होम American Journal of Kidney Diseases Oxalate absorption and endogenous oxalate synthesis from ascorbate in calcium oxalate stone formers...

Oxalate absorption and endogenous oxalate synthesis from ascorbate in calcium oxalate stone formers and non-stone formers

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खंड:
44
साल:
2004
भाषा:
english
पृष्ठ:
10
DOI:
10.1053/j.ajkd.2004.08.028
फ़ाइल:
PDF, 125 KB
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आप पुस्तक समीक्षा लिख सकते हैं और अपना अनुभव साझा कर सकते हैं. पढ़ूी हुई पुस्तकों के बारे में आपकी राय जानने में अन्य पाठकों को दिलचस्पी होगी. भले ही आपको किताब पसंद हो या न हो, अगर आप इसके बारे में ईमानदारी से और विस्तार से बताएँगे, तो लोग अपने लिए नई रुचिकर पुस्तकें खोज पाएँगे.
Oxalate Absorption and Endogenous Oxalate Synthesis
From Ascorbate in Calcium Oxalate Stone Formers and
Non–Stone Formers
Weiwen Chai, MS, Michael Liebman, PhD, Susan Kynast-Gales, PhD, and Linda Massey, PhD
● Background: Increased rates of either oxalate absorption or endogenous oxalate synthesis can contribute to
hyperoxaluria, a primary risk factor for the formation of calcium oxalate– containing kidney stones. This study
involves a comparative assessment of oxalate absorption and endogenous oxalate synthesis in subpopulations of
stone formers (SFs) and non–stone formers (NSFs) and an assessment of the effect of ascorbate supplementation
on oxalate absorption and endogenous oxalate synthesis. Methods: Twenty-nine individuals with a history of
calcium oxalate kidney stones (19 men, 10 women) and 19 age-matched NSFs (8 men, 11 women) participated in two
6-day controlled feeding experimental periods: ascorbate-supplement (2 g/d) and no-supplement treatments. An
oxalate load consisting of 118 mg of unlabeled oxalate and 18 mg of 13C2-oxalic acid was administered the morning
of day 6 of each experimental period. Results: Mean 13C2-oxalic acid absorption averaged across the ascorbate and
no-supplement treatments was significantly greater in SFs (9.9%) than NSFs (8.0%). SFs also had significantly
greater 24-hour post– oxalate load urinary total oxalate and endogenous oxalate levels with both treatments.
Twenty-four– hour urinary total oxalate level correlated strongly with both 13C2-oxalic acid absorption (SFs, r ⴝ 0.76;
P < 0.01; NSFs, r ⴝ 0.62; P < 0.01) and endogenous oxalate synthesis (SFs, r ⴝ 0.95; P < 0.01; NSFs, r ⴝ 0.92; P <
0.01). Conclusion: SFs are characterized by greater rates of both oxalate absorption and endogenous oxalate
synthesis, and both these factors contribute to the hyperoxaluric state. The finding that ascorbate supplementation
increased urinary total and endogenous oxalate levels suggested that this practice is a risk factor for individuals
predisposed to kidney stones. Am;  J Kidney Dis 44:1060-1069.
© 2004 by the National Kidney Foundation, Inc.
INDEX WORDS: Kidney stones; oxalate absorption; endogenous oxalate; ascorbic acid.

A

PPROXIMATELY 75% of all kidney stones
are composed primarily of calcium oxalate.1 Urinary oxalate, which originates from a
combination of absorbed dietary oxalate and
endogenous synthesis from such oxalate precursors as ascorbic acid and glyoxylate, is a primary
determinant of calcium oxalate saturation.2
Changes in urinary oxalate levels have a greater
impact on the risk for calcium oxalate stone
formation than proportional changes in urinary
calcium levels.3 The amount of urinary oxalate
generally is greater in populations of stone formers (SFs) than non–stone formers (NSFs).4

From the Department of Food Science and Human Nutrition, Washington State University, Spokane, WA; and Department of Family and Consumer Sciences (Nutrition), University of Wyoming, Laramie, WY.
Received June 28, 2004; accepted in revised form August
16, 2004.
Supported in part by the Attorney General of Washington,
Vitamins Settlement Fund.
Address reprint requests to Michael Liebman, PhD, Department of Family and Consumer Sciences (Nutrition),
Dept 3354, 1000 E University Ave, University of Wyoming,
Laramie, WY 82071. E-mail: liebman@uwyo.edu
© 2004 by the National Kidney Foundation, Inc.
0272-6386/04/4406-0012$30.00/0
doi:10.1053/j.ajkd.2004.08.028
1060

Increased endogenous production of oxalate
and increased oxalate intake or absorption are
the major causes of hyperoxaluria.5 Although the
majority of urinary oxalate has been suggested to
be of endogenous origin,2 intestinal absorption
of dietary oxalate also may have an important
role in patients with calcium oxalate–containing
kidney stones. Results from previous studies
using either carbon 14–labeled oxalate or 13C2oxalate to compare oxalate absorption between
stone-forming and non–stone-forming subpopulations are equivocal.6-12 Some studies found
increased oxalate absorption in SFs,6-9 whereas
others reported no differences between the 2
subpopulations.10,11 One study reported that SFs
absorbed less labeled oxalate than NSFs.12 With
respect to endogenous oxalate production, there
are few published studies that directly compared
stone-forming and non–stone-forming subpopulations.
The question of whether high-dose ascorbic
acid ingestion is a risk factor for kidney stone
formation is important in light of the high prevalence of kidney stones and extensive use of
ascorbate supplementation in the United States.
The potential effect of ascorbate supplementation on endogenous oxalate levels and kidney
stone risk is uncertain at the present time. Studies

American Journal of Kidney Diseases, Vol 44, No 6 (December), 2004: pp 1060-1069

OXALATE ABSORPTION AND ENDOGENOUS SYNTHESIS

of NSFs are equivocal in that ascorbate-induced
increases in urinary oxalate levels have been
reported in some studies,13-16 but not others.17-19
Studies that addressed this issue in SFs consistently showed that the ingestion of high-dose
ascorbate supplements significantly increases urinary oxalate levels.15,16,19,20
The primary objective of the present study is
to assess the effect of ascorbate supplementation
on kidney stone risk in subpopulations of SFs
and NSFs. However, the experimental design
also allowed a comparative assessment of oxalate absorption and endogenous oxalate synthesis in these 2 subpopulations, which is presented
in this report.
METHODS

Subjects
The study was approved by the Human Subjects Review
Committee at Washington State University (Spokane, WA),
and written informed consent was obtained from all participants.
Twenty-nine individuals with a history of calcium oxalate
kidney stones (19 men, 10 women) and 19 age-matched
NSFs (8 men, 11 women) participated in this study. SFs were
recruited from a pool of subjects who previously had participated in similar studies at Washington State University and
responders to advertisements published in local newspapers.
Selection criteria included a self-reported positive history of
calcium-containing kidney stones, age of at least 18 years,
absence of complicating medical conditions or medication
use, no use of calcium supplements greater than 100 mg/d,
and no extreme levels of physical activity. Specific medical
or dietary conditions that precluded participation were kidney disease, urinary tract infection, uncontrolled high blood
pressure, uncontrolled diabetes, diverticulitis or diverticulosis, colitis, irritable bowel syndrome, diarrhea or constipation, recent bed rest, thyroid disease, parathyroid disease,
osteoporosis, broken bones, gout, lactose intolerance, milk
allergy, and special dietary concerns. Subjects were not
screened for a past history of antibiotic treatment. Qualified
volunteers who were taking daily prescription medications
that do not affect calcium or oxalate metabolism were
allowed to continue taking these medications at identical
doses during the 2 experimental periods. To increase confidence that medication use would not affect study results,
only individuals who had been taking the same dose of
medication for at least 3 months were included in the study.
Habitual daily multiple vitamin-mineral use was allowed to
continue if total intake from supplements was no more than
100 mg of calcium, and all other nutrient levels were no
greater than 100% of the recommended dietary allowances.
Other nutrient supplement or medication use, including
aspirin, antacids, or laxatives, was prohibited during the
study period.
The 19 control subjects were recruited from spouses,
relatives, and friends of the subpopulation of SFs. These

1061

individuals met all qualifying conditions of SFs, with the
exception of never having formed a kidney stone.
The age of SFs ranged from 25 to 76 years, with a mean of
49.8 years. For NSFs, age ranged from 23 to 75 years, with a
mean of 50.8 years. For SFs, mean body weights and body
mass indices were 89.0 ⫾ 18.9 (SD) kg and 29.5 ⫾ 5.5
kg/m2 in men and 88.4 ⫾ 14.6 kg and 32.5 ⫾ 5.7 kg/m2 in
women. For NSFs, the corresponding values were 84.7 ⫾
15.0 kg and 30.9 ⫾ 8.1 kg/m2 in men and 75.1 ⫾ 16.1 kg and
28.9 ⫾ 5.5 kg/m2 in women.

Experimental Design
Each subject participated in two 6-day experimental periods. Because a primary objective is to assess the effect of
ascorbate supplementation on endogenous oxalate synthesis,
a randomized crossover design of 2 g of ascorbate supplement versus no supplement was used. For the 6-day ascorbate treatment period, subjects consumed a 1-g ascorbate
supplement with the morning meal during the 5 adaptation
days and at 7:00 AM with 175 g of applesauce during the
oxalate load day; the other 1-g ascorbate supplement was
taken with the evening meal at approximately 6:00 PM. The
other week, no supplement was given.
Each experimental period was divided into a 5-day freeliving adaptation period, followed by a 1-day metabolic-unit
study. Monday and Tuesday of each experimental period,
subjects consumed self-selected diets, but were asked to
avoid 10 of the foods containing the highest amounts of
oxalate (spinach, rhubarb, beets, beans, wheat or soy bran,
nonherbal teas, nuts, berries, chocolate, and soy products).
Diet records were kept by all subjects during this period.
Wednesday, Thursday, and Friday, participants consumed
only foods provided by the investigators, thereby ensuring
that both a known and limited amount of oxalate was
consumed. Participants completed 24-hour urine collections
during these 3 days, as well as the preceding Monday and
Tuesday. One hundred milliliters of 3 N of hydrochloric acid
was added to urine collection containers as a preservative.
Urinary ascorbate levels during this period were used as a
compliance check for the ascorbate-supplementation and
no-supplement treatments. Urinary oxalate for the 3 days
immediately preceding the 1-day metabolic unit study was
assumed to be primarily of endogenous origin because of the
low dietary oxalate content of foods (⬃40 mg/d) provided
by the investigators during this period.
Saturday, immediately after the 5 adaptation weekdays of
each experimental week, participants lived in the Human
Metabolic Unit on the Washington State University campus
so that both food consumption and urine collection could be
controlled. Diets were identical between the two 1-day
metabolic-unit periods because they included the same foods
purchased from the same lots or suppliers. All foods were
given in weighed portions on metabolic-unit days, and
subjects were required to consume the entire portions provided.
Participants fasted for a minimum of 12 hours before arriving at the Human Metabolic Unit Saturday morning. After
completing Friday’s 24-hour urine collection at 7:00 AM, subjects consumed an oxalate load administered in capsule form.
Capsules, consumed with a three-quarter cup serving of applesauce, contained 136 mg of oxalic acid, of which 18 mg was

1062

provided as the stable isotope 13C2-oxalic acid (Cambridge
Isotope Laboratories, Andover, MA). All subjects were administered the same dose of oxalic acid. The first of 7 timed urine
collections was performed 2 hours after ingestion of the oxalate
load and followed by a low-oxalate breakfast consisting of a
bagel, grape jam, cream cheese, yogurt, and apple juice. All
metabolic-unit meals were low in oxalate, providing a total of
7.0 to 8.0 mg, and met dietary nutritional recommendations.
Metabolic-unit diets were designed to contain moderate amounts
of calcium, approximately 800 mg/d, divided among the 3
meals. Deionized water was consumed ad libitum during the
metabolic stay, with the goal of each subject achieving a total
urine volume of 2 to 2.5 L/24-h period. The 24-hour controlled
urine collection period was divided into four 2-hour collections
(designated S-2 to S-8, with the number corresponding to the
number of hours after oxalate ingestion), followed by two
3-hour collections (designated S-11 and S-14), followed by an
overnight 10-hour collection period (designated S-24).

Sample Preparation and Analyses
In the metabolic unit, urine collection procedures were
standardized to prevent losses of oxalate by precipitation
and spontaneous conversion of ascorbate to oxalate. Urine
was pooled immediately in graduated cylinders containing 3
N of hydrochloric acid (20 mL/collection period) to maintain urine pH at less than 2.0. At the end of each timed
collection period, urine volumes were recorded and 10-mL
aliquots were filtered through no. 2 Whatman paper, then
frozen at ⫺20°C for subsequent analysis.
Urine samples and all plant foods from the meals provided in the metabolic unit were analyzed for oxalate using
an oxalate kit (based on the oxalate oxidase method) purchased from Trinity Biotech (Jamestown, NY). The acid
extraction method described by Ohkawa21 was used to
extract oxalate from the food samples before oxalate analysis. Labeled oxalate (13C2-oxalic acid) was analyzed in the
laboratories of Metabolic Solutions Inc (Merrimack, NH)
using a gas chromatograph-mass spectrometer after conversion of oxalate to its dimethyl ester. Urinary creatinine was
analyzed using the picric acid method.22 Urinary ascorbate
was analyzed by means of the 2,4-dinitrophenylhydrazine
method.23
Oxalate absorption was assessed using 2 methods: (1) recovery of administered 13C2-oxalic acid (direct method), and (2)
determination of the total amount of urinary oxalate in samples
collected after oxalate ingestion, with correction made for
endogenous urinary oxalate (indirect method). With the latter
method, the increment in oxalate excretion during the 24-hour
post–oxalate load period was determined by subtracting the
mean of three 24-hour preload (Wednesday to Friday) basal
values from the total 24-hour postload urinary oxalate level.
The utility of using urinary oxalate as an indicator of oxalate
absorption and synthesis is based on the finding that little
oxalate catabolism occurs after absorption and more than 90%
of an injected dose of radioisotope-labeled oxalate can be
recovered in urine within 24 to 36 hours.24
The 24-hour postload endogenous oxalate level was approximated by subtracting the computed oxalate absorbed
from the oxalate load ([recovered 13C2-oxalic acid/administered 13C2-oxalic acid] ⫻ total oxalate ingested) from total

CHAI ET AL

urinary oxalate excreted during 24 hours after oxalate ingestion.

Statistical Analysis
Differences in oxalate absorption, postload 24-hour total
urinary oxalate excretion and endogenous oxalate level, and
13
C2-oxalic acid excretion between the 2 subpopulations
(SFs and NSFs), as well as between the ascorbate-supplement and no-supplement treatments, were tested by means
of a 2 ⫻ 2 factorial analysis of variance. When this analysis
indicated a subpopulation or treatment effect, differences
between subpopulations for each treatment or differences
between treatments for each subpopulation were assessed
further by using either 2-group t-tests or paired t-tests.
Two-group t-tests also were used to test for sex differences
within each subpopulation (SFs and NSFs), as well as for
subpopulation differences within each sex group. Chisquared tests were used to test for differences in the distribution of 13C2-oxalic acid absorption levels between the subpopulations of SFs and NSFs. Pearson’s correlation
coefficients (r) were computed to assess the strength of
associations of post–oxalate load total urinary oxalate levels
with both endogenous oxalate and oxalate absorption and
the strength of the association between the mean of the 3
(Wednesday, Thursday, and Friday) preload 24-hour total
urinary oxalate levels and postload total urinary oxalate
levels. Pearson’s correlation coefficients also were computed to assess whether there was an association between the
2 estimates of oxalate absorption determined by means of
the direct and indirect methods described previously. Analysis of covariance was used to determine whether differences
between the 2 subpopulations in endogenous oxalate levels
could be explained in part by subpopulation differences in
the amount of lean body mass. Twenty-four–hour urinary
creatinine excretion, an index of amount of lean body mass,
was used as the covariate in this analysis. Pearson’s correlation coefficients also were computed to assess the strength of
the association between endogenous oxalate and 24-hour
creatinine excretion levels. SAS software (SAS Institute,
Cary, NC) was used for all statistical analyses. All P values
less than 0.05 are considered to designate statistical significance.

RESULTS

Compliance with the ascorbate supplementation protocol was confirmed in all study participants, evidenced by markedly greater urinary
ascorbate levels during supplementation compared with the no-supplement experimental periods (data not shown). Compliance with the maintenance of a low-oxalate diet during the
adaptation weekdays was suggested by the finding that the mean of the 3 preload 24-hour
urinary total oxalate levels correlated significantly with postload urinary total oxalate levels
for both the ascorbate (r ⫽ 0.35; P ⬍ 0.01) and
no-supplement treatments (r ⫽ 0.50; P ⬍ 0.01).

OXALATE ABSORPTION AND ENDOGENOUS SYNTHESIS

1063

Table 1. Mean 24-Hour Oxalate Absorption Levels Between SFs and NSFs for Ascorbate and
No-Supplement Treatments
Method
13

C2-Oxalic acid absorption (%)

Indirect method (%)

Treatment

SFs (n ⫽ 29)

NSFs (n ⫽ 19)

Ascorbate
No supplement
Average of treatments
Ascorbate
No supplement
Average of treatments

10.7 ⫾ 0.6*† (5.9–18.5)
9.0 ⫾ 0.5 (4.0–14.3)
9.9 ⫾ 0.4*
13.2 ⫾ 2.3 (0.0–58.0)
12.3 ⫾ 1.4 (0.7–29.1)
12.7 ⫾ 1.3

7.6 ⫾ 0.6 (3.3–15.3)
8.5 ⫾ 0.6 (3.9–14.6)
8.0 ⫾ 0.4
10.6 ⫾ 1.5 (1.8–22.5)
11.3 ⫾ 1.2 (3.8–19.4)
11.0 ⫾ 0.9

NOTE. Values expressed as mean ⫾ SE (range). The indirect method involved a computation of oxalate absorption using
the mean of the three 24-hour preload basal oxalate excretion levels as an estimate of endogenous oxalate excretion.
*Means are significantly different between SFs and NSFs, P ⬍ 0.05.
†Means are significantly different between ascorbate and no-supplement treatments, P ⬍ 0.05.

Mean 24-hour oxalate absorption levels in SFs
and NSFs for the 2 experimental treatments (ascorbate and no supplement) are listed in Table 1.
Overall, SFs had significantly greater 24-hour 13C2oxalic acid absorption averaged during the 2 treatments than NSFs (9.9% versus 8.0%). However,
the difference between the 2 subpopulations reached
statistical significance only for the ascorbate treatment. A similar trend was observed when oxalate
absorption was computed using the mean of the
three 24-hour preload basal oxalate excretion levels
as an estimate of endogenous oxalate (indirect
method), although no statistically significant differences were found. There was a significant correlation between oxalate absorption levels determined
using these 2 methods (r ⫽ 0.55; P ⬍ 0.01 and r ⫽
0.64; P ⬍ 0.01) for the ascorbate and no-supplement treatments, respectively.
Individuals who absorb greater than 10% of
administered labeled oxalate doses have been
described as high oxalate absorbers.7,8 Study
participants were subdivided into high and low
oxalate absorbers relative to this criterion. In this

analysis, average oxalate absorption for the 2
treatments was used for each subject. Thirtyeight percent of SFs, but only 11% of NSFs, had
a 13C2-oxalic acid absorption greater than 10%
(P ⬍ 0.05).
There was a significant effect of treatment on
13
C2-oxalic acid absorption (ascorbate greater
than no-supplement treatment) in SFs, but not
NSFs. There was no significant treatment effect
on oxalate absorption as estimated by the indirect method in either subpopulation.
Post–oxalate load 24-hour total urinary oxalate and endogenous oxalate levels in the stoneforming and non–stone-forming subpopulations
by treatment are listed in Table 2. SFs had
significantly greater total and endogenous urinary oxalate levels during the 24-hour post–
oxalate ingestion period compared with NSFs for
both experimental treatments. A previously reported association between total oxalate excretion levels and amount of lean body mass25 most
likely was mediated by an association between
endogenous oxalate level and lean body mass.

Table 2. Post–Oxalate Load Mean 24-Hour Total and Endogenous Oxalate Excretion Levels Between SFs and
NSFs for Ascorbate and No-Supplement Treatments
Parameter

Total oxalate (mg)

Endogenous oxalate (mg)

Treatment

SFs

NSFs

Ascorbate
No supplement
Average of treatments
Ascorbate
No supplement
Average of treatments

58.8 ⫾ 2.2*† (46.8–103.5)
51.1 ⫾ 1.7* (37.8–68.4)
54.9 ⫾ 1.5*
44.2 ⫾ 1.8*† (36.5–79.3)
38.9 ⫾ 1.3* (28.6–52.3)
41.5 ⫾ 1.1*

48.1 ⫾ 3.5 (19.8–79.2)
45.4 ⫾ 2.1 (24.3–62.1)
46.8 ⫾ 2.0
37.8 ⫾ 2.9 (15.3–58.4)
33.9 ⫾ 1.7 (19.0–50.2)
35.8 ⫾ 1.7

NOTE. Values expressed as mean ⫾ SE (range). To convert oxalate (MW ⫽ 90) in mg to ␮mol, multiply by 11.1.

*Means are significantly different between SFs and NSFs, P ⬍ 0.05.

†Means are significantly different between ascorbate and no-supplement treatments, P ⬍ 0.05.

1064

CHAI ET AL

Table 3. Mean Endogenous Oxalate Levels Averaged
Across Kidney SFs and NSFs for Ascorbate and
No-Supplement Treatments
Endogenous Oxalate (mg/h)

Sample No.*

Ascorbate Treatment

No-Supplement
Treatment

S-2
S-4
S-6
S-8
S-11
S-14
S-24

1.7 ⫾ 0.1
2.2 ⫾ 0.1
2.5 ⫾ 0.3†
2.2 ⫾ 0.2†
1.5 ⫾ 0.1
1.6 ⫾ 0.1
1.5 ⫾ 0.1

1.6 ⫾ 0.2
1.9 ⫾ 0.1
1.9 ⫾ 0.1
1.5 ⫾ 0.1
1.4 ⫾ 0.1
1.5 ⫾ 0.1
1.5 ⫾ 0.1

NOTE. Values expressed as mean ⫾ SE. To convert
oxalate (MW ⫽ 90) in mg to ␮mol, multiply by 11.1.
*S-2 to S-24 refer to the 7 urine samples sequentially
collected after 13C2-oxalate consumption: S-2, S-4, S-6,
and S-8 were collected at 2-hour intervals; S-11 and S-14
were collected at 3-hour intervals; and S-24 represents a
10-hour collection.
†Means are significantly different between ascorbate
and no-supplement treatments, P ⬍ 0.05.

Thus, there is the possibility that the greater
proportion of men (66%) in SFs than NSFs
(42%) could have partially accounted for the
greater excretion of endogenous oxalate. However, the subpopulation difference in endogenous
oxalate levels persisted even when this parameter was adjusted for 24-hour urinary creatinine
excretion, an index of amount of lean body mass.
There were no significant correlations between
levels of endogenous oxalate and 24-hour urinary creatinine for either the ascorbate (r ⫽ 0.01)
or no-supplement (r ⫽ 0.28) treatment.
There also was a significant main effect of
treatment on both total 24-hour oxalate and endogenous oxalate levels. However, paired t-tests
indicated that ascorbate supplementation was
associated with significantly greater levels of
these 2 parameters only in SFs (Table 2). To
further investigate the effect of ascorbate supplementation on endogenous oxalate levels, this
parameter was averaged across the 2 subpopulations (ie, for all study participants) for each of the
7 timed urine samples collected during the metabolic-unit day (Table 3). These data (expressed
as milligrams of oxalate per hour) indicated that
although endogenous synthesis was greater in
the first 5 samples, the most pronounced effect of
ascorbate supplementation on endogenous oxalate occurred in the S-6 and S-8 urine samples.

These samples were collected between 4 and 8
hours after the ingestion of the 1-g ascorbate
supplement with applesauce.
13
C2-Oxalic acid absorption and post–oxalate
load 24-hour urinary total and endogenous oxalate levels for men and women by treatment and
subpopulation are listed in Table 4. For SFs,
women had significantly greater 13C2-oxalic acid
absorption than men for the ascorbate treatment.
For NSFs, men had significantly greater 13C2oxalic acid absorption for the no-supplement
treatment and significantly greater total and endogenous urinary oxalate levels for the ascorbate
treatment. In addition, 13C2-oxalic acid absorption and urinary total and endogenous oxalate
levels were significantly greater in female SFs
than female NSFs for the ascorbate treatment.
To assess the relative predictive values of percentage of oxalate absorption compared with endogenous oxalate on total oxalate excretion, Pearson’s
correlation coefficients were determined between
post–oxalate load 24-hour total urinary oxalate excretion levels and each of these contributors to
oxalate excretion. Associations were determined
using data from only the no-supplement treatment
to assess the relationship between basal levels of
endogenous oxalate and total urinary oxalate excretion. Ascorbate is a major oxalate precursor, and the
greater levels of endogenous oxalate during the
ascorbate-supplement period would be expected to
enhance the ability of endogenous oxalate to predict total urinary oxalate levels. Post–oxalate load
24-hour total urinary oxalate excretion levels were
associated strongly with both 13C2-oxalic acid absorption (r ⫽ 0.76; P ⬍ 0.01) and postload 24-hour
endogenous oxalate excretion (r ⫽ 0.95; P ⬍ 0.01)
in the subpopulation of SFs. The corresponding
correlation coefficients for the subpopulation of
NSFs were 0.62 (P ⬍ 0.01; total oxalate versus
13
C2-oxalic acid absorption) and 0.92 (P ⬍ 0.01;
total oxalate versus endogenous oxalate).
To further investigate the effect of subpopulation on oxalate absorption, 13C2-oxalic acid excretion levels were averaged across the 2 treatments for each study participant (Table 5).
Averaged 13C2-oxalic acid levels were significantly greater at S-4, S-8, and S-11 in SFs than
NSFs. Data also indicate that the majority of
labeled 13C2-oxalic acid was absorbed and excreted during the first 6 hours post–oxalate ingestion (77% and 79% of the total amount excreted

6.7 ⫾ 0.4 (3.3–8.8)
7.2 ⫾ 0.6 (3.9–9.1)
41.4 ⫾ 3.5 (19.8–63.9)
42.4 ⫾ 2.7 (24.3–59.4)
32.3 ⫾ 3.0 (15.3–53.0)
32.6 ⫾ 2.1 (19.0–47.0)

Women (n ⫽ 11)

DISCUSSION

Endogenous oxalate (mg)

Total oxalate (mg)

Ascorbate
No supplement
Ascorbate
No supplement
Ascorbate
No supplement
C2-oxalic acid absorption (%)
13

The overall data indicated significantly greater
C2-oxalic acid absorption in the stone-forming
than non–stone-forming subpopulation. This finding is consistent with the majority of previous
studies6-9 that assessed oxalate absorption using
an isotope label, either carbon 14–labeled oxalate or 13C2-oxalate. Mean 24-hour 13C2-oxalic
acid absorptions averaged across the ascorbate
and no-supplement treatments for SFs and NSFs
were 9.9% and 8.0%, respectively. These values
were within the ranges previously reported by
most studies for these 2 subpopulations (SFs, 9%
to 17% versus NSFs, 6% to 14%).6-11 In the
present study, high oxalate absorbers are defined
as those who absorbed greater than 10% of
administered 13C2-oxalic acid. The greater proportion of high oxalate absorbers observed in
SFs (38%) than NSFs (11%) supports the assertion that SFs are more likely to have high oxalate
absorption. The enhancing effect of ascorbate
supplementation on 13C2-oxalic acid absorption
was observed only in SFs.
It should be acknowledged that, consistent
with previous reports,26,27 a greater percentage
of SFs than NSFs may have been characterized
by the absence of colonic oxalate-degrading bacteria, a condition that could increase predisposition to hyperoxaluria and urolithiasis. However,
the clinical significance of this factor with respect to the pathogenesis of hyperoxaluria has
not been established definitively. Because of the
colonic location of these bacteria, oxalate absorption and subsequent urinary excretion may not
always be affected greatly because the upper
gastrointestinal tract appears to be the primary
site of oxalate absorption in individuals free of
gastrointestinal disorders.28
In addition to the labeled oxalate method,
oxalate absorption also was estimated by using
the mean of the three 24-hour preload basal
oxalate excretion levels as an estimate of endogenous oxalate (indirect method). This method is
based on the assumption that subjects maintained
a low-oxalate diet during the three 24-hour preload and 24-hour post–oxalate load collection
periods, such that with the exception of oxalate
absorbed from the oxalate load, the remaining
13

NOTE. Values expressed as mean ⫾ SE (range). To convert oxalate (MW ⫽ 90) in mg to ␮mol, multiply by 11.1.
*Means are significantly different between males and females within the stone-forming or non–stone-forming subpopulation, P ⬍ 0.05.
†Means are significantly different between female SFs and female NSFs, P ⬍ 0.05.

8.9 ⫾ 1.1 (5.4–15.3)
10.2 ⫾ 1.1* (5.7–14.6)
57.4 ⫾ 5.5* (31.5–79.2)
49.5 ⫾ 3.2 (36.9–62.1)
45.2 ⫾ 4.4* (24.1–58.4)
35.6 ⫾ 3.0 (28.7–50.2)
12.8 ⫾ 1.2† (7.2–18.5)
8.9 ⫾ 0.8 (6.0–14.3)
62.3 ⫾ 5.1† (46.8–103.5)
50.2 ⫾ 3.0 (37.8–64.8)
44.8 ⫾ 4.0† (36.7–79.3)
38.1 ⫾ 2.5 (28.6–51.3)
9.6 ⫾ 0.6* (5.9–17.0)
9.0 ⫾ 0.6 (4.0–14.3)
57.0 ⫾ 2.1 (47.7–82.8)
51.5 ⫾ 2.1 (38.7–68.4)
43.9 ⫾ 1.8 (36.5–68.1)
39.3 ⫾ 1.5 (30.0–52.3)

Men (n ⫽ 8)
Women (n ⫽ 10)
Men (n ⫽ 19)
Treatment

1065

24 hours postingestion for SFs and NSFs, respectively).

NSFs
SFs
Parameter

Table 4.

13

C2-Oxalic Acid Absorption and Post–Oxalate Load Mean 24-Hour Total and Endogenous Oxalate Excretion Levels Between Men and Women for
Ascorbate and No-Supplement Treatments

OXALATE ABSORPTION AND ENDOGENOUS SYNTHESIS

1066

CHAI ET AL
Table 5. Mean 13C2-Oxalic Acid Excretion Averaged Across Ascorbate and No-Supplement
Treatments in SFs and NSFs
SFs

Sample No.*

S-2
S-4
S-6
S-8
S-11
S-14
S-24
Total

13

C2-Oxalic Acid (mg)

NSFs
Cumulative (%)

0.39 ⫾ 0.02
0.61 ⫾ 0.03†
0.37 ⫾ 0.03
0.16 ⫾ 0.01†
0.10 ⫾ 0.01†
0.05 ⫾ 0.01
0.09 ⫾ 0.01
1.77 ⫾ 0.07†

22
56
77
86
92
95
100

13

C2-Oxalic Acid (mg)

0.38 ⫾ 0.03
0.46 ⫾ 0.02
0.30 ⫾ 0.04
0.11 ⫾ 0.01
0.07 ⫾ 0.01
0.05 ⫾ 0.01
0.08 ⫾ 0.01
1.45 ⫾ 0.08

Cumulative (%)

26
58
79
86
91
94
100

NOTE. Values expressed as mean ⫾ SE. To convert 13C2-oxalic acid (MW ⫽ 92) in mg to ␮mol, multiply by 10.9.
*S-2 to S-24 refer to the 7 urine samples sequentially collected after 13C2-oxalate consumption: S-2, S-4, S-6, and S-8
were collected at 2-hour intervals; S-11 and S-14 were collected at 3-hour intervals; and S-24 represents a 10-hour
collection.
†Means are significantly different between SFs and NSFs, P ⬍ 0.05.

urinary oxalate was primarily of endogenous
origin. The validity of the indirect method used
to assess oxalate absorption is suggested by the
relatively high correlation between the corresponding absorption estimates.
Mean post–oxalate load 24-hour total urinary
oxalate and endogenous oxalate levels were significantly greater in the stone-forming than non–
stone-forming subpopulation. These findings are
in agreement with previous studies that showed
similar subpopulation differences in both total
urinary oxalate10-12,29 and endogenous oxalate
levels.12
It also should be acknowledged that the use of
basal oxalate excretion levels to estimate endogenous oxalate overestimates the contribution of
this fraction to total urinary oxalate levels because it does not account for the contribution of
absorbed dietary oxalate from the low-oxalate
diet. Holmes et al30 showed that even with diets
providing only 10 to 50 mg/d of oxalate, the
contribution of absorbed dietary oxalate to total
urinary oxalate was approximately 25%, which
corresponded to 5 to 7 mg of the total 24-hour
urinary oxalate level. Because the low-oxalate
diets used in the present study during the controlled-feeding 3-day pre–metabolic-unit period
and during the 1-day metabolic-unit period provided oxalate in a range of approximately 10 to
40 mg/d, it could be assumed that approximately
5 to 7 mg of the total 24-hour urinary oxalate
levels measured during these days was of exogenous origin (ie, oxalate absorbed from the low-

oxalate diets). Endogenous oxalate, computed as
the difference between total and exogenous oxalate, thus would have been overestimated by
this same amount. Although SFs and NSFs may
have showed differences in oxalate absorptive
efficiency from the low-oxalate diets, it appears
unlikely that the relatively small contribution of
this unaccounted-for exogenous oxalate to total
oxalate excretion would have confounded the
interpretation of the mentioned finding of greater
endogenous oxalate excretion in SFs compared
with NSFs.
The present study was unique in that it allowed a
direct comparison of both oxalate absorption and
endogenous oxalate synthesis between SFs and
NSFs on totally controlled low-oxalate diets. Gastrointestinal absorption of oxalate has been given a
great deal of attention in previous labeled-oxalate
studies. However, comparative assessment of endogenous urinary oxalate in SFs and NSFs also is
important for determining the relative predictive
values of percentage of oxalate absorption and
endogenous oxalate synthesis on total oxalate excretion in these 2 subpopulations. Potential mechanisms to explain enhanced endogenous oxalate
synthesis in SFs currently are uncertain, although
altered activity of the renal enzymes involved in
oxalate synthesis has been suggested.31 Data presented by Lemann et al25 supported the assertion
that endogenous oxalate synthesis is associated
with amount of lean body mass. Such an association was not found in the present study.

OXALATE ABSORPTION AND ENDOGENOUS SYNTHESIS

Both SFs and NSFs had greater urinary total and
endogenous oxalate levels for the ascorbate than
no-supplement treatment, although statistically significant differences were observed only in SFs.
This finding in SFs is consistent with results from
previous studies.15,16,19,20 Ascorbate-induced increases in mean endogenous oxalate levels during
the 24-hour timed urine collection were slightly
greater in SFs (5.3 mg) than NSFs (3.9 mg). Previously reported increments in total 24-hour urinary
oxalate levels in SFs after ingesting 1 or 2 g of
ascorbate ranged from 10 to 19 mg,15,16,20 whereas
corresponding increments in NSFs ranged from 2
to 14 mg.13-18 The clinical significance of the
ascorbate-induced increases in endogenous oxalate
levels may be greater in SFs than NSFs because
SFs appear to have greater levels of oxalate absorption and basal endogenous oxalate synthesis.
Although the 1-g ascorbate supplement was
administered 2 hours before the morning meal
and at the evening meal of the metabolic-unit
day, an increase in endogenous oxalate was most
pronounced between 4 to 8 hours after the ingestion of the early-morning 1-g ascorbate supplement. This suggests that the effect of ascorbate
supplementation on endogenous oxalate levels
might be most pronounced during a time frame
when maximal ascorbate absorption would be
expected. Padayatty et al32 found that plasma
ascorbate levels after a 1-g oral dose were elevated for 6 hours. Lack of an effect after the
evening ascorbate supplementation dose could
be attributed partially to a diurnal variation in
endogenous oxalate production or slowed ascorbate absorption caused by the large volume of
the meal. There also is the possibility that collection of 3-hour and 10-hour urine samples (S-14
and S-24) after the evening supplement may
have made it more difficult to detect significant
increases in endogenous oxalate levels, which
could have occurred within a 4- to 8-hour period.
The prevalence of kidney stones is approximately 3-fold greater in men than women.33 The
question arises about whether there are sex differences in oxalate absorption and urinary total and
endogenous oxalate levels that could provide at
least a partial explanation for the greater prevalence of kidney stones in men. Significantly
greater urinary total oxalate levels in men have
been reported within subpopulations of SFs8 and
NSFs.34,35 Although the present results indicate

1067

specific sex differences in 13C2-oxalic acid absorption and total and endogenous oxalate levels
within each subpopulation, there were no consistent sex differences across the 2 treatments in
any of these parameters. In contrast to previous
reports of either significantly greater 13C2-oxalic
acid absorption8 or total urinary oxalate levels35
in male SFs than male NSFs, neither parameter
differed between the 2 subpopulations of men in
the present study. However, present results show
a consistent trend of greater 13C2-oxalic acid
absorption and total and endogenous oxalate
levels in female SFs than female NSFs, and the
subpopulation differences were statistically significant for the ascorbate treatment. Additional
studies are required to elucidate the nature of sex
differences in these key oxalate-related parameters.
Hyperoxaluria is considered to be the most
important risk factor for the formation of calcium oxalate–containing kidney stones because
a small increase in urinary oxalate concentration
markedly increases the level of calcium oxalate
saturation.4 Causes of the increase in oxalate
excretion levels in SFs are not fully understood.
Hodgkinson29 reported that fasting abolished the
difference in mean oxalate-creatinine ratios between subpopulations of SFs and NSFs, suggesting that the moderate increase in total urinary
oxalate excretion observed in SFs was caused
mainly by increased oxalate absorption. Conversely, Schwille et al12 reported that the excess
urinary oxalate in SFs after ingesting an oxalatefree test meal containing various oxalate precursors was caused primarily by increased endogenous oxalate synthesis.
Post–oxalate load 24-hour total urinary oxalate excretion levels correlated significantly with
both 13C2-oxalic acid absorption and post–
oxalate load 24-hour endogenous oxalate excretion levels. Although Hatch36 postulated that
increased urinary oxalate levels in SFs were
caused by either increased absorption or increased endogenous synthesis (ie, 2 separate
subpopulations), the present data suggest that at
least some SFs could have both conditions. The
10 individuals with the greatest rates of total
oxalate excretion (5 SFs, 5 NSFs) also had both
high rates of endogenous synthesis and percentage of oxalate absorption. Thus, many individuals who excrete high amounts of urinary oxalate

1068

CHAI ET AL

are likely to have high rates of oxalate absorption
and endogenous oxalate synthesis. Siener et al37
assessed dietary intake and urine composition of
186 calcium-oxalate SFs and reported that urinary oxalate excretion was associated positively
with dietary ascorbate. Overall data were consistent with the assertion that hyperoxaluria may
result from a combination of increased endogenous synthesis and high oxalate absorption.
The majority of administered 13C2-oxalic acid
was absorbed and excreted during the first 6 hours
after oxalate ingestion for both SFs (77%) and
NSFs (79%). Because the majority of ingested
oxalate would not be expected to have reached the
colon within this period, these results support the
assertion that the upper gastrointestinal tract is the
major site for oxalate absorption.28
Because increased oxalate absorption and increased endogenous oxalate synthesis are 2 major contributors to hyperoxaluria, dietary restriction of both oxalate and oxalate precursors would
reduce the risk for kidney stone formation in
susceptible individuals. Concomitant ingestion
of calcium or magnesium also may be of help
because the intraluminal presence of both calcium38 and magnesium34 has been shown to
reduce oxalate absorption.
In conclusion, SFs had greater 13C2-oxalic
acid absorption and endogenous urinary oxalate
excretion and, consequently, greater post–oxalate load 24-hour total urinary oxalate levels
compared with NSFs. The strong associations
between total oxalate excretion and both 13C2oxalic acid absorption and endogenous oxalate
excretion suggest that hyperoxaluria can be
caused by a combination of increased oxalate
absorption and increased endogenous oxalate
synthesis.
Ingestion of a 1-g ascorbate supplement twice
a day increased urinary total and endogenous
oxalate levels in both the stone-forming and
non–stone-forming subpopulations. These data
suggest that high-dose ascorbate consumption is
a risk factor for individuals predisposed to the
formation of kidney stones.
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