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Urol Res (2007) 35:15–21
DOI 10.1007/s00240-006-0076-y
123
ORIGINAL PAPER
Vitamin B6 deWciency augments endogenous oxalogenesis after
intravenous
L-hydroxyproline loading in rats
Y. Ogawa · R. Z. Hossain · T. Ogawa · K. Yamakawa ·
H. Yonou · Y. Oshiro · S. Hokama · M. Morozumi ·
A. Uchida · K. Sugaya
Received: 15 August 2006 / Accepted: 1 December 2006 / Published online: 3 January 2007
© Springer-Verlag 2007
Abstract The eVects of an intravenous hydroxypro-
line load on endogenous oxalogenesis were compared
in rats fed a standard diet or a vitamin B6-deWcient diet.
Twelve male Wistar rats were randomized to two
groups and were fed either a standard diet (control
group) or a vitamin B6-deWcient diet for 3 weeks. Then
the animals were intravenously administered 100 mg
(762.6 mol)/ml hydroxyproline. In the control group,
infusion of hydroxyproline increased the 5-h urinary
oxalate and glycolate excretion above baseline to 0.27%
(2.02 § 1.11 mol) and 0.32% (2.43 §1.60 mol) of the
administered dose (mol/mol), while it was respectively
2.01% (15.24 § 2.13 mol) and 0.00% (¡0.02 §
0.19 mol) of the dose in the vitamin B6-deWcient
group. Therefore, vitamin B6 deWciency augmented
endogenous synthesis of oxalate from hydroxyproline
by 7.56-fold (15.24/2.02) compared with that in the con-
trol group. Urinary citrate excretion was signiWcantly
lower at baseline and all other times in the vitamin B6-
deWcient group compared with the control group. In
conclusions,
L-hydroxyproline loading augmented
endogenous oxalogenesis in the vitamin B6-deWcient
group without causing hyperglycolic aciduria, and also
led to signiWcant hypocitraturia. These Wndings suggest
that hydroxyproline is not metabolized to oxalate via
glycolate, but rather via the 4-hydroxyglutamate to gly-
oxylate pathway (usually requiring vitamin B6-depen-
dent enzymes) even in the presence of vitamin B6
deWciency.
Keywords Hydroxyproline · Vitamin B6-deWcient
diet · Endogenous oxalogenesis · Urinary oxalate ·
Capillary electrophoresis
Introduction
In humans and animals the chief immediate metabolic
precursor of oxalate is glyoxylate, which is formed
from glycolate in the peroxisomes and from hydroxy-
proline in the mitochondria. The glyoxylate pathway is
associated with gluconeogenesis, and it is believed to
be the major metabolic pathway for the synthesis of
oxalate, particularly in persons with primary hyperox-
aluria [1]. Among the various precursors of oxalate,
glycolate and glyoxylate appear to be the most impor-
tant substances that promote oxalogenesis [2, 3],
whereas administration of either glycine or ascorbate
does not increase urinary oxalate excretion in rats
[4–7]. A number of experimental models have been
developed to study calcium oxalate urolithiasis, and
virtually all of them employ hyperoxaluria as the basic
abnormality. In all the experimental models renal
injury is associated with crystal deposition mainly on
the intraluminal side of renal tubules and preferentially
attached to renal papillary tips and fornices. And rat
renal calcium oxalate urolithiasis is similar to calcium
oxalate stone disease in human [8]. A recent study of
pigs fed a 10% hydroxyproline diet revealed the devel-
opment of hyperoxaluria and calcium oxalate crystallu-
ria, suggesting that omnivores or herbivores (which
usually do not have a good glyoxylate-detoxifying sys-
tem) may be suitable as a model of hyperoxaluria after
a hydroxyproline load [9, 10]. We previously reported
that intravenous infusion of hydroxyproline increased
Y. Ogawa (&) · R. Z. Hossain · T. Ogawa · K. Yamakawa ·
H. Yonou · Y. Oshiro · S. Hokama · M. Morozumi ·
A. Uchida · K. Sugaya
Department of Urology, University of the Ryukyus,
207 Uehara, Nishihara, Okinawa 903-0215, Japan
e-mail: ogawa@eve.u-ryukyu.ac.jp
16 Urol Res (2007) 35:15–21
123
urinary oxalate in close relation with urinary glycolate
in rats [11, 12]. In addition, Ribaya and GershoV [13–
15] reported that urinary oxalate excretion was
increased in vitamin B6-deWcient rats or control rats
fed with a diet containing 5.2% hydroxyproline, while a
diet containing 3% glycine and 5.2% hydroxyproline
caused an increase of renal oxalate and glyoxylate lev-
els, but decreased the glycolate level, in vitamin B6-
deWcient rats compared with controls. These Wndings
prompted us to study the urinary excretion of oxalate
and glycolate after administration of
L-hydroxyproline
to vitamin B6-deWcient rats.
Materials and methods
Twelve male Wistar rats weighing 174.95 § 5.13 g
(mean § SD) were acclimatized at the Animal Center,
and then were randomized to two groups of six animals
each. One group was fed a standard (CE-2) diet (con-
trols), while the other rats were fed a vitamin B6-deW-
cient diet for 3 weeks. Both groups of rats had free
access to drinking water. After 3 weeks, the rats were
anesthetized with an intraperitoneal injection of ure-
thane (1.2 g/kg body weight) and were hydrated with
physiological saline via the femoral vein at a rate of
2.5–3.5 ml/h. Then the animals in both groups were
administered 1 ml of a 100 mg/ml hydroxyproline solu-
tion as a slow intravenous infusion over 10 min. Infu-
sion of saline was continued at a rate of 3.5 ml/h
throughout the experiment.
The hydroxyproline solution was prepared by dis-
solving 100 mg (762.6 mol) of hydroxyproline (molec-
ular weight: 131.13; Wako Pure Chemicals, Osaka,
Japan) in 1 ml of pure water. The bladder was emptied
1 h before hydroxyproline infusion and hourly urine
specimens were collected by bladder puncture at base-
line and every hour until 5 h after infusion. Measure-
ment of the urine volume was done at each time of
collection. The urine specimens were immediately
stored at ¡80°C until assay.
Thawed urine specimens were Wltered through a dis-
posable 0.2 m Wlter (Millex-LG syringe-driven unit,
Millipore, Bedford, MA, USA), were diluted 20- to 40-
fold with Milli-Q level pure water that was obtained
using a water puriWcation system (Millipore), and were
injected into a capillary tube at 50 mbar (5,000 Pa) for
4 s (approximately 20 nl). Then measurement of the
urinary glycolate and citrate levels was done by capil-
lary electrophoresis (Agilent CE, Germany) using an
organic acids buVer (pH 5.6) for high performance cap-
illary electrophoresis (HPCE) that contained 5 mM
2,6-pyridinedicarboxylic acid and 0.5 mM cetyltrime-
thylammonium bromide (CTAB) (Agilent Technolo-
gies, Germany) [16, 17]. In addition, aliquots of the
thawed urine specimens were acidiWed to <pH 2 with
6 N HCl and then were diluted with water in the same
fashion to measure urinary oxalate by capillary electro-
phoresis with a pyromellitic acid electrolyte buVer (pH
7.7) for anion HPCE (Fluka, Switzerland) [16, 17].
Urinary levels of oxalate, glycolate, and citrate at 0 h
were deWned as the baseline values for excretion of
each substance. The cumulative increment of urinary
oxalate and glycolate excretion above baseline (recov-
ery of excretion) after infusion of hydroxyproline was
calculated for each group by subtracting the baseline
oxalate and glycolate values. Hourly urinary excretion
levels for oxalate, glycolate, and citrate were compared
with the respective baseline values using the Wilcoxon
signed ranks test (2-tailed), while the hourly and total
urinary excretion values were compared between
groups using the Mann–Whitney U test. Data are
reported as the mean § SD and statistical signiWcance
was set at P < 0.05 for all comparisons.
Results
The rats showed signiWcant weight gain over time from
176.33 §5.28 g (mean §SD) and 173.56 § 5.04 g at
baseline to 394.38 § 8.40 g and 309.10 § 15.61 g after
3 weeks in the control and vitamin B6-deW
cient groups,
respectively (P < 0.05 for baseline vs. 3 weeks and
between the two groups at 3 weeks). Weight gain in the
vitamin B6-deWcient group occurred at a rate of
approximately 6.45 g/day, which was lower than in the
control group (approximately 10.38 g/day), and this
diVerence was statistically signiWcant (P < 0.01).
Baseline urinary oxalate excretion was signiWcantly
higher in the vitamin B6-deWcient group (1.14 §
0.27 mol) than in the control group (0.27 § 0.09 mol)
(P < 0.01) (Figs. 1, 2). Hourly urinary oxalate excretion
peaked within 2–3 h after hydroxyproline infusion in
both groups (Fig. 1). In the vitamin B6-deWcient group,
hourly urinary oxalate excretion was signiWcantly
higher than baseline from 1 to 5 h. In the control
group, it was higher from 1 to 3 h after hydroxyproline
infusion (P < 0.05) (Fig. 1). The total (0–5 h) urinary
oxalate excretion was signiWcantly higher in the vita-
min B6-deWcient group (22.07 § 1.85 mol) than in the
control group (3.65 § 0.74 mol) (P <0.01, between
groups). The 5-h cumulative increment of urinary
oxalate excretion above baseline (recovery rate)
accounted for 0.27% (mol/mol) (2.02 § 1.11 mol) ver-
sus 2.01% (15.24 § 2.13 mol) of the administered
dose of hydroxyproline in the control group and the
Urol Res (2007) 35:15–21 17
123
vitamin B6-deWcient group, respectively (P <0.01,
between groups). In the vitamin B6-deWcient group,
total urinary oxalate excretion was increased by
approximately 6.04-fold (22.07 vs. 3.65) and the 5-h
increment above baseline was 7.56-fold greater than in
the control group (15.24 vs. 2.02) (Fig. 1).
Baseline urinary glycolate excretion was signiW-
cantly higher in the control group (0.25 § 0.07 mol)
than in the vitamin B6-deWcient group (0.09 §
0.06 mol) (P < 0.01). Hourly urinary glycolate excre-
tion peaked at 2 h in the control group, but remained
low without any change in vitamin B6-deWcient group.
In the control group, urinary glycolate excretion was
signiWcantly higher than baseline from 1 to 4 h
(P < 0.05), but it remained low from 1 to 5 h in the vita-
min B6-deWcient group (Fig. 2). Infusion of hydroxy-
proline increased urinary glycolate excretion to 0.32%
(2.43 § 1.60 mol) versus 0.00% (0.02 § 0.19 mol) of
the administered dose in the control and vitamin B6-
deWcient groups, respectively (P < 0.01, between
groups). The 5-h cumulative increment of urinary
glycolate excretion above baseline (recovery rate)
accounted for 0.32% (2.43 § 1.60 mol) of the admin-
istered dose of hydroxyproline in the control group.
Total urinary glycolate excretion was signiWcantly
lower in the vitamin B6-deWcient group (0.54 §
0.22 mol) than in the control group (3.90 § 1.56 mol)
(P < 0.01, between groups).
Baseline urinary citrate excretion was signiWcantly
lower in the vitamin B6-deWcient group (0.13 §
0.07 mol) than in the control group (2.03 § 0.52 mol)
(P < 0.01). In the control group, hourly urinary citrate
excretion peaked at 1 h and was signiWcantly lower
than baseline at 2, 3, and 5 h after hydroxyproline infu-
sion (P <0.05) (Fig.3). In the vitamin B6-deWcient
group, however, it remained low at all times and only
Fig. 1 Urinary oxalate (Ox)
excretion after administration
of
L-hydroxyproline to vita-
min B6-deWcient rats and con-
trol rats. Total = total
cumulative urinary oxalate
excretion from 0 to 5 h.
Increment = cumulative
increment of urinary oxalate
excretion above baseline from
1 to 5 h after hydroxyproline
loading
0.00
1.00
2.00
3.00
4.00
5.00
6.00
0 h 1 h 2 h 3 h 4 h 5 h
Time (hours)
Control rats
Vit-B6 deficient rats
mol/hour
Urinary oxalate excretion
Fi
g.
2 U
r
i
nary g
l
yco
l
ate
(Glc) excretion after adminis-
tration of
L-hydroxyproline to
vitamin B6-deWcient rats and
control rats. Total = total
cumulative urinary glycolate
excretion from 0 to 5 h.
Increment = cumulative
increment of urinary glycolate
excretion above baseline from
1 to 5 h after hydroxyproline
loading
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
0 h 1 h 2 h 3 h 4 h 5 h
Time (hours)
Control rats
Vit-B6 deficient
rats
(
mol/hour)
18 Urol Res (2007) 35:15–21
123
showed a slight increase at 1–3 h after hydroxyproline
infusion (P <0.05) (Fig.3). Total cumulative urinary
citrate excretion and the 5-h cumulative increment of
citrate excretion was 8.55 § 2.85 and ¡3.61 §
4.35 mol versus 1.35 § 0.85 and 0.57 § 0.57 mol in
the control group and the vitamin B6-deWcient group,
respectively (P < 0.05, between groups).
Discussion
Glyoxylate, glycolate, and hydroxyproline are impor-
tant precursors of oxalate that are mainly metabolized
in the liver, while ascorbate is another precursor that
produces oxalate under special conditions [7]. In herbi-
vores, a major endogenous source of glyoxylate is oxi-
dation of glycolate by glycolate oxidase in the
peroxisomes, the peroxisomal localization of ser-
ine:pyruvate/alanine:glyoxylate aminotransferase (SPT/
AGT or AGT-1) may be important for removal of gly-
colate-derived glyoxylate. Two forms of alanine:glyoxy-
late aminotransferase (AGT-1 and AGT-2) have been
reported in the mitochondria of rat liver but we do not
know how AGT-1 and AGT-2 share the role of metab-
olizing glyoxylate in mitochondria. In carnivores, the
largely mitochondrial localization of SPT/AGT is
important for the formation of glyoxylate, which is syn-
thesized directly from 4-hydroxyproline-2-ketoglutarate
in the mitochondria [10, 11]. The intermediate step
involves transamination of 4-hydroxyglutamate to 4-
hydroxy-2-ketoglutarate and this enzymatic reaction
requires aspartate aminotransferase (AspAT) and vita-
min B6 as a cofactor [13, 14, 18]. Despite being a
vitamin B6-dependent reaction, the conversion of
hydroxyproline to oxalate (via glyoxylate) was promi-
nent in our vitamin B6-deWcient animals, so further
study of vitamin B6-dependent AspAT is warranted.
Urinary oxalate excretion was reported to be high in
rats given glyoxylate, glycolate, hydroxypyruvate, and
hydroxyproline, showing an increase in this order [2, 3].
In rats, the urinary oxalate excretion as a percentage of
the administered dose (mol/mol: recovery rate) was
approximately 22% for glyoxylate, 6.1% for glycolate,
0.49% for ethylene glycol, 0.4% for hydroxypyruvate,
0.26% for hydroxyproline, and 0.02% for pyruvate [12].
Little or no increment of oxalate excretion was
observed in rats given glycine, xylitol, or ascorbate [12].
Urinary glycolate excretion as a percentage of the
administered dose (mol/mol: recovery rate) was
approximately 2.92% for glyoxylate, 8.9% for glycolate,
0.66% for ethylene glycol, 0.32% for hydroxypyruvate,
0.2% for hydroxyproline, 0.017% for glycine, and
0.005% for xylitol [12]. No increase of glycolate was
observed after dosing with sodium pyruvate or ascor-
bate [12]. Pyruvate was previously identiWed by Chow
et al. [19] as preventing the deposition of calcium oxa-
late in the kidneys. They also reported that pyruvate
decreases urinary oxalate excretion in an experimental
model of urolithiasis and suggested that pyruvate could
prevent urolithiasis by decreasing oxalate synthesis [19].
We subsequently concluded that pyruvate salts inhibit
calcium oxalate crystal formation in the kidneys by
increasing the urinary citrate concentration rather than
by decreasing oxalate synthesis [20], and we showed
that pyruvate administration led to a minimal increase
of oxalate that was unrelated to glycolate [12
].
Vitamin B6 deWciency results in increased excretion
of glycolate as well as oxalate in cats and rats [21–23].
Hydroxyproline has been suggested to cause the
formation of calcium oxalate stones in rats [24], or to
Fig. 3 Urinary citrate (Cit)
excretion after administration
of
L-hydroxyproline to vita-
min B6-deWcient rats and con-
trol rats. Total = total
cumulative urinary citrate
excretion from 0 to 5 h.
Increment = cumulative
increment of urinary citrate
excretion above baseline from
1 to 5 h after hydroxyproline
loading
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 h 1 h 2 h 3 h 4 h 5 h
Time (hours)
(
mol/hour)
Control rats
Vit-B6 deficient
rats
Urol Res (2007) 35:15–21 19
123
promote excessive urinary oxalate excretion in vitamin
B6-deWcient rats [14, 25]. Hydroxyproline-induced
hyperoxaluria was also demonstrated in 16 infants who
were given 1 g of hydroxyproline [26]. We previously
reported that the increase of urinary oxalate and glyco-
late after administration of
L-hydroxyproline (630 mg)
was 0.24 and 0.06% of the dose, respectively [11]. Then
we conWrmed similar results and showed that
L-
hydroxyproline caused an increase of urinary oxalate
along with urinary glycolate in normal rats [12]. In vita-
min B6-deWcient rats, hyperoxaluria is both due to
decreased transamination by alanine:glyoxylate amino-
transferase (AGT) and enhancement of glycolate oxi-
dase activity [27–29].
As reported previously, the urinary glycolate level
corrected for creatinine was lower at baseline in the
control rats than the rats fed a vitamin B6-deWcient
diet for 4 weeks [29]. Low serum creatinine levels and
high urinary creatinine levels along with less weight
gain suggested mild malnutrition, dehydration, and
cachexia due to 4 weeks of vitamin B6 deWciency.
However, it is unclear whether changes of catabolic,
acidotic, or glycolytic metabolism lead to altered glyco-
late production. Although urinary glycolate excretion
at baseline shows slight increase in relation to weight
gain and the duration of vitamin B6 deWciency (1, 2, or
3 weeks), these changes were very subtle (unpublished
data) and could be too small to be worth studying. In
addition, we do not know anything about the possible
induction of glycolate-related enzymes over time due
to vitamin B6 deWciency. Therefore, we shortened the
feeding period to 3 weeks for this study, but growth
retardation was still not negligible. The mean weight of
the animals in this study diVered between the groups
and we compared hourly glycolate excretion, while cre-
atinine-corrected urinary glycolate excretion was
assessed in the previous study [29]. There are no deW-
nite factors that explain the discrepant or conXicting
results, and we are not sure whether these minor diVer-
ences are worth exploring further.
On the other hand, glycolate excretion was
increased signiWcantly over time by hydroxyproline
administration in the controls, but it did not increase at
all in the vitamin B6-deWcient animals. The immediate
metabolic precursors of glycolate are glycolaldehyde
(from hydroxypyruvate) and glyoxylate, so both sub-
stances are potential sources for production of glyco-
late [30]. In normal rats, glycolate is either derived
from glycolaldehyde (hydroxypyruvate) or from gly-
oxylate via glyoxylate reductase. This enzyme is depen-
dent on NADH rather than pyridoxal, but its redox
status and activity may be altered by changes of other
pyridoxal-dependent enzymes. Mitochondrial glycolate
production could be the principal source of hydroxy-
proline for oxalate synthesis in primary hyperoxaluria
1 [31]. Glycolate was reported to be largely derived
from metabolism after meals [32], but the source was
not indicated clearly. The authors may have meant that
glycolate was not directly ingested, but was derived
from glyoxylate or other precursors. However, better
evidence based on loading with both isotopic sub-
stances is required. So far, we know that two sub-
stances which increase urinary glycolate excretion in
vitamin B6-deWcient rats are hydroxypyruvate and eth-
ylene glycol (unpublished data). Peroxisomal glycolate
production could be the source of these substances for
oxalate synthesis.
Metabolism of hydroxyproline to oxalate does not
involve the peroxisomal glycolate pathway and glyoxy-
late is produced in the mitochondria. The reverse reac-
tion that transfers glyoxylate to glycolate is catalyzed
by cytosolic glyoxylate reductase in normal rats. The
association of hyperoxaluria with hypoglycolic aciduria
in vitamin B6-deWcient rats may suggest that glyoxy-
late reductase (which usually converts glyoxylate to
glycolate in the liver) also malfunctions due to B6
deWciency, so that glyoxylate produced from
hydroxyproline cannot be detoxiWed to glycolate by
this enzyme and therefore is metabolized to oxalate
by LDH. Vitamin B6 deWciency appears to produce
a combination of AGT and glyoxylate reductase/
hydroxypyruvate reductase (GRHPR) knockout or
knockdown, but this needs further investigation
(Fig. 4).
The intake of animal protein is associated with a sig-
niWcant increase of urinary oxalate excretion in
humans [
33–35]. There are two explanations for this
hyperoxaluria associated with meat intake. The intake
of oxalogenic precursors in meat and increased dietary
oxalate absorption by the presence of animal fat in the
Fig. 4 Proposed pathways of glyoxylate metabolism in vitamin
B6 deWciency
Peroxisome
Glyoxylate
Gl
y
ci
n
e
G
l
ycolate
Gly
oxy
late
Oxalate
Glycolate
Cytosol
Oxala
te
GRHP
R
LD
H
GO
GO
A
G
T
Glycolaldehyde
Hydroxypyruvate
U
r
i
n
e
PH T
ype
T
y
p
e
Vit B6
Hydro
xyproli
n
e
Ascorbate
D-glycerate
Ser
i
ne
P
yr
uv
a
te
Ethyleneglycol
Xylitol
X
X
X
AGT2
G
l
ycine
X
20 Urol Res (2007) 35:15–21
123
bowel are implicated in the genesis of hyperoxaluria
[36, 37]. Collagen accounts for approximately 7% of all
animal protein (on a weight basis) [38] and the
hydroxyproline content of collagen is as high as 13%.
Thus, the hydroxyproline content of 100 g meat is
approximately 1 g. If our present results obtained in
rats can be applied to humans, the absorption of about
30% of dietary hydroxyproline (300 mg = 2.3 mmol)
may result in conversion to 4 mg of oxalate
(2.3 £ 0.02 £90) in patients with vitamin B6 deW-
ciency. Therefore, dietary protein intake may contrib-
ute to hyperoxaluria in humans due to hydroxyproline-
associated endogenous oxalogenesis and fat-associated
hyperabsorption of exogenous oxalate.
Hypocitraturia is another important Wnding associ-
ated with vitamin B6 deWciency [29] and this was con-
Wrmed in the present study. Chronic metabolic acidosis
causes hypocitraturia and was reported to increase
renal cortical ATP citrate lyase activity in rats [39].
Adipose tissue and liver ATP citrate lyase activity was
reported to be decreased by vitamin B6 deWciency [40].
Tosukhong et al. [41, 42] reported an inverse relation-
ship between the activity of leukocyte ATP citrate
lyase (a potential predictor of enzymatic activity in
renal tubular cells) and urinary citrate excretion.
If this holds true, low ATP citrate lyase activity asso-
ciated with vitamin B6 deWciency should lead to hyper-
citraturia. In reality, however, this may occur the other
way around. That is, vitamin B6 deWciency may impair
the TCA cycle and decrease citrate production, so that
the citrate level remains low in blood and renal tissue
despite a decrease of ATP citrate lyase activity, but this
point needs further investigation.
In conclusion, the infusion of
L-hydroxyproline
increased urinary oxalate excretion in normal rats,
and caused an exaggerated increase (7.5-fold) in the
increment of urinary oxalate excretion that persisted
for more than 5 h without hyperglycolic aciduria in
vitamin B6-deWcient rats, suggesting that endogenous
oxalogenesis occurred via mitochondrial glyoxylate
rather than via peroxisomal glycolate. Vitamin B6
deWciency was also associated with hypocitraturia.
Therefore, the risk of increased oxalate excretion
associated with hypocitraturia must be kept in mind
after intake of meat by persons with low vitamin B6
levels due to malnutrition, long-term antibiotic ther-
apy, hyperalimentation, or bowel disease, among
other causes.
Acknowledgments This work was supported in part by a grant-
in-aid for ScientiWc Research from the Japanese Ministry of Edu-
cation, Culture, Sports, Science, and Technology (B213470338).
We are grateful for the technical assistance of Ms. Tomoko Mae-
da and Mr. Masami Oda.
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