk (1967).

HUMAN VASCULAR TISSUE

Analytical Procedure

In the aldose reductase investigations (Kirk, 1967) supernatant aliquots of centrifuged aqueous homogenates (corresponding to 25 or 50 mg fresh tissue) were incubated at 37°C with D-glyceraldehyde (Mann Co., New York) and NADPH, and the rate of oxidation of NADPH was measured spectrophotometrically at 340 mµ.

The final millimolar concentrations employed in the test (total volume, 3.1 ml) were: D-glyceraldehyde, 16.0; NADPH, 0.12; and phosphate buffer, pH 7.4, 50.0. After 5 minutes’ preincubation of the tissue supernatant sample with buffer and NADPH, the enzymic reaction was started by addition of the D-glyceraldehyde substrate. Optical density readings were then made for 20 minutes at 5-minute intervals; the reaction curve was usually linear over this period, and very satisfactory proportionality was found between amount of tissue incubated and recorded values. A tissue control sample in which the D-glyceraldehyde substrate was replaced with an equal volume of distilled water and a reagent blank were run with each test. The final 16.0 mM D-glyceraldehyde concentration used for measurement of aldose reductase activity was chosen because a Km value of 4.25 × 10−3 M was found for this substrate.

Results

The mean aldose reductase values observed for various types of human blood vessels (Table I-9) show an average activity for arterial samples of about 0.007 mmole of substrate metabolized/gm wet tissue/hour. Although this enzymic rate with the 3-carbon substrate D-glyceraldehyde is appreciable, a much lower activity will be achieved when glucose or other sugars with longer chain length are incubated with aldose reductase. It should also be pointed out that the activity of human arterial tissue is only about one-tenth of that reported in the literature for the nucleus of the calf lens.

TABLE I-9

MEAN ALDOSE REDUCTASE ACTIVITIES OF HUMAN VASCULAR TISSUEa,b

aValues expressed as millimoles of substrate metabolized per gram wet tissue and per gram. tissue nitrogen per hour.

bFrom Kirk, 1967.

cThoracic descending aorta.

As seen from Tables I-9 and I-10 significantly lower aldose reductase values were recorded for the vena cava inferior than for the aorta, whereas moderately higher mean activities were found in the pulmonary and coronary arteries. In both arterial and venous tissues, a tendency toward decrease in activity with age was observed (Table I-11). When expressed on the basis of tissue nitrogen content, comparison of arteriosclerotic and normal tissue portions (Table I-12) revealed slightly higher aldose reductase activity in pathological arterial samples.

TABLE I-10

MEAN ALDOSE REDUCTASE ACTIVITIES OF PULMONARY ARTERY, NORMAL CORONARY ARTERY, AND VENA CAVA INFERIOR SAMPLES EXPRESSED IN PERCENT OF ACTIVITIES OF NORMAL AORTIC TISSUE FROM THE SAME SUBJECTa

aFrom Kirk, 1967.

TABLE I-11

COEFFICIENTS OF CORRELATION BETWEEN AGE AND ALDOSE REDUCTASE ACTIVITYa

aFrom Kirk, 1967.

bThoracic descending aorta.

TABLE I-12

MEAN ALDOSE REDUCTASE ACTIVITIES OF HUMAN ARTERIOSCLEROTIC TISSUE EXPRESSED IN PERCENT OF ACTIVITIES OF NORMAL TISSUE PORTIONS FROM THE SAME ARTERIAL SAMPLEa

aFrom Kirk, 1967.

bThoracic descending aorta.

REFERENCES

Hers, H. G. Le mécanisme de la transformation de glucose en fructose par les vésicules séminales. Biochim. Biophys. Acta. 1956; 22:202–203.

Kirk, J. E. Transaldolase and aldose reductase activities of human vascular tissue. J. Lab. Clin. Med.. 1967; 70:889–890. [(abstract)].

Ritz, E., Kirk, J. E. The phosphofructokinase and sorbitol dehydrogenase activities of arterial tissue in individuals of various ages. J. Gerontol.. 1967; 22:433–438.

Lactic Dehydrogenase (l-Lactate : NAD Oxidoreductase; 1.1.1.27)

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This chapter focuses on lactic dehydrogenase, which mediates the reversible conversion of l-lactic acid to pyruvic acid by means of the transfer of hydrogen to and from NAD. The equilibrium of the reaction is in favor of the conversion of pyruvate to lactate. In alkaline medium and at high lactate and NAD concentrations, it is reversed notably to the conversion of lactate to pyruvate. The lactic dehydrogenase activity of human vascular tissue was determined by a macromodification of Strominger and Lowry’s procedure. The lactic dehydrogenase activity in human vascular tissue reveals a notably high level of this enzyme in the vessel walls. When expressed per gram wet tissue weight lower lactic dehydrogenase activity was exhibited by arteriosclerotic than by normal aortic samples derived from the 50- to 89-year age group, no significant difference was recorded when the enzymic values were calculated on the basis of tissue nitrogen content. The lactic dehydrogenase activities reported for bovine aortic and venous tissue are of the same order of magnitude as in human vascular tissue, when the values are expressed at the same temperature of enzymic assay. A tendency to decrease in activity with aging was noted.

Lactic dehydrogenase mediates the reversible conversion of l-lactic acid to pyruvic acid by means of the transfer of hydrogen to and from NAD:

The equilibrium of the reaction is in favor of the conversion of pyruvate to lactate; however, in alkaline medium and at high lactate and NAD concentrations it is reversed notably to the conversion of lactate to pyruvate. It was demonstrated by Kirk et al. (1954) that the rate of lactic acid formation in human aortic tissue is predominant and that high glycolysis occurs both under anaerobic and aerobic conditions. On the basis of these observations it has been suggested by Lehninger (1959) that the rather extensive lactic acid production in arterial tissue from original neutral compounds may result in a relatively low pH of the tissue which can be expected to be a factor of importance in preventing the formation of insoluble calcium phosphate compounds in the arterial wall. Because lactic dehydrogenase is directly involved in lactic acid production, the functioning of this enzyme in vascular tissue is of great significance. The activity of lactic dehydrogenase has been assayed both in human and animal arterial specimens, and extensive studies have recently been reported by Lojda and Frič (1966a,b) on the presence of lactic dehydrogenase isoenzymes in the aortic wall.

HUMAN VASCULAR TISSUE

Analytical Procedure

The lactic dehydrogenase activity of