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[14C]oxalate formation from [U-14C]glucose and [U-14C]xylitol in rat liver homogenate
BIOCHEMICAL
MEDICINE
21, 55-61
(1979)
[ 14C]Oxalate Formation from [U-L4C]Glucose and [U-14ClXylitoI in Rat Liver Homogenate S. Institute
for
HAUSCHILDT
Physiological
Chemistry, Federal
K.
AND
of Erlangen-Nuremberg,
University
qf
Republic
Received
BRAND
August
Germon?
3. 1978
Pathological condition such as kidney stone formation, ethylene glycol toxicity (I), and primary hyperoxaluria (2) have been shown to be associated with increased endogenous oxalate synthesis and formation of calcium oxalate crystals. The finding of calcium oxalate crystals in patients following infusion with high doses of xylitol (3-5) led to the suggestion that xylitol administration was also related to an increased oxalate production. Hannet et al. (6) have shown that pyridoxine-deficient rats infused with [U-‘4C] xylitol excreted more [‘“Cl oxalate when compared with other labeled infusates and more recently Rofe et al. (7) reported an enhanced oxalate production from xylitol in liver cells when compared with glucose, sorbitol, or fructose. Although in previous studies we could not show an increased oxalate formation after xylitol infusion in human subjects (8) we found an increased urinary excretion of glycolic acid a precursor of oxalic acid. This observation led us to investigate the effect of xylitol on oxalate formation in rat liver homogenate under conditions which would favor oxalate production from precursors possibly generated by xylitol breakdown (74. MATERIALS
AND METHODS
[U- 14C] Xylitol and [I/- 14C] glucose were purchased from Amersham Buchler (KG, Braunschweig, FRG), oxalate decarboxylase grade II from Coliybia Velutipes was obtained from Sigma Company Ltd. (Taufkirchen, FRG). Lewatit S 1080 (100-200 mesh) and Lewatit M 5080 (100-200 mesh) were purchased from E. Merck (Darmstadt, FRG) and Hepes 55
ooo6-2944/79/OlooS5-07$02.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
HAUSCHILDT
AND
BRAND
(N- 2- hydroxyethylpiperazine-N’2- ethanesul fonic acid) was from Serva (Heidelberg, FRG). The buffer used consisted of 0.05 M Hepes dissolved in Krebs-Ringer phosphate buffer, pH 7.6. The Krebs-Ringer phosphate buffer (free of calcium ions and glucose) contained 123 mM NaCl: 4.9 mM KCI; 1.24 mM MgSO,; 16 mM NaHPO,. Before use the labeled substrates were purified by ion-exchange chromatography, Male Sprague-Dawley rats (body weight 250-300 g) were fasted 24 hr before being sacrificed. Under ether anesthesia the liver was quickly excised and homogenized in buffer pH 7.6 to give a 33% homogenate. Zncubarion. The reaction mixture contained in a total volume of 3 ml: xylitol (8 mM) or glucose (6.6 mrvf), NAD (6.6 mM), phenazine methosulfate (PMS) (0.04 mM), oxygenated buffer pH 7.6 containing 40.0 mM Hepes, [U-14C] xylitol (0.083 &ilpmol) or [V-14C] glucose (0.1 &il pmole) and 1 ml liver homogenate. The incubation was carried out in an atmosphere of 95% O,-5% CO, in a stoppered Erlenmeyer flask with a center well containing a vial for CO, collection. The reaction was started by adding the appropriate substrate and was allowed to proceed in a shaking water bath for 1 hr at 37°C. The reaction was stopped by injecting 0.2 ml of 2.2~ perchloric acid into the medium and 0.7 ml of 2-phenylethylamine into the vial for i4C0, collection. After acidifying, shaking was continued for an additional hour for quantitative 14C0, liberation. Blanks contained all reactants and were treated like the samples except that 0.2 ml of 2.2 M perchloric acid was added at zero time. Uptake of substrate and formation of products were calculated by subtracting values measured in the blanks from those measured in the samples. Analytical procedure. The acidified incubation mixture was centrifuged and the pellets washed twice with 0.2~ perchloric acid. Acid supernatants plus washings were combined and brought to pH 3 with 50% KOH. After centrifugation the supernatant (5 ml) was used for 14C-oxalate anaiysis. i4C-Oxalate assays were carried out in 50 ml Erlenmeyer flasks closed with rubber caps containing a vial for i4C0, collection filled with 0.7 ml of 2-phenylethylamine. The assay mixture contained in a volume of 5 ml: critrate buffer pH 3(0.2 M), potassium oxalate (0.5 mrvf), EDTA (1.2 mM), supernatant 2.5 ml, oxalate decarboxylase 0.4 U. The reaction was started by adding oxalate decarboxylase. After an incubation time of 3 hr at 37°C in a shaking water bath the reaction was stopped by injection of 1 ml of 4 M perchloric acid into the medium. Shaking was continued for another hour. Blank values were determined by adding all reactants except oxalate decarboxylase to the reaction mixture. The 14C0, trapped by 2-phenylethylamine was counted in a Packard liquid scintillation counter
[W]
OXALATE
FORMATION
57
using the solvent of Bray. In the neutralized perchloric acid extracts not utilized substrates were determined by isotopic techniques as described by Katz et al. (9). An aliquot of the perchloric acid extract was passed through tandem columns of Lewatit S 1080 in the H2+ form followed by a column of Lewatit M 5080 in the acetate form. The columns were eluted with 6 x 10 ml of water. This fraction consists of neutral compounds. From the radioactivity found in the eluate of the incubation mixture in the blanks and in the samples the amount of substrate uptake was calculated. RESULTS
When incubating rat liver homogenate with xylitol or glucose as substrate in the absence of oxidants only 3.7 @mole glucose/g liver and 0.74 pmole xylitol/g liver were utilized. The 14C02 production was accordingly small-O.55 pmole/g liver from glucose and 0.19 pmole/g liver from xylitol. There was no oxalate formation. In order to enhance substrate uptake and metabolic processes in rat liver homogenate oxidants like NAD and phenazine methosulphate were added to the incubation mixture. As can be seen from Tables 1 and 2 substrate uptake and 14C02 release were increased. Glucose (22.6 pmole/g liver) and 45.9 pmole xylitol/g liver were metabolized corresponding to 37% of initially added glucose and 63% of initially added xylitol. Oxalate production from glucose (0.06 pmole/g liver) and xylitol (0.2 pmole/g liver) was induced by the addition of oxidants, the oxalate production being 1.6 times higher from xylitol than from glucose when referred to substrate uptake (Table 3). Relating the oxalate formation to the ‘4c0, production from the substrates a ratio of 2 was obtained (Table 3). This indicates that 14C02 release seems to be a fairly good indicator for substrate utilization. DISCUSSION
As previous studies on human subjects (8) failed to reveal a direct relationship between xylitol adminstration and oxalate accumulation in urine and plasma we decided to investigate the effect of xylitol administration on intracellular hepatic oxalate production. The studies were carried out in rat liver homogenate because in this system oxidants like NAD can readily enter metabolic pathways. The first step in the metabolism of xylitol is catalytic oxidation to D-xylulose by D-xylulose reductase. This step generates NADH and at substrate concentration above 1 mM causes an elevation in the intraceIlular NADH/NAD ratio (7,lO). Yet to stimulate oxalate biosynthesis from glyoxylate or glycollate which was found to be a metabolite of xylitol metabolism (7,s) the NADH/NAD ratio must be lowered to produce an oxidized cellular redox state. This was achieved by adding oxidants like
2
r
are
SEM
45.9
2 2.1
52.9
47.9 41.6 49.5 44 39.2
1
uptake
AND [TIOXALATE
TO,
Xylitol
as micromoles
given
2 3 4 5 6
Experiment No.
” Values
x; t SEM
17.1 22.6 + 4.7
6
uptake
23.5 17.5 26.4 20.6 30.5
Glucose
AND [T]OXALATE
I 2 3 4 5
Experiment No.
TO,
(w/w)
55.4 67 62.3 64.6 f
62.1 69.7 71.6
2.4
released
PRODUCTION NAD
gram
“CO,
per
per
I
TABLE
26.1
2
0.09 0.06 r 0.007
0.06 0.05 0.05 0.04
0.08
[ “C]Oxalate formed
100
100
112 152 158 142 2 9.6
117 I45 172
xylitol
released
utilized
pm01
“CO1
0.35 !I 0.03 0.30
0.18
0.20 0.33 0.40
“CO,
0.56 5 0.08
0.24 0.22 0.2 t 0.03
0.45
+mol released
0.35
100
~~~
0.20 0.54
xylitol
-.
0.014
“CO.
0.10
utilized
fimol
formed
OF
t
0.15 0.11 0.12 0.21
0.14 0.14
pm01 released
0.15
100
0.24 0.48 0.69
100
[ “C]Oxalate
OF
formed
IN THE PRESENCE
0.24 0.13 0.52 0.29 t 0.006
0.34 0.34 0.18
pm01 glucose utilized
[“C]Oxalate
IN THE PRESENCE
0.13 0.23 0.29
formed
[‘*C]Oxalate
IN RAT LIVER HOMOGENATE FROM [U-“C]XYLITOL AND PHENAZINE METHOSLJLFATE (PMS)”
60 min.
247 196.5 t
248 118 220 112
pm01 glucose utilized
released
234
100
‘*CO,
55.1
3.5
TABLE
IN RAT LIVER HOMOGENATE FROM [U-14C]G~~~~~~ AND PHENAZINE METHOSULFATE (PMS)”
43.4 31.4 45.5 34.2
released
42.3 41.9 t
“CO,
PRODUCTION NAD
2
% 5
%
0
5
5
I > c
ks
[‘Tc]
COMPARISON
OXALATE
TABLE 3 FORMATION FROM GLUCOSE IN RAT LIVER HOMOGENATE”
OF OXALATE
59
FORMATION
AND XYLITOL
AS SUBSTRATE
Substrate
Xylitol
Xylitoli Glucose
0.29
0.45
1.6
0.15
0.30
2.0
Glucose [“C]Oxalate
formed
100 pmol substrate utilized-~ [‘4C]Oxalate formed 100 pmol
‘“CO,
” Mean
values
released of Table
I and 2 are used for calculating
the xylitol/glucose
ratio.
NAD and phenazinemethosulfate (PMS) to the incubation medium. NAD may also promote oxalate biosynthesis being cosubstrate of LDH which catalyzes glycollate oxidation while PMS which is known to accept electrons from flavoproteins (11) may act directly on glycollate oxidase, a flavin-linked enzyme also catalyzing glycollate oxidation. Combination of the two substances stimulated substrate uptake (glucose six-fold, xylitol 62-fold) and thereby CO, production (glucose 76fold, xylitol 340-fold). Glucose uptake was half that of xylitol uptake (Tables 1 and 2). Only 0.0% of initially added glucose and 0.27% of initially added xylitol were converted to oxalate. The observed low oxalate formation is consistent with results reported by Oshinsky ef ul. (12) who found in infusion experiments that oxalate production from xylitol is negligible in rabbits. These values do increase from 0.09 to 0.2% for glucose and from 0.27 to 0.45% for xylitol when referring the amount of oxalate formed to the amount of substrate actually utilized (Tables 1 and 2, Column 6). Values of oxalate production from xylitol in comparison to glucose reported in the literature (7) may actually be lower when referred to substrate uptake. Although the amount of oxalate derived from xylitol is only 1.6 times higher than from glucose (Table 3) speculations on the origin of oxalate from xylitol arise. As glycollate has been shown to be a possible metabohte of xylitol metabolism (7,g) a mechanism whereby glycolaldehyde, an established precursor of glycollate, is oxidized to glycollate via the transketolase reaction cannot be excluded. In plants it has been shown (13) that superoxide radicals can serve as oxidants in the transketolase-catalyzed formation of glycollate from the a$dihydroxyethyl thiaminepyrophosphate complex. On the other hand Krause et al. (14) conclude from their results that glycollate synthesis from xylulose-5-phosphate by intact isolated chloroplasts is primarily the
60
HAUSCHILDT
AND
BRAND
result of ribulose-I.5bisphosphate oxygenase activity and no substantial role can be attributed to the oxidation of the a,@dihydroxyethyl thiaminepyrophosphate complex. However, as long as ribose-5in the phosphate is present as acceptor for the “active glycolaldehyde” transketolase reaction it is very unlikely that this C,-compound is liberated from the a,&dihydroxyethyl thimainepyrophosphate complex and oxidized to glycollate instead of being transferred to the acceptor forming sedoheptulose-7-phosphate. Oxalate synthesis from intermediates of the glycolytic pathway, i.e., from 3-phosphoglycerate via hydroxypyruvate (14, Fig. 1) is another alternative but would not account for increased formation of oxalate from xylitol since both carbohydrates share this pathway. Whatever the pathways are which lead to oxalate formation from glucose or xylitol remains to be clarified. Our results, however, clearly indicate that only in the presence of oxidants like NAD and phenazine methosulfate small amounts of oxalate are formed from glucose and xylitol as substrates. The abnormal shift in the NADH/NAD ratio to the oxidized state which is required to produce oxalate synthesis from these substrates is unlikely G ucose 1 G&P
rc"2 ,&PC
------<
S-glycerate-2-P
Glycolysis
FIG.
I
Possible
pathways
of glucose
and
xylitol
leading
to
oxalate.
[“Cc] OXALATE
FORMATION
61
to occur within the cell. This is particularly true for xylitol since extra NADH is generated by its metabolism. From our results it therefore appears that xylitol application does not favor oxalate synthesis under physiological conditions. SUMMARY When incubating rat liver homogenate with [,-I4 C] glucose or [U-‘4Cl xylitol as substrates in the absence of oxidants no oxalate was formed. Addition of NAD and phenazine methosulfate to the incubation medium led to an increased substrate uptake and CO, production. Under these conditions oxalate formation was observed from both substrates the oxalate production from xylitol being 1.6 times higher than that from glucose. ACKNOWLEDGMENTS We are greatly indebted to Mrs. Miinekhoff for her skilled techincal assistance. Part of this work was generously supported by grants of the Jacques-Pfrimmer-Gedkhtnisstiftung, Erlangen, Germany for which we are most grateful.
REFERENCES 1. Richardson, K. E., Toxicol. Appl. Pharmacol. 24, 530 (1973). 2. Williams, H. E., and Smith, L. H., Jr., Amer. J. Med. 45, 715 (1968). 3. Thomas, D. W., Edwards, J. B., Gilligan, J. E., Lawrence, J. R., and Edwards, R. G., yed J. Aust. 3, 1238 (1972). 4. Evans, G. W., Phillips, G., Mukhejee, T. M.. Snow, M. R., Lawrence, J. R., and Thomas, D. W., J. Clin. Puthol. 26, 32 (1973). 5. Schroder, R. W., De Lacroix, W. F., Franzen, U., Klein, P. J., and Miiller, W., Acto Neuropathol. 27, 181, (1974). 6. Hannett, D., Thomas, D. W., Chalmers, A. H., Rofe, A. M., Edwards, J. B.. and Edwards, R. G., J. Nutr. 107, 458 (1977). 7. Rofe, A. M., Thomas, D. W., Edwards R. G., and Edwards, J. B., Biochem. Med. 18, 440 (1977). 8. Hauschildt, S., Chalmers, R. A., Lawson, A. M., Schultis, K., and Watts, R. W. E., Amer. J. Clin. Nutr. 2, 258 (1976). 9. Katz, J., Brand, K., Goldberg, S., and Rubinstein, D., Cancer Res. 34, 872 (19741. 10. Woods, H. F., and Krebs, H. A., B&hem. J. 134,437 (1973). 1I. Takemor, S., and King, T. E., J. Biol. Chem. 239, 3546 (1964). 12. Oshinsky, R. J., Wang, Y. M., and Van Eyp, J., J. Nurr. 107, 792 (1977). 13. Asami, S., and Akazawa, T., Biochemisfry 16, 2202 (1977). 14. Krause, G. H., Thorne, S. W., and Lorimer, G. H.. Arch. Biochem. Biophys. 183,471 (1977). 15. Liao, L. L., and Richardson, K. E., Biochim. Biophys. Acra 538, 76 (1978).
MEDICINE
21, 55-61
(1979)
[ 14C]Oxalate Formation from [U-L4C]Glucose and [U-14ClXylitoI in Rat Liver Homogenate S. Institute
for
HAUSCHILDT
Physiological
Chemistry, Federal
K.
AND
of Erlangen-Nuremberg,
University
qf
Republic
Received
BRAND
August
Germon?
3. 1978
Pathological condition such as kidney stone formation, ethylene glycol toxicity (I), and primary hyperoxaluria (2) have been shown to be associated with increased endogenous oxalate synthesis and formation of calcium oxalate crystals. The finding of calcium oxalate crystals in patients following infusion with high doses of xylitol (3-5) led to the suggestion that xylitol administration was also related to an increased oxalate production. Hannet et al. (6) have shown that pyridoxine-deficient rats infused with [U-‘4C] xylitol excreted more [‘“Cl oxalate when compared with other labeled infusates and more recently Rofe et al. (7) reported an enhanced oxalate production from xylitol in liver cells when compared with glucose, sorbitol, or fructose. Although in previous studies we could not show an increased oxalate formation after xylitol infusion in human subjects (8) we found an increased urinary excretion of glycolic acid a precursor of oxalic acid. This observation led us to investigate the effect of xylitol on oxalate formation in rat liver homogenate under conditions which would favor oxalate production from precursors possibly generated by xylitol breakdown (74. MATERIALS
AND METHODS
[U- 14C] Xylitol and [I/- 14C] glucose were purchased from Amersham Buchler (KG, Braunschweig, FRG), oxalate decarboxylase grade II from Coliybia Velutipes was obtained from Sigma Company Ltd. (Taufkirchen, FRG). Lewatit S 1080 (100-200 mesh) and Lewatit M 5080 (100-200 mesh) were purchased from E. Merck (Darmstadt, FRG) and Hepes 55
ooo6-2944/79/OlooS5-07$02.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
HAUSCHILDT
AND
BRAND
(N- 2- hydroxyethylpiperazine-N’2- ethanesul fonic acid) was from Serva (Heidelberg, FRG). The buffer used consisted of 0.05 M Hepes dissolved in Krebs-Ringer phosphate buffer, pH 7.6. The Krebs-Ringer phosphate buffer (free of calcium ions and glucose) contained 123 mM NaCl: 4.9 mM KCI; 1.24 mM MgSO,; 16 mM NaHPO,. Before use the labeled substrates were purified by ion-exchange chromatography, Male Sprague-Dawley rats (body weight 250-300 g) were fasted 24 hr before being sacrificed. Under ether anesthesia the liver was quickly excised and homogenized in buffer pH 7.6 to give a 33% homogenate. Zncubarion. The reaction mixture contained in a total volume of 3 ml: xylitol (8 mM) or glucose (6.6 mrvf), NAD (6.6 mM), phenazine methosulfate (PMS) (0.04 mM), oxygenated buffer pH 7.6 containing 40.0 mM Hepes, [U-14C] xylitol (0.083 &ilpmol) or [V-14C] glucose (0.1 &il pmole) and 1 ml liver homogenate. The incubation was carried out in an atmosphere of 95% O,-5% CO, in a stoppered Erlenmeyer flask with a center well containing a vial for CO, collection. The reaction was started by adding the appropriate substrate and was allowed to proceed in a shaking water bath for 1 hr at 37°C. The reaction was stopped by injecting 0.2 ml of 2.2~ perchloric acid into the medium and 0.7 ml of 2-phenylethylamine into the vial for i4C0, collection. After acidifying, shaking was continued for an additional hour for quantitative 14C0, liberation. Blanks contained all reactants and were treated like the samples except that 0.2 ml of 2.2 M perchloric acid was added at zero time. Uptake of substrate and formation of products were calculated by subtracting values measured in the blanks from those measured in the samples. Analytical procedure. The acidified incubation mixture was centrifuged and the pellets washed twice with 0.2~ perchloric acid. Acid supernatants plus washings were combined and brought to pH 3 with 50% KOH. After centrifugation the supernatant (5 ml) was used for 14C-oxalate anaiysis. i4C-Oxalate assays were carried out in 50 ml Erlenmeyer flasks closed with rubber caps containing a vial for i4C0, collection filled with 0.7 ml of 2-phenylethylamine. The assay mixture contained in a volume of 5 ml: critrate buffer pH 3(0.2 M), potassium oxalate (0.5 mrvf), EDTA (1.2 mM), supernatant 2.5 ml, oxalate decarboxylase 0.4 U. The reaction was started by adding oxalate decarboxylase. After an incubation time of 3 hr at 37°C in a shaking water bath the reaction was stopped by injection of 1 ml of 4 M perchloric acid into the medium. Shaking was continued for another hour. Blank values were determined by adding all reactants except oxalate decarboxylase to the reaction mixture. The 14C0, trapped by 2-phenylethylamine was counted in a Packard liquid scintillation counter
[W]
OXALATE
FORMATION
57
using the solvent of Bray. In the neutralized perchloric acid extracts not utilized substrates were determined by isotopic techniques as described by Katz et al. (9). An aliquot of the perchloric acid extract was passed through tandem columns of Lewatit S 1080 in the H2+ form followed by a column of Lewatit M 5080 in the acetate form. The columns were eluted with 6 x 10 ml of water. This fraction consists of neutral compounds. From the radioactivity found in the eluate of the incubation mixture in the blanks and in the samples the amount of substrate uptake was calculated. RESULTS
When incubating rat liver homogenate with xylitol or glucose as substrate in the absence of oxidants only 3.7 @mole glucose/g liver and 0.74 pmole xylitol/g liver were utilized. The 14C02 production was accordingly small-O.55 pmole/g liver from glucose and 0.19 pmole/g liver from xylitol. There was no oxalate formation. In order to enhance substrate uptake and metabolic processes in rat liver homogenate oxidants like NAD and phenazine methosulphate were added to the incubation mixture. As can be seen from Tables 1 and 2 substrate uptake and 14C02 release were increased. Glucose (22.6 pmole/g liver) and 45.9 pmole xylitol/g liver were metabolized corresponding to 37% of initially added glucose and 63% of initially added xylitol. Oxalate production from glucose (0.06 pmole/g liver) and xylitol (0.2 pmole/g liver) was induced by the addition of oxidants, the oxalate production being 1.6 times higher from xylitol than from glucose when referred to substrate uptake (Table 3). Relating the oxalate formation to the ‘4c0, production from the substrates a ratio of 2 was obtained (Table 3). This indicates that 14C02 release seems to be a fairly good indicator for substrate utilization. DISCUSSION
As previous studies on human subjects (8) failed to reveal a direct relationship between xylitol adminstration and oxalate accumulation in urine and plasma we decided to investigate the effect of xylitol administration on intracellular hepatic oxalate production. The studies were carried out in rat liver homogenate because in this system oxidants like NAD can readily enter metabolic pathways. The first step in the metabolism of xylitol is catalytic oxidation to D-xylulose by D-xylulose reductase. This step generates NADH and at substrate concentration above 1 mM causes an elevation in the intraceIlular NADH/NAD ratio (7,lO). Yet to stimulate oxalate biosynthesis from glyoxylate or glycollate which was found to be a metabolite of xylitol metabolism (7,s) the NADH/NAD ratio must be lowered to produce an oxidized cellular redox state. This was achieved by adding oxidants like
2
r
are
SEM
45.9
2 2.1
52.9
47.9 41.6 49.5 44 39.2
1
uptake
AND [TIOXALATE
TO,
Xylitol
as micromoles
given
2 3 4 5 6
Experiment No.
” Values
x; t SEM
17.1 22.6 + 4.7
6
uptake
23.5 17.5 26.4 20.6 30.5
Glucose
AND [T]OXALATE
I 2 3 4 5
Experiment No.
TO,
(w/w)
55.4 67 62.3 64.6 f
62.1 69.7 71.6
2.4
released
PRODUCTION NAD
gram
“CO,
per
per
I
TABLE
26.1
2
0.09 0.06 r 0.007
0.06 0.05 0.05 0.04
0.08
[ “C]Oxalate formed
100
100
112 152 158 142 2 9.6
117 I45 172
xylitol
released
utilized
pm01
“CO1
0.35 !I 0.03 0.30
0.18
0.20 0.33 0.40
“CO,
0.56 5 0.08
0.24 0.22 0.2 t 0.03
0.45
+mol released
0.35
100
~~~
0.20 0.54
xylitol
-.
0.014
“CO.
0.10
utilized
fimol
formed
OF
t
0.15 0.11 0.12 0.21
0.14 0.14
pm01 released
0.15
100
0.24 0.48 0.69
100
[ “C]Oxalate
OF
formed
IN THE PRESENCE
0.24 0.13 0.52 0.29 t 0.006
0.34 0.34 0.18
pm01 glucose utilized
[“C]Oxalate
IN THE PRESENCE
0.13 0.23 0.29
formed
[‘*C]Oxalate
IN RAT LIVER HOMOGENATE FROM [U-“C]XYLITOL AND PHENAZINE METHOSLJLFATE (PMS)”
60 min.
247 196.5 t
248 118 220 112
pm01 glucose utilized
released
234
100
‘*CO,
55.1
3.5
TABLE
IN RAT LIVER HOMOGENATE FROM [U-14C]G~~~~~~ AND PHENAZINE METHOSULFATE (PMS)”
43.4 31.4 45.5 34.2
released
42.3 41.9 t
“CO,
PRODUCTION NAD
2
% 5
%
0
5
5
I > c
ks
[‘Tc]
COMPARISON
OXALATE
TABLE 3 FORMATION FROM GLUCOSE IN RAT LIVER HOMOGENATE”
OF OXALATE
59
FORMATION
AND XYLITOL
AS SUBSTRATE
Substrate
Xylitol
Xylitoli Glucose
0.29
0.45
1.6
0.15
0.30
2.0
Glucose [“C]Oxalate
formed
100 pmol substrate utilized-~ [‘4C]Oxalate formed 100 pmol
‘“CO,
” Mean
values
released of Table
I and 2 are used for calculating
the xylitol/glucose
ratio.
NAD and phenazinemethosulfate (PMS) to the incubation medium. NAD may also promote oxalate biosynthesis being cosubstrate of LDH which catalyzes glycollate oxidation while PMS which is known to accept electrons from flavoproteins (11) may act directly on glycollate oxidase, a flavin-linked enzyme also catalyzing glycollate oxidation. Combination of the two substances stimulated substrate uptake (glucose six-fold, xylitol 62-fold) and thereby CO, production (glucose 76fold, xylitol 340-fold). Glucose uptake was half that of xylitol uptake (Tables 1 and 2). Only 0.0% of initially added glucose and 0.27% of initially added xylitol were converted to oxalate. The observed low oxalate formation is consistent with results reported by Oshinsky ef ul. (12) who found in infusion experiments that oxalate production from xylitol is negligible in rabbits. These values do increase from 0.09 to 0.2% for glucose and from 0.27 to 0.45% for xylitol when referring the amount of oxalate formed to the amount of substrate actually utilized (Tables 1 and 2, Column 6). Values of oxalate production from xylitol in comparison to glucose reported in the literature (7) may actually be lower when referred to substrate uptake. Although the amount of oxalate derived from xylitol is only 1.6 times higher than from glucose (Table 3) speculations on the origin of oxalate from xylitol arise. As glycollate has been shown to be a possible metabohte of xylitol metabolism (7,g) a mechanism whereby glycolaldehyde, an established precursor of glycollate, is oxidized to glycollate via the transketolase reaction cannot be excluded. In plants it has been shown (13) that superoxide radicals can serve as oxidants in the transketolase-catalyzed formation of glycollate from the a$dihydroxyethyl thiaminepyrophosphate complex. On the other hand Krause et al. (14) conclude from their results that glycollate synthesis from xylulose-5-phosphate by intact isolated chloroplasts is primarily the
60
HAUSCHILDT
AND
BRAND
result of ribulose-I.5bisphosphate oxygenase activity and no substantial role can be attributed to the oxidation of the a,@dihydroxyethyl thiaminepyrophosphate complex. However, as long as ribose-5in the phosphate is present as acceptor for the “active glycolaldehyde” transketolase reaction it is very unlikely that this C,-compound is liberated from the a,&dihydroxyethyl thimainepyrophosphate complex and oxidized to glycollate instead of being transferred to the acceptor forming sedoheptulose-7-phosphate. Oxalate synthesis from intermediates of the glycolytic pathway, i.e., from 3-phosphoglycerate via hydroxypyruvate (14, Fig. 1) is another alternative but would not account for increased formation of oxalate from xylitol since both carbohydrates share this pathway. Whatever the pathways are which lead to oxalate formation from glucose or xylitol remains to be clarified. Our results, however, clearly indicate that only in the presence of oxidants like NAD and phenazine methosulfate small amounts of oxalate are formed from glucose and xylitol as substrates. The abnormal shift in the NADH/NAD ratio to the oxidized state which is required to produce oxalate synthesis from these substrates is unlikely G ucose 1 G&P
rc"2 ,&PC
------<
S-glycerate-2-P
Glycolysis
FIG.
I
Possible
pathways
of glucose
and
xylitol
leading
to
oxalate.
[“Cc] OXALATE
FORMATION
61
to occur within the cell. This is particularly true for xylitol since extra NADH is generated by its metabolism. From our results it therefore appears that xylitol application does not favor oxalate synthesis under physiological conditions. SUMMARY When incubating rat liver homogenate with [,-I4 C] glucose or [U-‘4Cl xylitol as substrates in the absence of oxidants no oxalate was formed. Addition of NAD and phenazine methosulfate to the incubation medium led to an increased substrate uptake and CO, production. Under these conditions oxalate formation was observed from both substrates the oxalate production from xylitol being 1.6 times higher than that from glucose. ACKNOWLEDGMENTS We are greatly indebted to Mrs. Miinekhoff for her skilled techincal assistance. Part of this work was generously supported by grants of the Jacques-Pfrimmer-Gedkhtnisstiftung, Erlangen, Germany for which we are most grateful.
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