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University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600
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Studies on the metabolism of oxalate, glyoxylate, glycolate and glycine by peroxisomes and mitochondria from rat liver
Sutherland, Steven Thomas, Ph.D.
The Ohio State University, 1991
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
STUDIES ON THE METABOLISM OF OXALATE, GLYOXYLATE,
GLYCOLATE AND GLYCINE BY PEROXISOMES AND
MITOCHONDRIA FROM RAT LIVER
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Steven Thomas Sutherland, B.S.
*****
The Ohio State University
1991
Dissertation Committee: Approved by
K.E. Richardson j / / c> (P * / /? G.P. Brierley Adviser Department of Medical A.J. Merola Biochemistry •To Mom and Dad
ii ACKNOWLEDGEMENTS
I wish to express sincere appreciation to Dr. Keith E.
Richardson for his guidance and patience throughout the research. Thanks go to the members of my advisory committee, Drs. Gerald P. Brierley and A. John Merola.
Special thanks go to Dr. Richard H. Nuenke for his assistance. VITA
September 14, 1956 .... Born - Takoma Park, Maryland
1978 ...... B.S., Andrews University Berrien Springs, Michigan
1979-1980 ...... Graduate Teaching Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio
1982-1985 ...... Assistant Chemist and Associate Chemist, Loma Linda Foods, Mt. Vernon, Ohio
1986-1988 ...... Graduate Research Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio
1988-1990 ...... Biochemistry Tutor, College of Dentistry, The Ohio State University, Columbus, Ohio
FIELDS OF STUDY
Major Field: Medical Biochemistry
iv TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii
VITA ...... iv
LIST OF TABLES vi
LIST OF FIGURES ...... viii
LIST OF ABBREVIATIONS ...... xi
INTRODUCTION ...... 1
CHAPTER I LITERATURE REVIEW ...... 3
CHAPTER II MATERIALS AND METHODS ...... 52
CHAPTER III RESULTS AND DISCUSSION ...... 71
CONCLUSIONS ...... 125
APPENDIX A: DATA FOR THE METABOLISM OF GLYOXYLATE AND GLYCOLATE IN RAT LIVER MITOCHONDRIA AND PEROXISOMES ...... 130
APPENDIX B: DATA FOR THE METABOLISM OF GLYOXYLATE AND GLYCOLATE IN RAT LIVER MITOCHONDRIA AND PEROXISOMES—II ...... 134
APPENDIX C: DATA FOR THE EFFECTS OF ALANINE, GLUTAMATE AND ORNITHINE ...... 138
APPENDIX D: DATA FOR THE EFFECTS OF DL-PHENYL- LACTATE ...... 142
LIST OF REFERENCES ...... 129
V LIST OF TABLES
1. Purity of [l-,4C]glyoxylate. Identity of contaminants...... 73
2. Specific activities of marker enzymes ...... 75
3. Peroxisomal uptake of oxalate, glyoxylate, glycolate and glycine. Radioactivity retained with peroxisomes...... 83
4. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Metabolic products ...... 102
5. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-ll. Metabolic products ...... 106
6. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Metabolic products ...... Ill
7. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Metabolic products...... 116
8. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of the initial incubations ...... 131
9. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations . . 132
10. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions ...... 133
vi 11. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of the initial incubations ...... 135
12. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of the AG 50W-X8 column separations . . 136
13. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of glycolate, glyoxylate and oxalate determinations of formate fractions ...... 137
14. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the initial incubations...... 139
15. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column s e p a r a t i o n s ...... 140
16. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions ...... 141
17. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the initial incubations...... 143
18. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations...... 144
19. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions ...... 145
vii LIST OF FIGURES
1. Precursors of urinary oxalate ...... 9
2. Structures of oxalate, glyoxylate and glycolate . 10
3. Pathways of oxalate biosynthesis ...... 11
4. Peroxisomal, mitochondrial and cytosolic compartmentalization of glyoxylate metabolism. . . 16 5. Pathway of oxalate biosynthesis from ascorbic acid in m a n ...... 25
6. Pathway for the biosynthesis of oxalate from ascorbic acid in animals other than man and other primates...... 28
7. Pathways of oxalate biosynthesis from various carbohydrates ...... 33
8. Pathways for the conversion of xylitol to oxalate 3 6
9. Metabolic lesions of primary hyperoxaluria type I and type I I ...... 42
10. Pathway for the metabolism of ethylene glycol . . 48
11. Modified Lowry protein determination standard c u r v e ...... 59
12. Phosphorus standard curve ...... 65
13. 14C quench curve for the Beckman LS 7000 liquid scintillation counter ...... 70
14. Chromatogram of [1-I4C] glyoxylate using a radioactive flow detector ...... 72
viii 15. Swelling of rat liver mitochondria in iso-osmotic solutions of ammonium glycine, ammonium glyoxylate and ammonium glycolate ...... 78
16. Mechanism of glyoxylate, glycolate and glycine uptake by mitochondria ...... 79
17. Swelling of rat liver mitochondria in iso-osmotic solutions of ammonium oxalate ...... 80
18. Mechanism of oxalate uptake by mitochondria . . . 81
19. Uptake of oxalate by peroxisomes...... 84
20. Uptake of glyoxylate by peroxisomes ...... 85
21. Uptake of glycolate by peroxisomes ...... 86
22. Uptake of glycine by peroxisomes...... 87
23. The enzymatic reactions in glyoxylate metabolism occurring in mammalian systems ...... 89
24. Subcellular location of enzymes ...... 92
25. Time study. Incubation of oxalate with mitochon dria ...... 93
26. Time study. Incubation of glycolate with mito chondria ...... 94
27. Time study. Incubation of glycine with mitochon dria ...... 95
28. Time study. Incubation of oxalate with peroxi somes ...... 96
29. Time study. Incubation of glycolate with peroxi somes ...... 97
30. Time study. Incubation of glycine with peroxi somes ...... 98
31. Pathways of glyoxylate metabolism in mitochondria 103
32. Pathways of glycolate and glyoxylate metabolism in peroxisomes...... 105
33. The metabolism of glycolate and glyoxylate in preparations containing both peroxisomes and mitochondria ...... 108
ix 34. The effects of alanine, glutamate and ornithine on the metabolism of glyoxylate in mitochondria 112
35. The effects of alanine, glutamate and ornithine on the metabolism of glyoxylate in peroxisomes . 114
36. The effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in preparations containing both peroxisomes and mitochondria ...... 115
37. The effects of DL-phenyllactate on the metabolism of glycolate and glyoxylate in peroxisomes . . . 117
38. The effects of DL-phenyllactate on the metabolism of glyoxylate in mitochondria ...... 122
39. The effects of DL-phenyllactate on the metabolism of glycolate and glyoxylate in preparations containing both peroxisomes and mitochondria . . 124
40. Pathways for the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes ...... 129
X LIST OF ABBREVIATIONS
ADH ...... Alcohol dehydrogenase
AGT ...... Alanine:glyoxylate transaminase
CCCP ...... Carbonyl cyanide m-chlorophenylhydrazone
C i ...... Curie
C o A ...... Coenzyme A
CPM ...... Counts per minute
DPM . . . . . Disintegrations per minute
DTT ...... Dithiothreitol
EG ...... Ethylene glycol
ER ...... Endoplasmic reticulum
FAD ...... Flavin adenine dinucleotide
FMN ...... Flavin mononucleotide
FW ...... Formula weight
GAD ...... Glycolic acid dehydrogenase
GAO ...... Glycolic acid oxidase
GGT ...... Glutamate:glyoxylate transaminase
HPLC ...... High performance liquid chromatography
LDH ...... Lactate dehydrogenase
NAD ...... Nicotinamide adenine dinucleotide (oxidized)
NADH ...... Nicotinamide adenine dinucleotide (reduced)
NEM ...... N-ethylmaleimide PHI ...... Primary hyperoxaluria type I
PHII ...... Primary hyperoxaluria type II
TCA ...... Trichloroacetic acid
U ...... Unit of enzyme activity
XO ...... Xanthine oxidase
xii Introduction
Oxalate biosynthesis is directly involved in a number of serious pathological conditions in man. Recent studies in the United States and Western Europe indicate that 5 to
10% of the total adult population suffers from kidney stones. About 0.1% of Americans are hospitalized each year for treatment of this condition (Boyce et al., 1965).
Calcium oxalate is a constituent of more than two-thirds of all kidney stones (Nordin and Hodgkinson, 1967; Elliot,
1968; Prien and Prien, 1968). The toxicity of ethylene glycol (EG), which accounts for some 40 deaths each year and serious injury to the renal, cardiopulmonary and central nervous systems of many others, is related to the formation and deposition of oxalic acid in these tissues (Berman et al., 1957) and to the production of glycolate, a precursor of oxalate (Chou and Richardson, 1978). Further, primary hyperoxaluria type I and type II, rare metabolic inborn errors of oxalate metabolism characterized by large amounts of oxalate in the urine, result in kidney stone formation, nephrocalcinosis and frequently death at an early age from uremic poisoning (O'Keeffe et al., 1973).
1 Past studies of oxalate biosynthesis in the rat have used the intact rat, isolated perfused liver and kidney, and hepatocytes. The liver has been shown to be the major site of these pathways. The enzymes involved are distributed among the cytosol, mitochondria and peroxisomes.
In order to better understand the oxalate biosynthetic pathways, the relative contributions of the peroxisomes and the mitochondria to these pathways were investigated. To accomplish this purpose the permeability of the membranes of these organelles to oxalate and several of its precursors was studied. The metabolism of these precursors in isolated mitochondria, isolated peroxisomes, and reconstituted mixtures of both organelles was investigated. Finally, studies on the effects of the cosubstrates of several aminotransferases involved in these pathways and of an inhibitor of glycolic acid oxidase, a central enzyme, were conducted. Chapter I
LITERATURE REVIEW
Oxalic acid, HOOC-COOH, is a relatively strong dicarboxylic acid (pK^l.27, pK2=4.27 for the dihydrate). It crystallizes from water as the dihydrate (m.p. 101-102°C); two anhydrous forms also exist. Anhydrous oxalic acid is soluble in water to 8.7 g/100 g, while the dihydrate is more soluble, 14 g/100 g. Oxalate salts are common in nature and can be found in rocks, microorganisms, plants and animals.
High concentrations are present in leafy plants such as spinach, rhubarb, parsley, cocoa and tea, although their biochemical function has not been established. Oxalate is a constituent in normal human urine (20 to 40 mg/24 hours).
A wide variety of oxalate salts exists including neutral and acid forms. Most oxalate salts are sparingly soluble in water except those of the alkali metals (Li, Na, K), ammonium and iron(III). The least soluble common oxalates are those of calcium and lead, calcium being the most important biologically.
Calcium oxalate exists as the mono-, di- and trihydrate with the mono- and dihydrate being the most common. Calcium
3 oxalate is practically insoluble at neutral or alkaline pH.
The solubility is 0.67 mg/100 g at pH 7 and 13°C and is
greatly affected by ionic strength. The precipitation of
calcium oxalate is inhibited by a number of compounds,
including: urea, citrate, lactate, sulfate, and
pyrophosphate; certain metal ions such as aluminum, copper, iron, magnesium, tin, and zinc; certain colloids and low
molecular weight polypeptides; and glycosaminoglycans,
especially chondroitin sulfate and heparin (Fleisch and
Bisaz, 1964a,b; Elliot and Eusebio, 1965; Sutor, 1969; Sutor
and Wooley, 1970; Welshman and McGeown, 1972; Robertson et
al., 1973; Meyer and Smith, 1975). The large number of
inhibitors found in urine is believed to prevent the
precipitation of calcium oxalate, which is frequently
supersaturating under physiologic conditions.
Calculus disease of the urinary tract has been known
since antiquity and was described by Hippocrates, Celsus,
Galen and Morgagni (Hagler and Herman, 1973). Calcium
oxalate crystals in plant cells were among the first things
observed under the optical microscope (Leeuwenhoek, 1675;
Malpighi, 1686), and by the middle of the 19th century the widespread occurrence of oxalate salts in nature was
recognized. In the early 19th century calcium oxalate was
identified in urinary and renal stones (Wollaston, 1797;
1810) and then in human urine (Donne, 1838) where its presence was thought to be caused by certain diseases. Bird (1851) showed that calcium oxalate crystals occur in both normal and pathological urines and distinguished two main types of these crystals, the mono- and the dihydrate.
Wohler synthesized oxalate from inorganic substances in 1824, four years before his synthesis of urea which ultimately put the lie to the vital force theory. August
Almen, in 1868, appears to be the first to describe calcium oxalate crystals in animal tissues (Hodgkinson, 1977).
The widespread finding of oxalic acid and its salts in living organisms led to interest in the origins of these oxalates. Wollaston suggested the importance of dietary oxalate as a source of oxalate in the urine (Hodgkinson
1977). Dunlop (1896) and others studied intestinal absorption of oxalates. Early suggestions for oxalate precursors included sugar (Bird, 1851) and uric acid (Bird,
1851; Lehmann, 1851). Initially, it was concluded that no oxalate is formed by animal metabolism (Bunge, 1889; Dunlop,
1896; Gaglio, 1887), but work by Auerbach (1879), Mills
(1885) and Salkowski (1899) on dogs and by Lommel (1899) on adult males, using improved analytical techniques, corrected this error. Gaglio (1887), Pohl (1896) and Dunlop (1896) determined that oxalate is not catabolized by animals.
In 1946, calcium oxalate crystal deposition in the kidneys, urinary tract, and the engorged vessels of the brain and the meninges after EG ingestion was reported (Pons and Custer, 1946). Primary hyperoxaluria was first described by Lepoutre in 1925. The first detailed report of a case of oxalosis, the general deposition of calcium oxalate crystals in renal and extrarenal tissues, was in
1950 (Davis et al., 1950) following a post-mortem examination. The term oxalosis was introduced by Chou and
Donohue in 1952. Primary hyperoxaluria was first diagnosed in 1953 (Newns and Black, 1953), when a child with recurrent kidney stones was found to have increased levels of urinary oxalate. The report of hyperoxaluria and oxalosis in identical twins dying from chronic renal failure (Aponte and
Fetter, 1954) brought to light the familial nature of the disease. In the 1960s primary hyperoxaluria and its relationship to oxalosis became better understood. Oxalate is an end product of metabolism in humans and other mammals. Labeled oxalate injected into rats was excreted unchanged in the urine and feces, and respiratory
C02 and urinary hippurate were not significantly labeled
(Weinhouse and Friedmann, 1951; Curtin and King, 1955;
Brubacher et al., 1956). The presence of labeled hippurate would have indicated oxalate conversion to glyoxylate and glycine. In humans, more than 95% of intravenously injected
[14C] oxalate was recovered unchanged in the urine within 36 hours, and no labeled respiratory C02 was detected (Elder and Wyngaarden, 1960). In normal humans, essentially all of the absorbed and endogenously synthesized oxalate is balanced by urinary excretion. Urinary oxalate is directly derived from three major sources— ascorbate, which contributes about 40% (Atkins et al., 1964; Baker et al., 1966); the two-carbon metabolites glyoxylate and glycolate, from which another 40-50% is produced (Crawhall et al.,
1959; Rofe et al., 1980); and dietary oxalate itself, which is 5-10%, occasionally up to 15%, of the urinary oxalate
(Earnest et al., 1974).
Estimates of oxalate in a typical diet range from 97 to
930 mg per day (Zarembski and Hodgkinson, 1962; Archer et al., 1957a). Intake may show seasonal and geographic variation. In developing countries like India where large amounts of oxalate-containing vegetables are consumed, dietary oxalate intake may go up to 2 g per day (Singh et al., 1972).
Dietary oxalate is poorly absorbed under normal conditions mainly due to the low solubility of most oxalates. The majority of the oxalate found in leafy vegetables is highly insoluble calcium oxalate which is not absorbed. Nearly 5% of dietary oxalate is absorbed and excreted in the urine (Marshall et al., 1972; Chadwick et al., 1973). Oxalate absorption is a passive simple diffusion process in rat and rabbit ileal mucosa. It is not energy-dependent since 2,4-dinitrophenol and ouabain have no effect (Binder, 1974). Pinto and Paternain (1978) have shown that oxalate absorption is a non-energy-dependent process in human and rabbit brush border cells. They have also demonstrated the presence of an oxalate binding protein
in the cytosolic fraction of the brush border cells of human
ileum. The significance of this oxalate binding protein is
unknown. Oxalate absorption is highest in the jejunum and
lowest in the colon (Madorsky and Finlayson, 1977).
Sources of urinary oxalate in man are the dietary
oxalate absorbed from the digestive tract and the endogenous
oxalate synthesized from precursors (Figure 1) . The two major endogenous precursors of oxalate in man are ascorbic
acid and glycine. Other sources include glycolic acid,
aromatic amino acids, hydroxyproline, serine, hydroxypyruvic
acid and xylitol. Glyoxylate, glycolate and probably
ascorbate are the only known immediate precursors of
oxalate. Glyoxylate and glycolate (structures shown in
Figure 2) serve as intermediates in the pathways of oxalate
synthesis from all other precursors except ascorbate. The pathways of oxalate biosynthesis are shown in Figure 3.
The role of glyoxylate as a precursor of oxalate was
suspected when it was reported that the ingestion of glyoxylate by rats results in hyperoxaluria (Roncato and
Mascarello, 1937). Confirmation was provided in rats
(Weinhouse and Friedmann, 1951) and man (King and Wainer,
1968) by the administration of 14C-labeled glyoxylate. The oxidation of glyoxylate to oxalate is catalyzed by three
enzymes— xanthine oxidase (XO) (Booth, 1938), glycolic acid 9
7// 'z Ascorbic Acid
li££j Glycine
H i Dietary Oxalate I 1 Hydroxyproline
Serine
Glycolate
Aromatic Amino Acids
Figure 1. Precursors of urinary oxalate. 10
COOH CHO CHoOH I I I COOH COOH COOH
Oxalate Glyoxylate Glycolate
Figure 2. Structures of oxalate, glyoxylate and glycolate. 11
D - glycerata L-gly carafe
hydroxypyruvate «z*-
glycolaidohyde serine
EJhyfcne Glycol efhanolamine CX
glycine
CO, Hydraiyprolme T y r o s i n e GLYCOLATE ■ M0 CLDH? » 6DOXYLATE Tryplophon Phqiyhionine— l d h '"GAO Curate ? GAD xo LDH OXALATE NOH-EHZYKC
ASCORBATE GLYOXYLATE
Figure 3. Pathways of oxalate biosynthesis. (Richardson and Farinelli, 1981) 12
oxidase (GAO) (Richardson and Tolbert, 1961) and lactate
dehydrogenase (LDH) (Banner and Rosalki, 1967).
Xanthine oxidase catalyzes the oxidation of
hypoxanthine to xanthine and xanthine to uric acid. It is
a flavoprotein (FAD) containing iron and molybdenum. The
contribution of XO to in vivo oxalate synthesis is probably
not significant: patients with hereditary xanthinuria, a genetic deficiency of XO, had normal oxalate excretion, and the administration of the XO inhibitor allopurinol to gout patients at levels which completely inhibit purine synthesis did not alter their urinary oxalate excretion significantly
(Gibbs and Watts, 1966).. Also, in vitro inhibition of XO with cyanide did not significantly affect oxalate synthesis in rat liver homogenates (Liao and Richardson, 1972).
Lactate dehydrogenase has been proposed as the major enzyme in controlling oxalate synthesis from glyoxylate in humans (Williams and Smith, 1983). It has been identified as a major enzyme of oxalate synthesis in the 100,000 x g fractions of human liver and heart tissues (Gibbs and Watts,
1973) and in leukocytes and erythrocytes (Smith et al.,
1971). Oxalate synthesis from glyoxylate is maximally inhibited by oxalate monohydrazide and hydroxymethane sulfonate in erythrocyte hemolysates and liver supernatants
(Smith et al., 1972), leading the authors to conclude that
LDH accounts for all of the oxalate synthesis from glyoxylate in tissues other than liver. Lactate dehydrogenase is found ubiquitously in animal
tissues. The generally accepted cellular location of
Lactate dehydrogenase is only in the cytosol. However,
recently researchers have found a relatively small but
significant LDH activity in mitochondria (Kline et al.,
1986). Lactate dehydrogenase catalyzes the reversible
conversion of lactate to pyruvate and requires the coenzyme
NAD for activity. The reversible conversion of glyoxylate
to glycolate and the oxidation of glyoxylate to oxalate are
also catalyzed by LDH. The equilibrium of the
glyoxylate-glycolate interconversion is far in the direction
of the reduced substrate. Dismutation reactions of
glyoxylate to glycolate and oxalate may be accounted for by
LDH.
The isozyme LDH-I, (heart type) has a K,, of 5 mM and
LDH-Ij (muscle type) a Km of 30 mM for glyoxylate oxidation
(Banner and Rosalki, 1967) . The affinity of LDH for
glyoxylate is much less than for lactate. The pH optima are
6.9 for the reduction reaction and 9.3 for the oxidation
reaction (Warren, 1970). The oxidation of glyoxylate to
oxalate is competitively inhibited by oxalate and
noncompetitively inhibited by oxamate (Warren, 1970) . It is
stimulated by pyruvate (Romano and Cerra, 1969) and hydroxypyruvate (Smith et al., 1971). These enhancers may
exert their effect by reoxidizing NADH, the coenzyme of LDH.
Oxalate is a noncompetitive inhibitor of the LDH-catalyzed 14 reduction of glyoxylate; oxamate is a competitive inhibitor
(Banner and Rosalki, 1967).
Glycolic acid oxidase belongs to a class of enzymes called L-a-hydroxy acid oxidases which are found in the liver and kidney peroxisomes. Glycolic acid oxidase is localized in the liver and has a specificity for short chain hydroxy acids. The kidney L-a-hydroxy acid oxidase is specific for long chain acids (Masters and Holmes, 1977) .
Glycolic acid oxidase catalyzes the oxidation of glycolate to glyoxylate and of glyoxylate to oxalate.
Glycolic acid oxidase was identified and partially purified from rat liver by Kun et al. (1954). It was shown to contain FMN and require molecular oxygen for activity.
Schuman and Massey (197la,b) purified pig liver GAO and showed that it was identical to the rat liver enzyme in its activity and requirements. The affinity of rat liver GAO is glycolate » L-lactate > glyoxylate (Richardson and Tolbert,
1961).
Fry and Richardson (1979a) were the first to isolate and characterize GAO from human liver. The authors found that GAO activity in man is limited to the liver. The enzyme has a broad specificity for a-hydroxy acids. FMN is required for enzyme activity. Glycolate is the most effective substrate with a K,, of 3.3 x 10"* M and a pH optimum of 8.8. Glyoxylate is also a substrate for GAO with a K,,, of 3.54 x 10'3 M and a broad pH optimum between 8.4 and 15
8.8. The relative rate of oxidation of glyoxylate compared to glycolate is much lower than that reported for rat liver
GAO (Ushijima, 1973).
The conversion of glyoxylate to oxalate is not the major pathway of glyoxylate metabolism; most of the glyoxylate is oxidized to carbon dioxide (Dean et al., 1967;
King and Wainer, 1968) . Two pathways produce C02 from glyoxylate. Glyoxylate can be condensed with a-ketoglutarate by glyoxylate:a-ketoglutarate carboligase
(Crawhall and Watts, 1962). One of the carbons of glyoxylate is released as C02, and 5-hydroxy-4-ketovalerate is formed (Schlossberg et al., 1968). Carboligase activity is associated' with the mitochondrial a-ketoglutarate dehydrogenase complex (Schlossberg et al., 1970; Saito et al., 1971; O'Fallon and Brosemer, 1977). In the second pathway (Figure 4) , glyoxylate can be combined with pyruvate by mitochondrial 4-hydroxy-2-ketoglutarate aldolase to form
4-hydroxy-2-ketoglutarate (Dekker and Maitra, 1962). This reaction is reversible and will be discussed again in connection with the metabolism of hydroxyproline. The a-ketoglutarate dehydrogenase complex can oxidize
4-hydroxy-2-ketoglutarate to C02 and malyl-CoA (Gupta and
Dekker, 1980). Malate formed by the action of citrate synthase on malyl-CoA can enter the cytosol where it is acted on by malic enzyme to form C02 and pyruvate. This 16
Glycolate Glycolate * Glycolate LDH / % a o \ CAD Glyoxylate <— Glyoxylate Glyoxylate Ji;ay-»0xalate GKC H y d ro iy - C0a Glycine k e to - Pyruvate g lu ta ra te Glycine C 0 g < t KGDH m e ^ COg Malate - ► Malate
Glycine
Cvtosol Mitochondrion Peroxisome
ENZYMES
A Aldolase DAO D-Amino acid oxidase GAD Glycolic acid dehydrogenase GAO Glycolic acid oxidase GKC Glyoxylate:alpha-ketoglutarate carboligase GR Glyoxylate reductase KGDH Alpha-ketoglutarate dehydrogenase LDH Lactate dehydrogenase ME Malic enzyme TA Transaminase
Figure 4. Peroxisomal, mitochondrial and cytosolic compartmentalization of glyoxylate metabolism. 17 sequence of reactions is called the glyoxylate oxidation cycle (Dekker and Gupta, 1979) .
Glyoxylate can be converted to glycine by a number of pyridoxal phosphate-requiring aminotransferases.
Glutamate:glyoxylate (Nakada, 1964; Thompson and Richardson,
1966), ornithine:glyoxylate (Strecker, 1965), alanine:glyoxylate (Thompson and Richardson, 1967), serine:glyoxylate (Noguchi et al., 1978) and aromatic amino acid:glyoxylate (Harada et al., 1978), aminotransferases have been reported. All of them are irreversible under physiological conditions and favor the formation of glycine.
Two enzymes catalyze the conversion of glyoxylate to glycolate. Glyoxylate reductase, or D-glycerate dehydrogenase, uses NADH and is located in the peroxisomes of both liver and kidney (Vandor and Tolbert, 1971). LDH, the other enzyme, has been discussed earlier. The peroxisomal, mitochondrial and cytosolic compartmentalization of glyoxylate metabolism is shown in
Figure 4.
The existence of an alternate pathway of oxalate synthesis from glycolate in which free glyoxylate is not an intermediate was suggested by many studies. Runyan and
Gershoff (1965) found that conversion of glycolate, ethanolamine, and EG to oxalate increased 18-, 14-, and
10-fold, respectively, in vitamin B6-deficient rats, while there was no major increase in the conversion rate of 18 glyoxylate and glycine to oxalate. It was suggested by Cook and Henderson (1969) that glyoxylate is not an intermediate in the synthesis of oxalate from aromatic amino acids.
Studies by Liao and Richardson using isolated perfused rat liver showed that glycolate and ethanolamine are more efficiently converted to oxalate than is glyoxylate (Liao and Richardson, 1972), and that glycolate and hydroxypyruvate inhibit glyoxylate conversion to oxalate while stimulating total oxalate synthesis (Liao and
Richardson, 1978) .
These observations are inconsistent with the metabolism of glycolate to oxalate via glyoxylate, but are easily explained by the direct conversion of glycolate to oxalate.
In fact, glycolate may be oxidized directly to oxalate by the enzyme glycolic acid dehydrogenase (GAD) without the formation of glyoxylate or any other detectable free intermediates. GAD was first reported in the liver of the rat (Richardson and Fry, 1977), and, subsequently, in the liver of the cow, pig and man (Fry and Richardson, 1979b) and the kidneys of the rat (Murthy et al., 1982).
Fry and Richardson (1979b) isolated and characterized
GAD from human liver. In man, enzyme activity is found only in the liver, predominantly in the supernatant fraction.
(Peroxisomes were not resolved in this procedure and were found in the supernatant fraction.) GAD is specific for glycolate; it exhibits no activity towards glyoxylate or 19 glycine. The Michaelis constant for glycolate is 6.3 x 10‘5
M, lower than that of GAO. GAD is inhibited by glyoxylate and DL-phenyllactate. No requirement for oxygen or for cofactors has been demonstrated.
Harris and Richardson (1980) report essentially 100% absorption of orally administered [1-14C]glycolate in the rat, with 3% being recovered as urinary oxalate within 48 hours. They suggest that the oxidation of glyoxylate to oxalate is a rate limiting step in the biosynthesis of oxalate, since the percentage of glycolate converted to glyoxylate remained constant with the administration of higher doses of glycolate, while the percentage converted to oxalate decreased. Significant amounts of glycolate are present in many fruits and vegetables, while much lower amounts are present in meats and milk (Harris and
Richardson, 1980).
Although less than 0.5% of glycine is converted to oxalate (Elder and Wyngaarden, 1960), experiments with isotopically labeled glycine show that 30-40% of urinary oxalate may come from this source (Crawhall et al., 1959).
Two metabolic pathways are possible for the oxidation of glycine to oxalate:
(A) glycine —* glyoxylate — » oxalate
and
(B) glycine —* serine —* * ,ethanolamine . or_ , __„—* ' 3 J hydroxypyruvate glycolaldehyde — >glycolate — * glyoxylate —* oxalate. The conversion of glycine to glyoxylate in pathway A may be catalyzed by D-amino acid oxidase, a flavin-dependent enzyme-/ or by any of several vitamin B6-requiring aminotransferases. However, the high KM of glycine oxidase for glycine, 0.1 M, makes a significant contribution to glyoxylate formation by this enzyme seem unlikely, and the aminotransferase reactions appear to convert glyoxylate to glycine irreversibly (Thompson and Richardson, 1966; 1967).
Liao and Richardson (1972) investigated the conversion of [1-,4C]glycine, [2-l4C]glycine and [U-14C]glycine to oxalate by isolated perfused rat liver. Pathway A would incorporate both carbon atoms from glycine into oxalate, but pathway B would incorporate only the alpha carbon from glycine since the carbonyl carbon would be lost as C02. If both pathways are operative, the contribution of each could be determined from the amounts of 14C recovered in the oxalate from each of the 14C-labeled glycine precursors. The incorporation of radioactivity into oxalate was essentially identical for all three radioisotopes of glycine, a result explained by pathway A only with no significant contribution from pathway
B.
A limited conversion of [3-14C] serine to [14C] oxalate has been demonstrated in the rat (Runyan and Gershoff, 1965).
Gambardella and Richardson (1978) report that less than 4% of serine is metabolized by pathways that contribute to oxalate synthesis in the rat. Serine may be oxidized to 21 oxalate by essentially the same pathways proposed for glycine:
(A) serine — ► glycine — * glyoxylate — » oxalate
and
(B) serine — ’ glYcolaldehyde — > glycolate — * glyoxylate — ►oxalate.
Experiments by Liao and Richardson (1972) show that
[3-14C] serine is a better precursor of oxalate in isolated perfused rat liver than [ 1-I4C]serine, suggesting that the major pathway of oxalate synthesis from serine does not involve glycine as an intermediate. The authors report that at low concentrations of serine pathway B accounts for more than 90% of the [14C]oxalate produced. However, at higher concentrations of serine pathway B accounts for only 75% of the [14C]oxalate, as pathway A plays a larger role. Pathway
B probably proceeds through hydroxypyruvate and not ethanolamine (Gambardella and Richardson, 1978).
Liao and Richardson (1978) demonstrated that
[3-14C]hydroxypyruvate, but not [1-14C]hydroxypyruvate, is oxidized to [I4C]oxalate in isolated perfused rat liver.
Using isotope dilution techniques, they determined the most significant pathway of oxalate synthesis from hydroxypyruvate to be:
hydroxypyruvate — ► glycolaldehyde — ► glycolate
— » glyoxylate — ► oxalate. 22
However, the pathway catalyzing the oxidation of hydroxypyruvate to oxalate through serine, glycine and glyoxylate was shown to be operative in the intact rat
(Gambardella and Richardson, 1978).
The role of hydroxyproline in oxalate biosynthesis is uncertain. Hydroxyproline injected into rats resulted in calcium oxalate deposition in the kidneys (Thomas et al.,
1971; Tawashi, et al.,1980). Also, the feeding of large amounts of hydroxyproline to rats resulted in a greater degree of hyperoxaluria than was induced by vitamin B6 deficiency. However, no increase in oxalate excretion was observed in patients with hydroxyprolinuria, and the administration of large doses of hydroxyproline to humans failed to increase their excretion of oxalate (Williams and
Smith, 1983).
Hydroxyproline is converted to pyrroline-3-hydroxy-5-carboxylate , and then on to
4-hydroxyglutamate (Adams and Goldstone, 1960). This compound is transaminated to form 4-hydroxy-2-ketoglutarate
(Goldstone and Adams, 1962) which is cleaved by a specific mitochondrial aldolase to form glyoxylate and pyruvate
(Dekker and Maitra, 1962). [3,5-,4C]hydroxyproline is a better precursor of [,4C]oxalate than is [2-14C]hydroxyproline
(Ribaya and Gershoff, 1981).
The role of aromatic amino acids as endogenous precursors of oxalate was first suggested by Gershoff and Prien (1960) and Faber and coworkers (1963) who showed that the urinary excretion of oxalate increased for 1-2 days in human subjects following the ingestion of 10 grams of
DL-tryptophan. Many investigators (Doy, 1960; Schwarz,
1961; Pitt, 1962; Faber et al., 1963; Holcomb et al., 1965;
Cook and Henderson, 1969) suggested that the ketoacids phenylpyruvate, hydroxyphenylpyruvate and indolepyruvate are intermediates in oxalate synthesis from phenylalanine, tyrosine and tryptophan, respectively. It was demonstrated in the rat that oxalate derives generally from the side chain of tryptophan (Faragalla and Gershoff, 1963), specifically from C, and C2 (Cook and Henderson, 1969).
However, Cook and Henderson (1969) concluded from hippuric acid recovery studies that glyoxylate is not an intermediate in oxalate synthesis from tryptophan.
The pathways of oxalate formation from phenylalanine, tyrosine and tryptophan were studied by Gambardella and
Richardson (1977). They detected [,4C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine and serine in the urine of rats injected with the L-[ 1-,4C]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of GAO and GAD, reduced the amount of [l4C]oxalate recovered in the urine of rats given the L- [ 1-14C]-labeled aromatic amino acids, but increased the amount of [14C] glycolate from
L-[1-14C]phenylalanine and L-[l-14C] tyrosine and the amount of
[14C]glyoxylate from L-[1-I4C]tryptophan. The investigators 24
concluded that the conversion of phenylalanine and tyrosine
to oxalate proceeds via glycolate which is oxidized directly
to oxalate by GAD, while tryptophan is metabolized to
glyoxylate which is oxidized directly to oxalate by GAO.
However, the pathways of glycolate synthesis from the
ketoacids of phenylalanine and tyrosine and glyoxylate
formation from indolepyruvate remain unknown.
The contribution of ascorbic acid to urinary oxalate
was first demonstrated in the guinea pig (Burns et al.,
1951) and then in the rat (Curtin and King, 1955) and the
rhesus monkey (Abt et al., 1962). Lamden and Chrystowski
(1953) showed that adult males given 9 g of ascorbic acid
for two days had significantly higher urine oxalate
levels— more than twice normal. Heilman and Burns (1955)
reported radioactivity in oxalate from the urine of two
subjects given [ 1-14C]ascorbic acid. Ascorbic acid oxidation
produces 3 5 to 50% of the oxalate excreted in urine (Baker
et al., 1962; Atkins et al., 1964).
The metabolism of ascorbic acid has been extensively
studied, but the mechanism of oxalate synthesis remains
unclear. In vitro, ascorbic acid autoxidizes to give oxalic
acid. The process is shown in Figure 5. It is this
sequence of reactions, possibly enzyme catalyzed, that many
assume to account for oxalate synthesis in animals and man.
Ascorbic acid is converted by the loss of an electron to monodehydroascorbic acid, a semiquinone-like free radical 25
OXALIC ACID COOH
HO- • 0—c --- COOH 9 L <
HO—| \HO-C XJU — o H- ■ > H-i ' s - t H-C-OH HO-C-H HO—C—H :h 2o h CH,OH CH2OH L-ASCORBIC MONODEHYDRO- DEHYDRO DIKETO- THREONIC ACID ASCORBIC ASCORBIC GULONIC ACID ACID ACID ACID
Figure 5. Pathway of oxalate biosynthesis from ascorbic acid in man. 26 which has been detected by electron spin resonance (Yamazaki et al., 1960; Lagercrantz, 1964). The loss of a second electron produces dehydroascorbic acid, a stable lactone, which is hydrolyzed to 2, 3-diketo-L-gulonic acid (Levandoski et al., 1964; Saari et al., 1967). 2,3-Diketogulonic acid can be cleaved between carbons 2 and 3 to yield L-threonic acid and oxalic acid. Oxygen, multivalent cations, alkaline pH, light, high temperature, and low ionic strength enhance ascorbic acid oxidation and ring rupture.
Ascorbic acid is metabolized extensively to C02 in the guinea pig (Burns et al., 1951) and rat (Curtin and King,
1955) . In Macaque monkeys 90-95% of an oral dose of
[1-,4C] ascorbic acid appeared as respiratory 14C02 in 24 hours, while little or no I4C02 appeared after an injected dose (Baker et al., 1975). No 14C02 is detected after labeled ascorbic acid is administered in man (Heilman and
Burns, 1955; 1958; Baker et al., 1962). Baker and colleagues (1966) reported that oxidation impurities resulting from the instability of ascorbic acid under certain shipping conditions and in solution may explain reports of C02 production in man (Atkins et al., 1964) and other primates (Abt et al., 1962).
Ascorbic acid metabolites appearing in human urine include dehydroascorbic acid and oxalate (Heilman and Burns,
1958), and a number of other compounds, of which only ascorbate-2-sulfate has been identified (Baker et al., 27
1971). Unmetabolized ascorbic acid is excreted, but no diketogulonic acid.
The major pathway for ascorbic acid catabolism in animals, except man and other primates, is shown in Figure
6. The conversion of ascorbate to dehydroascorbate may be enzymic or nonenzymic. No specific ascorbic acid oxidase has been reported in animals. The enzymic delactonization of dehydroascorbate has been reported by Kagawa and coworkers (1961; 1962). Enzyme activity is present in guinea pig, rat, rabbit, and ox livers, but essentially absent in man and other primates. The absence of this enzyme is consistent with the absence of respiratory I4C02 production and urinary 2,3-diketogulonate from
[1-,4C]ascorbate in man. The formation of L-lyxonate and
L-xylonate from diketogulonate is catalyzed by
2,3-diketogulonate decarboxylase (Kanfer et al., 1959;
Kagawa, 1962), a widely distributed enzyme probably present in all animals, including man.
The pathway responsible for oxalate synthesis represents 4-10% of the essential catabolism of ascorbic acid in man (Tolbert et al., 1975). Banay and Dimant (1962) used double labelling to show that in guinea pigs oxalate derives from C, and C2 of ascorbic acid. It is commonly suggested that oxalate is derived from 2,3-diketogulonic acid (Saari et al., 1967), since it is an intermediate in the formation of oxalate from spontaneous decomposition of 28
L-ASCORBIC ACID > L-DEHYDROASCORBIC ACID
2,3-DIKETO-L-GULONATE > L-XYLONATE — > L-XYLOSE L-LYXONATE — > L-LYXOSE C02
— > L-XYLULOSE — > L-XYLULOSE-5-P — > L-RIBULOSE-5-P
> XYLITOL -----> C02 (pentose phosphate pathway).
Figure 6. Pathway for the biosynthesis of oxalate from ascorbic acid in animals other than man and other primates. 29
ascorbic acid solutions. Yet in man there is significant
urinary excretion of oxalate without apparent diketogulonate
formation, so this suggestion is probably erroneous. And,
again, the lack of C02 production is not consistent with
this pathway, or with glyoxylate or glycolate as
intermediates. Gambardella and Richardson (1977)
demonstrated that glyoxylate and glycolate are not
intermediates in oxalate formation from ascorbate by isotope
dilution studies. Baker et al. (1966) suggest a mechanism
in which the cleavage of the C2-C3 bond of ascorbic acid is
catalyzed enzymatically and dehydroascorbic acid exists only
as an enzyme-bound intermediate; however, so far no such
enzyme has been reported in any animal tissue.
Since ascorbic acid is a potential precursor of
oxalate, the megadoses of ascorbic acid, up to 10 g/day,
recommended by Linus Pauling (1970; 1974; 1976) and others
has led to concern on the part of health care professionals
over the potential dangers of large doses. In general,
studies on lower levels of supplementation report no
significant effect on urine oxalate excretion. Reports on
higher levels of supplementation are more difficult to
interpret.
Studies by Kallner et al. (1979) of ascorbic acid metabolism in man indicate that under normal physiological
intakes of vitamin C, the pathway for formation of urinary
excretion products is saturated at levels of 40 to 50 mg/day. This suggests that increased oxalate formation from excess ascorbate intake is minimal. Hughes et al. (1981) reported that urinary oxalate levels increased two-fold after the administration of 1 g of ascorbic acid, with no further increase in oxalate excretion with the administration of up to 9 g. Work by Knappwost and Ruhe (1979) showed a dose-dependent increase in urinary oxalate up to 3 g/day of ascorbic acid. Schmidt and co-workers
(1981) suggest that the fact that urinary oxalate levels in subjects plateaued after the third dose demonstrates a saturable system for the metabolism of ascorbic acid.
Studies by Lamden and Chrystowski (1954) indicate that significant increases in 24-hour urine oxalate levels of 45 mg and 68 mg followed daily oral administration of 8 g and
9 g of ascorbic acid, respectively. Doses of less than 4 g of ascorbic acid gave insignificant increases in urine oxalate levels. Takenouchi et al. (1966) found that oxalate excretion rose 25 mg after an ascorbic acid dose of 9 g/day for three days, but noted no significant increase when 3 g/day were administered. The long-term effects on oxalate excretion of daily 1 and 2 g doses of ascorbic acid were reported to be insignificant by Takiguchi et al. (1966) and
Tiselius and Almgard (1977). Takiguchi followed the oxalate levels of subjects ingesting 1 and 2 g/day for 90 to 180 days; Tiselius used subjects ingesting 1 g/day for three weeks. 31
Costello (1979) reported only a 10 mg increase in
24-hour urine oxalate during the daily administration of a
Redoxon tablet containing 8 g of ascorbate, but the level rose to 30 mg above normal one week after the cessation of supplementation. Furthermore, the daily oral administration of 8 g of pure ascorbic acid resulted in no increase in the urine oxalate level during or following the period of ingestion; rather, a slight decrease was observed. Hatch et al. (1980) also observed urine oxalate increases mostly during the post-ascorbate supplementation period. They suggested a lowered renal clearance of oxalate during ascorbic acid administration and an increase when the treatment ends. Schmidt and co-workers (1981) reported an increase in urine oxalate of 37 mg/day during the period of administration of 10 g/day of ascorbic acid for five days and observed a return to normal levels within one day after cessation.
Neither high nor low doses of ascorbic acid contribute significantly to urine oxalate levels in hyperoxaluric patients. McLaurin and associates (1961) reported that the urinary oxalate levels of a hyperoxaluric patient and a normal patient remained within their control ranges during the daily administration of 4 g of ascorbate for three days.
Studies by Atkins et al. (1963) show that ascorbic acid accounts for only 5% of oxalate excreted in hyperoxaluric patients. Oxalate production from various carbohydrates was
studied by Rofe et al. (1980) in isolated perfused rat
hepatocytes. The order of oxalate production from 10 mM
concentrations of the carbohydrates is fructose > glycerol
> xylitol > sorbitol > glucose. Thom et al. (1981) showed that the urinary excretion of oxalate is increased by high
intakes of sucrose in humans. Studies by Ribaya-Mercado and
Gershoff (1984) demonstrated that rats fed diets containing galactose or lactose excreted greater amounts of oxalate in the urine than those fed glucose, fructose or sucrose.
Also, the [14C] oxalate detected in the urine of rats decreased as they were injected with D-[U-I4C]galactose >
D-[U-,4C]fructose > D-[U-14C]glucose. Figure 7 provides an overview of oxalate biosynthesis from various carbohydrates.
In Australia, in 1969, some patients receiving parenterally administered xylitol developed adverse reactions, including calcium oxalate deposition in the brain and kidneys (Evans et al., 1973). Sixteen deaths resulted.
Approximately 22 deaths associated with xylitol infusions were reported in Germany by the early 1970s (Conyers et al.,
1985). Most of the adverse reactions to the parenteral use of xylitol could be readily explained, however, the finding of oxalate deposition was completely unexpected (Conyers et al., 1985). No pathway for oxalate synthesis from xylitol was known at that time. 33
LACTOSE SUCROSE
GALACT05E FRUCTOSE
XYUTOL SORBITOL
CLYCEROL
S crim
POLYSOKOATE tE .F tro l) PROTEIN Glycine*
UICHLOROACETATE
METHQXYFLURANE ASCORDATE
Figure 7. Pathways of oxalate biosynthesis from various carbohydrates. (Conyers et al., 1990) Hannett et al. (1977) reported that urinary excretion
of oxalate was increased significantly in vitamin
B6-deficient rats infused with xylitol compared to rats given glucose, fructose or sorbitol. Furthermore, only in those rats infused with [U-14C] xylitol were significant amounts of [,4C]oxalate excreted. Rofe and coworkers (1977) confirmed the production of labeled oxalate from
[U-14C] xylitol both in vivo and in isolated hepatocytes.
Also, they found that in hepatocytes from pyridoxine-deficient rats, maximal conversion of xylitol to oxalate occurs at a xylitol concentration of 1 mM. At this concentration, the conversion of five [U-,4C]-labeled sugars to [14C]oxalate by hepatocytes from pyridoxine-def icient rats
is in the order: xylitol > fructose > glycerol > sorbitol » glucose (Rofe et al., 1977).
Urinary excretion of oxalate and glycolate have been reported to increase in patients infused with nontoxic amounts of xylitol (Hauschildt et al., 1976; Ogawa, (1981);
Bar and Lohlein, 1987) and in volunteers consuming xylitol,
1 g/kg body weight/day, in their diets (Bar, 1985). In six-month xylitol-feeding studies in mice, the 200 g/kg xylitol diet resulted in increased occurrences of nephrocalcinosis and bladder calculi (primarily calcium oxalate), and increased the excretion of glycolate and oxalate (Bar, 1985). 35
Two pathways have been proposed for the conversion of xylitol to oxalate (see Figure 8). Xylitol is metabolized to D-xylulose by polyol dehydrogenase or D-xylulose reductase and then phosphorylated by xylulokinase to
D-xylulose-5-phosphate. It has been suggested that, in unusual circumstances such as an excess of xylulose-5-phosphate, the transketolase reaction is overloaded, and active glycolaldehyde formed during the xylulose-5-phosphate-transketolase reaction is released as free glycolaldehyde (Thomas et al., 1972).
Xylulose is also converted to xylulose-l-phosphate by fructokinase. Xylulose-l-phosphate can be cleaved by aldolase to form dihydroxyacetone phosphate and glycolaldehyde. The conversion of D-xylulose to glycolaldehyde has been demonstrated using bovine liver fructokinase and rat muscle aldolase (Barngrover et al.,
1981) and fructokinase and aldolase purified from human liver (James et al., 1982). The of the fructokinase reaction with D-xylulose is about 65% of that with fructose, which shows that D-xylulose can be a good substrate for fructokinase (Barngrover et al., 1981). However, this pathway of xylitol metabolism is a quantitatively minor pathway (Dills et al., 1985).
Considerable evidence exists to suggest that the liver is the major site of oxalate biosynthesis and that GAO and
GAD are the major enzymes contributing to oxalate xylitol
1
V D-xylulose
D-xylulose D-xylulose 5-phosphate 1-phosphate glyceraldehyde glycolaldehyde dihydroxyacetone 3-phosphate phosphate 6
V glycolate
7
V oxalate
Figure 8. Pathways for the conversion of xylitol to oxalate. The enzymes catalyzing the reactions are: 1) D-xylulose reductase or polyol reductase, 2) xylulokinase, 3) fructokinase, 4) transketolase, 5) aldolase, 6) aldehyde dehydrogenase, and 7) glycolic acid dehydrogenase or glycolic acid oxidase. 37 biosynthesis. Richardson (1964) discovered a sex difference in GAO levels in adult rats— levels in female rats are
30-40% lower than those in males. Furthermore, hyperoxaluria in male rats correlates with an increased level of liver GAO (Richardson, 1965; 1967) . No increase in liver LDH was observed. Partial hepatectomy markedly decreased the toxicity of ethylene glycol and glyoxylate and increased the toxicity of glyoxylate in the rat (Richardson,
1973). Since GAO and GAD are primarily limited to the liver and LDH is ubiquitous, the marked effect of partial hepatectomy identifies GAO and GAD as the major contributing enzymes.
Liao and Richardson (1973) observed that the oxidation of glycolate or glyoxylate by GAO partially purified from human or rat liver is completely inhibited by 0.02 M
DL-phenyllactate or n-heptanoate; the oxidation of glyoxylate by LDH or XO is not inhibited by 0.067 M
DL-phenyllactate or n-heptanoate. They also reported the inhibition of oxalate synthesis from glyoxylate in isolated perfused rat liver at an inhibitor:substrate ratio of 2:1 was 100% and 92.5% with DL-phenyllactate and n-heptanoate, respectively, thus confirming the major role of GAO in oxalate biosynthesis in this system. Finally, they demonstrated that DL-phenyllactate effectively inhibits oxalate synthesis in vivo. DL-phenyllactate reduced urinary oxalate levels more than 50% in male rats fed sufficient 38 ethylene glycol to cause 50% mortality within 48 hours.
None of the rats given DL-phenyllactate died within 48 hours.
The liver is capable of accounting for all of the oxalate synthesized in the rat, while no oxalate synthesis could be demonstrated in isolated perfused rat kidney
(Richardson, 1973). James et al. (1985) studied the production of oxalate from xylitol in five tissues, the liver, kidney, brain, heart and muscle. They concluded that the liver is the only tissue in which a significant amount of oxalate is synthesized from xylitol; oxalate is formed in the liver and transported to other tissues.
Finally, total hepatectomy significantly altered the metabolism of glycolate and glyoxylate in the rat (Farinelli and Richardson, 1983) . The production of C02 and unidentified metabolites, presumed to be primarily glycine, serine and ethanolamine, from both substrates was reduced by hepatectomy. Oxalate production from glycolate was reduced, while oxalate production from glyoxylate remained essentially unchanged. These results point strongly to the liver as the major site of glyoxylate and glycolate metabolism and of oxalate production.
Primary hyperoxaluria is the term applied to two rare genetic disorders associated with abnormalities in glyoxylate metabolism. It was proved to be an inborn error of metabolism through detailed study of several families in which the traits of recurrent calcium oxalate urolithiasis
and hyperoxaluria were segregated (Archer et al., 1957b;
Archer et al., 1958; Scowen et al., 1959). Type I (PHI),
also called glycolic aciduria, is distinguished by excessive
amounts of oxalic, glycolic and glyoxylic acids in the urine
(Hockaday et al., 1965). In type II (PHII), termed
L-glyceric aciduria, increased amounts of oxalic and
L-glyceric acids are excreted, but excretion of glycolic and glyoxylic acids is normal (Williams and Smith, 1968). These disorders are characterized clinically by recurrent calcium
oxalate nephrolithiasis and nephrocalcinosis, leading to chronic renal failure and death before the age of 20 from uremia. Calcium oxalate deposits may be found in extrarenal tissues, a pathologic condition termed oxalosis.
In the early 1960s, several in vivo studies (Frederick et al., 1963; Smith et al., 1964; Hockaday et al., 1964,
1965) suggested that PHI might be caused by a defect in the transamination of glyoxylate to glycine. The reports that pyridoxine deficiency leads to hyperoxaluria in man (Faber et al., 1963) and experimental animals (Gershoff et al.,
1959; Runyan and Gershoff, 1965) supported this conclusion, since the pyridoxine derivative, pyridoxal phosphate, is required for transamination reactions. However, in vitro studies (Crawhall and Watts, 1962; Dean et al., 1967;
Williams et al., 1967) gave less clear results. Then Koch and colleagues (1967) reported a deficiency of cytosolic 40 a-ketoglutarate: glyoxylate carboligase activity, but not the mitochondrial enzyme, in preparations from liver, spleen and kidney in five patients with the type I disorder. They suggested that the low activity of the carboligase leads to the accumulation of glyoxylate with its subsequent conversion to glycolate and oxalate. The hypothesis that cytosolic carboligase deficiency, rather than abnormal transamination, was the cause of PHI was commonly accepted into the early 1980s (Williams and Smith, 1983).
Schlossberg et al. (1970) and Saito et al. (1971) showed that the carboligase is identical to mitochondrial a-ketoglutarate dehydrogenase in the rat. These reports were confirmed by O'Fallon and Brosemer (1977) who showed that in the rat and rabbit carboligase activity is associated with the a-ketoglutarate dehydrogenase complex and is exclusively mitochondrial. These findings were thought to represent species differences in the subcellular location of the enzyme (Williams and Smith, 1983). Bourke et al. (1972) reported normal dehydrogenase activity in skeletal muscle from a primary hyperoxaluric (type I) patient, but suggested that this was evidence of further biochemical heterogeneity within PHI.
Danpure et al. (1986) investigated the subcellular distribution of the carboligase in normal human and rat livers and in the liver of a primary hyperoxaluric (type I) patient subjected to various degrees and types of trauma. 41
They concluded that the carboligase is probably wholly
mitochondrial and that the cytosolic form is an artifact due
to mitochondrial damage. This led Danpure and Jennings
(1986) to reinvestigate glyoxylate transamination in PHI,
and they found a substantial decrease in alanine:glyoxylate
aminotransferase (AGT) activity in the livers of two
patients with the disease. Subfractionation of one of the
livers revealed a total absence of peroxisomal AGT (the
affected pathway is shown in Figure 9) . Danpure and
coworkers (1987) reported AGT activity was reduced to 11-47%
of the mean control value in six patients with PHI, and the
degree of residual activity appeared to be related to the
clinical severity of the disease. Glutamate:glyoxylate
aminotransferase (GGT) activity was normal in these
patients, and in the most severe cases residual AGT activity was thought to be a result of crossover reactivity from GGT.
The authors suggest using a percutaneous hepatic needle
biopsy and assaying AGT activity to diagnose PHI and
determine its severity.
Three clinical phenotypes— infantile, juvenile and
adult variants— can be distinguished on the basis of the age
of onset of stone formation and/or renal failure. Of
these, the juvenile, onset between 2 and 18 years, is the most common. PHI exhibits genetic heterogeneity.
Pyridoxine sensitive and pyridoxine resistant variants of
PHI have been reported (Watts et al., 1985). 42
TVPE I CHjOH COOH Glycolate A
\ / ^ CHjjHHg ACT x CHO x COOH COOH V ~ 7 \ COOH / COOH Glycine ' ^Glyoxylate Oxalate
TVPE II
CHaOH CH2OH CH2OH \ I D-QDH \ c h n h 2^= — 0= 0 V / CHOH COOH COOH / COOH / Serine HydroxypyruvateD-Glycerate
L-Glycerate Oxalate
Figure 9 Metabolic lesions of primary hyperoxaluria type I and type II. (AGT = alanine:glyoxylate aminotransferase, D-GDH = D-glycerate dehydrogenase) PHII is the result of a different metabolic lesion
(Figure 9), and a distinct pattern of urinary organic acids is seen. Williams and Smith (1968) reported a total absence of D-glycerate dehydrogenase activity in the leukocytes of four patients with L-glyceric aciduria. D-glycerate dehydrogenase is an important enzyme in serine metabolism, catalyzing the reversible conversion of hydroxypyruvate to
D-glycerate. Increased reduction of hydroxypyruvate to
L-glycerate by LDH creates the L-glyceric aciduria. Three mechanisms have been postulated to explain the resulting hyperoxaluria: (1) the conversion of accumulating hydroxypyruvate to L-glycerate increases the NAD/NADH ratio which stimulates the oxidation of glyoxylate to oxalate by
LDH (Williams and Smith, 1971); (2) the excess hydroxypyruvate is converted to oxalate enzymically via glycolaldehyde and glycolate (Liao and Richardson, 1978; Fry and Richardson, 1979b); and, (3) unmetabolized hydroxypyruvate could accumulate and spontaneously produce oxalate (Raghavan and Richardson, 1983b).
Williams and Smith (1971) observed that hydroxypyruvate stimulates the oxidation of glyoxylate to oxalate by rat liver preparations, human leukocytes and erythrocytes, and purified LDH. Furthermore, they showed that
[1-14C] hydroxypyruvate was a precursor of urinary
L-glycerate, but not of oxalate, and [ 1-,4C]glyoxylate a precursor of urinary oxalate, but not of L-glycerate, in a 44 subject with L-glyceric aciduria. They concluded that hydroxypyruvate is not a precursor of oxalate and proposed that the increased NAD/NADH ratio from the reduction of excess hydroxypyruvate to L-glycerate stimulates the oxidation of glyoxylate to oxalate by LDH. This hypothesis is contraindicated by studies using isolated perfused rat liver (Liao and Richardson, 1978), rabbit muscle and beef heart LDH (Raghavan and Richardson, 1983a), and a purified preparation of GAO from human liver, a human liver extract, a lobe of rat liver and LDH (Raghavan and Richardson, 1983b) which show that hydroxypyruvate inhibits the oxidation of
[1-,4C]glyoxylate to [l4C]oxalate. Raghavan and Richardson
(1983a) report that hydroxypyruvate brought about the nonenzymic decarboxylation of glyoxylate and inhibited the
LDH-catalyzed oxidation of glyoxylate to oxalate. They suggest that the nonenzymic decarboxylation of glyoxylate might explain the results obtained by Williams and Smith
(1971).
Liao and Richardson (1978) found that hydroxypyruvate inhibits the oxidation of glyoxylate to oxalate in the isolated perfused rat liver, but stimulates total oxalate and glycolate synthesis and the conversion of glycolate to oxalate. They also showed that [3-14C]hydroxypyruvate, not
[l-l4C]hydroxypyruvate, is a precursor of [I4C]oxalate via glycolaldehyde and glycolate. The authors proposed that the hyperoxaluria is caused by the conversion of some of the 45 accumulated hydroxypyruvate to oxalate. The last step, the oxidation of glycolate to oxalate, may be catalyzed by GAD
(Fry and Richardson, 1979b) which is not inhibited by hydroxypyruvate. And since the normal pathways of glycolate and glyoxylate metabolism are not altered in PHII, there is normal excretion of these compounds in the urine.
Raghavan and Richardson (1983b) provide radioactive isotope dilution and high performance liquid chromatography evidence of the autoxidation of hydroxypyruvate to oxalate on standing in buffered solution at pH 7.4. The authors suggest that accumulating hydroxypyruvic acid may saturate enzyme systems, build up in the tissue and produce oxalate upon aging. This does not explain initial hyperoxaluria in the disease, but may account for part of the excess urinary oxalate as the process advances.
Treatment of primary hyperoxaluria may follow several • different courses— prevention of calcium oxalate precipitation and stone formation, limitation of oxalate precursors and glyoxylate conversion to oxalate, stimulation or replacement of the missing enzyme activity, and treatment of renal failure. In the most severe cases, treatment has not prevented most of the consequences of the disease, including renal failure. Treatment of primary hyperoxaluria has recently been reviewed by Smith (1986).
Efforts to prevent stone formation have followed the treatment of other disorders involving calcium oxalate stone 46 formation and are covered in the discussion of kidney stones. These treatments are not effective in preventing renal failure in PHI. This topic has been reviewed by Butz et al. (1986).
Dietary modification to restrict oxalate and its precursors has been of limited value. Hemodialysis can remove large quantities of oxalate and its precursors in cases of renal failure (Saxon, 1973). Attempts to prevent the oxidation of glyoxylate to oxalate have been not met with much success.
The enzyme deficiency in PHI involves a pyridoxine-dependent reaction, and supplementation with large doses of pyridoxine decreases oxalate excretion in some milder cases of PHI (Faber et al., 1963). This is consistent with the partial cofactor dependency seen in other disorders involving pyridoxal phosphate-requiring enzymes. Finally, organ transplantation has shown promise both as a means of treating renal failure and for enzyme replacement (Deohar et al., 1969; Saxon et al., 1974;
Leumann et al., 1978; Watts et al., 1988). Renal transplantation by itself is not sufficient because of the rapid accumulation of calcium oxalate in the transplanted kidney (Klauwers et al., 1969; Koch et al., 1972;
Halverstadt and Wenzl, 1974). However, a combined liver-kidney transplant has been shown to result in substantial reduction of urinary and plasma oxalate to 47
levels just above normal 8 months after the surgery (Watts
et al., 1987).
EG intoxication is a significant medical and veterinary
problem. EG is the "poor man's" substitute for alcohol and
a popular agent for suicide. Its warm, sweet taste and common use in antifreeze and household cleaning solutions
contribute to the pediatric risk.
The pathway for EG metabolism is shown in Figure 10.
The first reaction, the conversion of EG to glycolaldehyde,
is catalyzed by alcohol dehydrogenase (ADH), a cytosolic
enzyme. The conversion of glycolaldehyde to glycolate is
catalyzed by the mitochondrial enzyme aldehyde
dehydrogenase. Glycolate oxidation to glyoxylate and
oxalate has already been described.
EG toxicity has been attributed to the direct effect of
EG on the central nervous system (Pons and Custer, 1946).
However, studies have shown that EG itself is about as toxic
as ethanol on the central nervous system (Beasley and Buck,
1980; Brown et al., 1983). Numerous authors have proposed
that the toxicity of EG is due to its metabolic
products— glycolaldehyde, glycolate, glyoxylate and oxalate
(Milles, 1946; Bachmann and Goldberg, 1971; Richardson,
1973; Chou and Richardson, 1978). Partial hepatectomy on male rats increased the toxicity of glyoxylate, decreased
the toxicity of glycolate and ethylene glycol, and increased
the urinary oxalate of rats fed glyoxylate, but not those 48
ch 2oh I Ethylene Glycol CH2OH
CHOi | Glycolaldehyde CH2OHi COOH | Glycolic Acid CH2OH 3 V COOH Glyoxylic Acid CHO COOH COOH CHHHa CH2NH Glycine CH2OH COOH Serine Oxalic Acid COOH
Figure 10. Pathway for the metabolism of ethylene glycol. 49 fed glycolate, glycine or ethylene glycol (Richardson,
1973). The relative toxicity is EG < glycolate < glycolaldehyde < glyoxylate. These results suggest that the toxicity of EG is due to the formation of metabolic products such as glyoxylate and oxalate. Studies by McChesney et al.
(1971, 1972), however, have shown that oxalate synthesis is no more than a minor contributing factor.
Chou and Richardson (1978) report that there are two rate-limiting steps in the metabolism of EG. The first is the initial oxidation catalyzed by ADH, and the other is the conversion of glycolate to glyoxylate by GAO. They found that mortality from EG in the rat correlated directly with the concentration of glycolate recovered in the urine and inversely with the concentration of EG, demonstrating that glycolate is the toxic mediator. The concentrations of glycolaldehyde and glyoxylate in the urine of rats metabolizing EG were relatively small and constant.
Finally, Chou and Richardson (1978) demonstrated that glycolaldehyde, glycolate and glyoxylate are themselves toxic, and their toxicity is not due to metabolic intermediates produced.
EG is rapidly absorbed and distributed evenly throughout the body tissues (McChesney et al., 1971). It is metabolized primarily in the liver. Its metabolites are potent inhibitors of respiration and glucose metabolism in slices of rat liver, brain and myocardium (Ruffo et al., 50
1962) . EG toxicity has been divided into three clinical stages (Berman et al.f 1957; Brown et al., 1983): Stage 1 (0 to 12 hours post ingestion)— central
nervous system, gastrointestinal, and
metabolic manifestations,
Stage 2 (12 to 24 hours post ingestion)—
cardiorespiratory manifestations,
Stage 3 (1 to 3 days post ingestion)— renal
manifestations.
Calcium oxalate crystal deposition occurs in the kidneys, urinary tract, and the engorged vessels of the brain and the meninges after EG ingestion (Pons and Custer, 1946).
Treatment of EG poisoning is generally supportive and symptomatic as the physician endeavors to rectify the metabolic abnormalities observed (Winek et al., 1978).
Administration of ethanol, a competitive substrate for ADH, is the most effective treatment, inhibiting the metabolism of EG and allowing it to be excreted unchanged in the urine
(Borden and Bidwell, 1968; Beckett and Shields, 1971). This treatment is only beneficial if begun within four to six hours of EG ingestion (Parry and Wallach, 1974; Peterson et al., 1981). Gastric lavage is effective only if the stomach still contains EG. Hemodialysis is frequently used to decrease serum levels of EG and its metabolites (Collins et al., 1970; Peterson et al., 1981; Jacobsen et al, 1982).
Treatment is usually unsuccessful in cases of excessive EG 51 ingestion or where treatment has been delayed and EG has been extensively metabolized. For delayed treatment to be effective, the toxic mediator, glycolate, must be detoxified or removed.
Urolithiasis is one of the oldest diseases known to man. Until this century, calculi were found most frequently in the bladders of male children. They consisted mostly of calcium oxalate, uric acid and urates. Urinary calculi of the lower urinary tract (bladder and ureters) still predominate in developing countries such as India, the
Middle and the Far East, while stones of the upper urinary tract (kidneys) are found in the highly developed countries of North America and Western Europe.
Only 10-20% of the stone-forming patients have a predisposing disease entity— anatomical defect, metabolic or genetic disorder, renal or bowel disease, etc. All others who form stones with no known underlying cause are termed
"idiopathic stone formers".
Rao et al. (1982) investigated 392 stone formers and found increased urinary excretion of calcium, oxalate or uric acid in 40% and more than one abnormality in 16% of the patients. Robertson et al. (1979) observed a direct correlation between stone disease and increased consumption of calcium, sugar and animal protein. Chapter II
MATERIALS AND METHODS
Determination of Radiochemical Purity
The radiochemical purity of I4C-labeled compounds was determined by HPLC using the method of Marshall (1982) . An aliquot of the sample was chromatographed using an Aminex
HPX-87 (Bio-Rad Laboratories, Richmond, CA) anion exchange column (7.8 x 300 mm) at 40°C. The eluant was 0.0065 M H2S04 at a flow rate of 0.6 ml/min. The effluent was monitored for radioactivity by a Flow-One Beta, Model IC, Flow Through
Detector (Radiometric Instruments and Chemical Company,
Tampa, FL) using Flow-Scint III (Radiometric Instruments) as the scintillation cocktail.
Isolation of Mitochondria for Swelling Studies
Rat liver mitochondria were isolated using the procedure of differential centrifugation (Johnson and Lardy,
1967) with some modification by Strzelecki and Menon (1986).
A male Wistar rat weighing approximately 250 g was decapitated. The liver was removed and placed in an ice-cold solution of 225 mM mannitol, 75 mM sucrose, 0.05 mM
52 EGTA and 5 mM MOPS buffer, pH 7.2 (isolation medium). The liver was minced with a scalpel and homogenized in isolation medium using a Potter-Elvehjem homogenizer. Unbroken cells and nuclei were sedimented at 600 x g for 10 min at 4°C in a Sorvall centrifuge. The supernatant was decanted and saved. The pellet, which contains some mitochondria, was resuspended in isolation medium and centrifuged at 600 x g for 10 min. The supernatant fractions were combined; the pellets were discarded. Mitochondria were sedimented from the supernatant by centrifugation at 15,000 x g for 5 min.
The supernatant was discarded and the pellet was washed and finally resuspended in isolation medium to a concentration of 30-60 mg of protein per ml.
Swelling of Mitochondria
Studies on the swelling of mitochondria were performed as described by Strzelecki and Menon (1986) in solutions of
100 mM ammonium oxalate, ammonium glyoxylate, ammonium glycolate and ammonium glycine plus 5 mM MOPS buffer adjusted to pH 7.2 with NH40H. Five millimolar
W-ethylmaleimide (NEM) , 5 mM phosphate and 0.2 juM carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added as indicated. The absorbance of the mitochondrial suspension
(0.3 mg of protein/ml) was measured at 610 nm at room temperature. 54 Studies of Solute Uptake bv Peroxisomes
Peroxisomal uptake was studied by a simplification of
the method of Van Veldhoven et al. (1983, 1987).
Peroxisomes were purified as described below for the
metabolic studies. They were then incubated with
radioactive solutes and then separated from their incubation
medium through an organic layer of brominated hydrocarbons.
Three solutions of varying densities were layered in
Eppendorf tubes (1.5 ml). The bottom layer was 0.5 ml of
homogenization medium made 0.396 M in sucrose (d = 1.051).
The middle layer was 0.4 ml of a mixture of bromododecane
and bromodecane (8:2, v/v; d = 1.044). The upper layer,
0.55 ml, consisted of 0.24 M sucrose, 20 mM Tricine, pH 7.2,
5 mM DTT and 0.1% (v/v) ethanol. This layer also contained
the purified peroxisomes and 0.5 mM of labeled solute used without dilution of the radioactivity, which amounted to
approximately 1.6 to 1.8 juci/ml. Incubations were started
by the addition of 0.2 to 0. 3 mg of peroxisomal protein. At
the specified times the tubes were centrifuged in an
Eppendorf 5414 table centrifuge at 9980 x g for 7 min. The upper layer and organic middle layer were aspirated. The bottom layer containing the sedimented peroxisomes was mixed with 0.1 ml of 0.2% Triton X-100 and then a portion was
solubilized with Soluene and counted for radioactivity. 55
Preparation of the Mitochondrial and Peroxisomal Fractions for Metabolic Studies
Methods described by de Duve et al. (1955) were used with slight modifications to obtain mitochondrion- and peroxisome-enriched fractions. These fractions were further purified using the methods of Ghosh and Hajra (1986) for peroxisomes and Declercq et al. (1984) and Mannaerts et al.
(1982) for mitochondria.
Male Wistar rats weighing about 250 g were fasted overnight and decapitated. Their livers were quickly removed and placed in tared beakers containing ice-chilled homogenization buffer (0.25 M sucrose, pH 7.3, containing 5 mM DTT, 1 mM diNaEDTA, 2 mM HEPES and 0.1% ethanol). The weight of the liver and the remaining carcass of each rat were determined. The livers were minced on a watch glass using a scalpel and then homogenized in 3 vol (ml/g) homogenization buffer in a glass Potter-Elvehjem homogenizer with a Teflon pestle (Arthur Thomas Co.) using one pass of the pestle at 1,500 rpm. The homogenate was centrifuged at
600 x g for 10 min at 4°C using an SS-34 rotor in a Sorvall
RC2-B centrifuge. The supernatant was saved and the residue rehomogenized and centrifuged as above. This was repeated one more time. The final residue was discarded, and the combined supernatants were centrifuged at 3,300 x g for 10 min at 4°C. The supernatant was saved and the mitochondrion-enriched pellet was washed (3,300 x g, 10 min) 56 with homogenization buffer. The pellet was resuspended in homogenization buffer to a total volume corresponding to 1 ml per 4 g original liver weight.
One and three-quarters milliliters of this suspension were layered on 20 ml of 30% Percoll solution (prepared by mixing 9 vol of Percoll with 1 vol of 2.5 M sucrose and diluting this 3.3-fold with homogenization buffer) in a 30 ml polycarbonate tube (Beckman Cat. No. 340382) and centrifuged at 57,000 x g for 3 0 min at 4°C in a Beckman
Model L5-65 Ultracentrifuge. The bottom 6 ml which contained most of the mitochondria were carefully removed and diluted 5-fold with homogenization buffer to lower the
Percoll concentration. The mitochondria were sedimented by centrifugation at 3,300 x g for 10 min at 4°C and, finally, resuspended in homogenization buffer to approximately 1 ml per 6 g original liver weight.
The 3,300 x g supernatant that was saved above was centrifuged at 25,000 x g for 10 min at 4°C. The resulting supernatant, including as much of the pink fluffy layer as possible, was removed by aspiration. The peroxisome-enriched pellet was washed once (25,000 x g, 10 min) in homogenization buffer and then resuspended in enough buffer to obtain 1 ml per 1 g original liver weight.
Two milliliters of this suspension were carefully layered on the top of 15 ml of 30% Nycodenz (w/v) , 10 mM
TES, 1 mM diNaEDTA solution (pH 7.3, density 1.15 g/ml) and 57 centrifuged at 57,000 x g for 10 min at 4°C. After centrifugation the upper turbid solution was removed with gentle suction and the peroxisomes, present as a small green pellet at the bottom of the tube, were collected after resuspending them in the remaining Nycodenz solution. The
Nycodenz suspension was diluted 5-fold with homogenization buffer, and the peroxisomes were sedimented by centrifugation at 25,000 x g for 10 min at 4°C. The peroxisomes were resuspended in a minimal amount of homogenization buffer.
Modified Lowrv Protein Assay
Protein concentration was determined by the Lowry method (Lowry et al., 1951) as reported by Cooper (1977).
Samples were pretreated as described by Bensadoun and
Weinstein (1976) to remove interfering substances.
The protein solution to be assayed was adjusted to a final volume of 3 ml with distilled HzO in a test tube. Two percent deoxycholate (0.025 ml) was added. The solution was mixed vigorously and allowed to stand for 15 min. One milliliter of 24% trichloroacetic acid (TCA) was added and the solution mixed thoroughly. The test tube contents were spun at 3300 x g for 30 min and the supernatant carefully removed with a Pasteur pipet attached to a water-vacuum line. The last bit of solution was removed by tilting the test tube while keeping the pipet tip in contact with the 58
side of the tube. The pellet was adjusted to 3 ml with
distilled H20, and the procedure was repeated. Two protein
precipitations were needed to remove interference by
Nycodenz. The pellet from the second precipitation was used
for the Lowry protein determination.
Reagents used for the Lowry protein assay were:
reagent A— 100 g Na2C03 to 1 liter with 0.5 N NaOH
reagent B— 1 g CuS04- 5H20 to 100 ml with H20
reagent C— 2 g potassium tartrate to 100 ml with H20
Folin-phenol reagent, 2 N
The protein sample was brought to 1 ml with H20. One milliliter of a mixture of reagents A,B and C (20:1:1,v/v) was added, and the sample was vortexed to mix the contents
completely. The sample was incubated for 15 min at room
temperature. Three milliliters of Folin-phenol reagent (5 ml diluted with 50 ml of H20) were forcibly added, and the
sample was vortexed immediately. The sample was incubated
for 45 min at room temperature and the absorbance measured
at 540 nm. Bovine serum albumin (Sigma) was used for the
standard curve (Figure 11).
Determination of the Purity of the Mitochondrial and
Peroxisomal Fractions
The assay of marker enzymes was used to determine the degree of enrichment of the mitochondrial and peroxisomal iue 1 Mdfe or rti eemnto standard determination protein Lowry Modified 11. Figure Absorbance (540 nm) .5 .6 .7 .8 .2 1 3 4 0 0 curve. 50 rti (Micrograms) Protein 0 10 0 20 300 250 200 150 100
59 60 fractions, the cross-contamination, and the contamination by lysosomes and endoplasmic reticulum (ER). Catalase, urate oxidase and GAO were used as peroxisomal markers.
Mitochondrial markers were cytochrome c oxidase and succinate-cytochrome c reductase. Glucose-6-phophatase was used for ER and acid phophatase for lysosomes.
Catalase was assayed using a micromodification of the method of Baudhuin et al. (1964) by Peters et al. (1972).
Buffered substrate was prepared by dissolving 50 mg of bovine serum albumin in 5 ml of 0.2 M imidazole buffer pH
7.0, adding 5 ml of 2% (w/v) Triton X-100 and 0.1 ml of 30% hydrogen peroxide, and bringing to a total volume of 50 ml with distilled water. Titanium sulfate solution was prepared as described by Leighton et al. (1968). Titanium sulfate, 0.27 g, was dissolved in 40 ml of boiling 2 N H2S04, allowed to cool, filtered, and the filtrate diluted with 20 ml of 2 N H2S04. This was diluted 1:1 (v/v) with distilled water before use.
One-tenth of a milliliter of suitably diluted sample was incubated for 30 min at 25°C with 0.1 ml of freshly prepared buffered substrate. The reaction was stopped and the remaining H202 estimated by the addition of 2 ml of the titanyl sulfate solution. The absorbance of the solution was measured at 405 nm. One unit of enzyme is defined as the amount of enzyme destroying 90% of the H202 present in a
50 ml reaction volume in one minute. 61
Urate oxidase was assayed as described by Leighton et al. (1968) . Three milliliters of 42 fiH sodium urate in 30 mM potassium phosphate buffer pH 7.4 containing 1 mM EDTA and 1% Triton X-100 was added to 0.1 ml of enzyme. The reaction was run at 37°C. Urate oxidase was measured by following the decrease in absorbance at 292 nm. One unit of enzyme is equivalent to a decrease in absorbance of 3.94 per minute.
GAO activity was assayed by the method of Baker and
Tolbert (1966) based on the measurement of glyoxylate phenylhydrazone formation. To a 1 cm cuvette were added:
2.0 ml Potassium phosphate buffer, 0.1 M, pH 8.3
0.1 ml Cysteine*HCl, 0.1 M, pH 6 (prepared
fresh)
0.1 ml Phenylhydrazine*HC1, 0.1 M, pH 6
0.1 ml Sodium glycolate, 0.04 M, about pH 8
0.6 ml Water
The reaction was initiated by the addition of 0.1 ml of enzyme-containing solution, and the progress was followed spectrophotometrically at 324 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the production of one micromole of glyoxylate per minute. For this assay, an increase in absorbance of 0.057 per minute equals 0.01 units. 62
Succinate-cytochrome c reductase was assayed by the
method of Sottocasa et al. (1967) . The following were added to a 3 ml cuvette:
2.7 ml Potassium phosphate buffer, 50 mM, pH 7.5
0.1 ml Potassium cyanide, 9 mM, in buffer
0.1 ml Potassium succinate, 90 mM, in buffer
0.1ml Cytochrome c , 3 mM
0.1 ml enzyme
The reaction was started by the addition of the substrate.
The reaction was run at 30°C, and the reduction of
cytochrome c was measured at 550 nm.
Glucose-6-phosphatase was assayed by the method of
Nordlie and Arion (1966). The following solutions were
prepared:
sodium cacodylate buffer, 0.10 M, pH 6.5
sodium glucose-6-phosphate, 0.15 M, pH 6.5
trichloroacetic acid, 10% (w/v)
To a test tube were added 0.6 ml of cacodylate buffer, 0.2 ml of glucose-6-phosphate solution, and sufficient distilled water to bring the volume to 1.5 ml after the addition of
sample. The test tube and its contents were then preincubated for 5 min at 30°C. The reaction was initiated by the addition of appropriately diluted sample. One milliliter of TCA solution was added to a "zero time" control before the sample. The test tube contents were
incubated for 10 min at 30°C in a shaker bath and then 1.0 63 ml of TCA solution was added to terminate the reaction. The test tube was centrifuged for 8 min at 1800 rpm in an
International Model CL clinical centrifuge to sediment the protein. An aliquot, usually 0.5 or 1.0 ml, was assayed for inorganic phosphate.
Acid phosphatase was assayed by the method of Baudhuin
(1974). Two-tenths of a milliliter of suitably diluted sample were added to 1.8 ml of buffered substrate. 0.2 ml of diluent was used for the blank. The diluent was Triton
X-100 (0.1 g/liter), EDTA (1 mM) and sodium bicarbonate (90 mg/liter); the buffered substrate was 56 mM sodium
0-glycerophosphate in 56 mM acetate buffer, pH 5.0.
Thiomersalate (0.1 g/liter) was added to the buffered substrate to prevent bacterial growth during the long incubation. After incubating the sample for 20 h at 37°C in a shaker bath, it was assayed for phosphorus.
Phosphorus was assayed by the method of Fiske and
Subbarow (1925) as reported by Leloir and Cardini (1957).
The reducing reagent was prepared as described by Flynn et al. (1954).
Reducing reagent was prepared as follows. Twenty nine grams of sodium bisulfite and 1 g of sodium sulfite were dissolved in 200 ml of water. l-Amino-2-naphthol-4-sulfonic acid, 0.5 g, was ground with a little sulfite-bisulfite solution in a mortar. It was washed with the remainder of 64
the solution and filtered. The reagent was prepared fresh
weekly.
To a suitable aliquot of sample 1.0 ml of 5 N sulfuric
acid and 1.0 ml of 2.5% ammonium molybdate were added and
mixed thoroughly. Reducing reagent (0.4 ml) was added, and
the total volume was brought to 10 ml with distilled water.
The contents were mixed well and let sit for 10 min at room
temperature. The absorbance of the solution was measured at
660 nm.
A phosphorus standard was prepared by dissolving 1.3613
g of KH2P04 (FW = 136.09) in 1 liter of water. This was
diluted 1:10 for a working standard containing 1 n m o l P/ml.
The standard curve obtained is shown in Figure 12).
Metabolic Studies
The incubation buffer used was a 130 mM KC1, 20 mM TES,
5 mM K2HP04/ 5 mM NH4C1, 1 mM MgCl2/ 0.1 mM EDTA and 3%
dextran solution adjusted to pH 7.2. Mitochondria,
peroxisomes and mitochondria plus peroxisomes were diluted
with incubation buffer to concentrations which will be given with the results of each experiment. Substrates were
diluted separately with incubation buffer. Solutions of
organelles and substrates were brought to 37°C. Equal
volumes of an organelle-containing solution and a substrate
solution were added to each flask. A center well containing
a piece of filter paper (1 x 2.5 cm) and 0.25 ml of Figure 12. Phosphorus standard curve. standard Phosphorus 12. Figure Absorbance (660 nm) .1 2 0.0 3 4 0^
0.2 hshrs (Micromoles) Phosphorus 0.4 0.6 0.8 1.0 65 66 phenethylamine:toluene:methanol (2:1:1, v/v) was added to each flask to trap C02. Each flask was sealed and its contents thoroughly mixed. Flasks were incubated at 37°C for a specified amount of time and then boiled for 15 min to terminate any enzymatic activity.
Analysis of radioactive metabolites
Carbon dioxide. The incubation flask was acidified by the injection of 0.2 ml of 12 M H2S04 and incubated for 4 h to assure the complete absorption of the ,4C02. The well containing the filter paper with the absorbed ,4C02 was transferred to a counting vial containing 10 ml of scintillation cocktail (0.1 g POPOP, 5 g PPO, 300 ml methanol and 700 ml toluene) and the radioactivity determined in a liquid scintillation counter.
Total amount of rl4Clmetabolites in solution. An aliquot of the solution remaining in the flask, 0.1 ml, was transferred to a counting vial containing 3 ml of Thrift
Solve and counted.
Separation of r14Clmetabolites. The method of
Varalakshmi and Richardson (1983) for the isolation of glycolate, glyoxylate and oxalate from urine was applied and extended here. An aliquot of sample, 1 ml, was applied to a cation exchange column (AG 50W-X8, hydrogen form, 100-200 mesh, 1 x 3 cm) and eluted with 50 mM formate buffer, pH
3.5. Glycolate, glyoxylate and oxalate were completely 67 removed by elution. The eluate was diluted to a known volume, and a small aliquot was taken to determine the total radioactivity of the formate fraction. The column was further eluted with 1.0 M NaCl, 50 mM MOPS, pH 7.0. Glycine and serine were quantitatively removed by this step. This fraction was diluted to a known volume, and a small aliquot was taken to determine the total radioactivity of the salt fraction. All of the radioactivity placed on the column could be accounted for in these two fractions.
Glvoxvlate. Glyoxylate was assayed by the method of
Varalakshmi and Richardson (1983). An aliquot of the formate fraction, 1 ml, was added to 3 ml of 100 mM sodium phosphate buffer, pH 7.4, and placed in a doubly sealed flask with a center well containing filter paper (1 x 2.5 cm) and 0.2 ml of phenethylamine/toluene/methanol (2:1:1, v/v) for trapping C02. Three percent H202 (0.2 ml) was injected through the rubber seal by hypodermic needle, and the sample was incubated for 8 h at 37°C. Under these conditions, H202 completely decarboxylates glyoxylate, but has no effect on glycolate or oxalate (Raghavan and
Richardson, 1983a). After the 8 h incubation, the contents of the flask were acidified by the injection of 0.2 ml of 12
M H2S04 and incubated for another 4 h to ensure complete trapping of C02. The well was then removed and placed in a vial containing 10 ml of scintillation cocktail and counted. Oxalate. Oxalate was assayed essentially by the procedure of Fry and Richardson (1979b). An aliquot of the formate fraction, 1 ml, was added to a flask containing 3 ml of 0.2 M sodium citrate buffer, pH 3.0. A plastic center well holding a strip of filter paper (1 x 2.5 cm) and 0.2 ml phenethylamine:toluene:methanol (2:1:1, v/v) was inserted to trap C02, and the flask was sealed. Oxalate decarboxylase
(0.1 U) was injected through the stopper and the flask incubated for 8 h at 37 °C to ensure complete decarboxylation. 12 M H2S04 (0.2 ml) was added to release dissolved C02 and the incubation continued for 1 h. The center well was transferred to a vial containing 10 ml of scintillation cocktail and counted.
Glvcolate. Glycolate was estimated by subtracting the radioactivity of [14C] oxalate and [1-14C]glyoxylate from the total radioactivity of the formate fraction.
Glycine. An attempt was made to determine the
[14C]metabolites present in the salt fraction by paper chromatography on Whatman No. 1 filter paper. The mobile phase employed was n-butanol:propionic acid:H20 (24:11:15, v/v) after Benson et al., 1950. The method resolved glycine and serine standards. Salt fractions were chromatographed with unlabeled glycine and serine. Only one spot, corresponding to glycine, was found to be radioactive on chromatograms of the salt fractions. Therefore, all 69 radioactivity present in the salt fractions was assumed to be from glycine.
Determination of Counting Efficiency
The 14C-counting efficiency of the Beckman LS 7000 liquid scintillation counter was determined by plotting percent counting efficiency versus H-number for a standard quench series (Figure 13) . A standard quench series of
51,000 DPM (New England Nuclear Research Products, Boston,
MA) was counted using program #4— channel #1 0-655, channel
#2 397-655, two sigma counting error of two, and counting time of ten minutes. (The shape of the resulting quench curve was verified using another standard quench series.)
The graph was divided into three regions which were fitted to three lines by linear regression: for H#'s 0-99 Percent Efficiency = -0.0557(H#) +96.7 (1)
99-155 = -0.0768(H#) + 98.8 (2)
155-200 = -0.185(H#) + 115.6 (3)
Statistics
Statistical analysis was performed using the one-way analysis of variance option of the Statgraphics program
(Statistical Graphics Corporation). iue1. unhcre o h eka S 00 liquid 7000 LS forBeckman the curve quench 13. Figure Counting Efficiency (%) 100 0 scintillation counter.scintillation 50 100 H-Number 150 200 250 70 Chapter III
RESULTS AMD DISCUSSION
Radiochemical Purities of r14C10xalate. n - 14C IGlvoxvlate and
n - 14C1 Glvcolate
Glyoxylate is the least stable of the radiochemicals
used in these studies. The radiochemical purity of a
freshly prepared aqueous solution of [1-I4C] glyoxylate was
determined by HPLC employing a radioactive flow detector.
The chromatogram is shown in Figure 14. One major peak and
four smaller ones are present. Solutions of [14C]oxalate and
[l-14C]glycolate were chromatographed to determine their
retention times. These two chromatograms showed essentially
single peaks, 100% radiochemical purity. The average
retention times for oxalate, glyoxylate (assumed to be the
major peak) and glycolate were 9.7 min, 11.4 min and 14.2 min, respectively. These correspond to peaks 2, 3 and 4 on
the glyoxylate chromatogram (Table 1). The identities of peaks 1 and 5 were not determined. [1-,4C]Glyoxylate is
approximately 90% radiochemically pure, may be 3.5%
glycolate and 2% oxalate, and contains two undetermined
impurities containing 5% of the radioactivity.
71 Figure 14. Chromatogram of [l-^C] using [l-^C] glyoxylate of Chromatogram 14.Figure NET CPM 14-C Scale: 150 o o o O a radioactive flow detector.flow radioactive a o 1 * Ifl Ai o
72 Table 1. Purity of [1-14C]glyoxylate. Identity of contaminants.
Retention Percent of Identity of Time (min} Radioactivitv Comoound
7.9 2.5 Unknown
9.8 1.9 Oxalate (?)
11.4 89 .7 Glyoxyiate
14.3 3.6 Glycolate(?)
15.2 2.3 Unknown 74
The [l4C]oxalate contamination of [1-,4C] glyoxylate was determined by measuring the 14C02 produced during incubation with oxalate decarboxylase. Oxalate decarboxylase is specific for oxalate and does not catalyze C02 production from glyoxylate. The results for the solution chromatographed indicate that 3.8% of the radioactivity in
[1-14C]glyoxylate was [14C]oxalate. This is in good agreement with the 3.6% figure obtained for the peak eluting at 9.8 min on the chromatogram. The oxalate contamination of each glyoxylate incubation medium used was evaluated using the oxalate decarboxylase assay. The results of the experiments were corrected for this contamination which varied between solutions from 0.2 to 3.8%. The possible glycolate contamination was not further evaluated, since the error in such a determination is larger than the amount of glycolate indicated by HPLC.
Purity of Mitochondrial and Peroxisomal Preparations
The mitochondrial and peroxisomal preparations were checked for cross-contamination and for contamination by lysosomes and endoplasmic reticulum by assaying for marker enzymes. Representative results are given in Table 2.
Mitochondria isolated for use in uptake studies were not as pure. Since other organelles do not swell, further purification to remove peroxisomes and lysosomes from the mitochondrial fraction was not required. 75
Table 2. Specific Activities of marker enzymes. Values are reported for two preparations. GAO = glycolic acid oxidase (peroxisomes), SCR = succinate cytochrome c reductase (mitochondria), G6P = glucose-6-phosphatase (ER), AP = acid phosphatase (lysosomes). Specific activities are reported in nmol min-1 mg protein--1- •
Fraction GAO SCR G6P AP liver homogenate 1.6 30.3 76.1 261 2.7 35.5 84.3 270 mitochondria ND 81.7 9.1 39.2 76.2 10.2 41.5 peroxisomes 37.4 3.3 28.1 ND 83.9 3.0 35.9
ND = not detected 76
No peroxisomal contamination of the mitochondrial
preparations could be detected. Mitochondrial contamination
of peroxisomal preparations was less than 4% based on a
comparison of the specific activity of succinate-cytochrome
c reductase found in each fraction. The peroxisomal
preparations were free from lysosomal contamination but
contained some ER. Mitochondrial preparations had a small
degree of contamination by both lysosomes and ER.
Uptake of Oxalate. Glvoxvlate. Glvcolate and Glycine by Rat
Liver Mitochondria— Mitochondrial Swelling Studies
Mitochondrial suspensions make very good osmometers.
The influx of a permeable solute causes an increase in the
matrix volume which can be measured as a decrease in
absorbance on a spectrophotometer. To observe osmotic
swelling in mitochondria both the cation and the anion must
permeate the membrane and the overall charge balance across
the membrane must be maintained. The latter requirement means that the cation and anion must enter by the same mode,
be it electroneutral or electrical. The ammonium ion
penetrates the inner membrane as neutral ammonia. If mitochondrial swelling is to be observed in an ammonium salt
solution, the anion must also penetrate the membrane in a neutral form. A number of low molecular weight acids are
able to penetrate the membrane in their uncharged forms.
Glyoxylate, glycolate and glycine appear to fit into this group. 77
Iso-osmotic solutions of ammonium glycine, ammonium
glycolate and ammonium glyoxylate caused rat liver mitochondria to swell (Figure 15). Phosphate, NEM and CCCP
had no effect on the swelling. These findings are
consistent with the electroneutral uptake of glyoxylate,
glycolate and glycine by rat liver mitochondria (see Figure
16) .
Rat liver mitochondria exhibited minimal swelling when
suspended in an iso-osmotic solution of ammonium oxalate.
The addition of a small amount of phosphate caused swelling
of the mitochondria (Figure 17) . N-ethylmaleimide, a fairly
specific inhibitor of phosphate transport, completely
inhibited the swelling induced by phosphate. These findings
are similar to those reported by Strzelecki and Menon
(1986). Phosphate is required for mitochondrial swelling
to occur in iso-osmotic solutions of ammonium oxalate.
Strzelecki and Menon (1986) concluded that oxalate is transported across the inner membrane by the dicarboxylate transporter in exchange for phosphate. NEM inhibits the phosphate transporter preventing phosphate uptake and, therefore, phosphate-oxalate exchange (Figure 18).
The mitochondrial inner membrane has a relatively restricted permeability. It was important to determine whether or not the projected substrates and metabolites of the contemplated metabolic studies could cross this membrane
for several reasons. First, no metabolic study would be Figure 15. Swelling of rat liver mitochondria in mitochondria liver rat of Swelling 15. Figure Absorbance (610 ran) Mito Mitochondria (final concentration 0.3 mg of 0.3mg (final concentration Mitochondria was the same in each solution.each in same the was protein/ml) were suspended in a solution ain suspended were protein/ml) glyoxylate or glycolate with 10 mM MOPS,10mM with glycolate or glyoxylate containing 100 mM ammonium glycine, ammonium 100mM containing glycolate. ammonium and glyoxylate ammonium glycine, ammonium of solutions iso-osmotic (gm oeoe p . a 5C Swelling (xg/ml1 25°C. at7.2 pH rotenone, O.D. 0.01 Time 1min
78 79
IHNER MEMBRANE out m
NHiJ—s:------>HH3- 'V T H+ H+
R-COO' >R-COOH- -»R-COOH-Ji >R_COO'
R = -CHO glyoxylate -CH2OH glycolate -CHaHHa glycine
Figure 16. Mechanism of glyoxylate, glycolate and glycine uptake by mitochondria. Figure 17. Swelling of rat liver mitochondria in mitochondria liver rat of Swelling 17. Figure Absorbance (610 nm) marked P^_ NEM (5 mM) was used as (5 used was mM) NEM P^_ marked OS 1(ig/ml MOPS, 25°C. at7.2 pH rotenone, protein/ml) were added to a solution ato added were protein/ml) 0.3mg (final concentration Mitochondria indicated’by the dashed line. dashed the indicated’by arrow the at (5 added was mM) Phosphate otiig10m moimoaae 10mM oxalate, ammonium 100 mM containing iso-osmotic solutions of ammonium oxalate. ammonium of solutions iso-osmotic O.D. 0.01 Mito Time 1min NEM
80 81
OUTSIDE INNER INSIDE MEMBRANE
OH" 4 OH' Phosphate Transporter
H2POh“ > HgPOu"
HpPOii” 4 Dicarboxylate Transporter
HOOCCOO' ^ HOOCCOO'
Figure 18. Mechanism of oxalate uptake by mitochondria. 82 necessary for any proposed substrate that could not cross.
Second, the correct interpretation of a metabolic study, particularly one involving a mixture of mitochondria and peroxisomes, may require knowledge of which metabolites can traverse the membrane and which cannot.
Permeability of Rat Liver Peroxisomes to Oxalate.
Glvoxvlate. Glycolate and Glycine
The uptake of oxalate, glyoxylate, glycolate and glycine by isolated rat liver peroxisomes was observed.
Labeled solutes were incubated with peroxisomes for 0, 5,
10, 20 and 30 min and the quantity of radiactivity remaining with the pelleted peroxisomes was determined. The results are presented in Table 3.
The approximately 25,000 cpm pelleted with the peroxisomes from the 0 min incubations is due to peroxisomal uptake during the 7 min centrifugation, solute adhering to the peroxisomes, and solute that penetrates into the bottom layer during centrifugation. The last factor contributes only about 800 cpm. If the radioactivity from the 0 min incubations is subtracted from the other results, the data very closely fit the curve of a rectangular hyperbola
(Figures 19-22) .
Oxalate, glyoxylate, glycolate and glycine are taken up by peroxisomes. The results for all four solutes are very similar, indicating a common mechanism of uptake, probably 83
Table 3. Peroxisomal uptake of oxalate, glyoxylate, glycolate and glycine. Radioactivity retained with peroxisomes.
Incubation CPM Pelleting with Peroxisomes Time Labeled Solute (minutes) Oxalate Glyoxylate Glycolate Glycine
0 23493 24596 25381 27436
5 39762 42073 41540 44698
10 44078 46281 45629 48079
20 46121 48119 47257 51052
30 46675 48561 47841 51666 Figure 19. Uptake of oxalate by peroxisomes. by oxalate of Uptake 19. Figure CPM with Peroxisomes 20,000 25,000 10,000 15,000 5,000 0 0 10 ie (Minutes) Time 20 30 84 Figure 20. Uptake of glyoxylate by peroxisomes. by glyoxylate of Uptake 20. Figure CPM with Peroxisomes 20,000 25,000 10,000 15,000 5,000 0 0 10 ie (Minutes) Time 20 30 85 Figure 21. Uptake of glycolate by peroxisomes. by glycolate of Uptake 21. Figure CPM with Peroxisomes 10,000 15,000 20,000 25,000 5,000 0 0 10 ie (Minutes) Time 20 30 86 Figure 22. Uptake of glycine by peroxisomes. by glycine of Uptake 22. Figure CPM with Peroxisomes 10,000 15,000 20,000 25,000 5,000 0 0 10 ie (Minutes) Time 20 30 87 88 simple diffusion. This is supported by the equilibrium in the uptake of radioactivity reached between 10 and 15 min of incubation.
Time Studies of the Metabolism of r14C) Oxalate. n - 14ClGlvcolate and fl-14ClGlvcine in Rat Liver Peroxisomes and Mitochondria
A knowledge of the enzymes involved in the metabolism of glycolate, glyoxylate and glycine in the rat may be helpful in interpreting the results of the following experiments. Glycolate may be converted to glyoxylate by
GAO or LDH. GAO is found in peroxisomes, while LDH is located primarily in the cytosol. However, a very low level of LDH activity is found in mitochondria. Glycolate may also be converted to oxalate, either directly by GAD or indirectly through glyoxylate by GAO.
Glyoxylate is a precursor for numerous compounds
(Figure 23) including carbon dioxide, glycine, glycolate and oxalate. Carbon dioxide may be formed by any of three metabolic pathways found in mitochondria— the carboligase reaction, the glyoxylate oxidation cycle, and the glycine cleavage system (glycine synthase) after conversion to glycine. Glyoxylate can be converted to glycine by a number of aminotransferases— alanine:glyoxylate aminotransferase, glutamate:glyoxylate aminotransferase and ornithine:glyoxylate aminotransferase. Alanine:glyoxylate 89
Glyoxylate + [0] ---> Oxalate
b6 Glyoxylate + L-Glutamate <===> Glycine + alpha- Ketoglutarate
B6 Glyoxylate + L - O r m t h m e <===> Glycine + Glutamic-gamma- semialdehyde
b 6 Glyoxylate + Other Ammo Acids <===> Glycine + Keto acids
Glyoxylate <===> Glycolate
TPP Glyoxylate + alpha-Ketoglutarate ---> alpha-Hydroxy-beta- ketoadipate + CO2
Glyoxylate + pyruvate <===> 2-Keto-4-hydroxyglutarate
FMN Glyoxylate + CoASH ---> Formyl-S-CoA + C02
Figure 23. The enzymatic reactions in glyoxylate metabolism occurring in mammalian systems (Williams and Smith, 1983). aminotransferase appears to make the most important contribution to glyoxylate metabolism. Alanine:glyoxylate aminotransferase is found predominately in mitochondria
(Rowsell et al., 1972; Noguchi et al., 1978), but significant activity is also found in peroxisomes (Noguchi et al., 1978). The effects of the amino acid cosubstrates, alanine, glutamate and ornithine, on the metabolism of glyoxylate, glycolate and glycine were investigated in this study. Glyoxylate may be converted to glycolate by glyoxylate reductase (D-glycerate dehydrogenase) and by LDH.
Glyoxylate reductase is a peroxisomal enzyme (Vandor and
Tolbert, 1971) . The conversion of glyoxylate to oxalate has already been discussed.
GAO catalyzes the conversion of glycolate to glyoxylate and glyoxylate to oxalate. DL-phenyllactate is a potent inhibitor of GAO, but not of LDH (Liao and Richardson,
1973) . The effect of DL-phenyllactate on the metabolism of glycolate and glyoxylate in preparations of mitochondria, peroxisomes, and mitochondria plus peroxisomes has been investigated here.
The major pathways of glycine catabolism include its direct cleavage to form methylene-tetrahydrofolate, carbon dioxide and ammonia. This is catalyzed by the reversible glycine synthase, a mitochondrial enzyme. Serine may be formed from glycine by the action of serine hydroxymethyltransferase which is localized in both the 91 mitochondria and cytosol. Glycine is also converted to glyoxylate by D-amino acid oxidase, a peroxisomal enzyme.
A listing of the key enzymes is given in Figure 24.
In this study, mitochondria (4.0 mg protein) or peroxisomes (0.30 mg protein) were incubated with
[14C]oxalate, [1-,4C]glycolate or [1-,4C]glycine. Incubations were carried out as described in the methods except that carbon dioxide was not trapped. Instead, 0.5 ml aliquots were removed at 0, 5, 10, 20, 30 and 60 min. These were boiled to terminate enzyme activity. An aliquot was counted. Data obtained on the radioactivity remaining in the solutions are plotted graphically in Figures 25 through
30.
Any loss of radioactivity from the solutions over time was attributed to the production of carbon dioxide. The incubation of oxalate with preparations of mitochondria or peroxisomes resulted in no loss of radioactivity in solution. This was expected, since oxalate is an end-product of rat metabolism.
In mitochondrial preparations, essentially 100% of the radioactivity remained in solution after 60 min when glycine was the substrate and 45% when glycolate was the substrate.
In peroxisomal preparations, approximately 55% of the radioactivity in glycine-containing solutions remained after
60 min and 80% in glycolate-containing solutions. Little or 92
PEROXISOMAL ENZYMES D-amino acid oxidase Alanine:glyoxylate aminotranferase (AGT) Glutamate:glyoxylate aminotranferase (GGT) Glycolic acid dehydrogenase (GAD) Glycolic acid oxidase (GAO) Glycolate reductase
MITOCHONDRIAL ENZYMES Alanine:glyoxylate aminotransferase (AGT) Glycine synthase Glyoxylate:"-ketoglutarate carboligase Lactate dehydrogenase (LDH) Serine hydroxymethyltransferase
Figure 24. Subcellular location of enzymes. Figure 25. Time study. Incubation of oxalate with oxalate of Incubation study. Time 25. Figure Percent of Radioactivity Remaining in Solution 100 mitochondria. ie (minutes) Time 60 93 Figure 26. Time study. Incubation of glycolate with glycolate of Incubation study. Time 26. Figure Percent of Radioactivity Remaining in Solution 100 20 40 80 60 0 0 mitochondria. 10 20 ie (minutes) Time 30 40 50 60 94 Figure 27. Time study. Incubation of glycine with glycine of Incubation study. Time 27. Figure Percent of Radioactivity Remaining in Solution 100 20 40 80 60 0 0 mitochondria. 10 20 ie (minutes) Time 30 40 50 60 95 Figure 28. Time study. Incubation of oxalate with oxalate of Incubation study. Time 28. Figure Percent of Radioactivity Remaining in Solution 100 l Q 1 2 3 4 5 60 50 40 30 20 10 0 ______peroxisomes. I i ______1 i ______ie (minutes) Time I . I i i I _ i ______96 Figure 29. Time study. Incubation of glycolate with glycolate of Incubation study. Time 29. Figure Percent of Radioactivity Remaining in Solution 100 20 40 60 80 0 0 peroxisomes. 10 20 ie (minutes) Time 30 40 50 60 97 Figure 30. Time study. Incubation of glycine with glycine of Incubation study. Time 30. Figure Percent of Radioactivity Remaining in Solution 100 20 40 80 60 0 peroxisomes. ie (minutes) Time 40 50 60 98 99 no carbon dioxide was released during the incubation of glycine with mitochondria. This is surprising since a major pathway of glycine catabolism is its cleavage by the mitochondrial enzyme glycine synthase to form C02, ammonia and methylene-tetrahydrofolate. It is possible that one or more of the cofactors, pyridoxal phosphate, tetrahydrofolate and lipoic acid were lost during isolation of the mitochondria.
The large decrease in the radioactivity of solutions containing [1-14C]glycolate and mitochondria (Figure 26) was unexpected. Carbon dioxide lost during the metabolism of glycolate in mitochondial preparations probably involved the conversion of glycolate to glyoxylate. This reaction may be catalyzed by mitochondrial LDH or peroxisomal GAO contamination. The production of C02 from glyoxylate probably involves glyoxylate:a-ketoglutarate carboligase.
A possible role for glycine synthase is ruled out by the lack of carbon dioxide formation from glycine. Other experiments indicate that C02 production from glycolate by mitochondria is small, ranging from 1-3% in 15 min incubations, and can account for only a fraction of the loss of radioactivity observed in this experiment. The volatility of glycolate may explain the rest of the loss, since the reaction flasks were not sealed in the time studies. The volatility of glycolate has been observed 1 0 0
during paper chromatography (Tolbert, unpublished
observation).
Carbon dioxide was produced from glycine by peroxisomal
preparations, yet no pathway explaining this has been
described. This cannot be due to mitochondrial
contamination, since mitochondrial preparations did not
produce C02 from glycine. Carbon dioxide was also formed
from glycolate in peroxisomal preparations. This presumably
occurs through oxidation of glycolate to glyoxylate by GAO
and transamination of glyoxylate to glycine.
The Metabolism of r l-14C1Glvoxvlate. r l-14ClGlvcolate and
n - 14ClGlvcine in Rat Liver Peroxisomes and Mitochondria
The metabolism of glycolate, glyoxylate and glycine in
rat liver peroxisomes and mitochondria was investigated
first. Mitochondrial preparations (4.0 mg protein) and
peroxisomal preparations (0.30 mg protein) were used in a 2
ml incubation volume. Samples were run in triplicate and
the results reported as the average. The samples were
incubated for 15 min. Enzyme activity was terminated by
boiling the samples for 5 min.
Data was obtained from two separate mitochondrial and
two separate peroxisomal preparations. The patterns of
substrate metabolism were very similar in the different preparations of each organelle, although there were some 101 quantitative differences. The results are summarized in
Table 4. The data can be found in Appendix A.
Very little glycolate was metabolized in mitochondrial preparations. However, glyoxylate was extensively metabolized. Twenty five percent of the glyoxylate was recovered as carbon dioxide, 19% as glycine and 13% as glycolate. Almost no glycine metabolism, including serine formation, was detected in mitochondrial preparations.
GAO activity is absent in mitochondria and the low levels of LDH activity present apparently did not oxidize glycolate to glyoxylate significantly. However, LDH may be responsible for catalyzing the reduction of glyoxylate to glycolate in mitochondrial preparations. Aminotransferase activity is present in mitochondria and probably accounts for the glycine production. Since little carbon dioxide was produced from glycine metabolism, the contribution of glycine synthase to C02 synthesis in mitochondrial preparations must be small. The carboligase reaction is probably the major source of C02 in these experiments. The results are explained in terms of mitochondrial metabolic pathways in Figure 31.
The major product of glycolate metabolism in peroxisomes was glyoxylate (14%). Carbon dioxide (2.6%) was also formed. Glyoxylate was metabolized to glycolate (17%),
C02 (7-11%) and glycine (1%). Glycine was not metabolized by peroxisomes. 102
Table 4. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Metabolic products.
ORGANELLE SUBSTRATE Percentage of radioactivity recovered as % Recovery COj Glycolate Glyoxylate Oxalate Glycine
Mitochondria Glycolate 0.99 97.24 0.44 0.11 3.93 102.7 Glyoxylate 25.56 13.06 39.61 0.26 19.36 97.85 Glycine 0.99 0.20 0.02 ND 102.3 103.5 Peroxisomes Glycolate 2.64 87.40 14.49 0.13 0.28 104.9 Glyoxylate 11.12 17.43 74.09 0.23 0.93 103.8 Glycine 0.02 0.17 0.04 ND 100.6 100.8 ND— not detected 103
Glycolate A
Glyoxylate GKC / \ TA
Figure 31. Pathways of glyoxylate metabolism in mitochondria. (LDH = lactate dehydrogenase, GKC = alpha-ketoglutarate, TA = transaminase) 104 Peroxisomal GAO is responsible for glycolate oxidation
to glyoxylate. Aminotransferases present in the peroxisomes
likely account for glycine formation. The author is not
aware of a pathway that would explain the C02 formation in
peroxisomes. Carbon dioxide production may be the result of
mitochondrial contamination, but the magnitude of C02
production makes this seem unlikely. Also, higher levels of
glycine synthesis would be expected to result from
mitochondrial contamination. Therefore, it appears that a
previously undetected pathway for C02 formation from
glyoxylate must exist in peroxisomes. Figure 32 presents
the pathways of glycolate and glyoxylate metabolism observed in peroxisomes.
A follow-up experiment was conducted which employs two
modifications of the previous study. Equal concentrations
of each substrate were used and the metabolism of substrates
in a prepared mixture of mitochondria and peroxisomes was
studied. The 2 ml incubation mixture contained preparations
of mitochondria (4 mg protein), peroxisomes (0.3 mg
protein), or mitochondria (24.6 mg protein) plus peroxisomes
(0.3 mg protein). A substrate, glycolate (0.39 /xCi, 54.5
nmol), glyoxylate (0.42 p C i , 56.7 nmol), or glycine (0.41 fj.Ci, 56.1 nmol), was added. Reaction time was 15 min. The results are summarized in Table 5. The data are presented in Appendix B. 105
Glycolate
GlyoxylateA 0-G > Oxalate
Figure 32. Pathways of glycolate and glyoxylate metabolism in peroxisomes. (GAD = glycolic acid dehydrogenase, GAO = glycolic acid oxidase, GR = glyoxylate reductase, TA = transaminase) 106
Table 5. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Metabolic products.
ORGANELLE SUBSTRATE Percentage of radioactivity recovered as % Recovery C02 Glycolate Glyoxylate Oxalate Glycine
Mitochondria Glycolate 2.67 95.67 0.85 0.04 1.00 100.2 Glyoxylate 31.57 12.24 45.15 0.23 6.11 95.30 Glycine 1.59 0.19 0.05 ND 99.15 101.0 Peroxisomes Glycolate 8.68 58.34 35.56 0.13 0.41 103.1 Glyoxylate 12.67 14.38 69.20 0.27 1.19 97.71 Glycine 0.02 0.23 ND ND 103.4 103.6 Mitochondria Glycolate 15.86 34.38 2.99 0.06 46.46 99.75 and Peroxisomes Glyoxylate 21.58 9.39 10.97 0.17 52.91 95.02 Glycine 8.01 0.26 ND ND 95.45 103.7 ND— not detected 107 The results obtained with these mitochondrial and peroxisomal preparations were consistent with those already discussed. Substrates incubated with a mixture of mitochondria and peroxisomes produced interesting results.
When glyoxylate was incubated with a mixture of mitochondria and peroxisomes, the products were glycine (53%), carbon dioxide (22%) and glycolate (11%). The products of glycolate metabolism in the mixture of both organelles were glycine (46%), C02 (16%) and glyoxylate (3%). Finally, 8% of the glycine was recovered as carbon dioxide.
Since all of the detected metabolites are permeable to both membranes, a mixture of mitochondrial and peroxisomal preparations would be expected to offer a synthesis of the pathways present in each organelle. This appears to be the case (see Figure 33) . The pattern of products observed with glycolate as substrate parallels what was observed with glyoxylate. The major metabolite of glycolate in peroxisomal preparations was glyoxylate. In fact, it appears to be the only immediate metabolite of glycolate in these organelles. So glycolate is only one step away from glyoxylate and then metabolism is the same. The major enzyme catalyzing the oxidation of glycolate to glyoxylate in this system is peroxisomal GAO. Very little glycolate was metabolized in mitochondria. However, glyoxylate metabolism appears to be largely mitochondrial. Large increases in C02 and glycine production over what was 108
G lycolate G lycolate
LDH G R// ^ A 0 X ^ G A D G lyoxylate Glyoxylate >Oxalate \ i!TA Glycine— 2—>C02
Mitochondrion P eroxisom e
Figure 33. The metabolism of glycolate and glyoxylate in preparations containing both peroxisomes and mitochondria. (GAD = glycolic acid dehydrogenase, GAO = glycolic acid oxidase, GKC = glyoxylate:alpha-ketoglutarate carboligase, GR = glyoxylate reductase, LDH = lactate dehydrogenase, TA = transaminase) 109 observed with separate organelles resulted from glycolate metabolism in mitochondria when peroxisomes were also present. An explanation for this finding is that C02 production from glycolate proceeds via glyoxylate.
Peroxisomal GAO converts the glycolate to glyoxylate which mitochondrial carboligase metabolizes to C02. Minimal C02 would be generated from glycolate by peroxisomes which have a limited ability to metabolize glyoxylate to C02 and by mitochondria which may have a limited capacity to convert glycolate to glyoxylate.
Glycine synthesis from glycolate also appears to involve glyoxylate as an intermediate. Peroxisomes contain the glyoxylate-producing enzyme and mitochondria the more active glycine-producing system. The combination of both organelles gives increased glycine production from glycolate.
Finally, in peroxisomal preparations metabolizing glycolate, 36% of the radioactivity was recovered as glyoxylate, 58% as glycolate and 0.4%as glycine. When mitochondria were also present in the preparation, only 3% of the label was recovered as glyoxylate and 9% as glycolate, but 53% was recovered as glycine. Glyoxylate has been shown to inhibit the GAO-catalyzed oxidation of glycolate (Fry and Richardson, 1979a). The build up of glyoxylate in the metabolism of glycolate by peroxisomal preparations may inhibit further conversion of glycolate. 110 In a mixture of mitochondria and peroxisomes, the glyoxylate may be rapidly removed by transamination to glycine and the inhibition of GAO may be eliminated.
The Effects of Alanine. Glutamate and Ornithine on the
Metabolism of f l-14C1Glvoxvlate. n-^CIGlvcolate and ri-14ciGlvcine in Rat Liver Peroxisomes and Mitochondria
This experiment was conceived to show the effects of the transaminase cosubstrates, alanine, glutamate and ornithine on the flux of glycolate and glyoxylate metabolism. Concentrations used in the 2 ml incubation mixtures were as follows: organelles— 0.15 mg protein/ml for peroxisomes, 7.3 mg protein/ml for mitochondria; substrates— 0.21 /xCi/ml, 28 nmol/ml for glycolate, 59 nCi/ml, 28 nmol/ml for glyoxylate; amino acids were 70 nmol/ml. The results obtained from this experiment are reported in Table 6. The data from the incubations and column separations are shown in Appendix C.
Alanine, and to a lesser extent glutamate and ornithine, enhanced glycine synthesis from glyoxylate and glycolate in mitochondrial and peroxisomal preparations. In mitochondrial preparations, C02 production was decreased indicating that glycine formation and C02 synthesis are competing pathways for available glyoxylate (see Figure 34) .
In peroxisomes, C02 production is increased by these amino Table 6. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Metabolic products.
ORGANELLE SUBSTRATE1 Percentage of radioactivity recovered as % RECOVERY CO2 Glycolate Glyoxylate Oxalate Glycine
Mitochondria Glycolate 2.71® 96.34® 0.97® 0..05®'° 2.99® 103.1 +Ala 0.78“ 96.54® 0.52° 0.05®'° 6.28° 104.2 +Glu 1.82° 94.31® 0.48° 0.06® 4.63° 101.3 +Orn 2.11“ 97.02® 0.50° 0.04° 4.47° 104.1 Glyoxylate 29.97® 15.44® 37.14® 0.26® 16.29® 99.1 +Ala 13.66b 19.23° 24.86° 0.13° 42.97° 100.8 +Glu 25.51° 12.80° 34.72° 0.15° 23.24° 96.4 +Orn 26.87° 12.57° 35.89°'° 0.16° 20.06° 95.6 Peroxisomes Glycolate 4.67® 50.41® 50.37® 0.06® 0.45® 106.0 +Ala 6.53“ 46.29® 52.81® 0.03° 1.18° 106.8 +Glu 5.83“ '° 45.80® 50.17® 0.03°'° 0.97° 102.8 +Orn 5.12®'° 48.27® 50.92® 0.04° 0.63° 105.0 Glyoxylate 11.29® 16.77® 71.24® 0.25® 1.13® 100.7 +Ala 14.76° 17.43® 67.12® 0.13° 1.52° 101.0 +G1U 13.20° 16.24® 68.99® 0.16° 1.43° 100.0 +Orn 13.17° 19.10° 70.47® 0.17° 1.10® 104.0 Mitochondria Glycolate 17.16® 22.48® 20.61® 0.71® 43.69® 104.6 and +Ala 15.20° 11.05° 12.88° 0.30° 60.11° 99.5 Peroxisomes +Glu 16.92® 18.39° 13.74° 0.32° 52.93° • 102.3 +Orn 17.24® 23.18® 16.93° 0.32° 46.01® 103.7 Glyoxylate 23.47® 11.14® 24.26® 0.18® 53.23® 100.4 +Ala 17.40° 9.06° 2.03° 0.08° 73.62° 102.2 +Glu 19.85°'° 11.43® 6.73° 0.06° 65.80° 103.9 +Orn 21.22®'° 8.85° 6.21° 0.08° 66.22° 102.6 1 +Ala ■> substrate plus alanine +Glu = substrate plus glutamate +Orn = substrate plus ornithine a,b,c & d superscript letters are used to show significant differences within vertical groups of three or four (95% confidence level) 112
Glycolate
ld h Glyoxylate GKC TA
Glycine
Figure 34. The effects of alanine, glutamate and ornithine on the metabolism of glyoxylate in mitochondria. A darker arrow indicates increased activity of the pathway; a dashed arrow decreased activity. (GKC = glyoxylate:alpha- ketoglutarate carboligase, LDH = lactate dehydrogenase, TA = transaminase) 113 acids. This is consistent with the pathway of C02 production in peroxisomes going through glycine. These amino acids also decreased oxalate production probably by channelling glyoxylate toward glycine and away from oxalate synthesis. The general order of effectiveness of these amino acids was alanine > glutamate > ornithine. The effects of the aminotransferase cofactors on glycolate and glyoxylate metabolism in peroxisomes is shown in Figure 35.
Figure 36 presents the effects of the cofactors in preparations containing both mitochondria and peroxisomes.
The Effect of DL-Phenvllactate on the Metabolism of ri-14C1Glvoxvlate and ri-14Cl Glycolate in Rat Liver
Peroxisomes and Mitochondria
For this experiment, the 2 ml incubation mixture contained peroxisomes (0.20 mg protein), mitochondria (4.73 mg protein), or peroxisomes (0.20 mg protein) plus mitochondria (4.74 mg protein). Glycolate (0.34 #xCi, 52 nmol) or glyoxylate (0.38 ( iC i, 61 nmol) was the substrate.
Alanine and DL-phenyllactate, when present, were 140 nmol and 0.2 M, respectively. The results are summarized in
Table 7. The complete data are available in Appendix D.
The effects of DL-phenyllactate on metabolism in peroxisomes is shown in Figure 37. The metabolism of glyoxylate in peroxisomes resulted in the formation of glycolate (20%), carbon dioxide (4%), oxalate (2%) and 114
Glycolate
Glyoxylate > 0 x a la te
Figure 35. The effects of alanine, glutamate and ornithine on the metabolism of glyoxylate in peroxisomes. A darker arrow indicates increased activity of the pathway; a dashed arrow decreased activity. (GAD = glycolic acid dehydrogenase, GAO = glycolic acid oxidase, GR = glyoxylate reductase, TA = transaminase) 115
G lycolate G lycolate "1 // GV A O N' ' GAD Glyoxylate f Glyoxylate----->Oxalate GKC TA VA, Glycine Glycine— >C0e
Mitochondrion Peroxisome
Figure 36. The effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in preparations containing both peroxisomes and mitochondria. A darker arrow indicates increased activity of the pathway; a dashed arrow decreased activity. (GAD = glycolic acid dehydrogenase, GAO = glycolic acid oxidase, GKC = glyoxylate:alpha-ketoglutarate carboligase, GR = glyoxylate reductase, LDH = lactate dehydrogenase, TA = transaminase) 1 1 6
Table 7. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Metabolic products.
ORGANELLE SUBSTRATE1 Percentage of radioactivity recovered as % RECOVERY CO2 Glycolate Glyoxylate Oxalate Glycine Mitochondria Glycolate 1.70® 95.10® i.l3® 0.04® 1.44® 99.4 +PL 0.11" 98.77" 0.96®'° 0.10° 0. 03 100.0 +Ala 0.17" 95.42® 0-61b V. 0.06® 3.97" 100.2 +PL,Ala 0.12" 102.36" 0.93®'" 0.05® 0.21b 103.7 Glyoxylate 32.91® 16.49® 35.20® 2.37® 8.27® 95.2 +PL 1.50" 18.57" 74.72" 1.67b 2 . 70b 99.2 +PL,Ala 1 . 48 21.69" 72.38° 1.90" 2.90b 100.4 Peroxisomes Glycolate 6.15® 25.59® 65.37® 0.38® 0.47® 98.0 +PL 0.05b 103.86" 0.93" 0.04" 0.24®'b 105.1 +Ala 8.77° 22.84® 67.27® 0.47" 1.15" 100.5 +PL,Ala 0.05" 101.50" 0.95" 0.02" ND 102.5 Glyoxylate 4.14® 18.39® 79.37® 1.88®'b 1.55® 105.3 +PL 0 . 52 20.97® 78.98® 2.44b 1.09® 104.0 +PL,Ala 0.46 21.30® 80.11® 1.57® 0.60® 104.0 Mitochondria Glycolate 29.29® 19.57® 36.63® 0.50® 9.30® 95.3 and +PL 0.15" 95.00". 1.67" 0.04b NDb 96.9 Peroxisomes +Ala 14.69° 12.77" 11.05" 0.20" 59.02" 97.7 +PL,Ala 0.12" 98.63" 1.30" 0.04b 0.56b 100. 6 Glyoxylate 34.35® 14.89® 34.44® 2.37® 10.52® 96.6 +PL 1.12b 22.27" 72.76" 2.07® 3.06" 101.3 +PL,Ala 1.09" 23.33" 73.44" 1.82® 3.14b 102.8 1 +PL =■ substrate plus DL-phenyllactate +Ala = substrate plus alanine +PL,Ala = substrate plus DL-phenyllactate plus alanine a,b,c S d superscript letters are used to show significant differences within vertical groups of three or four (95% confidence level) ND— not detected 117
Glycolate
GlyoxylateOxalate
Figure 37. The effects of DL-phenyllactate on the metabolism of glycolate and glyoxylate in peroxisomes. (GAD = glycolic acid dehydrogenase, GAO = glycolic acid oxidase, GR = glyoxylate reductase, TA = transaminase) 118
glycine (1.5%). About 80% of the glyoxylate was recovered
unaltered. The reduction of glyoxylate to glycolate may be
catalyzed by glyoxylate reductase. GAO may be responsible
for the conversion of glyoxylate to oxalate. Glycine may be
formed by the transamination of glyoxylate. The pathway for
C02 formation from glycolate in peroxisomes is unclear. The
only significant change in glyoxylate metabolism caused by
the presence of DL-phenyllactate was the 87% reduction in
C02 synthesis. The mechanism of this inhibition is unknown.
The major product of the metabolism of glycolate in peroxisomes was glyoxylate (65%). A small amount of
radioactivity was recovered as carbon dioxide (6%), and trace amounts of glycine (0.5%) and oxalate (0.4%) were detected. About one quarter of the glycolate was recovered unchanged.
Glyoxylate is formed as a result of GAO activity on glycolate. This was confirmed by the almost complete
inhibition of glycolate metabolism by the GAO inhibitor
DL-phenyllactate. Oxalate and glycine are likely formed via glyoxylate. Conversion of glyoxylate to oxalate may be catalyzed by GAO; glycine formation may be the result of transamination by any of several aminotransferases. The mode of C02 production is unknown. The almost complete inhibition of glycolate metabolism by DL-phenyllactate supports the role of glyoxylate as an intermediate in the production of oxalate, glycine and C02. 119
Alanine increased the formation of glycine 145%, carbon dioxide 43% and oxalate 24%. Alanine is a substrate (amino donor) for alanine:glyoxylate aminotransferase and its addition increased transamination of glyoxylate to glycine.
The increased C02 resulting from the presence of alanine indicates an intermediate role for glycine in C02 production. Alanine had no effect on DL-phenyllactate inhibition of glycolate metabolism, indicating that the location of alanine action is more remote than that of
DL-phenyllactate.
The products of the metabolism of glyoxylate in mitochondria were carbon dioxide (33%), glycolate (16.5%), glycine (8%) and oxalate (2%). Glyoxylate:a-ketoglutarate carboligase, glycine synthase and/or glyoxylate oxidation cycle activity may be responsible for the conversion of glyoxylate to C02 in mitochondria. Aminotransferase activity is present in mitochondria and probably accounts for the glycine synthesis. Mitochondrial LDH may reduce glyoxylate to glycolate and may be responsible for the oxidation of glyoxylate to oxalate.
The effect of DL-phenyllactate on glyoxylate metabolism in mitochondria is dramatic. Carbon dioxide formation is reduced by 95%. Glycine production is reduced 67% and oxalate 25%. Glycolate synthesis is increased slightly.
The amount of unmetabolized glyoxylate was doubled. 120
Glycolate was not extensively metabolized in mitochondria; 95% was recovered unchanged. The detected metabolites were carbon dioxide (1.7%), glycine (1.4%), glyoxylate (1.1%) and oxalate (0.04%). Limited oxidation of glycolate to glyoxylate by mitochondrial LDH or contaminating peroxisomal GAO may account for the C02, glycine and oxalate produced from glycolate.
Alanine increased glycine synthesis 176% and decreased
C02 production 90%. This indicates that the major route of
C02 formation is not via glycine and the glycine synthase system, at least not at low glyoxylate concentrations.
Transamination of glyoxylate to form glycine may have decreased the availability of glyoxylate for carboligase-catalyzed C02 production. Glyoxylate was reduced 46% by alanine.
The differing results of alanine on carbon dioxide formation in mitochondria and peroxisomes demonstrate the existence of different pathways for C02 production in these organelles. Glycine and C02 were increased in peroxisomes by alanine, while in mitochondria glycine was also increased but C02 was reduced. The pathway for C02 production appears to be through glycine in peroxisomes, but not to involve glycine in mitochondria.
The effects of DL-phenyllactate on glycolate metabolism in mitochondria were almost complete inhibition of glycine and C02 synthesis and a slight increase in unmetabolized 121
glycolate. Figure 38 illustrates the effects of DL-
phenyllactate on the metabolism of glycolate and glyoxylate
in mitochondria.
The results obtained when glyoxylate was incubated with
a mixture of peroxisomes and mitochondria are very similar
to those for glyoxylate incubated with mitochondria alone.
This indicates that the contribution of peroxisomes to
glyoxylate metabolism is negligible.
The metabolism of glycolate by a mixture of peroxisomes
and mitochondria is comparable to that of glyoxylate.
Metabolites detected were glyoxylate (37%), carbon dioxide
(29%), glycine (9%) and oxalate (0.5%). Peroxisomal GAO can
convert glycolate to glyoxylate and then mitochondrial
enzymes can further metabolize glyoxylate to C02 and
glycine.
Alanine caused a striking shift in metabolism. Glycine
production was increased 535%, while carbon dioxide was
decreased 50%, glyoxylate 70% and oxalate 60%. Alanine
probably exerts its effect through greatly increased
transamination of glyoxylate to glycine by
alanine:glyoxylate aminotransferase thus reducing the
glyoxylate available for C02 and oxalate synthesis. The mitochondria appear to be the major organelle for
alanine:glyoxylate aminotransferase, since glycine production from glycolate in peroxisomes was much lower.
Also, the conclusion that mitochondrial C02 production is 122
Glycolate T G lyoxylate
Figure 38. The effects DL-phenyllactate on the metabolism of glyoxylate in mitochondria. A darker arrow indicates increased activity of the pathway; a dashed arrow decreased activity; a dark line across the arrow indicates virtually no activity. (GKC = glyoxylate:alpha- ketoglutarate carboligase, LDH = lactate dehydrogenase, TA = transaminase) 123 opposed to glycine formation is supported, since a concomitant increase in C02 was not observed with this large increase in glycine.
DL-phenyllactate inhibited virtually all metabolism of glycolate in this mixture of organelles (Figure 39). This is most likely through the inhibition of GAO. The addition of alanine had no effect on DL-phenyllactate inhibition.
The mitochondrion appears to be the major site of C02 production, although the peroxisome does exhibit a limited ability to produce C02. The ability of mitochondria to metabolize glycolate is very limited. However, it seems to be the major site of glyoxylate metabolism.
The addition of alanine to organelles metabolizing glycolate increased glycine formation by 2.4-, 2.8-, and
6.3-fold in peroxisomes, mitochondria, and mitochondria plus peroxisomes, respectively. This supports the role of alanine:glyoxylate aminotransferase as a major enzyme in glycine synthesis from glyoxylate. Alanine inhibits mitochondrial C02 formation and enhances peroxisomal C02 formation; DL-phenyllactate inhibits C02 production in both organelles. How DL-phenyllactate inhibits glyoxylate:a-ketoglutarate carboligase in mitochondria and
C02 production from glyoxylate in peroxisomes is unclear. 124
G lycolate G lycolate L 0 H fi 6 GR^/^GAO '^GAD Glyoxylate * G lyoxylate O xalate w/ \ t a . Glycine Glycine ?-)CQ
Mitochondrion Peroxisome
Figure 39. The effects of DL-phehyllactate on the metabolism of glycolate and glyoxylate in preparations containing both peroxisomes and mitochondria. A darker arrow indicates increased activity of the pathway; a dashed arrow decreased activity; a dark bar across an arrow indicates virtually no activity. (GAD = glycolic acid dehydrogenase, GAO = glycolic acid oxidase, GKC = glyoxylate:alpha-ketoglutarate carboligase, GR = glyoxylate reductase, LDH = lactate dehydrogenase, TA = transaminase) Conclusions
The main goal of this project was to assess the relative contributions of the mitochondrion and the peroxisome to pathways associated with oxalate biosynthesis.
To accomplish this the uptake of oxalate, glycolate, glyoxylate and glycine by rat liver mitochondria and peroxisomes was studied. The metabolism of glycolate, glyoxylate and glycine was investigated in preparations of rat liver peroxisomes and mitochondria. The effects of
DL-phenyllactate, an inhibitor of GAO, and of alanine, glutamate and ornithine, cosubstrates of the relevant aminotransferases, were also examined.
First it was vital to identify which intermediates of these pathways are able to traverse the membranes of the mitochondria and peroxisomes in order to know if the reactions contained in each organelle work independently of the other organelle or in concert with it. The transport of oxalate and glycine across the mitochondrial membrane has been studied, but mitochondrial uptake of glyoxylate and glycolate and peroxisomal uptake of oxalate, glyoxylate, glycolate and glycine have not been reported. This study demonstrated that oxalate, glycolate, glyoxylate and glycine readily cross the membranes of both mitochondria and
125 126
peroxisomes. Oxalate uptake by mitochondria required
phosphate, but the other compounds were taken up as ammonium
salts. Glycine is known to cross the membrane in its uncharged form, but it is also transported. These studies
suggest that glyoxylate and glycolate uptake is
electroneutral, crossing the lipid bilayer in their
uncharged forms. The peroxisomal membrane is not a barrier
to the uptake of any of these compounds by that organelle.
No transporter has been reported in peroxisomes.
The peroxisomes are the major sites of glycolate
conversion to glyoxylate. These studies showed extensive
metabolism of glycolate in peroxisomes with glyoxylate the
major product. Glycolate was recovered largely
unmetabolized in mitochondria. However, the mitochondria
are the major organelles of glyoxylate metabolism. When the
organelles were compared in proportions equivalent to those
found in the liver, both carbon dioxide and glycine
synthesis from glycolate were greater in mitochonrial
preparations than in peroxisomal preparations. And when mitochondria were added to peroxisomes, carbon dioxide and
glycine synthesis from glycolate greatly increased.
Oxalate synthesis was very limited under all conditions
used in this study. This is not surprising given the
extremely low substrate concentrations used. Oxalate
synthesis was limited to peroxisomes and peroxisomes plus mitochondria. Oxalate formation was reduced approximately 127 50% by alanine, glutamate and ornithine and 90% by
DL-phenyllactate.
Peroxisomes appear to contain a pathway for C02 production, likely through glycine. The time study showed
C02 released from glycine incubated with peroxisomes but not with mitochondria. Other experiments exhibited C02 production from glyoxylate and glycolate incubated with peroxisomes, although less than when incubated with mitochondria. Mitochondrial C02 synthesis was mostly by way of the carboligase reaction and not by glycine formation and then cleavage by glycine synthase. This conclusion is supported by the findings that very little C02 was generated during a 60 min incubation of glycine with mitochondria and that alanine, glutamate and ornithine increased glycine synthesis but decreased C02 formation.
Alanine caused a substantial increase in glycine production from glyoxylate in each organelle and an enormous increase when both organelles are present. Glutamate and ornithine show similar though less pronounced results.
These results fit the role of these amino acids as cosubstrates for aminotranferases forming glycine. Finally, DL-phenyllactate strongly inhibits the metabolism of both glycolate and glyoxylate in both organelles. Inhibition of glycolate metabolism by
DL-phenyllactate may logically follow from its inhibition of 128
GAO. However, this would not explain the effect on C02 production from glyoxylate metabolism in both organelles.
Since the mechanism of C02 production seems to be different in each organelle, this finding is even more puzzling.
Figure 40 depicts the pathways observed in peroxisomes and mitochondria for the metabolism of glyoxylate and glycolate. To summarize:
1) The peroxisomal membrane is permeable to oxalate,
glyoxylate, glycolate and glycine.
2) Electroneutral uptake of glycolate and glyoxylate
occurs in mitochondria.
3) Glycolate is metabolized in peroxisomes to
glyoxylate which can then be metabolized in
mitochondria to yield carbon dioxide and glycine
as the major products.
4) Moderate conversion of glyoxylate to glycolate
occurs in both organelles.
5) Carbon dioxide is a product of glycolate and
glyoxylate metabolism in peroxisomes though no
pathway for C02 production in peroxisomes has been
reported.
This research project poses two very interesting questions. First, what is the pathway for carbon dioxide production in mitochondria? Second, what is/are the mechanism/s for DL-phenyllactate inhibition of carbon dioxide synthesis? These are questions for future study. 129
G lycolate G lycolate
Glyoxylate <■ Glyoxylate >0xalate
Mitochondrion P eroxisom e
Figure 40. Pathways for the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. APPENDIX A
DATA FOR THE METABOLISM OF GLYOXYLATE AND GLYCOLATE IN RAT LIVER MITOCHONDRIA AND PEROXISOMES
130 131
Table 8. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of the initial incubations.
ORGANELLE SUBSTRATE DPM in Flask DPM in Well % RECOVERY
Mitochondria Glycolate 996057(10082)1 10012 (173)1 99.50
Glyoxylate 628437(12260) 229491(5669) 95.54
Glycine 868188(11723) 868 (547) 100.6
Peroxisomes Glycolate 1014842(9058) 26717(1587) 103.0
Glyoxylate 815322(16822) 99888(6938) 101.9
Glycine 912846(5543) 157 (2) 104.8 mean of 3 samples (S.E.M.) 132
Table 9. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations.
ORGANELLE SUBSTRATE DPM in Formate DPM in Salt % REC< Fraction Fraction
Mitochondria Glycolate 449447 (6702)1 18070(329)x 103.3
Glyoxylate 216049 (5538) 79023(1500) 103.3
Glycine 861(67) 405100(4514) 102.9
Peroxisomes Glycolate 468845(896) 1302(354) 101.9
Glyoxylate 370989(4619) 3776(193) 101.1
Glycine 836(200) 398226(6142) 96.18
mean of 3 samples (S.E ■ M.) 133
Table 10. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions.
ORGANELLE SUBSTRATE DPM recovered as Glycolate Glyoxylate Oxalate
Mitochondria Glycolate 446944 (6878)1 2008 (234)1 494 (28) 1
Glyoxylate 53307(5620) 161665(532) 1076(28)
Glycine 780(53) 81(20) 0
Peroxisomes Glycolate 401689(1668) 66578(1379) 579(106)
Glyoxylate 71138(12108) 302423(6426) 931(83)
Glycine 655(188) 169(88) 12(12) 1 mean of 3 samples (S.E.M.) APPENDIX B
DATA FOR THE METABOLISM OF GLYOXYLATE AND GLYCOLATE IN RAT LIVER MITOCHONDRIA AND PEROXISOMES-II
134 Table 11. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of the initial incubations.
ORGANELLE SUBSTRATE DPM in Flask DPM in Well % RECOVERY
Mitochondria Glycolate 815431 (2715)1 22738(1084)1 93.61
Glyoxylate 574048(16085) 292083(12970) 98.58
Glycine 890996(6572) 14259(997) 101.0
Peroxisomes Glycolate 791692(8967) 73779(5085) 101.8
Glyoxylate 788515(18486) 117245(19617) 97.89
Glycine 949688(6543) 161(8) 105.9
Mitochondria Glycolate 712575(11165) 134865(3691) 99.67 and Peroxisomes Glyoxylate 672207(17009) 199669(9129) 94.23
Glycine 828373(8692) 71788(2472) 100.4
1 mean of 3 samples (S.E.M.) 136
Table 12. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of the AG 50W-X8 column separations.
ORGANELLE SUBSTRATE DPM in Formate DPM in Salt % RECOV] Fraction Fraction
Mitochondria Glycolate 373973(5803)1 3859(277)1 101.9
Glyoxylate 242312 (7161) 25704(247) 102.7
Glycine 982(90) 404141 (3839) 100.0
Peroxisomes Glycolate 363404(2211) 1579(2) 101.4
Glyoxylate 352707(6895) 5016(98) 99.81
Glycine 1009(156) 421253 (4221) 98.21
Mitochondria Glycolate 144648(4586) 179538(7763) 100.1 and Peroxisomes Glyoxylate 86676(5892) 222547(6656) 101.2
Glycine 1137(26) 389041(5435) 103.6
mean of 3 samples (S.E.M.) 137
Table 13. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of glycolate, glyoxylate and oxalate determinations of formate fractions.
ORGANELLE SUBSTRATE DPM recovered as Glycolate Glyoxylate Oxalate
Mitochondria Glycolate 369732(9699) 1 3419(310)x 157 (24)1
Glyoxylate 51459(1183) 189906(5980) 947 (221)
Glycine 785(81) 198(19) 0
Peroxisomes Glycolate 225453(5571) 137430(3631) 521 (26)
Glyoxylate 60492(5560) 291062(6624) 1153(58)
Glycine 945(147) 64(15) 0
Mitochondria Glycolate 132862(2220) 11551(2881) 235 (51) and Peroxisomes Glyoxylate 39494(1583) 46124(5343) 710(5)
Glycine 1068 (11) 69(27) 0
mean of 3 samples (S.E.M.) APPENDIX C
DATA FOR THE EFFECTS OF ALANINE, GLUTAMATE AND ORNITHINE
138 139
Table 14. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the initial incubations.
ORGANELLE S0BSTRATEJ DPM in Flask DPM in Well % RECOVERY
Mitochondria Glycolate(3)3 925,294(5,125)3 24,876(1,333)3 103.5 +Ala (3) 941,078(3,425) 7,160(240) 103.3 +Glu(3) 922,593(3,807) 16,707(730) 102.3 +Orn(3) 921,665(3,776) 19,369(579) 102.6 Glyoxylate(3) 187,013(2,803) 78,801(3,613) 101.1 +Ala(3) 231,762(4,261) 35,917(1,284) 101.8 +Glu(3) 189,353(7,803) 67,074 (2,840) 97.5 +Orn(3) 184,502 (3,013) 70,650(3,068) 97.0
Peroxisomes Glycolate(3) 899,813(6,377) 42, 868(1,981) 102.7 +Ala(3) 877,207 (7,097) 59,942(4,756) 102.1 +Glu(3) 867,242(5,134) 53,516(1,669) 100.3 +Orn(3) 890,128(4,675) 46,999(3,426) 102.1 Glyoxylate (3) 241,901(1,787) 29, 685 (1,243) 103.3 +Ala (3) 233,592(1,871) 38,809(1,718) 103.6 +Glu(3) 241,358 (4,497) 34,707(630) 105.0 +Orn (3) 235,947(3,254) 34,628(1,672) 102.9 Mitochondria Glycolate(3) 778,780(8,097) 157,520(3,958) 102.0 and +Ala(3) 834,502(10,617) 139,528(2,854) 106.1 Peroxisomes +Glu (3) 780,333(10,258) 155,317(4,137) 101.9 +Orn (3) 747,284 (6,341) 158,255(3,769) 98.6
Glyoxylate(3) 190,374 (9,112) 61,710(4,323) 95. 9 +Ala(3) 212,097 (6,088) 45,750(3,126) 98.1 +Glu(3) 204,727(5,444) 52,192(2,496) 97.7 +Orn(3) 198,158(6528) 55,794(1,244) 96.6 +Ala = substrate plus alanine +Glu = substrate plus glutamate +Orn = substrate plus ornithine number of samples mean(S.E.M.) 140
Table 15. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations.
ORGANELLE SUBSTRATE1 DPM in Formate DPM in Salt % RECOVERY Fraction Fraction
Mitochondria Glycolate (3)^ 406,222(10,217)3 12,474(469)3 99.6 +Ala(3) 405,202(9,389) 26,199(1,184) 100.9 +Glu(3) 395,746(10,302) 19,323(1,089) 99.0 +Orn (3) 407,092(9,684) 18,652(658) 101.6 Glyoxylate (3) 63,159(1,019) 19,474(1,733) 97.2 +Ala(3) 52,841(1,618) 51,361(1,328) 98.9 +Glu(3) 56,972(2,276) 27,772(1,466) 98.5 +Orn(3) 58,103(1,233) 23,978(1,946) 97.9 Peroxisomes Glycolate(3) 420,768(2,470) 1,880(68) 103.3 +Ala(3) 413,628(3,003) 4,925(100) 105.0 +Glu (3) 400,571(11,994) 4,048 (83) 102.6 +Orn(3) 414,008(14,424) 2,626(83) 103.0 Glyoxylate (3) 105,496(4,545) 1,350(54) 97.2 +Ala(3) 101,208(2,656) 1,821(95) 97.0 +Glu(3) 102,047(2,528) 1,706(79) 94.6 +Orn(3) 107,257(2,199) 1,315(51) 101.2 Mitochondria Glycolate(3) 182,763(6,388) 182,310(7,377) 103.1 and +Ala(3) 101,106(2,995) 250,821(5,010) 92.3 Peroxisomes +Glu(3) 135,383(4,482) 220,850(9,237) 100.4 +0rn(3) 168,700(1,214) 191,971(8,726) 106.2 Glyoxylate(3) 42,526(1,558) 63,612(3,482) 122.7 +Ala(3) 13,354(351) 87,985(1,817) 105.1 +Glu(3) 21,767(488) 78,637(6,756) 107.9 +Orn(3) 18,095(586) 79,142(5,370) 108.0 1 +Ala = substrate plus alanine +Glu = substrate plus glutamate +Orn = substrate plus ornithine ~ number of samples 3 mean(S.E.M.) 141
Table 16. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions.
ORGANELLE SUBSTRATE-1 DPM recovered as Glycolate Glyoxylate Oxalate
Mitochondria Glycolate(3)2 401,966(10,114)3 4,045(143)3 210 (18)3 +Ala(3) 402,818(9,379) 2,162(61) 222 (28) +Glu(3) 393,488(10,386) 2,003(111) 255 (28) +Orn(3) 404,835(9,628) 2,082(72) 175 (7) Glyoxylate(3) 18,457(317) 44,392(11,930) 310(25) +Ala(3) 22,978(986) 29,708(905) 155(9) +Glu(3) 15,301(1,168) 41,493(1,191) 178(7) +Orn(3) 15,024(587) 42,890(660) 189(14)
Peroxisomes Glycolate(3) 210,343(7,658) 210,173(9,857) 252(2) +Ala(3) 193,165(4,403) 220,337(5,244) 125(8) +Glu(3) 191,092(11,240) 209,343(778) 137(10) +Orn(3) 201,398(9,101) 212,461(6,788) 149(13)
Glyoxylate(3) 20,048(776) 85,147(3,790) 301 (10) +Ala(3) 20,827(231) 80,221(2,614) 160 (6) +Glu (3) 19,405(274) 82,450(2784) 192 (23) +Orn(3) 22,830(1,322) 84,219(3,013) 208(7)
Mitochondria Glycolate(3) 93,794(2,015) 85,998(4,461) 2,971(133) and +Ala(3) 46,109(1,048) 53,739(1,945) 1,258(45) Peroxisomes +Glu (3) 76,731(4,749) 57,330(1,463) 1,322(90) +Orn (3) 96,722(1,620) 70,641(2,336) 1,337 (67)
Glyoxylate(3) 13,315(653) 28,991(1,812) 220(9) +Ala(3) 10,830(189) 2,424(212) 100(15) +Glu(3) 13,658(422) 8,038(191) 71(9) +Orn(3) 10,576(776) 7,423 (231) 95(18)
+Ala = substrate plus alanine +Glu = substrate plus glutamate +Orn = substrate plus ornithine number of samples mean(S.E.M.) APPENDIX D
DATA FOR THE EFFECTS OF DL-PHENYLLACTATE
142 143
Table 17. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the initial incubations.
ORGANELLE SUBSTRATE3 DPM in Flask DPM in Well % RECOVERY
Mitochondria Glycolate(3)^ 752,488(7,305}3 12,711 (693)3 102.4 +PL(3) 776,050(6,690) 831(143) 104.0 +Ala(3) 757,944(3,160) 1,295(87) 101.6 +PL,Ala(3) 752,510 (4,740) 934(172) 100.8 Glyoxylate(3) 545,314 (44,082) 279,367(44,625) 97.1 +PL (3) 860,970(11,554) 12,773(97) 102.9 +PL,Ala(3) 842,270(4,534) 12,568(201) 100.7
Peroxisomes Glycolate(3) 691,702(10,129) 45,938(2,852) 98.7 +PL(3) 758,494 (4,056) 360(44) 101.5 +Ala(3) 677,886(11,060) •65,555(10,244) 99.5 +PL,Ala(2) 768,086(5,852) 374(0) 102.8
Glyoxylate(3) 830,038(18,287) 35,176(7,411) 101.9 +PL(3) 852,419(66,588) 4,405 (99) 100.9 +PL,Ala(1) 863,742 3,865 102.2
Mitochondria Glycolate(3) 524,304(22,855) 218,913(24,881) 99.4 and +PL(3) 776,160 (1,215) 1,090(44) 104.0 Peroxisomes +Ala(3) 645,084 (3,113) 109,760(81) 101.0 +PL,Ala(3) 766,040(11,954) 933(201) 102.6
Glyoxylate(3) 515,834 (34,941) 291,631(52,292) 95.1 +PL(3) 852,126(10,337) 9,549(831) 101.5 +PL,Ala(1) 858,462 9255 102.2
+PL = substrate plus DL-phenyllactate +Ala = substrate plus alanine +PL,Ala = substrate plus DL-phenyllactate plus alanine number of samples mean(S.E.M.) 144
Table 18. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations.
ORGANELLE SUBSTRATEJ DPM in Formate DPM in Salt % RECOVERY Fraction Fraction
Mitochondria Glycolate(3)^ 327,046(2,646)3 4, 900(1,091)3 97.0 +PL(3) 339,117(2,254) 100(321) 96.2 +Ala(3) 328,554(1,968) 13,500 (491) 99.3 +PL,Ala(3) 343,839(7,948) 700 (450) 100.7
Glyoxylate(3) 208,609(19,204) 31,900(348) 97.0 +PL(3) 366,440(6,027) 10,400(696) 96.3 +PL,Ala(3) 370,372(1,658) 11,200(696) 99.7
Peroxisomes Glycolate(3) 304,710(11,252) 1,600(473) 97.4 +PL(3) 356,090(6,275) 800(379) 103.5 +Ala(3) 307,720(10,456) 3,900 (291) 101.1 +PL,Ala(2) 348,110(1,400) 0(250) 99.7
Glyoxylate(3) 382,410(8,572) 6,000(2,979) 102.9 +PL(3) 395,080(5,875) 4,200(961) 103.1 +PL,Ala(1) 397,390 2,300 101.8
Mitochondria Glycolate(3) 192,640(15,179) 31,600(1,325) 94.1 and +PL(3) 328,518(5,399) 0(203) 93.1 Peroxisomes +Ala(3) 81,595(819) 200,500 (4,599) 96.2 +PL,Ala(3) 339,589(3,634) 1, 900 (451) 98.1
Glyoxylate(3) 180,480(19,571) 40,600(1,193) 94.3 +PL(3) 375,269(5,239) 11, 800 (371) 99.9 +PL,Ala(1) 380,438 12,100 100.6 1 +PL = substrate plus DL-phenyllactate +Ala = substrate plus alanine +PL,Ala = substrate plus DL-phenyllactate plus alanine j? number of samples ^ mean(S.E.M.) 145
Table 19. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions.
ORGANELLE SUBSTRATE DPM recovered as Glycolate Glyoxylate Oxalate
Mitochondria Glycolate (3)3 323,059(2,088)3 3,836(951)3 141(6)3 +PL(3) 335,515(2,062) 3,264(445) 350 (44) +Ala(3) 324,135(14) 2,079(543 191(25) +PL,Ala(3) 347,735(5,863) 3,150(70) 182(38)
Glyoxylate(3) 63,630(325) 135,827(19,480) 9,136(660) +PL(3) 71,658(5,402) 288,336(3,095) 6,455(260) +PL,Ala(3) 83,718(1,300) 279,314(2,927) 7,341(285) Peroxisomes Glycolate(3) 86,947(7,881) 222,082(19,728) 1,300 (133) +PL(3) 352,816(6,490) 3,164(439) 141 (42) +Ala(3) 77,586(7,782) 228,508(2,948) 1,605(203) +PL,Ala(2) 344,803(1,277) 3,227(130) 77(7)
Glyoxylate(3) 70,980(2,842) 306,299(5,718) 7,250(753) +PL(3) 80,906(8,106) 304,773(5,560) 9,423(632) +PL,Ala(1) 82,194 309,155 6,041
Mitochondria Glycolate(3) 66,488(4,710) 124,432(15,178) 1,695(160) and +PL(3) 322,740(6,933) 5,664(1,732) 132(28) Peroxisomes +Ala(3) 43,384(154) 37,550 (754) 664 (81) +PL,Ala(3) 355,050(3,741) 4,409(970) 127(23)
Glyoxylate(3) 57,445(605) 132,909(826) 9,155(1,305) +PL(3) 85,939(4,305) 280,777(7,384) 7,986(459) +PL,Ala(1) 90,041 283,386 7023 +PL = substrate plus DL-phenyllactate +Ala = substrate plus alanine +PL,Ala = substrate plus DL-phenyllactate plus alanine number of samples mean(S.E.M.) LIST OF REFERENCES
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