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Xerox University Microfilms 300 North Zeab Road Ann Arbor, Michigan 4S106 75-26,557 CHERNQFF, Harvey Norman, 1936- THE EFFECT OF PHENYLKETONURIA ON OXALATE BIOSYNTHESIS. The Ohio State University, Ph.D., 1975 Chemistry, biological

Xerox University Microfilmst Ann Arbor. Michigan 48106 THE EFFECT OF PHENYLKETONURIA ON OXALATE BIOSYNTHESIS

Presented in Partial Fulfillment of the Requirements for

the Degree of Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Harvey Norman Chemoff, B.Sc., M.Sc. * * * *

The Ohio State University

1975

Reading Committee; Approved By

G. F. Grannis, Ph.D. H.-D. Grueraer, M.D. K. E. Richardson, Ph.D. 1 0 1 A/T- - f\\ \ } d L & i d L J. S. Rieske, Ph.D. /Adviser H. W. Sprecher, Ph.D. Department of Physiological Chemistry ACKNOWLEDGEMENTS

To Jane, whose support and encouragement throughout graduate school were invaluable, and without whose scientific contribution this project would not have been completed. She made the long hours and short nights seem worthwhile. Special thanks to Dr. Keith Richardson, who made the project possible and whose support will always be remembered and appreciated. Thanks to Drs. Sprecher and Gruemer, who contributed much of their time and scientific expertise to this project. Thanks also to

Drs. Rieske and Grannis for their contribution to this project. A spe­ cial thanks to Dr. Boutwell for his personal encouragement and interest in this training and to the Center for Disease Control for their support throughout.

ii VITA

May 3, 1936 ...... B o m - Douglas, Georgia

1958 ...... B.S., The University of Georgia, Athen s, Georgia

1958 - 1960 ...... Managed Family Store, Rainbows Department Store, Broxton, Georgia

1960 - 1961 ...... U. S . Army Active Duty. Then in U. S. Army Reserves at Fort McPherson, Atlanta, Georgia

1962 - 1963 ...... Analytical Chemist, Food and Drug Administration, Atlanta, Georgia

1963 — 1967 ...... Research Associate, Biochemistry Department, The University of . Georgia, Athens, Georgia

1967 ...... M.S., The University of Georgia, Athens, Georgia

1967 - 1972 ...... Supervisory Chemist, Clinical Chemistry Unit, Center for Disease Control, Atlanta, Georgia

PUBLICATIONS

"A Stable Liquid Urine Preparation", Clin. Chem., 17, 634 (1971).

"Effect of Phenylketonuria on Oxalate Synthesis", Clin. Chem., in press.

iii FIELDS OF STUDY

Major Field: Clinical Chemistry (Pathology)

Studies in: Pathology, Professor H.-D. Gruemer

Biochemistry, Professor Keith E. Richardson

iv TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... 11

VITA...... Ill

LIST OF TABLES...... vl

LIST OF FIGURES. . '...... vii

ABBREVIATIONS...... viii

Chapters

I STATEMENT OF THE PROBLEM...... 1

II LITERATURE REVIEW...... '...... 3

III MATERIALS AND METHODS...... 29

Phenyllactate...... 30

O-hydroxyphenylacetate • • • • 31

Oxalate..... 31

Glycolic Acid...... 31

Glyoxylic Acid...... 32

IV RESULTS...... 34

V DISCUSSION...... 46

VI SUMMARY...... 52

BIBLIOGRAPHY...... 53

v LIST OF TABLES

Table Page

1. Urinary Excretion of Creatinine, Oxalate, Glyoxylate, Glycolate, Phenyllactate and O-Hydroxyphenyl- acetate...•..•...... 35

2. Correlation Coefficients for 24 Hr. Excretion of Oxalate, Oxalate Precursors and Phenylketonuric Metabolites with Creatinine...... 39

3. Urinary Oxalate, Glyoxylate, Glycolate, Phenyllactate and O-Hydroxyphenylacetate Excreted Per Gram Creatinine...... 41

4. Correlation Coefficients for Oxalate Metabolites (Acid/g. Creatinine) and Phenylketonuric Metabolites (Acid/g. Creatinine)...... 43

vi LIST OF FIGURES

Figure No. Title Page

1. Metabolic Pathways of Oxalate Biosynthesis...... 12

2. Probable Pathway of L-Ascorbic Acid Metabolism to Form Oxalic Acid..... 14

3. Pathways of Serine Metabolism In Mammalian Systems 17

4. Pathways of Glyoxylate Metabolism...... 21

5. Disorders of Oxalate Metabolism in Man...... 23

6.- Linear Regression of Phenyllactate and Oxalate...... 44

7. Linear Regression of Phenyllactate and Glycolate 45

vii ABBREVIATIONS

EDTA: Ethylenediaminetetraacetic acid

Na: Sodium

K: Potassium

Li: Lithium

Carbon 2 and Carbon 3 C2"C3: Cl40„: Carbon 14 (radioactive) and carbon dioxide

3-p-glycerate: 3-phosphoglycerate

2-P-glycerate: 2-phosphoglycerate

3-carbon: Beta carbon

FH4: Tetrahydrofolate 5 10 N , N -methylene FH^: N**, methylene tetrahydrofolate

Oxygen ° 2* Hydrogen peroxide H 2 ° 2 : NH3: Ammonia

H20: Water

a-keto: Alpha-keto

a-keto-y-hydroxyglutamate Alpha-keto-gamma—hydroxyglutamate

a-hydroxy-3-keto: Alpha-hydroxy-b e ta-keto

NADH2: Nicotinamide adenine dinudeotlde, reduced

Vitamin (Thiamine) Br V Vitamin Bg (Pyridoxine) viii • LDH: Lactic dehydrogenase

NADPH: Nicotinamide adenine dinucleotide phosphate, reduced

MgO: Magnesium oxide

PLA: Phenyllactic acid

OHPAA: O-hydroxyphenylacetic acid

FeCl^: Ferric chloride

viv CHAPTER I

STATEMENT OF THE PROBLEM

Kidney stones are a common problem in man. Since about 70 percent of these stones contain oxalate in quantities that cannot be accounted for by dietary oxalate absorption, the endogenous synthesis of oxalate is an Important factor in kidney stone formation. Consequently, inhibit­ ing the formation of oxalate should be an effective means of treating ■ patients having these kidney stones.

Therapeutic measures have been directed primarily to decreasing oxalate synthesis. An early approach to this problem was to reduce the available glycine by restricting protein intake. Since glycine is a precursor of oxalate, reduction of this amino acid was should decrease the amount of oxalate formed. Another approach was used in patients with primary hyperoxaluria. This disease was observed to be associated with a vitamin Bg deficiency. Since this vitamin is a cofactor in the transamination of glyoxylate, an oxalate precursor, to glycine, administration of vitamin Bg should enhance the conversion of glyoxylate to glycine and reduce the oxidation of glyoxylate to oxalate. A third attempt has been directed at inhibiting the enzyme that oxidizes glycolaldehyde to the oxalate precursor, glycolic acid. Calcium carbamide has been used to depress oxalate synthesis by inhibiting aldehyde dehydrogenase. Many other approaches to kidney stone therapy

1 have been attempted but all of the present therapeutic approaches are inadequate.

The present research is designed to investigate a new and promising approach to kidney stone therapy. This work is directed at inhibiting

the major enzyme in oxalate synthesis, glycolic acid oxidase. DL- phenyllactate has been shown to inhibit this enzyme in "in vitro" and

"in vivo" studies with rats. Oxalate synthesis was dramatically reduced

in these rat systems. Since rat liver glycolic acid oxidase and human liver glycolic acid oxidase are very similar, DL-phenyllactate should decrease oxalate synthesis in man. Patients with phenylketonuria are known to produce high levels, of L-phenyllactate and should also have a

decreased level of oxalate. In addition, these patients should have

elevated levels of oxalate precursors such as glyoxylate and glycolate.

This work would evaluate the feasibility of regulating oxalate synthesis

by a natural metabolite of man and the usefulness of administration of

this metabolite in kidney stone therapy. CHAPTER II

LITERATURE REVIEW

Urinary tract calculi, or stones, are a common problem in man.

These calculi may occur anywhere in the urinary tract, but the bladder

and kidneys are the most common sites. In addition to the different

locations, these calculi also differ in their frequency and chemical

composition. The bladder stones, which consist primarily of oxalates

and urates, account for only about 5 percent of all the.urinary tract

calculi in western countries (1, 2, 3). These calculi are probably the

result of a nutritional disease, or at least poor nutrition is of major

importance. However, kidney stones, which consist primarily of calcium

oxalate and calcium phosphate, are the most common urinary tract calculi

(4, 5). Oxalate, the compound of interest to the author, is found in

approximately 70 percent of all kidney stones (6). Since renal calculi

are the major problem in western countries and frequently contain

oxalate, only renal oxalate calculi will be discussed.

The formation of renal calculi is confusing and at best poorly

understood (7). Anderson (8) has suggested five etiological factors which singularly or in combination may play a part in stone formation.

These are:

1. Supersaturation of crystalline salts in the urine.

2. Nidus of solid material with urinary salts.

3 3. Urinary reactions to keep urinary salts in solution.

This largely determines the composition of the stone.

4. Urinary obstruction causing stagnation of urine and

resulting in crystallization.

5. Hyperparathyroidism causing increased urinary excretion

of calcium and the tendency of calcium salts to de­

position.

While the etiological factors are generally accepted, no single explana­ tion of the pathogenesis appears tenable.

For example, Randall (9) in 1937 proposed that renal calculi develop from damage to the renal papilla. This results in calcium .deposition in

the injured tissue. This calcium deposition, which becomes exposed after the surrounding tissue is lost by ulceration, serves as a nidus on which urinary salts crystallize. The stone enlarges and eventually . breaks away from its mooring to become a free renal stone. This early hypothesis of Randall has been replaced with newer hypotheses as our knowledge about renal calculi has increased. Current hypotheses of renal stone formation fall into three broad groups-— the "matrix theory"

(10, 11, 12, 13), the "crystallization inhibition theory" (14, 15, 16,

17, 18, 19), and the "hyperexcretion-crystallization theory" (20, 21,

22, 23). All of these theories require the supersaturation of urine with stone-forming salts, but the degree of supersaturation varies. For

example, the degree of supersaturation required for the Inhibition and hyperexcretion models is greater than for the matrix model. Despite the variability in supersaturation required, the deposition of supersaturated stone-forming salts from the urine and the formation of a nidus appear

to be the two common features of all models for renal calculi formation.

Urine from normal subjects is generally supersaturated with stone-

forming salts (24). This urine is regarded as being a metastable super­

saturated solution which means that the urine can exist in this super­

saturated state for long periods without precipitation or stone

formation. The metastable supersaturation of urine can be explained by

ionic strength effects and by the various soluble complexes formed be­

tween the major ionizable components— calcium, phosphate, and oxalate.

For example, the solubility of calcium oxalate is increased in the

presence of EDTA, urea, citrate, lactate, potassium, sodium, sulfate,

inorganic pyrophosphate, chloride, and magnesium (25, 26). Up to 50

percent of urinary calcium may be complexed with citrate, phosphate, and

sulfate, .thus reducing free calcium levels and preventing formation of

calcium oxalate crystals despite supersaturation of calcium and oxalate.

Urinary stone formation is clouded by the fact that increasing cation

concentration decreases the solubility of both calcium and calcium magnesium oxalate thus enhancing renal stone formation (26). The forma­

tion of a nidus is closely associated with the deposition of super­

saturated stone-forming salts from the urine. Normal urine is not over­

saturated; therefore, spontaneous homogenous nucleation does not normally occur. The actual formation of the nidus is still unclear, but once it

is formed, diurnal variation of calcium oxalate appears to be important.

Hodgkinson et al. (27) demonstrated that urines from patients with recur­ rent idiopathic calcium stones were more highly saturated and had shorter periods of undersaturation than did urines from normal patients. Thus, once the nidus is formed, patients with idiopathic calcium stone forma­ tion have longer periods of deposition of supersaturated stone-forming salts and shorter periods of dissolution.

Pathogenesis of kidney stones cannot be studied by "in vivo” experiments; therefore, most of our present knowledge is obtained through the use of urine. There is, for example, widespread acceptance that urinary calcium is increased in individuals with calcium oxalate kidney stones. Recently Hodgkinson (28) demonstrated that these calcium oxalate kidney stone individuals also excreted elevated levels of oxalate. The calcium and oxalate levels were both elevated by approxi­ mately 20 percent as compared to controls. In addition, adults showed no significant variation in oxalate excretion with age (28, 29). There was no correlation between oxalate excretion and body weight, but oxalate and creatinine excretion were closely correlated. While analysis of urine is not the ideal way of studying the pathogenesis of urinary calculi, urinary oxalate investigations do contribute to an understanding of kidney stone diseases.

The source of the urinary oxalate is of major importance. Oxalate is present in many foods and is found in high concentration in some foods such as cocoa, chocolate, tea, rhubarb, spinach, chard, parsley, and beet tops (30). However, the amount of oxalate in a normal diet cannot be clearly established. One problem is the lack of accurate and reproducible analytical methods for determining oxalate. In addition, there are variations in the oxalate content of food due to seasonal changes, age of the plant, different parts of the plant, climate, soil conditions, and fertilizers (31). With these variables in mind, the oxalate content of a normal diet has been estimated to range from

70 - 150 rag/day to 850 - 980 mg/day (32, 33). The oxalate in the diet is important because it influences the availability of dietary calcium and oxalate as well as contributing to the level of oxalate excreted.

Increased levels of dietary oxalate will significantly decrease calcium and magnesium absorption through the formation of insoluble oxalates.

Thus low oxalate diets are recommended for patients with calcium defi­ ciency states. This reduced oxalate diet may not be as critical for patients who normally have a high intake of oxalate sinCe there may be adaptations by these individuals to the high oxalate diets. Despite this apparent paradox, oxalate does influence calcium and magnesium absorp­ tion as well as kidney stone formation.

Only 5 percent or less of dietary oxalate is absorbed; therefore, oxalates in the diet are not normally toxic (30). There are, however, cases of oxalate intoxication reported in the literature. These cases are reportedly due to Increased exogenous intake of oxalate. An example of oxalate intoxication is "rhubarb gluttony" (25, 31, 34), but this gluttony has not been sufficiently documented to confirm the diagnosis

(30). Despite the lack of adequate documentation, oxalate intoxication may exist as diets high in oxalate and low in calcium favor the excre­ tion of urinary oxalate that may lead to kidney stone formation and/or oxalate intoxication. This has been demonstrated in animals; a high incidence of kidney stones was observed in animals on high oxalate, low magnesium, and low vitamin diets.

The importance of dietary conditions in oxalate stone formation has been emphasized by the high Incidence of oxalate stones in certain areas of the world. The British soldiers in India, for example, were found to have a high incidence of oxalate stones in association with hyperoxal- uria. This condition was attributed to the large intake of oxalate from tea plus the dehydrating effect of the climate (35). After World War I, the wave of oxalate stones in Europe was attributed to excessive use of oxalate-rich green vegetables (36), but this may have been in part due to borderline nutritional status and decreased intake of foods containing calcium. Nordin and Hodgkinson (1) suggest that in a hot, dry climate, the passage of concentrated urine and the use of well water with a high calcium content play a role in kidney stone disease. In any event, endemic stone disease and the wave of stones following both World Wars are probably related to increased dietary intake of oxalate and the decreased intake of calcium and vitamins. These dietary changes all favor oxalate excretion and exist in areas where stone disease Is endemic. Thus poor nutrition may be of importance in the etiology of some urinary calculi in humans.

The dietary effects of vitamins, especially vitamins B^, Bg, and

C, have been Investigated. Among other dietary compounds, tryptophan, glycine, and hydroxyproline have been studied. The effect of these compounds on the diet will be discussed in connection with the endogenous synthesis of oxalate.

The actual absorption of oxalate is not well understood. As pre­ viously noted, approximately 5 percent of Ingested oxalate is absorbed and this absorbed oxalate can be recovered in the urine (32). The soluble oxalates such as sodium oxalate are much more readily absorbed than the Insoluble salts such as calcium oxalate. In the stomach, the calcium oxalate is soluble due to the low pH, but in the alkaline environment of the intestine, calcium oxalate tends to precipitate.

The degree of precipitation depends on the ratio of calcium to oxalate and the presence or absence of magnesium (31). Despite these uncertain­ ties of absorption, the ingested oxalate that is eventually excreted accounts for only a small portion of urinary oxalate (32). The majority of urinary oxalate is, therefore, derived from endogenous synthesis.

The amount of oxalate synthesized has been calculated to be 24 to 46 mg. per day (37), and agrees well with the normal excretion of 17 to

43 mg. oxalate per day (38). Both absorption and endogenous synthesis contribute to urinary oxalate, but the major source is biosynthesis.

Before looking at the biochemical means by which oxalic acid is synthesized within the body, the chemistry of oxalic acid should be re­ viewed. Oxalic acid is a dicarboxylic acid (HOOC-COOH) which crystal­ lizes from aqueous solution to form a white dihydrate. The mono-, di-, and trihydrate of oxalic acid have been found in biological materials, but the dihydrate is most frequently found. The two molecules of water are lost when the crystals are heated to 100°C, and at 200°C the acid decomposes into carbon dioxide, carbon monoxide, formic acid, and water

(31). The acid is moderately soluble in water (8.7 g/100 g of water at

20°C) and is a relatively strong acid with pKa^ = 1.23 and pKa£ “ 4.19.

Oxalic acid is oxidized to carbon dioxide and water by ferric and eerie salts and potassium permanganate. This dicarboxylic acid forms two series of salts, neutral and acidic. The neutral salts are moderately soluble in water while the acid salts are relatively Insoluble. The acid potassium salt will combine with another molecule of oxalic acid to form 10 a salt known as quadroxalate (salt of Sorrell). Oxalic acid also forms mono- and di-esters as well as a monamide (oxamic acid) and a diamide

(oxamide). Oxalic acid is reduced to glyoxylic acid (0HC-C00H) and then glycolic acid (HOHgC-COOH) in the presence of zinc and hydrochloric acid.

The formation of salts is very important since this has a profound influence on the solubility of oxalate. Salts of the alkali metal ions

(Na, K, Li) and ferrous compounds are soluble; however, most other salts are only sparingly soluble in water (39, 40), The calcium salts of oxalic acid are practically insoluble at neutral or alkaline pH. Calcium oxalate usually precipitates from urine as the dihydrate but, to a much smaller extent, may also precipitate as the trihydrate. The solubility of calcium oxalate can be increased by decreasing the pH, but pH changes in the physiological range have only a small effect. The calcium oxalate

solubility can also be increased by the presence of urea, citrate, mag­ nesium, lactate,sulfate, sodium, potassium, and EDTA (41, 42). Heparin and inorganic pyrophosphate inhibit crystallization or precipitation of

calcium oxalate (43, 44). The role of these parameters and/or compounds in the biological system is still unclear; however, they are considered important.

Oxalates are known to be distributed in all body tissues and fluids with wide variation in-concentration between tissues. The wide distribu­

tion of oxalate in the body is easily understood because oxalate is

freely permeable to all mammalian membranes with the possible exception

of the mitochondrial membrane. The large variability in concentration between tissues is not easily understood. An example of this concentra­

tion variability is observed in blood. The plasma oxalate levels are 11 normally 13 pg/100 ml. (45); however, whole blood has higher oxalate values suggesting a higher concentration of oxalate in the red blood cells. Since oxalates do not bind to proteins at physiological pH (46), the proteins are probably not responsible for the unequal distribution of oxalate. The reason for this unequal distribution is not apparent or understood. There is no evidence that oxalate is utilized or catab— olized in mammalian tissues because in isotope dilution studies, 89 to

98 percent of the isotopically labeled oxalate given to experimental animals or humans was recovered from the urine in 36 hr. (47). The purpose of the wide distribution of oxalate into body tissues and fluids remains unknown.

The endogenous synthesis of oxalate occurs primarily in the liver

(48, 49, 50, 51). The major immediate precursors of oxalate are gly— oxylate and ascorbic acid; however, only glyoxylate has been clearly established as an immediate precursor. Ascorbic acid is known to be a principal source of urinary oxalate (52, 53); however, no pathway for the conversion of ascorbic acid to oxalate has been clearly identified.

In addition, tryptophan may be a precursor of oxalate. "In vivo" studies support tryptophan's role as a precursor to oxalate (54, 55); however, this acid contributes less to the urinary oxalate than gly­ oxylate and ascorbic acid. Other possible contributors to urinary oxalate include glycolate (56), glycine (57, 58), and hydroxyproline via o-keto-P-hydroxyglutarate (59). These compounds are all thought to contribute to oxalate synthesis through glyoxylate. Ethanolamine (60), ethylene glycol (61), and glycoaldehyde may be contributors to gly­ oxylate formation via glycolic acid. Glyoxylate, in addition to forming 12

GLUCOSE PYRUVATE t ♦ I I CHoO(P) CH2OtP) c h 2 o h CH2OH CH2OH I _ I I c = o — HOCH - (PJOCH HOCH HCOH I I I I I COOH COOH COOH COOH COOH 3-P-HO-Pyruvate 3-P-Glycerate 2-P-Glycerate D-Glycerate L-Glycerute

CH20(P) CH2OH CH2OH I HCNH2 HCNH2 c = o I I I COOH COOH COOH L-P-Serine L-Scrine CH2OH HO-Pyrovate V I co2 c h 2n h 2 co2 II. Ethanolamine h CH2NH2 c h 2o h a p h c h 2o h

COOH CHO CH2OH Glycine HO-B-Ketoadipatc Glycolaldehyde Ethylene gtycot I CHO LDH c h 2o h HO-Prolinc. 7-HO-Q-Ketoglutarate: t I COOH GAO ~ COOH Malate glyoxylate - glycolate LDH GAO Tryptophan— -'.ndoleoyruvaie - - p . - COOH Ascorhate Phenylalanine* "Pltcnylyyruvate —-*■ - i-l jr. COOH Tyrosine------HO-pheny I pyruvate*’ Oxalate

Figure 1. Metabolic pathways of oxalate biosynthesis. 13 oxalate, may be Irreversibly oxidized to carbon dioxide and formate

(61, 62, 63). These relationships are summarized in Figure 1.

The biosynthesis of oxalate will be reviewed emphasizing the current pathways and their role in abnormal oxalate synthesis. This discussion will be divided into four sections— (1) Ascorbic Acid, (2) Glycine,

(3) Glyoxylic Acid, and (4) Glycolic Acid.

1. Ascorbic Acid

L-ascorbic acid has been shown to be a precursor of urinary oxalate in several laboratory animals and man (64, 65, 66, 52). In addition, these "in vivo" studies with C^-ascorbate suggest that oxalate is de­ rived from carbon atoms 1 and 2. Although the metabolic pathway of ascorbic acid conversion to oxalate is not known with certainty, the probable pathway is outlined. (Figure 2). Ascorbate is oxidized to dehydroascorbate, then reduced to 2,3 diketog ulonic acid, and is subse­ quently converted to oxalate and threonic acid. There is, however, some evidence that 2,3 diketogluconic acid is not an Intermediate, but that the bond may be split enzymatically while the ascorbate lactone ring is still intact (67). Another uncertainty about the ascorbic acid pathway is whether or not glyoxylate is an intermediate. Smith suggests that glyoxylate is probably not involved in ascorbic acid metabolism 14 14 since there is no C 0^ formed after administration of ascorbate-C

(68); however, this lack of formation also suggests that 2,3 di- ketogluonic acid is not being converted to L-xyIonic and L-Lyxonic acids.

Despite the uncertainty of the metabolic pathway, ascorbic acid is apparently a precursor of oxalate. 0 0 II It C-OH C-OH 1 1 HOCH HCOH I I HCOH HCOH I HOCH HOCH C-OH I HCOH HCOH o = c H H HO-C o = c o = c L-Xylonic L-Lyxonic Acid Acid JI HCOH

HOCH HOCH HCOH •HO] OO ^ II 11 HCOH HCOH HCOH C-OH C-OH H 1 1 HCOH L-Ascorbic L-Dehydroascorbic 2,3-Diketo-L- C-OH Acid Acid Gulonic Acid 11 1 o HOCH Oxalic Acid I HCOH H L-Threonic Acid

Figure 2. Probable pathway of L-ascorbic acid metabolism to

form oxalic acid. 15

Under normal conditions 35-50 percent of urinary oxalate is derived from dietary ascorbate (46). L-ascorbate is, therefore, a major pre­ cursor of oxalate, but large ascorbate loads (4 gra.) do not materially

Increase urinary oxalate in normal or primary hyperoxaluric patients

(69, 70). Hockaday et al. (71) suggested that the rate of oxalate formation from L-ascorbate is limited by low enzyme activity or a metabolic control mechanism. In any event, the increased urinary oxalate in primary hyperoxaluric patients does not appear to be derived from the metabolism of ascorbic acid.

2. Glycine

Glycine is a major source of glyoxylate and oxalate in man. It is . estimated that 40 percent of oxalate is derived from glycine (72, 73), but this is not a major pathway for glycine metabolism as only 0.5 to

1.0 percent of C^-glycine can be accounted for by this pathway (73).

Glycine conversion into oxalate is not increased in the genetic disorder of primary hyperoxaluria, suggesting that there is no Impairment of glycine metabolism In this disorder. Since the conversion to glyoxylate and oxalate is a minor metabolic pathway of glycine, the effects of pri- mary hyperoxaluria on glycine may not be observable. Glycine's role in hyperoxaluria, therefore, is not clear.

Glycine is ingested in the diet and/or derived from serine in mammalian systems. The interrelationships between glycine, serine, and glycolysis (3-P-glycerate and 2-P-glycerate) are summarized in Figure 3

(74). Glycine is synthesized from serine with the latter amino acid losing its 6-carbon to tetrahydrofolate (FH^) forming N**, N^-methylene- 16 tetrahydrofolate and glycine. The reaction can be written as: 5 10 Serine + FH^ — -> Glycine + N N -methylene FH^

Glycine from the body glycine pool is then metabolized by several dif­ ferent pathways, one of which is to glyoxylate and oxalate.

Glycine is irreversibly converted to glyoxylate by the enzyme glycine oxidase. This reaction is an oxidative deamination of glycine and can be written as:

COOH Glycine C00H

L , O T , + °2 + H2°2 °Xld'Se| L o +NH3 +H 2° Glycine Glyoxylic Acid

This enzyme, glycine oxidase, is the same as D-amino acid oxidase. D- amino acid oxidase is found in highest concentrations in mammalian kidneys and livers (75, 76).

Glycine can also be converted to glyoxylate by reversible trans­ amination (77, 78, 79, 80). This reaction can be written as:

COOH o n COOH NH„ I " B6 I 11 CH.OT, + R-C'C00H CHO + R-c-C00H Glycine keto acid Glyoxylic Amino Acid Acid

Three enzymes known to catalyze this reaction are: (1) alanineigly- oxylate aminotransferase, (2) glutamate:glyoxylate aminotransferase, and

(3) ornithine:a-ketoglutarate aminotransferase. All three of these enzymes require pyridoxine (vitamin Bg) as a cofactor. Gershoff has demonstrated a relationship between vitamin Bg deficiency and Increased oxalate excretion (81, 82, 83, 84). This reaction has an equilibrium far in the direction of glycine synthesis; therefore, pyridoxine defi­ ciency does not appear to be an important cause of hyperoxaluria. I P -0 OH P -0 0 P -0 n h 2 I I I H I i H2C-(j-COOH- -H2C-C-COOH* ■ h 2c - c - c o o h

H OHNH2 H I | --Glycine 3-P-GIycerate P-Hydroxypyruvate P-Serine H2C- C-COOH<^, ■Pyruvate H 1 Serine OH O P * OH OH OHO 1 1 I 1 I II H2c~ (j-C 0 0 H -*- —► h 2c - c - c o o h - ■h 2c - c - c o o h 1 H H 2-P*Glycerate D-GIycerate Hydroxypyruvate OH H I I 1 H2C -C -C 0 0 H I OH L-Glycerate LDH = lactic dehydrogenase.

Figure 3. Pathways of serine metabolism in mammalian systems. [74] 3. Glycolate

The dietary intake and the absorption of glycolate have not been studied and remain uncertain (68, 70). Glycolate's conversion to gly­ oxylate has been studied, but other aspects of glycolate metabolism have not been studied. The lack of knowledge about the ingestion, ab­ sorption, and metabolism of glycolate limits our knowledge about the origin of glycolate excreted. It is, however, known that excessive

Ingestion of glycolic acid by rats may lead to generalized deposition of oxalate (85). Neither blood nor plasma levels of this acid have been determined in man, but the 24 hr. urinary excretion levels of nineteen adults were found to range from 18.2 to 60.7 mg. (86).

Glycolic acid has two known precursors in mammals. These pre­ cursors are glyoxylate and glycolaldehyde (see Figure 1). Glycolaldehyde is in turn formed from hydroxypyruvate, ethylene glycol, or serine by way of ethanolamine. The oxidation of glycolaldehyde to glycolic acid is catalyzed by both aldehyde oxidase and aldehyde dehydrogenase (87).

The formation of glycolic acid from glyoxylic acid is catalyzed by the

NADII^-linked enzyme glyoxylate reductase (88), by lactic dehydrogenase

(89), and possibly by a separate NADI^-linked glyoxylate reductase (90).

The reverse reaction, glycolic acid to glyoxylic acid, is catalyzed by glycolic acid oxidase (91). Glycolic acid oxidase also converts lactic acid to pyruvic acid and glyoxylic acid to oxalic acid (92). The affin­ ity of the enzyme for these substrates is glycolate^*L—lactate^> gly­ oxylate (92). After the initial conversion of glycolate to glyosqrlate, the proximity of glycolic acid oxidase to glyoxylate would suggest that glyoxylate would be particularly susceptible to further enzymatic 19 oxidation to oxalate. This did not occur "in vivo" and may be due to

feedback inhibition of oxalate synthesis (92, 49). Despite the apparent low affinity for glyoxylate, glycolic acid oxidase is probably the major enzyme in. the oxidation of glyoxylate to oxalate (93) and the only "in vivo" enzyme known to oxidize glycolate to oxalate. This enzyme is induced by testosterone (94, 95) which may explain the 30-40 percent lower liver glycolic acid oxidase levels found in mature female rats.

In addition, a lower rate of glycolic and glyoxylic acid oxidation may explain the mature female rat*s greater resistance to glycolic acid nephrotoxicity (96). The "in vivo" importance of glycolic acid oxidase has also been demonstrated with DL-phenyllactate, a specific nontoxic inhibitor of glycolic acid oxidase (93). This enzyme appears to be essential for the normal biosynthesis of oxalate and for the oxidation of glycolic acid to glyoxylate. The only known fate for glycolic acid is its conversion to glyoxylate and/or oxalate.

4. Glyoxylate

Glyoxylate is the only known immediate precursor of oxalate.

Approximately 40 percent of urinary oxalate is derived via glyoxylate.

Little is known about glyoxylate content of food and, consequently, about glyoxylate absorption.Glyoxylate is a very reactive compound and is known to form compounds with urea (97), cysteine (98), and some a-keto acids (99). The fact that glyoxylate is highly reactive and forms additional compounds may explain why blood levels of 12.5 pg/100 ml. were observed in 2 of 23 thiamine deficient patients tested (100).

By Isotope dilution technique, the mean urinary glyoxylate level was

3.9 mg./24 hr. with a range from 2.2 to 6.0 mg. These results were 20

obtained from 12 normal adults (71). The degree to which glyoxylate

forms irreversible products in urine is a source of uncertainty in all

glyoxylate measurements (86).

The major sources of glyoxylate are probably glycine and glycolate

(Figure 4). Both of these precursors have been previously discussed.

Another source of glyoxylate is from hydroxyproline via a-keto-Y-hydroxy-

glutamate. The significance of this reversible reaction is not known, but the contribution of hydroxyproline to urinary oxalate is probably

age and sex dependent. In any case, further study will be required before hydroxyproline's precise role in oxalate metabolism can be estab­

lished. Other possible sources of glyoxylate are: the diet, pyrimi­

dines, phenylalanine, tyrosine, and tryptophan (46). Cook and Henderson

suggested that glyoxylate is not a major intermediate in oxalate formed

from tryptophan (101). This pathway for oxalate formation is of only minor significance in rats and of unknown significance in humans; however, it may be physiologically important in humans in certain metabolic diseases such as phenylketonuria where the levels of aromatic a—keto acids are known to be increased. It is obvious that many com­ pounds are potential contributors to oxalate biosynthesis, but the

significance of these various precursors has not been well established.

Vitamins are not precursors of oxalate, but they have an Important role in the formation and degradation of glyoxylate and, consequently, oxalate. The importance of vitamins and Bg are seen in Figure 4.

The transamination of glyoxylate to glycine is vitamin Bg dependent.

Vitamin dependent reactions include the conversion of glyoxylate

to N-formy1-glutamate and of glyoxylate to a-hydroxy-8-keto adipic acid. Glycolic Acid

Hydroxyproline Glyoxylate Reductase Glycolic Acid Oxidase a-Keto*7 -Hydroxyglutamate LDH Glycine Oxidase (FAD) Formic Acid + CO2 \ Tranwminases^ Glyoxylic Acid Glycine N-Formyl-glutemate (B6) Glycolic Acid +C02 Oxidase f r olj3g a-Hydroxy-^-Keto _____ Reaction inhibited I r LDH U r ”lBei by Allinopurinol ” Xanthine Oxidase

5 Hydroxy*4-Keto 2,3 Dihydroxy Oxalic Acid Valeric Acid 4-Ketopimelic Acid

Figure 4. Pathways of glyoxylate metabolism 22

The role of these vitamins in disease states involving oxalate metabo­ lism is not clear.

The importance of understanding the biochemical pathways of oxalate

synthesis becomes readily apparent when disease states such as primary and secondary hyperoxaluria are considered. Both result in elevated urinary oxalate excretion, but these disease conditions are caused by

different abnormalities. Primary hyperoxaluria is genetically determined while secondary hyperoxaluria is nutritionally determined. (Figure 5.)

In primary hyperoxaluria, two variants of this hereditary disease are known and are classified as type I or glycolic aciduria and type II or

L-glyceric aciduria. Clinically both types of primary hyperoxaluria patients have nephrolithiasis, but differentiation of these two types of hereditary oxalate disorders is based on biochemical criteria. In

glycolic aciduria, oxalic, glyoxylic, arid glycolic acids are excreted

in excess in the urine. The metabolic basis for this disorder is a

deficiency of a-keto-gluturate: glyoxylate carboligase in the liver, kidney, and/or spleen (87). This enzyme converts glyoxylic acid to a-hydroxy-B-keto adipic acid (Figure 5); therefore, a deficiency of

this enzyme causes an accumulation of glyoxylic acid which Is then converted into glycolic and oxalic acids. This results in elevated urinary excretion of all three of these acids (68, 87).

In L-glyceric aciduria, type II hyperoxaluria, urinary oxalic and

glyceric acids are excreted in elevated amounts, but urinary glycolic acid is normal. This abnormality is thought to be due to a deficiency of the enzyme D-glyeerie dehydrogenase. This enzyme forms D-glyeerie acid from hydroxypyruvate, a product of serine transamination (Figure 3). ^ ,Cth ylene GLYCOLIC Clycol Primary Hyperoxaluria Type // Primary Hyperglycinemia ,Hyperoxaluria (HypO'Oxaluria}\ Type / « -K E T O -0 - ...... ______— GLYCINE GLYOXYLIC HYDROXY • ADIPIC B

Thiamine' Deficiency PyridoxineJ Deficiency Oxalic Acid OXALIC Poisoning

The open arrows indicate exogenous causes of hyperoxaluria. The arrows with open points indicate the known acquired and hereditary enzyme defects leading to disorders of oxalate metabolism

Figure 5. Disorders of oxalate metabolism in man. 134J 24

This enzymatic reduction of hydroxypyruvate requires either NADH or NADPH as a cofactor. However, hydroxypyruvate can also be enzymatically con­ verted to L-glyceric acid. This conversion may involve lactic dehydro­ genase. The only known metabolic pathway in mammals for the L-glyceric acid is its oxidation back to hydroxypyruvate. Therefore a deficiency of D-glyceric dehydrogenase results in an increased formation of L- glyceric acid and the subsequent urinary excretion of excess amounts of this acid (68, 87). The cause of the hyperoxaluria is not obvious from the enzymatic defect. D-glyceric dehydrogenase, however, may be the same as glyoxylate reductase (68). The deficiency of D-glyceric dehydro­ genase in this hereditary disease would account for a block in the reduction of glyoxylate to glycolate as well as the defect in the reduc­ tion of hydroxypyruvate to D-glycerate. This enzymatic defect in these two metabolic pathways would explaih the observed findings of L-glyceric aciduria and hyperoxaluria in the absence of hyperglycolic aciduria.

Richardson and Liao have proposed an alternate explanation for the in­ creased urinary excretion of L-glyceric acid and oxalate. These authors suggest that hydroxypyruvate accumulates because of a deficiency of D— glyceric dehydrogenase. The hyperoxaluria results from an increased amount of hydroxypyruvate being converted to oxalate (102) (see Figure 1)

Despite the various explanations for the metabolic defects, hyperoxaluria and L-glyceric aciduria are always found in type IX primary hyperoxaluric

Secondary hyperoxaluria, or acquired oxalate disorders, can result from either excessive ingestion of oxalate-forming compounds or de­ ficiency of vitamins and Bg which are associated with oxalate 25 formation (Figure 5). The excessive ingestion of compounds that can form oxalate is seen in oxalosis and oxalate poisoning. Oxalosis has been produced in laboratory animals by the administration of excess oxalate.

Oxalosis can also be produced in laboratory animals, and presumably in man, by excessive intake of oxalate precursors such as glycolate and ethylene glycol. In rats, the excessive intake of glycolic acid or ethylene glycol leads to oxalosis, uremia, and even death (87, 103) .

However, deficiencies resulting in secondary hyperoxaluria are usually deficiencies of vitamins. Vitamin B^, for example, is a co-factor in the enzymatic conversion of glyoxylate to a-keto-B-hydroxy adipic acid

(Figure 5). A deficiency of this vitamin produced symptoms similar to . those seen in primary hyperoxaluria type I. A deficiency of Vitamin B^, however, produces hyperoxaluria and oxalosis in experimental animals.

This vitamin deficiency reduces the transamination of glyoxylate to glycine (81) resulting in a progressive increase of urinary oxalate in man (54). While much is known about these various hyperoxaluria prob­ lems, the biochemistry and pathogenesis are not completely understood.

Early diagnosis and treatment of patients with primary hyper­ oxaluria is imperative as progressive renal failure eventually develops leading to an early demise. Therapeutic measures are directed toward

(1) Increasing calcium -oxalate solubility, (2) treating uremia, and

(3) decreasing oxalate excretion by inhibition of oxalate synthesis.

Many investigators have worked with these therapeutic measures, but none have developed a successful long term treatment for primary hyperoxaluria.

As a therapeutic measure, calcium oxalate solubility can be modestly increased by the maintenance of a large urine volume and by the 26 restriction of dietary calcium. Magnesium supplements have been used in increasing calcium oxalate solubility and with some success in inhibit­ ing the formation of calcium oxalate stones in rats (104, 105). Daily

MgO and pyridoxine have been used in human patients with modest thera­ peutic success (106). The magnesium therapy is promising, but no systematic study of magnesium therapy in primary hyperoxaluria has been reported. Another therapeutic approach is aimed at preventing the precipitation of oxalate and calcium by high phosphate intake (107,

108, 109). Support for this therapeutic measure has primarily come from rat studies, but a few human patients have been studied with limited success (87). These methods of keeping calcium and oxalate in solution have been proposed for patients that are not uremic. Once the patient becomes uremic, drastic measures are generally followed. For example, renal transplants have been used in uremic patients with primary hyper­ oxaluria. Very little success has been achieved as only one patient has been maintained for 6 months without evidence of renal insufficiency

(87). With this limited success, dialysis appears to be a better alternative. The problem of dialysis is similar to that in high phos­ phate and magnesium therapy, namely that there are no long term studies in patients with primary hyperoxaluria. Neither of these therapeutic approaches have been consistently successful.

The third approach to treating primary hyperoxaluria is the reduc­ tion of oxalate synthesis and excretion. This therapy has met with vary­ ing degrees of success. One approach Is to reduce the available glycine by restricting protein intake, but this has not been successful because glycine is readily synthesized. Another means is to trap the glycine 27 as hippurate with benzoate. This procedure has met with modest success, but reduction was only temporary; consequently, this means of therapy was not useful for long term treatment (110). Still another therapeutic approach has been directed at vitamin Bg because hyperoxaluria has been observed to accompany pyridoxine deficiency. The administration of pyri­ doxine to experimental animals is thought to increase the transamination of glyoxylate to glycine (111), and the administration of pyridoxine to normal and mentally deficient patients without hyperoxaluria has been reported to reduce urinary oxalate excretion (83, 112). In addition, primary hyperoxaluric patients given large doses of pyridoxine demon­ strated a decrease in oxalate excretion (112, 113).

The enzyme inhibitors constitute another group of compounds that are used to reduce oxalate synthesis and excretion. One such inhibitor is calcium carblmlde. This is a nontoxic inhibitor of aldehyde dehydrogen­ ase which catalyzes the conversion of glycolaldehyde to glycolate (114); however, Investigators have been unable to confirm a consistent reduction in urinary oxalate (115, 116). The usefulness of this inhibitor is, therefore, questionable. A more promising inhibitor is DL-phenyllactate.

This is a nontoxic inhibitor of glycolic acid oxidase which catalyzes the conversion of glyoxylate to oxalate. Two other enzymes, lactic dehydro­ genase and xanthine oxidase, also catalyze the conversion of glyoxylate to oxalate; however, Liao and Richardson (49) have demonstrated that these two enzymes do not catalyze the conversion of significant amounts of glyoxylate to oxalate. Therefore, inhibition of glycolic acid oxi­ dase is equivalent to inhibition of the catalysis of glyoxylate to oxalate. Liao and Richardson demonstrated a reduction of oxalate synthesis and excretion In perfused rat livers and a limited number of rats. The feasibility of regulating oxalate synthesis by a specific nontoxic inhibitor of glycolic acid oxidase, such as DL-phenyllactate, is very promising. CHAPTER III

MATERIALS AND METHODS

Urine was collected from 22 phenylketonuric (PKU) patients and 18 mentally deficient but non-PKU patients living at Orient State Hospital,

Orient, Ohio. Each patient was identified by an arbitrarily assigned number. The phenylketonuric patients were assigned numbers between

0 and 28 and the non-PKU patients were given numbers between 29 and 50.

Urine was collected for 48 hours from each of the 40 patients, usually as two 24 hr. specimens, but some as a single 48 hr. specimen. On the day terminating the 48 hr. collection period, each specimen was screened for pH, protein, glucose, ketones, bilirubin, and occult blood by Bili-

Labstix (Ames Co., Elkart, Ind. 46514)»and for phenylpyruvate by the ferric chloride test (117). In addition, the volumes were recorded, and each of the samples was assayed several times for creatinine (118).

The creatinine value used was a mean of the assayed values. This creat­ inine value was then utilized to calculate the total creatinine in each specimen. In the samples collected as duplicate 24 hr. specimens, only the 24 hr. specimen with the largest amount of creatinine was used for additional analysis, but all samples collected as a single 48 hr. speci­ men were used for additional analysis. The specimens thus chosen for further analysis had a 30 ml. aliquot removed, and these aliquots were stored in individual vials at 5°C. Samples for further analysis were taken from these aliquots. All the remaining urine specimens were then

29 30 assayed in duplicate for each of the following five urinary constitu­ ents.

(1) Phenyllactate: The PKU derivative, phenyllactate, was assayed by the gas chromatographic method of Hoffman and Gooding (119). In this method, the urine was acidified to pH 1 with 2M HCL, and this acidified urine was extracted three times with 10 ml. aliquots of ethyl acetate. The combined ethyl acetate extracts (30 ml.) were then evapor­ ated in a rotary evaporator using a vacuum of 10-20 mm. Hg (water as­ pirator) and a temperature of <40°C. Up to this point, each specimen was done in duplicate. The residue- of one of the extracts was combined with 0.5 ml. bis (trimethylsilyl) -trifluoroacetamide (BSTFA), and

1-3 yl. of this BSTFA-treated specimen was injected into the gas chro­ matograph. If this•specimen had no evidence of phenyllactate from the chromatogram and if the ferric chloride test was negative, the specimen was said to have <3 yg. phenyllactate/ml. urine, and no further analysis of phenyllactate was pursued. However, if the specimen did contain phenyllactate, the second residue was utilized. To this residue, 100 yl. of an internal standard containing 50 yg. tetradecane and 30 yg. tri— decane was added. This specimen plus internal standard was methylated with 0.5 ml. BSTFA, and 1-3 yl. of this methylated specimen was injected into the gas chromatograph. The resulting chromatograph was then used to quantitate the phenyllactate. The phenyllactate concentration was determined by calculating the ratio of the tetradecane internal standard peak height to the phenyllactate peak height. The concentration of phenyllactate was then determined from a standard curve. 31

(2) O-hydroxyphenylacetate; This acid was extracted and assayed simultaneously with phenyllactate (119). The concentration of 0- hydroxyphenylacetate is determined in exactly the same way as the phenyllactate concentration except O-hydroxyphenylacetate is used as the standard.

(3) Oxalate: Before assaying for oxalate, the dicarboxylate was converted to the free acid by treating 5 ml. of urine with 5 ml. of

Dowex 50 W- X8 (acid form). The urine and cation exchange resin were mixed well by shaking in a glass stoppered 15 ml. centrifuge tube.

After the mixture separated, aliquots of urine were taken from the supernatants for oxalate analysis. The oxalate was. then determined by . the method of Bernstein and Khan (120, 121). This method is based on the measurement of a decrease in absorbance caused by the displacement of uranium by oxalate from the red uranium (IV) -4(2-pyridilazo)— resorcinol complex (UO^PAR ). Removing the uranium from this colored complex leaves 4-(2-pyridilazo)—resorcinol (PAR), a colorless compound.

The most likely sequence of reactions is (116):

PAR ► PAR" + H+ PAR" + U09 ► U0 PAR U02PAR + C204 ------* U°2C2°4 + PAR”

All oxalate analyses were done in duplicate.

(4) Glycolic Acid: The glycolic acid content was determined by the method of Dagley and Rodgers (122). Chromatropic acid was added to the urine specimen which was then acidified with concentrated sulfuric acid. This solution develops a purple color upon heating in a boiling water bath. The heating of the acidified urine promotes the decomposi­ tion of glycolic acid (H0-CH2-C00H) into COg, H20, and HCHO. This acid 32 hydrolysis is quantitative if the specimen contains 0 to 150 pg. gly­ colic acid. The chromatropic acid combines specifically with formalde­ hyde to form a purple complex. By comparing the absorbance of this purple complex at 570 nm. with a standard curve, the glycolic acid con­ centration of the specimens was determined.

(5) Glyoxylic Acid: Glyoxylic acid was determined by the method of Lui and Roels (123). Urine containing glyoxylic acid was combined with phenylhydrazine at pH 7.0, 25°C and in a reaction mixture of high ionic strength (0.25 M sodium phosphate). The phenylhydrazone formed was then oxidized with potassium ferricyanide in the presence of strong acid. The reduced ferricyanide was quantitated by measuring the absorr bance at 517 nm. The absorbance of the unknown urinary glyoxylate was compared to a standard curve to determine the concentration of glyoxy­ lic acid in the urine specimen. The accuracy of this or any other urinary glyoxylate method is limited by the degree to which glyoxylate irreversibly forms compounds not detectable in the urine (86).

Statistical analysis of the data includes the indicators of central tendency — mean (x), and standard deviation (s). The data was refined by deleting any value greater than ± 3s, and this refined data was utilized for all further calculations. The amount of each acid excreted/

24 hr. was correlated with the 24 hr. creatinine excretion. A correla­ tion coefficient (r) and a "t" test for the significance of this r (t^) were determined for each acid. In addition, the amount of each acid excreted per gram (g.) of creatinine was correlated with the phenyl­ lactate and the O-hydroxyphenylacetate excreted per g. creatinine. An r and a t value were calculated for each of the correlations. Graphs statistically significant latter correlations were prepared, CHAPTER IV

RESULTS

The urine used in these experiments were screened for abnormalities in pH and for abnormal amounts of protein, glucose, ketones, bilirubin, blood, and phenylpyruvate. Among the phenylketonuric patients, one patient had abnormally high urinary pH (patient no. 25) and six patients

(patients no. 8, 14, 15, 16, 25, and 26) had negative test results for phenylpyruvate. These negative phenylpyruvate results may have been due to the -decomposition of this.labile phenylketonuric metabolite.

The control group, however, had two patients with abnormal values for urinary constituents. Patient no. 41 had elevated protein, and patient no. 48 had elevated protein and blood in the urine. As expected, all control patients had negative test results for phenylpyruvate.

The 24 hr. urinary excretions of oxalate, glycolate, glyoxylate, creatinine, phenyllactate, and O-hydroxyphenylacetate were determined as described in materials and methods (Table 1). The mean oxalate values for PKU and control patients were 39.2 mg./24 hr. and 31.1 mg./24 hr. respectively. These values compare favorably with the normal values of 17-43 mg./24 hr, (86). Oxalate excretion, however, varies consider­ ably between normal individuals (124). This variation between indivi­ duals was observed in both the control and phenylketonuric patients, but the variation was more pronounced in the phenylketonuric patients.

34 TABLE 1

Urinary Excretion of Creatinine, Oxalate, Glyoxylate, -Glycolate, Phenyllactate and O-Hydroxyphenylacetate

PHENYLKETONURIC CONTROL Patients* Patients2 Urinary Constituent Mean Standard Range Mean Standard Range Assayed (mg./24 hr.) Deviation (mg./24 hr.) (mg./24 hr.) Deviation (mg./24 hr.)

Oxalate 39.2 31.3 7.40 - 128 31.1 13.9 12.5 - 64.3

Glyoxylate 5.87 2.68 1.15 - 12.2 .3.69 1.84 1.38 - 8.58

Glycolate 126 46.4 43.7 - 201 166 107 38.4 - 465

Creatinine 704 218 261 - 1086 • 1149 324 534 - 1787

Phenyllactate2 602 404 71.4 - 1752 ' - - **

O-Hydroxyphenylacetate 3 174 94 10.3 - 375. - - -

1. Urines from 22 phenylketonuric patients were assayed for each constituent.

2. Urine from 18 mentally retarded but non-PKU patients was assayed for each constituent.

3. Urinary levels for these constituents were not detectable in control patients. 36

This pronounced variability of oxalate excretion between individuals was reflected in the phenylketonuric patients standard deviation (31.3) and their range of oxalate values (7.4 to 128 mg./24 hr.). The control patients had a considerably smaller standard deviation (13.9) and a smaller range of urinary oxalate values (13 to 64 mg./24 hr.). The phenylketonuric patients had more variability in the oxalate excreted per 24 hr. than did the control patients, but both the phenylketonuric and the control patients mean 24 hr. oxalate excretion was similar to that expected from normal patients.

The mean values of urinary glyoxylate in phenylketonuric and con­ trol patients were 5.87 and 3.69 mg./24 hr. respectively. The litera­ ture values for normal adults have a range from 2.2 to 6.0 mg./24 hr. with a mean value of 3.7 mg./24 hr. These normal values were determined by isotope dilution (71). Both of the groups studied here have mean values that fall in the normal range. In the phenylketonuric group approximately 41 percent (9 of 22 values) of the reported urinary gly— oxylates were elevated while only one patient from the control group had an elevated urinary glyoxylate. The standard deviations of the phenyl— ketonuric and control groups are 2.68 and 1.84 respectively; however, the relative standard deviation for these two groups is very similar (45.6 percent for phenylketonurics and 49.8 percent for control patients).

The relative variability of glyoxylate for these two groups is quite similar, but the mean values are dissimilar.

The glycolate mean values for these two groups of patients are elevated when compared to the normal glycolate values determined by isotope dilution (71). The values 724.8 and 934.2 mg./24 hr. (patients 37

no. 14 and 35 respectively) were deleted from the phenylketonuric and

control groups respectively due to the fact that these values were

greater than +3 standard deviations. After deleting these values, the

■ phenylketonuric patients had a mean glycolate of 125.9 mg./24 hr., and

the control group had a mean value of 165*7 rog./24 hr. The normal range

of values was 18.2 to 60.7 mg./24 hr. Only one value from each group

of patients falls within the normal range suggesting that these values

are abnormally high. Since all 40 patients were on the same diet and

both the phenylketonuric and control groups were similarly elevated,

diets may be responsible for these abnormally high glycolate values.

The variability about the mean, however, is smaller in the phenyl­

ketonuric group than in the control group. This is reflected in the

standard deviations for the phenylketonuric group and the control group

of 46.4 and 107.2 respectively. The glycolate values are abnormally

high with greater variability xii the control group.

The phenylketonuric patients mean creatinine value of 0.704 g./24 hr.

is slightly below the creatinine range of 0.8 to 2.0 g./24 hr. for

normal patients (118). The control group has a mean creatinine value of

1.15 g-./24 hr. The percent relative variation per standard deviation

unit is 31.0 percent for phenylketonuric patients and 28.2 percent for

control patients. These two groups of patients have relative vari­

abilities that are similar but mean values which are greatly different.

Phenyllactic and O-hydroxyphenylacetic acids were excreted at the

rate of 602 mg. and 174 mg./24 hr. respectively. This compares favor­

ably with the values of 813 and 125 mg./24 hr. for phenyllactate and

O-hydroxyphenylacetate respectively as reported by Hoffman and flooding 38

(119) . 1'hese authors reported values from a very small number of pa­

tients. Despite the small population, the literature mean values are similar to the mean values obtained in this larger study. This was ex­ pected because all patients in both studies had phenylketonuria.

The mean values from the 24 hr. urinary excretions of oxalate, glyoxylate, and creatinine coincide with the range of values for a normal population. Glycolate mean values from both phenylketonuric and control patients are elevated when compared to the normal population.

The phenylketonuric patients had mean values for phenyllactic and

O-hydroxyphenylacetlc acids which agree with literature values for these patients. The phenylketonuric and control patients have relatively normal values except for glycolate, and the phenylketonuric patients have the expected elevation of phenyllactate and O-hydroxyphenylacetate.

Both oxalate and creatinine levels in urine vary considerably between individuals, but the excretion of these constituents is rela­ tively constant within one individual (124). The possibility of a correlation between the excretion of creatinine and oxalate or one of its precursors exists. The urinary oxalate of the phenylketonuric patients had little correlation with creatinine, but the control pa­ tients had a high correlation which was statistically significant

(Table 2). The reverse was true for the oxalate precursors, glycolate and glyoxylate. These latter constituents both had a high correlation which was statistically significant when the urinary constituents from phenylketonuric patients were correlated with creatinine; however, low correlations were found between these same constituents in urine from control patients. The phenylketonuric metabolites, phenyllactate and 39

TABLE 2

Correlation Coefficients for 24 Hr. Excretion of Oxalate, Oxalate Precursors and Phenylketonuric Metabolites with Creatinine

CREATININE (24 HR.)

PKU Patients Control Patients

(t )2 . (r)1 . 2 (rl1 . r

Oxalate (24 hr.) 0.28 85.0 0.72 99.95

Glyoxylate 0.60 99.5 -0.26 85.0

Glycolate 0.76 99.95 !■ 0.23 80.0

-3 Phenyllactic Acid 0.38 90.0 ! • “ 3 . : O-Hydroxyphenylacetic Acid 0.36 95.0 _ 3 i!. . - 3 1 ...... !

1. r = Correlation coefficient.

2. t = Level of statistical significance expressed in percent as determined by ,,t" test.

3. Insufficient data. O-hydroxyphenylacetate, had low correlations with creatinine, but both

correlations were significant.

Creatinine is constantly being formed and this creatinine is rapid­

ly eliminated in the urine. The amount of urinary creatinine excreted

is remarkably constant (125). This consistency of creatinine provides

a basis for calculating the concentration of urinary constituents which

is especially useful when the collection of 24-hour urine specimens is not reliable. Since there is great difficulty in collecting all urine for a given period of time from mentally deficient patients, the con­ centration of oxalate, oxalate precursors, and phenylketonuric metabo­ lites were calculated per gram creatinine (Table 3). The mean (x) . oxalate and glyoxylate values were higher in the phenylketonuric pat­ ients than in the control patients, and the distribution of values was broader in the phenylketonuric patients than in the control patients.

This distribution of values is reflected in the respective standard deviations. The glycolate mean value is greater in the phenylketonuric patients, but the standard deviation is larger in the control patients.

Regrettably, no known literature values are reported in similar units.

The values from the analysis of phenyllactate and O-hydroxyphenylace- tate, are also contained in Table 3. The phenylketonuric patient^ mean

(x) value for urinary phenyllactate is 0.745 g. per g. creatinine. This agrees well with the few available literature values. The 0-hydroxy- s % phenylacetate mean (x) value of 0.208 g. per g. creatinine is essential­ ly the same as the mean of 0.22 g. per g. creatinine reported in the literature by Armstrong et al. (126). The individual patient values for phenyllactate and O-hydroxyphenylacetate are distributed over a TABLE 3

Urinary Oxalate, Glyoxylate, Glycolate, Phenyllactate and O-Hydroxyphenylacetate Excreted Per Gram Creatinine

PHENYLKETONURIC CONTROL PATIENTS1 PATIENTS2

Standard Standard Urinary Constituents Assayed Mean Deviation Range Mean Deviation Range

t Oxalate Cmg./g. Creatinine) 53.1 38.2 10.7- — 154 26.7 8.31 16.2 - 46.4

Glyoxylate (mg./g. Creatinine) 8.02 3.05 '4.41 - 15.4 3.22 1.31 1.78- 5.94

Glycolate (mg./g. Creatinine) 167 35.6 106 - 237 128 54.9 40.9 - 228

Phenyllactate (g./g. Creatinine)3 0.745 0.448 .086 - 1.77 - - -

O-Hydroxyphenyl- acetate (mg./g. Creatinine)3 208 79.9 116 - 412 - - -

1. Urines from 22 phenylketonuric patients were assayed for each constituent.

2. Urine from 18 mentally retarded but non-PKU patients was assayed for each constituent.

3. Urinary levels of these constituents were not detectable in control patients. 42

broad range, and this is reflected in the large standard deviations for

these two constituents. Since the concentrations per g. creatinine of

oxalate, oxalate precursors, and phenylketonuric metabolites are simi­

lar to literature values;and since these values are considered to be

more representative of the true concentration of these constituents,

correlation studies involving these constituents will utilize these

concentrations. . . .

Oxalate, glyoxylate, and glycolate were correlated with phenyl­

lactate and O-hydroxyphenylacetate from the urine of phenylketonuric

patients (Table 4). Oxalate has a negative correlation with phenyl­

lactate (r= -0.45) but does not correlate with O-hydroxyphenylacetate.

In addition, the correlation between oxalate and phenyllactate is.

statistically significant (t^ = 97.5 percent). Since glyoxylate assays

are unreliable, no significance is attached to these correlation

studies. The glycolate values, however, can be interpreted. There is

a positive correlation between glycolate and PLA which is significant

(t^ = 90 percent). There is essentially no correlation with OHPAA.

The oxalate and glycolate correlations with PLA are graphically dis­ played in Figures 6 and 7. TABLE A

Correlation Coefficients for Oxalate Metabolites (Acid/g. Creatinine) and Phenylketonuric Metabolites (Acid/g. Creatinine)

PHENYLKETONURIC PA T I E N T S Phenyllactlc Acid O-hydroxyphenylacetic No. of (g./g. Creatinine) Acid (mg./g. Creatinine) Patients Used r1 t 2 (%) r1 tr2 (%) r

Oxalate (g./g. Creatinine) 21 -0.45 97.5 -0.14 N.S.3

Glyoxylate (mg./g. Creatinine) 21 0.22. 80.0 0.24 85.0

Glycolate (mg./g. Creatinine) 20 0.32 90.0 -0.01 N.S.3

1. r is correlation coefficient,

2. t represents level of statistical significance expressed in percent as determined by the ,ft,r test.

3. N.S. represents level of significance less than 73 percent. Figure 6. Linear regression of phenyllactate and oxalate. and regressionphenyllactate Linear of 6. Figure Phenyllactate (g/g Creatinine) 2.00 o.so 0 .5 1 1.00 10 20 30 40 50 60 xlt (mg/gOxalateCreatinine) 70 80 SO 0 10 2 10 4 10 1G0 150 140 130 120 110 100 44 iue7 Lna ersino lcyleaeac lclt. ' ancl glycolate. regressionplicnylluetate Linear of 7. Figure Phenyllactate (g/g Creatinine) 50 .5 1 50 .5 0 1.00 100 Glycolate (mg/g Creatinine) (mg/g Glycolate 200 45 CHAPTER V

DISCUSSION

The urine collected from the phenylketonuric (PKU) and control patients was screened for abnormal urinary constituents. The pH, pro­ tein, and phenylpyruvate were of special importance. The first two, pH and protein, are known to influence the oxalate assay. Phenylpyruvate

(PPA), a precursor of phenyllactate (PLA) and O-hydroxyphenylacetate

(OHPAA), was used as an indicator for the presence of PLA and OHPAA.

The pH and protein were monitored to insure that these were not producing erratic oxalate values. The range of urinary pH values record: ed were found to be adequately buffered by this method to produce a final pH “ 4.7. This was the pH the author recommended; therefore, pH was not a problem. The other potential problem, protein, was found in greater than trace amounts in only two patients —— patients 41 and 48.

In these two specimens, the protein concentration was approximately

100 mg./lOO ml. which is about one half the concentration Bernstein and

Kahn (120) tested. These authors reported that protein concentrations of approximately 200 mg./lOO ml. had no effect on the oxalate assay; therefore, protein in our assay was not considered to be a problem. All the other Bili-Labstix (Ames Co., Elkhart, Ind.) tests were negative ex­ cept for the presence of glucose in patient 23 and blood in patient 48*

Neither of these constituents are known to interfere with any of the assay procedures. 46 47

The FeClg test for PPA was performed to Insure that the phenyl­ ketonuric patients were properly identified and were excreting elevated

levels of PKU metabolites and that control patients were not. The

FeClg test is a qualitative test for PPA; urines with >10 mg. PPA/100 ml.

give a positive test (117). Urinary concentrations of 50-100 mg./lOO ml. are commonly found in phenylketonuric infants (117); therefore, it was

surprising to find that 27 percent (6 of 22 PKU patients) of the urine

tested from PKU patients gave negative FeCl^ results. Negative FeCl^

results are observed in phenylketonuric patients who have very dilute

urines, low phenylpyruvic acid output, or low protein intake. Day-old urine from phenylketonuric patients may give negative test results be­

cause labile phenylpyruvic acid decomposes (119). Phenylpyruvate is,

therefore, labile but phenyllactate and O-hydroxyphenylacetate are

relatively stable’ (119). In 48-hr. urines, decomposition of phenyl­ pyruvate could occur while the phenyllactate and O-hydroxyphenylacetate are not affected. This apparently happened as the expected amounts of phenyllactate and O-hydroxyphenylacetate were found, but abnormally

low amounts of phenylpyruvate were found in phenylketonuric patients*

urine. This suggests decomposition of phenylpyruvate.

The amount of oxalate excreted from the phenylketonuric patients was not reduced when compared to the control patients or to the litera­

ture values for normal patients. The phenylketonuric patients excreted

39.2 mg./24 hr. (mean value) while the mean value for the control pa­

tients was 31.1 mg./24 hr.; the normal individual is expected to excrete

10 to 40 mg./24 hr. Decreased amounts of oxalate had been expected in

phenylketonuric patients because researchers have shown substantial reductions of oxalate in the presence of elevated amounts of phenyl­

lactate, a metabolite normally elevated in phenylketonuria (93). The

higher oxalate values from the phenylketonuric patients may be due to

increased oxalate formation by alternate pathways. This is not known

because there is no adequate basis for comparison between phenylketonuric

patients and phenylketonuric patients without abnormal amounts of phenyl­

lactate. All the factors contributing to the increased oxalate excretion

in the phenylketonuric population are not known; however, the important

fact is the phenyllactate-oxalate correlation rather than the absolute

values of the oxalate excreted from phenylketonuric patients.

Liao arid'Richardson (93) reduced urinary oxalate excretion by

approximately 52 percent in rats, by the administration of DL-phenyl-

lactate. These rats were force fed 300 mg. DL-phenyllactate every 4 hr.

for 24 hr. which is 6.95 g. DL-phenyllactate/kg. rat weight/24 hr. The phenylketonuric patients* maximum excretion of L-phenyllactate was

1.752-g./24 hr.; and assuming that this was a 70 kg. individual, this is

25.0 mg. L-phenyllactate/kg. human weight/24 hr. Assuming equal effects of phenyllactate per g. of human and rat body weights, the urinary

oxalate excretion in man would only be reduced approximately 0.187 percent. The source of the phenyllactate, however, is important. In

the rats, the DL-phenyllactate is force fed and must be absorbed before it can reach the site of endogenous oxalate synthesis. In man, however,

the L-phenyllactate Is synthesized in the liver which is also the site of endogenous oxalate synthesis. Even though the L-phenyllactate is

synthesized in the liver, the acid may be separated from the site of

endogenous oxalate synthesis. Despite the problems associated with 49 comparing the different sources of phenyllactate, the amount of L-phenyl- lactate produced by phenylketonuria patients is apparently inadequate in producing a dramatic decrease in endogenous oxalate synthesis in man.

Glycolic acid oxidase is known to catalyze the oxidation of gly­ colic acid to glyoxylic acid and the further oxidation of glyoxylic acid to oxalic acid (92). Liao and Richardson (93) have demonstrated that this oxidase is the major enzyme catalyzing the oxidation of glyoxylate

"in vitro"; therefore, glycolate oxidase is the primary enzyme in oxalate synthesis. The inhibition of this enzyme by the metabolite DL-phenyl­ lactate at nontoxic levels decreased the oxalate synthesis in perfused rat livers and in a limited number of rats fed ethylene glycol at LD 50 levels. The decrease in oxalate as phenyllactate increases in the phenyl­ ketonuric patients suggests that the L-phenyllactate is inhibiting the glycolic acid oxidase and, consequently, the oxalate synthesis is de­ creased. The negative correlation coefficient and its high level of significance (tr = 97.5) support this hypothesis. Since L-phenyllactate probably inhibits glycolic acid oxidase, glycolate and glyoxylate should also be affected. These metabolites should accumulate as the oxidation of glyoxylate and glycolate are both catalyzed by glycolic acid oxidase.

This enzyme, however, has a greater affinity for glycolic acid than it does for glyoxylic acid (92). The difference in affinity is probably important in normal individuals, but the depressed level of active gly­ colic acid oxidase has the effect of causing both glycolate and gly­ oxylate to accumulate. The "in vivo" mechanism of action of L-phenyl­ lactate confirms this effect on glycolate. The correlation between

L-phenyllactate and glycolate was positive and was statistically significant (t^ = 90.0). This increase in glycolate as L-phenyllactate increases suggests that the inhibition of glycolic acid oxidase causes a decrease in the catalytic oxidation of glycolate to glyoxylate and oxalate. In addition, glyoxylate may be reduced to glycolate due to the depression of active glycolic acid oxidase and the presence of active lactic dehydrogenase which is known to catalyze this reduction. Thus increased excretion of glycolate may be due to increased reduction of glyoxylate and/or oxidation of glycolate. These data confirm the feasi­ bility of regulating oxalate synthesis in man with specific nontoxic inhibitors of glycolic acid oxidase.

Cook and Henderson (101) described a pathway for the formation of oxalic acid from aromatic amino acids and implied that this pathway should be important in disease states such as phenylketonuria. These authors suggest that the aromatic a-keto analogs of tryptophan, tyro­ sine, and phenylalanine are intermediates in the pathway by which urinary oxalate is derived from the two terminal carbon atoms of the side chain of these amino acids in the intact animal. This pathway of oxalate formation was suggested to be important in humans with metabolic dis­ eases such as phenylketonuria where the levels of aromatic a-keto acids are known to be increased in the blood and urine. The data from this study does not support these authors* claim as the amount of oxalate excreted in the phenylketonuric and control group were not dramatically different. Mean values are 39.2 and 31.1 mg./24 hr. for the phenyl­ ketonuria and control groups respectively. Possibly more important was the occurrence of a highly significant negative correlation between oxalate and phenyllactate. This is exactly the reverse of what these 51 authors would have predicted. Therefore the formation of oxalate from aromatic amino acids does not appear to be physiologically important in humans.

The utilization of exogenous phenyllactate to control oxalate synthesis in humans has not been investigated. It Is known that phenyl­ lactate is a metabolite In normal humans, but in phenylketonuric pa­ tients it is abnormally elevated. The common neurological problems of mental deficiency occur only during the time the brain is developing.

Once the brain has completely developed, there are no known effects of the elevated metabolites in these patients. This suggests that phenyl­ lactate could be used in mature adults to control oxalate synthesis in conditions such as primary hyperoxaluria,' ethylene glycol poisoning, and kidney stone formation, in elevated concentrations, the normal metabolite, phenyllactate, demonstrates the potential for controlling oxalate formation by Inhibiting oxalate synthesis; however, the admini­ stration of 500 g. of phenyllactate per day to a patient is not reason­ able. Similar, but more potent, glycolic acid oxidase inhibitors would be a feasible means of treating recurrent kidney stones and of preventing the early demise of patients suffering from primary hyperoxaluria. CHAPTER VI

SUMMARY

A new approach to the problem of controlling oxalate synthesis in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone forma­ tion was investigated. DL-phenyllactate, which is elevated in phenyl­ ketonuric patients, inhibits an essential enzyme in oxalate biosynthesis.

This enzyme is glycolic acid oxidase. Studies correlating the produc­ tion of phenyllactate with oxalate, glyoxylate, and glycolate demon­ strated that increased phenyllactate concentrations were associated with decreased oxalate production. In addition, the oxalate precursors, glyoxylate and glycolate, increased as' phenyllactate increased. The glyoxylate increase with phenyllactate increase was not highly signifi­ cant statistically, but this may be due to the formation of stable urinary complexes with glyoxylate. The oxalate and glycolate correla­ tions with phenyllactate were highly significant statistically. This data supports the concept that Increased levels of phenyllactate depress oxalate biosynthesis and that similar, but more effective, inhibitors will be feasible therapeutic agents for primary hyperoxaluria, ethylene glycol poisoning, and recurrent kidney stones.

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