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The pattern of urinary citric acid cycle and related acids in experimental porphyria in the rat
SHORT COMMUNICATIONS BBA
647
23506
The pattern of urinary citric acid cycle and related acids in experimental porphyria in the rat* The biochemical defect in the acute intermittent type of porphyria both that produced experimentally in rats by certain chemical substances and that occurring clinically in man, has been established as an hepatic over-production of S-aminolevulinate synthetase 1-3. This enzyme is responsible for the condensation of glycine and succinyl-CoA which are the precursors of porphyrins 4. Succinyl-CoA is a member of the Krebs citric acid cycle and may represent a link between this cycle and the porphyrin biosynthetic pathway. The effect of an increased formation of hepatic S-aminolevulinate synthetase on the body levels of acids of the Krebs cycle has not been studied and is the basis of the present investigation. If there is indeed an overproduction of S-aminolevulinate synthetase, this fact could be reflected in a change of the urinary levels of at least some acids of the Krebs cycle, particularly succinate since the heme biosynthetic pathway may derive this precursor by the way of the cycle. Similarly, if succinate is derived from the pyruvate-malate-fumarate bypass, as suggested by the work of KURUMADA AND LABBE5, a study of the pattern of the Krebs cycle and related acids could indicate such an origin. With these possibilities in mind, the patterns of the Krebs cycle and certain related acids were determined in the urine of control rats and of rats in which porphyria, very similar to acute porphyria in man, was produced by the feeding of sedormid (allylisopropylacetylcarbamide). i2 male albino rats (Sprague-Dawley strain) weighing between 2oo and 24 ° g were placed in individual metabolism cages (Hoeltge) in which 24-h urine specimens can be obtained free of feces. The animals were fed commercial rat chow and were allowed to drink water ad libitum. Experimental porphyria was induced in 8 of the i2 rats by the feeding of sedormid via a stomach tube in a daily dose of 3o mg/Ioo g body wt. A sedormid suspension was prepared by mixing 5 g sedormid with IOO ml of propylene glycols. The remaining rats were used as controls with the vehicle, and were administered i ml of propylene glycol daily via stomach tube. In addition, each rat of course served as its own control during the period before porphyria was produced. The 24-h urine samples were collected every other day under toluene and analyzed for coproporphyrin~, ~-aminolevulinic acid-porphobilinogen8 and organic acids by a silica gel chromatographic method 9. Analyses were continued for 2 weeks after the urine specimens demonstrated the chemical characteristics of acute intermittent porphyria, i.e. elevation of $-aminolevulinic acid, porphobilinogen and coproporphyrin. The sedormid-treated animals excreted porphobilinogen, $-aminolevulinic acid and coproporphyrin in greater quantities than either the controls or the same rats before sedormid was administered, as shown in Table I. This finding is in agreement with those of other investigators, both in rats and in man 1°, 11. The quantity of urinary porphobilinogen in a 24-h urine specimen was approx. 13o times greater in the * Portions of the d a t a in this p a p e r are t a k e n from a dissertation s u b m i t t e d to the G r a d u a t e Division of W a y n e State University b y one of the a u t h o r s (C.B.V.) for the degree Doctor of Philosophy, J u n e 1965 .
Biochim. Biophys. Acga, x77 (1969) 647-649
648 TABLE
SHORT COMMUNICATIONS I
COPROPORPHYRIN
PORPHYRIC
AND
PORPHYRIN
INTERMEDIATES
IN
THE URINE OF CONTROL AND
RATS
V a l u e s a r e / z g / 2 4 h.
cS-Aminolevulinic acid Porphobilinogen Coproporphyrin
Control (z2 rats)
Sedormid treated (8 rats)
Average Range
Average Range
73 ii 15
273 1331 lO2
lO.1-151.2 6.5- 32.7 1. 5 - 36. 7
11.4- 987.0 6.8-2887.5 4.3- 212-7
Confidence
(P)
0.37 o.03 o.oi
sedormid-treated animals than during the pre-treatment period and in the control group. The difference, using the method of SWED AND EISENHART 12, proved to be statistically significant, P ~ 0.03. Differences in coproporphyrin excretion were also significant. A difference in ~-aminolevulinic acid excretion between the control group and the sedormid group was apparent, but not significant by statistical analysis because of individual variations between rats in their response to sedormid. Perhaps also the inherent complexity of the analytical procedure for the determination of ~-aminolevulinic acid may be complicating factor. There were no differences between the values obtained on the animals receiving propylene glycol and those of the same animals before receiving propylene glycol. A comparison of the excretion of the Krebs cycle and related acids in the urine of the control and sedormid-treated rats (Table II) demonstrated a decreased amount of succinate in the urine of the porphyric animals. This decrease was highly significant, P ~ o.oi. There was also a decrease in the amount of citrate (P ---- o.17) and possibly of fumarate (P---- 0.64) in urine of the treated animals. Variations in the levels of TABLE
II
ORGANIC ACIDS IN URINE OF CONTROL AND EXPERIMENTAL RATS V a l u e s a r e m g / 2 4 h.
Control (z2 rats)
Sedormid treated (8 rats)
Average Range
Average Range
Confidence
(P)
Non-I4rebs cycle acids Lactic Hydroxybutyric Pyruvic Acetoacetic Acetic
16 i 2 4 i
8.2-25.7 o . 3 - 3.8 o . 9 - 2.9 2.0- 5.8 o . i - 1.8
9 I 4 4 i
2.o-26.6 o . i - 3-5 o.2- 9.2 1.3-14.1 o . i - 1. 9
o.17 o.96 o.83 o.64 o.97
44 I I i IO 2 i
27.5-58.8 0. 7 - 2.1 0 . 8 - 2.2 o . 8 - 1. 4 2.9-12.6 i . o - 3.7 o . i - 1.4
21 2 2 i 3 i i
13.5-45.8 0 . 2 - 8.2 0 . 4 - 3.9 o. 3 - 2 . 6 o . i - 7.9 o . 2 - 1. 7 0 . 2 - I.O
o.17 o.17 o.36 0.83 o.oi 0.64
Krebs cycle acids Citric Aconitic Isocitric Ketoglutaric Succinic Fumaric Malic
Biochim. Biophys. Acta,
177 (1969) 6 4 7 - 6 4 9
0.8 3
SHORT COMMUNICATIONS
649
the other cycle acids and related acids with the exception of lactate and pyruvate, were variable and not significant. There was some decrease (P = o.17) in lactate and possibly an increase in pyruvate (P -~ 0.83). The results of the present investigation can be explained by the fact that the administration of sedormid produces an increase in the amount and/or activity of hepatic ~-aminolevulinate synthetase. An increase in this enzyme would increase the biosynthesis of 8-aminolevulinic acid from succinate and glycine thus diverting some succinate into the porphyrin biosynthetic pathway, decreasing the tissue level and, in turn, decreasing succinate loss in the urine, as was found. The diversion of succinate into porphyrin formation would lessen the amount of oxalacetate for citrate formation via the Krebs citric acid cycle and correspondingly decrease its level in the urine, as occurred. The decrease in lactate observed may be explained by the suggested conversion of pyruvate to succinate and thence into porphyrin formation s. The conversion of pyruvate to succinate requires NADH which would decrease its availability for the conversion of pyruvate to lactate. The fact that the urinary levels of subsequent acids of the Krebs cycle after citrate were not significantly altered also lends support to the interpretation that the diversion of metabolites into porphyrin biosynthesis is via pyruvate to -malate-fumarate-succinate s. Calculations made on the data in Tables I and II show that the decrease in succinate in the urine is considerably greater than the total average increase in 8-aminolevulinic acid, porphobilinogen and coproporphyrin, as manifest by their urinary excretion. It is rather difficult to quantitatively assess such values because unknown amounts of succinate may have been incorporated into other members of the porphyrin biosynthetic chain, not determined, including the end products heme and hemoglobin themselves. Also, some succinate may have been channeled into other metabolic pathways. Further investigations, using labeled precursors, are projected to study these questions. This work was supported by National Institutes of Health research grant H E lO569 and by a Post-Doctor Fellowship (C.B.V.) from the National Institutes of Health, 1962-1965 .
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Mich. (U.S.A.)
CLARENCE B. VAUGI-IX JAMES M. ORTEN
I S. GRANICK, J. Biol. Chem., 241 (1966) 1359. 2 D. P. TSCHUDY, M. G. PERLROTH, H. S. MARRER, A. COLLINS AND G. HUNTER, Proc. Natl. Acad. Sci. U.S., 53 (1965) 841. 3 H. S. MARRER, A. COLLINS, D. P. TSCHUDY AND M. RECHEIGL, J. Biol. Chem., 241 (1966) 4323, 4 D. SHEMIN AND S. KUMIN, J. Biol. Chem., 198 (1952) 828. 5 T. KURUMADA AND R. F. LABBE, Science, 151 (1966) 1228. 6 J. CASE, R. ALDRICH AND R. NEVE, Proc. Soc. Exptl. Biol. Med., 83 (1953) 566. 7 V. SARDESAI, S. A. DOEHR AND J. M. ORTEN, Am. J. Physiol., 208 (1965) 127o. 8 D. MAUZERALL AND S. GRANICK, J. Biol. Chem., 219 (1956) 435. 9 J.M. ORTEN,W. GAMBLE, C. B. VAUGHN AND K. C. SHRIVASTAVA, Microchem. J., 13 (i968) 183. i o A. GOLDBERG AND C. RIMINGTON, Diseases of Porphyrin Metabolism, Charles C. T h o m a s , . Springfield, nl., 1958, p. 6. i i A. 12qEUBERGER, J. SCOTT AND C. GRAY, Biochem. J., 58 (1954) X L I . I2 F. SWED ANn C. EISENHART, Ann. Math. Statistics, 14 (1943) 66.
Received February I4th , 1969 Biochim. Biophys. Acta, 177 (1969) 647-649,
647
23506
The pattern of urinary citric acid cycle and related acids in experimental porphyria in the rat* The biochemical defect in the acute intermittent type of porphyria both that produced experimentally in rats by certain chemical substances and that occurring clinically in man, has been established as an hepatic over-production of S-aminolevulinate synthetase 1-3. This enzyme is responsible for the condensation of glycine and succinyl-CoA which are the precursors of porphyrins 4. Succinyl-CoA is a member of the Krebs citric acid cycle and may represent a link between this cycle and the porphyrin biosynthetic pathway. The effect of an increased formation of hepatic S-aminolevulinate synthetase on the body levels of acids of the Krebs cycle has not been studied and is the basis of the present investigation. If there is indeed an overproduction of S-aminolevulinate synthetase, this fact could be reflected in a change of the urinary levels of at least some acids of the Krebs cycle, particularly succinate since the heme biosynthetic pathway may derive this precursor by the way of the cycle. Similarly, if succinate is derived from the pyruvate-malate-fumarate bypass, as suggested by the work of KURUMADA AND LABBE5, a study of the pattern of the Krebs cycle and related acids could indicate such an origin. With these possibilities in mind, the patterns of the Krebs cycle and certain related acids were determined in the urine of control rats and of rats in which porphyria, very similar to acute porphyria in man, was produced by the feeding of sedormid (allylisopropylacetylcarbamide). i2 male albino rats (Sprague-Dawley strain) weighing between 2oo and 24 ° g were placed in individual metabolism cages (Hoeltge) in which 24-h urine specimens can be obtained free of feces. The animals were fed commercial rat chow and were allowed to drink water ad libitum. Experimental porphyria was induced in 8 of the i2 rats by the feeding of sedormid via a stomach tube in a daily dose of 3o mg/Ioo g body wt. A sedormid suspension was prepared by mixing 5 g sedormid with IOO ml of propylene glycols. The remaining rats were used as controls with the vehicle, and were administered i ml of propylene glycol daily via stomach tube. In addition, each rat of course served as its own control during the period before porphyria was produced. The 24-h urine samples were collected every other day under toluene and analyzed for coproporphyrin~, ~-aminolevulinic acid-porphobilinogen8 and organic acids by a silica gel chromatographic method 9. Analyses were continued for 2 weeks after the urine specimens demonstrated the chemical characteristics of acute intermittent porphyria, i.e. elevation of $-aminolevulinic acid, porphobilinogen and coproporphyrin. The sedormid-treated animals excreted porphobilinogen, $-aminolevulinic acid and coproporphyrin in greater quantities than either the controls or the same rats before sedormid was administered, as shown in Table I. This finding is in agreement with those of other investigators, both in rats and in man 1°, 11. The quantity of urinary porphobilinogen in a 24-h urine specimen was approx. 13o times greater in the * Portions of the d a t a in this p a p e r are t a k e n from a dissertation s u b m i t t e d to the G r a d u a t e Division of W a y n e State University b y one of the a u t h o r s (C.B.V.) for the degree Doctor of Philosophy, J u n e 1965 .
Biochim. Biophys. Acga, x77 (1969) 647-649
648 TABLE
SHORT COMMUNICATIONS I
COPROPORPHYRIN
PORPHYRIC
AND
PORPHYRIN
INTERMEDIATES
IN
THE URINE OF CONTROL AND
RATS
V a l u e s a r e / z g / 2 4 h.
cS-Aminolevulinic acid Porphobilinogen Coproporphyrin
Control (z2 rats)
Sedormid treated (8 rats)
Average Range
Average Range
73 ii 15
273 1331 lO2
lO.1-151.2 6.5- 32.7 1. 5 - 36. 7
11.4- 987.0 6.8-2887.5 4.3- 212-7
Confidence
(P)
0.37 o.03 o.oi
sedormid-treated animals than during the pre-treatment period and in the control group. The difference, using the method of SWED AND EISENHART 12, proved to be statistically significant, P ~ 0.03. Differences in coproporphyrin excretion were also significant. A difference in ~-aminolevulinic acid excretion between the control group and the sedormid group was apparent, but not significant by statistical analysis because of individual variations between rats in their response to sedormid. Perhaps also the inherent complexity of the analytical procedure for the determination of ~-aminolevulinic acid may be complicating factor. There were no differences between the values obtained on the animals receiving propylene glycol and those of the same animals before receiving propylene glycol. A comparison of the excretion of the Krebs cycle and related acids in the urine of the control and sedormid-treated rats (Table II) demonstrated a decreased amount of succinate in the urine of the porphyric animals. This decrease was highly significant, P ~ o.oi. There was also a decrease in the amount of citrate (P ---- o.17) and possibly of fumarate (P---- 0.64) in urine of the treated animals. Variations in the levels of TABLE
II
ORGANIC ACIDS IN URINE OF CONTROL AND EXPERIMENTAL RATS V a l u e s a r e m g / 2 4 h.
Control (z2 rats)
Sedormid treated (8 rats)
Average Range
Average Range
Confidence
(P)
Non-I4rebs cycle acids Lactic Hydroxybutyric Pyruvic Acetoacetic Acetic
16 i 2 4 i
8.2-25.7 o . 3 - 3.8 o . 9 - 2.9 2.0- 5.8 o . i - 1.8
9 I 4 4 i
2.o-26.6 o . i - 3-5 o.2- 9.2 1.3-14.1 o . i - 1. 9
o.17 o.96 o.83 o.64 o.97
44 I I i IO 2 i
27.5-58.8 0. 7 - 2.1 0 . 8 - 2.2 o . 8 - 1. 4 2.9-12.6 i . o - 3.7 o . i - 1.4
21 2 2 i 3 i i
13.5-45.8 0 . 2 - 8.2 0 . 4 - 3.9 o. 3 - 2 . 6 o . i - 7.9 o . 2 - 1. 7 0 . 2 - I.O
o.17 o.17 o.36 0.83 o.oi 0.64
Krebs cycle acids Citric Aconitic Isocitric Ketoglutaric Succinic Fumaric Malic
Biochim. Biophys. Acta,
177 (1969) 6 4 7 - 6 4 9
0.8 3
SHORT COMMUNICATIONS
649
the other cycle acids and related acids with the exception of lactate and pyruvate, were variable and not significant. There was some decrease (P = o.17) in lactate and possibly an increase in pyruvate (P -~ 0.83). The results of the present investigation can be explained by the fact that the administration of sedormid produces an increase in the amount and/or activity of hepatic ~-aminolevulinate synthetase. An increase in this enzyme would increase the biosynthesis of 8-aminolevulinic acid from succinate and glycine thus diverting some succinate into the porphyrin biosynthetic pathway, decreasing the tissue level and, in turn, decreasing succinate loss in the urine, as was found. The diversion of succinate into porphyrin formation would lessen the amount of oxalacetate for citrate formation via the Krebs citric acid cycle and correspondingly decrease its level in the urine, as occurred. The decrease in lactate observed may be explained by the suggested conversion of pyruvate to succinate and thence into porphyrin formation s. The conversion of pyruvate to succinate requires NADH which would decrease its availability for the conversion of pyruvate to lactate. The fact that the urinary levels of subsequent acids of the Krebs cycle after citrate were not significantly altered also lends support to the interpretation that the diversion of metabolites into porphyrin biosynthesis is via pyruvate to -malate-fumarate-succinate s. Calculations made on the data in Tables I and II show that the decrease in succinate in the urine is considerably greater than the total average increase in 8-aminolevulinic acid, porphobilinogen and coproporphyrin, as manifest by their urinary excretion. It is rather difficult to quantitatively assess such values because unknown amounts of succinate may have been incorporated into other members of the porphyrin biosynthetic chain, not determined, including the end products heme and hemoglobin themselves. Also, some succinate may have been channeled into other metabolic pathways. Further investigations, using labeled precursors, are projected to study these questions. This work was supported by National Institutes of Health research grant H E lO569 and by a Post-Doctor Fellowship (C.B.V.) from the National Institutes of Health, 1962-1965 .
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Mich. (U.S.A.)
CLARENCE B. VAUGI-IX JAMES M. ORTEN
I S. GRANICK, J. Biol. Chem., 241 (1966) 1359. 2 D. P. TSCHUDY, M. G. PERLROTH, H. S. MARRER, A. COLLINS AND G. HUNTER, Proc. Natl. Acad. Sci. U.S., 53 (1965) 841. 3 H. S. MARRER, A. COLLINS, D. P. TSCHUDY AND M. RECHEIGL, J. Biol. Chem., 241 (1966) 4323, 4 D. SHEMIN AND S. KUMIN, J. Biol. Chem., 198 (1952) 828. 5 T. KURUMADA AND R. F. LABBE, Science, 151 (1966) 1228. 6 J. CASE, R. ALDRICH AND R. NEVE, Proc. Soc. Exptl. Biol. Med., 83 (1953) 566. 7 V. SARDESAI, S. A. DOEHR AND J. M. ORTEN, Am. J. Physiol., 208 (1965) 127o. 8 D. MAUZERALL AND S. GRANICK, J. Biol. Chem., 219 (1956) 435. 9 J.M. ORTEN,W. GAMBLE, C. B. VAUGHN AND K. C. SHRIVASTAVA, Microchem. J., 13 (i968) 183. i o A. GOLDBERG AND C. RIMINGTON, Diseases of Porphyrin Metabolism, Charles C. T h o m a s , . Springfield, nl., 1958, p. 6. i i A. 12qEUBERGER, J. SCOTT AND C. GRAY, Biochem. J., 58 (1954) X L I . I2 F. SWED ANn C. EISENHART, Ann. Math. Statistics, 14 (1943) 66.
Received February I4th , 1969 Biochim. Biophys. Acta, 177 (1969) 647-649,