III.
Nutritional
and
Metabolic
Studies
in a Patient
Thomas H. Shepard, II, M.D., Edwin G. Krebs, M.D., Lou-sein W. Lee, M.S., and Mary Louise Johnson, Ph.D.
Departments of Pediatrics (T.H.S.), Biochemistry (E.G.K., L.W.L.) and Home Economics (M.L.J.), School of IIedicine and College of Arts and Sciences, University of Washington
(Accepted September 15, 1959; submitted May 18.)
This study was supported by the U.S.S. Bremerton Fund, Bremerton, Washington.
ADDRESS: (T.H.S.) Seattle 5, Washington.
1008
Psnwrmcs, June 1960
P
RIMARY hyperoxaluria is an inheriteddis-order characterized by the presence of
urinary excretion of oxalate in excess of 50
mg per day. This disease is usually
associ-ated with calcium oxalate nephrocabcinosis,
and available data suggest that inheritance
may be due to a recessive’ or a dominant2
gene. There is a lack of basic knowledge
concerning the normal metabolism of oxalic
acid. The absorption of dietary oxabate, its
detoxification, its endogenous source or
sources and its excretion are not fully
understood. Oxalic acid, if ingested in
amounts over 2 gm has been reported to be
poisonous,3 and evidence has been
pre-sented that increased nitrogen catabolism46
and ingestion of ascorbic acid7 may cause
an increased excretion of oxalate in the
urine.
In two preceding papers the clinical and pathologic findings8 and the genetic studies2
pertaining to the child under study have
been described. The purpose of this paper
is to report a number of metabolic studies
carried out in an attempt to detect the
source of the elevated excretion of oxalate
in the urine.
METHODS AND MATERIALS
Oxahic acid in the urine was determined by
a colonimetric method after chromatographic
separation of most of the inorganic phosphate
and other interfering substances.#{176} The values
reported are for oxalic acid. The lower range
of sensitivity by this method is so close to the
0 Details of this method will be published in a
separate communication.
concentration of oxalate in the serum that
the previously reported values for oxalate
in serum9 may be unreliable. The 24-hour
unines, most of which were collected at home,
were voided directly into the glass container
that was delivered to the laboratory. Because of
the intelligence and co-operation of the mother and patient the accuracy of collection was be-lieved to be greater at home than on a hospital ward. The determination of nitrogens in urine
was done by a micro-Kjeldahl method using
direct Nesslenization.
The mother was trained by an experienced nutritionist to measure and record the patient’s
food intake. The dietary values for protein,
fat and carbohydrate were estimated from
tables.boui The values for oxalic acid were
cal-culated from the data summarized and
tabu-lated by Jeghens and Murphy.3
RESULTS
Effect of Reduced Intake of Oxalate
To observe the effect on urinary excretion of oxalate the patient was given a diet low
in oxalate, containing 50 mg or less of esti-mated oxalate per day. The results are re-corded in Table I and illustrated in Fig. 1,
and do not show any change. During the
preceding 5-day control period the dietary intake of oxalate was estimated to be 50 to 150 mg/day. A similar experiment carried
out for 7 days also revealed no change in
excretion of oxalate in the urine.
Effect of Carbohydrate and Protein
The effect of dietary carbohydrate and
pro-TABLE I
EFFECT OF DIET LOW IN OXALATE ON
OXALATE EXCRETION (May, 1957) C.T.E. 300 #{182}200 .-J 100
DAYS 12345 6 7 8 9 10 1112 13 1415 1617 18 19 2021
50 MGM. OXALATE DIET
FIG. 1. Urinary excretion of oxalate before and
after initiation of diet low in oxalate.
TABLE II Day of Period Volume (ml) Oxalate (mg/100 ml) Total Oxalate (mg) Total Nitrogen (gm) Total Calcium (mg) 1 920 2 1,140 3 875 4 840 5 810 6 875 7 695 8 798 9 720 10 1,523 11 1,275 12 1,525 13 990 14 1,525 15 1,070 16 1,480 17 1,780 12.5 11.8 14.4 13.6 25.2 14.9 12.8 15.0 15.2 15.2 14.3 14.3 17.0 15.6 15.5 18.5 6.6 Normal
Low protein, high carbohydrate
High protein, low carbohydrate
Urine
Day
-___________
of Total Total Diet
Period Volume Oxal ate (ml) (mg)
I 1,360 148)
3 ‘2,060 218k Normal
5 2,100 223
6 -
-8 1,960 251
11 1 980 273 Low oxalate
17 ‘2,280 238
19 2,120 226
21 ‘2,295 250
tein, high in carbohydrate for a 7-day
period and then abruptly reversing the diet
to one high in protein and low in
carbo-hydrate. During the low protein, high
carbohydrate diet he received an estimated
4 gm of nitrogen and 231 gm of
carbo-hydrate, and on the second diet an
esti-mated 9.5 gm of nitrogen and 95 gm of
carbohydrate. The results are tabulated in
Table II and shown graphically in Fig. 2.
The daily urinary excretion of calcium is
also shown.
The concentration of oxalate in the urine
did not change but the urine volume and
total output of oxalate increased during the
high protein, low carbohydrate period. The
blood urea nitrogen was 17 mg/100 ml at
EFFECT OF DIETARY PROTEIN AND CARBOHYDRATE ON OXALATE AND NITROGEN EXCRETION IN UIuNE
(March, 1956)
115 2.9 1.2
135 2.8 0.9
126 - 2.2
114 - 28.6
204 2.3 28.2
130 1.4 24.3
89 - 0.0
120 1.3 8.9
109 - 14.0
232 - 0.0
188 - 7.2
218 - 18.8
168 - 22.2
238 - 8.6
166 4.9 29.0
200 - 86.0
114 - 88.7
URINE
OXALATE
TOTAL
MGM.
C .T. E.
r30 URINE
g
CALCIUM0
Fic. 2. The effect of a high carbohydrate-low protein diet, and a low carbo-hydrate-high protein diet on urinary excretion of oxalate. Urinary excretion
of calcium per 24 hours is also shown.
the beginning and 13 mg/100 ml at the
end of the low protein, high carbohydrate period; at the end of the diet containing
high protein it was 42 mg/100 ml. The
fasting blood sugar, and concentrations of
calcium and phosphorus in the blood did
not change appreciably. There was no
sig-nificant change in body weight during these
periods. Later, during one of the surgical
procedures, the patient was maintained on
fluids administered parenterally and
re-ceived only 10% glucose as a source of
calories deficient in carbohydrate for 3 days.
During these 3 days the 24-hour urine was
collected by means of a catheter in the
bladder and the total content of oxalate
was 121, 156 and 109 mg/24 hours. The
total content of nitrogen in these 3
speci-mens was 6.1, 7.1 and 4.8 gm and the
oxalate content expressed as percent of
nitrogen was 2.0, 2.2 and 2.2 respectively.
Effect of Glycine
During another period the patient
in-gested 10 gm of glycine per day in addition to his regular diet. The resulting values are
shown in Table HI. On the 4th day after
discontinuance of the glycine, he developed frequency of urination, and two calculi in
the bladder were seen by roentgenogram.
This was the first definitive evidence of
calculi in the urinary tract. The day before the symptoms appeared the ratio of oxalate to nitrogen decreased (Table III). Although exact urine volumes during this period are
unavailable there was no large change
noted.
Effect of Cortisone
Cortisone (100 mg/day administered
in-tramuscularby) did not change the ratio of nitrogen to oxalate (Table IV). In Figure 3 the 24-hour content of oxalate is plotted
against the nitrogen content (ratio = + .66).
The patient gained 1.9 kg of weight during
this 7-day period; there were no untoward
symptoms.
Effect of Sodium Benzoate
It was believed that the administration of sodium benzoate to the patient was un-wise in view of the impending renal failure. The mother, who also had elevated urinary excretion of oxalate, volunteered to take 20
gm of sodium benzoate daily in four
di-vided doses; the results are shown in
* Normal diet given throughout tile study period.
TABLE III
EFFECT OF ADDITIONAL GLYCINE ON OXALATE AND NITROGEN EXCRETION IN URINE
(February, 1958)
,. Oxalate
. olume Total Oxalate Total A ltrogen
Day of Period I \ I \ Comments
1fl1) mgj gm NX1,000
1 1,560 249 6.8 3.7
i 1,800 262 8.4 3.1
3 1,260 223 6.9 3.2
4 1,800 316 8.5 3.7 Glycine 10 gm orally, daily
5 2,280 270 .1 3.3!
6 1,830 232 5.8 4.0J
7 1,980 346 8.1 4.3 Glycine stopped
8 1,830 199 6.0 3.3
9 - 219 10.1 2.2
10 - 219 10.1 2.2
11 - - - - Bladder stone symptoms
12 - 84.5 2.6 3.3
13 - - -
-14 - 243 8.7 2.8
* Normal diet given throughout the study perioi
of sodium benzoate was slightly lower than limits as studied by high voltage paper
in the control period, the decrease being electrophoresis. Allantoin in the urine was
of questionable significance (t ± 1.9, measured13 and values of 2 and 7 mg/b were
D.F., 6, P < 0.05). found as compared to a control value of
7.1 mg/i.
Pattern of Nitrogen Excretion
The excretion of nitrogen in the urine Effect of Ascorbic Acid
was studied by determining its individual The effect of normal dietary intake of
components (Table VI); the amino acid ascorbic acid was evaluated by comparing
pattern was found to be within normal the estimated daily intake with the urinary
TABLE IV
EFFECT OF CORTISONE ON OXALATE AND NITROGEN EXCRETION IN URINE
(February, 1958)
. Volume Total Oxalate Total Nitrogen OxalCie
Day of Period In
r
I tmg1 \ Igm1 \ .VXJ,000 Comments*1 2,880 430 13.9 3.1
2 1,440 180 5.3 3.4
3 1,800 173 6.3 2.8
4 1,440 137 5.2 2.6
5 1,800 216 8.1 2.7
6 1,200 182 6.2 2.9
7 1,680 186 7.5 2.5 Cortisone 100 mg
intra-S I,680 224 10 .I 2.2 muscularly, daily
9 2,160 324 10.0 3.2
400
300
200
I00
2
Oo
0
0
0
0
---‘---,---T--- 10 2 4
0
FIG. .3. The relationship between individual
de-terminations of oxalate and nitrogen in urine.
excretion of oxalate. No correlation was
noted. The day after a control 4-hour
cob-lection of urine the subject and a “control”
patient recovering from pneumonia were
given 100 mg of ascorbic acid intravenously and the urine collected during the
subse-TABLE V
* Normal (liet given throughout the study period.
quent 4-hour period. The results are shown
0 in Table VII. Both patients had not
re-ceived supplementary ascorbic acid during
the preceding 2 weeks. Of the 100 mg of
ascorbic acid administered, 15.5% and 7.1%
(estimated by subtracting the control values
from the test values) were recovered from
the urine of the patient and the control,
re-spectively. According to Ralli et this is
a low but not scorbutic response. The
in-dex of Kajdi et al.b6 was 112 for the patient
and 44 for the control (normal 10). At a
later time (April, 1958) the Iatiellt was given 3 gm of ascorbic acid daily (Table
VIII) and no untoward results were
oh-served during a 6-day period. Unfortunately only two 24-hour collections of urine were made while receiving ascorbic acid.
N gms/24 hrs
Effects of Riboflavin and Magnesium
Two additional dietary studies were
car-ned out, one with riboflavin and one with magnesium sulfate; the results are reported
in Table VIII. The determinations of oxa-late during these two periods were carried
out by another method.17 The only effect
observed when 4 gm of riboflavin was given was that the urine and stool turned yellow.
EFFE-F OF SoDIUM BENZOATE ON OXALATE AND NITROGEN EXCRETION IN URINE OF PATIENT’S MOTHER
Oxal ale
Day of Period Volume
(ml)
Total Oxalat (mg)
Total Nitrogen
(gm) NX 1,000
Comments*
1 1,080 105 6.4 1.6
2 1,620 185 10.1 1.8
3 2,280 101 6.4 1.6
4 2,040 35 3.6 1.0
5 2,460 138 8.2 1.7
6 2,401) 79 7.7 1.0
7
8
2,400
2,040
48
71
6.3
5.7
.8
.3 Sodium benzoate 20 gm
9 2,520 163 8.1 2.0 orally, daily
10 2,820 111 8.5 1.3
11 12 13
2,160
2,400
2,880
300
162
144
5.7
6.4
7.4
5.3
2.5
2.0)
14 2,400 90 5.8 1.6
15 2,520 79 8.4 .9
‘FABLE VI
P%ITEIIN (-O’ NITu0GFN EXCRETION IN URINE
$ub.stan(e Jatie,tt .‘Vormal11 (ni1rogen
(%
of total JV) (% of total V)I rca
(‘reat liii IIC (‘rcitirie
Uric A(i(I
IIIIfl1OIii;L
Recoveretl
92.4
5.3
.05
85 4.5
1.5 2.0
of foods high in oxalate content (rhubarb
and sorrel) has been reviewed critically by
J
eghers and Murphy.3 The symptoms arenot unlike “ptomaine poisoning” but may
be associated with hematuria.
The absorption of oxalate depends largely
on the cations present. The calcium salt is
remarkably insoluble and poorly absorbed,
whereas the sodium salt is soluble and
I.3 4.5 readily absorbed. Archer et al.18
demon-1OO..))
-
strated increases in the urine of normalsub-jects as high as 250 mg/day when sodium
oxalate was administered in amTlOunts up to
4 gui per day. They estimated that 2 to 5%
was recovered in the urine. However, when
the calcium salt of oxalate was given no rise in urinary excretion of oxalate occurred.
During a 2-week period, while receiving a diet containing less than 50 mg of
oxa-late per day the patient continued to
cx-crete larger amounts in the urine than were
in the diet. This suggests but does not prove
that exogenous sources of oxalate are
un-important. Other have been
tin-successful in reducing the output of oxa-late in hyperoxaluria by using diets low in
oxalate.
Is the absorption of oxalate facilitated in
the patient with hyperoxaluria? Archer et
418 have fed patients calcium and
so-dium oxalate and observed the same
in-TABLE VII
EFFECT OF ASCORBIC ACID ON OXALATE EXCRETION Niagnesium citrate, 2.5 gm fotmr times daily,
(li(l miot change the urinary excretion of
oxa-late. The findings illustrate the fluctuation
in daily Outl)ut of oxalate and the need for
a sufficient numTIl)er of observations before
the expenimnental study.
DISCUSSION
Dietary Source and Absorption of Oxalate
In the patient there was no evidence of
increased quantities of oxalate in the diet;
the estimated daily intake (50 to 150 mg)
was the approximate amount expected for a
boy of his age. Oxalate poisoning may
oc-cur from accidental ingestion3 or contact
with oxalic acid crystals.14 The range of
lethal dose varies from 2 to 30 gm.3 The
evidence that acute and chronic oxalate
poisoning may occur as a result of ingestion
Date
.
Time Urine loliinie (ml)
Ascor
--bic Acid
-- Total Oxalate
(mg) Total ( ri ne Blood
(mg) (mg/100 ml)
Subject
-8-I2AM
11-25-57 470 23.5 48
(control day)
11-26-57 8AM 1.4
(test day)
11-26-57 8-12 AM ‘3’2() 39 2.2 (Il AM) 23
Control Patient
11-25-57 8-12AM 57 4.2
(control (lay)
11-26-57 8AM 1.1
(test day)
Day of
Period
EFFE-r OF ASCORBIC ACID, RIBOFLAVIN AND MAGNESIUM ON OXALATE AND NITn0GEN
Volume Total Oxalate
(ml) (mg)
4 8
April 1958
115
86
131
80
AscorI)i( 1(i(I 3 gIll urn IIv
1 1,335 120
2 805 169
3 1,706 272
4 1,660 215
5 1,140 171
6 1,130 124
7 1,670 103
8 1,590 133
9 1,290 108
Riboflavin 4 GIll OrilIly, daily
April 1956
1 1,110 70
2 1,230 18
3 1,205 20
4 1,120 125
5 1,140 171
6
7
1,085
1,150
65
102
Magnesium citrate 100 gin orally, (lady
8 965 86
9 915 67
TABLE VIII
EXCRETION IN THE URINE
Oxal ate TotaIN
NX1,000
(am nu.n!s
1 1,920
2 1,860
3
2,100 I,560
4.0 2.9
3.1 2.8
4.9 2.7
3.6 2.2
June 1956
* Normal diet given throughout the study period.
crease in urinary excretion of oxalate as
was observed in normal control adults.
Amino Acid and Other Nitrogenous Sources
of Oxalate
The extensive and interesting studies by Archer et a!. of patients with primary hy-peroxaluria have led them to the
hypothe-sis that a defect in glycine-glyoxylate
me-tabolism leads to excessive accumulation of oxalate. Subsequent to the demonstration
that high protein diets and administration
Of glycine caused a rise in urinary
excre-tion of oxalate, they administered sodium
benzoate, which depletes the glycine pool
through the formation of hippuric acid,
and showed that the urinary oxalate
dropped. Scowen et 20 later administered
glycine-1-C’3 to the same patients and
re-covered C13 oxalate in the urine. It was
not possible to test their findings in the
present patient but comparable amounts of
sodium benzoate were administered to the
mother who also had hyperoxaluria.
Un-fortunately the results were not definitive. The patient received glycine (10 gm/day for 5 days) and showed no increase in un-nary excretion of oxalate although he did exhibit stones in the urinary bladder for the first time 4 days after receiving this rela-tively small dose of glycine. Archer et al. administered 50 gm of glycine per day and showed a probable increase in the urinary
con-firmed their findings. It is of interest in the present patient that one of the lowest ratios
of oxalate to nitrogen occurred on the day
preceding the appearance of bladder stones.
This serves to emphasize the fallacy of
as-sliming a decrease in the urine oxalate is
equivalent to decreased production of
oxa-late. It is probable that this decrease in the
ratio was due to deposition of calcium
oxa-late in the stones.
lated to formation of oxalate are outlined
in Figure 4. Only enzyme systems for which there is evidence in mammalian tissues are
shown. No evidence for oxidation of oxalic
acid has been obtained in mammals;21
therefore, no pathway involving destruc-tion of oxalate, is depicted, and the
sub-stance is viewed as being metabolically in-ert. Two enzymes are depicted as being in-volved in the conversion of glycine to
gly-GLYCINE
FORMATE+C02
VITAMIN
C
t
I#{188}, E6
E7
GLYCINE
GLYOXYLATEE5OXALATE
1’b
,/v E4
ETHANOLAMINE
I
‘I’
PENTOSE
METABOLISM
SERINE
Fic. 4. Certain metabolic pathways related to metabolism of oxalate.
A previously undescribed finding in
pri-mary hyperoxaluria is the constant
relation-ship between nitrogen and oxalate in the
urine. The content of oxalate varied
be-tween 2 to 4% of the nitrogen during very
wide fluctuations in the content of nitrogen.
When the content of carbohydrate in the
diet was high and the nitrogen content low,
the highest ratio of oxalate to nitrogen was
present. The total output of oxalic acid
during this time was unchanged. This
ob-servation might suggest that the oxalate
stilts from catabolism of endogenous
pro-teins rather than the metabolism of
exoge-nous proteins or that a portion comes from
carbohydrate. However, during periods
when increased protein catabolism might
be expected (high doses of cortisone and
during a period postoperatively) the patient
did not have an increased amount of
oxa-late in the urine.
Related Enzyme Pathways
As many inheritable metabolic diseases
have been shown to be due to a simple
en-zyme defect, the authors have attempted
to approach this problem with this in mind.
The various pathways of metabolism
re-oxylate. One of these (E1) is the glycine
oxidase of Ratner et al.22 which may be of
questionable physiologic significance.21 The
other enzyme (E2) is a transaminase that
has not been characterized exactly as to
specificity, but which is known to exist in
liver.24’25 Glyoxylate may arise from ethanol-amine (E3).26 In addition, it is probable
that glyoxylate may be derived from
car-bohydrates through pentose metabolism
(E4); no attempt will be made to review the literature involving this pathway.
Glyoxy-late is readily oxidized to oxalate (En),
espe-cially under conditions where the
concen-tration of glyoxylate becomes relatively
high.23 An alternate pathway (E) for
gly-oxylate utilization is its oxidation to
for-mate and carbon dioxide. Vitamin C has
also been shown to be a precursor of
oxa-late in mammals, but would seem to be an
unlikely source in oxabosis.
It would appear that glyoxylate occupies a key position in the formation of oxalate.
Its accelerated formation by any of the
pathways shown, or by other pathways that
may exist, could conceivably account for
in the conversion of glyoxylate to glycine
(
E3) or in its oxidation to formate and car-bon dioxide (E6). With tissues obtained atnecropsy from the patient in the present
study, preliminary experiments have shown
that the enzymic transamination of
glyoxy-late (Efl) occurs at a normal rate. In these
tests a combination of glutamine, glutamic
acid, asparagine and aspartic acid was used
as a source of the amine group. A block in
the conversion of glyoxylate to formate and carbon dioxide (E1) remains open as a pos-sible explanation for the defect in this type
of oxalosis.
No allowance has been made in this
dis-cussion for the single additional laboratory
finding of probable significance
metaboli-cally in the patient, i.e. the elevated
con-centration of uric acid in the blood.8 In
lower species it is known that oxalic acid
can arise from a breakdown of uric acid,
but this has not been reported for man. A
portion of the uric acid found in man
ap-pears to be degraded, however, by
unde-fined pathways.27
Attempts at Therapy
The administration of riboflavin had no
effect on urine oxalate.
Because of Hammarsten’s28 extensive
cx-perience with magnesium in the prevention
of oxalate stones in rats, a therapeutic
at-tempt was made using magnesium, also
un-successful; however, this period was
proba-bly too short to allow any conclusions to be
drawn.
Lamden and Chrystowski have shown
that radioactive ascorbic acid is excreted as
oxalate in guinea pigs and that adminis-tration of over 5 gm of ascorbic acid can increase the urinary excretion of oxalate in
man. The results of the ascorbic acid test intravenously are interpreted as being nor-mal and ascorbic acid, 3 gm daily added to
the diet, did not appreciably change the
excretion of oxalate in the present patient.
Treatment for other patients with
pri-mary hyperoxaluria, if a
similar
relation-ship between nitrogen and oxalate in the
urine can be demonstrated, should be
di-rected along further investigation of the
effects of diets low in nitrogen. The daily
intake of nitrogen in the diet should not be excessive. It would be of interest, in
pa-tients with oxalate stones and normal
amounts of oxalate in the urine, to test the effect of nitrogen loading. The initiation of
stone formation might be associated with
transient hyperoxaluria after intakes high
in protein.
SUMMARY
Dietary and metabolic studies carried out on a patient with primary hyperoxaluria are reported. There was no appreciable change in the urinary excretion of oxalate when he was given a diet low in oxalate, or ascorbic acid, magnesium or riboflavin. A diet high in protein and low in carbohydrate was as-sociated with increased excretion of oxalate, whereas increased catabolism of protein
in-duced by cortisone and the postoperative
state did not appreciably change the
un-nary excretion of oxalate.
Evidence that the source of increased
accumulation of oxalate might be related to the glycine-glyoxylate-oxalate pathway is the observation that the increased
un-nary excretion of oxalate in the mother was
slightly depressed by the administration of
sodium benzoate. Glycine (10 gm) did not
cause an appreciable increase in oxalate in the urine but may have been associated with formation of bladder stones in the pa-tient.
The ratio of oxalate to nitrogen in the urine remained relatively constant during wide fluctuations in excretion of nitrogen.
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