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Renal effects of dichloroacetate in vivo
265
Clinrco Chimicu Acto, 160 (1986) 26.5-211 Elsevier
CCA 03609
Renal effects of dichloroacetate Hiroko
Kodama
and (’Department
in vivo
a, Seiji Yamaguchi “, Ichiro Okabe a, Masaaki Tadao Orii b, Shigehiko Kamoshita a.* u Depurtment of Pediatrics, Jichi Medical School,
Kodama
‘,
h Depurtment of Pediatrics, School of Medicine, Gifu Universit, of Marine Biochemistry, School of Fisheries Sciences, Kitasato University (Jupun) (Received
14 April 1986; revision
Key words: Dichloroucetute;
Luck
23 July 1986)
acidosis: Urinuv
lactate; Urinq
pyruvnte
We have investigated the effect of dichloroacetate (DCA) on the urinary excretion of lactate and pyruvate in a patient with congenital lactic acidosis and in healthy human controls. DCA administered orally in doses of 50 mg/kg and 30 mg/kg decreased plasma lactate and pyruvate of both the patient and the controls, while the urinary excretion of lactate and pyruvate was increased. However, these urinary increments were too small to contribute to the decrease of plasma lactate and pyruvate.
Introduction Sodium dichloroacetate (DCA) has been used to treat lactic acidosis as a congenital condition, in hepatic disease, in renal disease and in diabetes mellitus [l-5], because it reduces blood lactate and pyruvate. This effect is thought to be due to activation of the pyruvate dehydrogenase (PDH) complex which in turn is initiated by inhibition of PDH kinase [6,7]. On the other hand, aliphatic and aromatic compounds inhibit reabsorption of monocarboxylic acid in the proximal renal tubule [8,9]. However, the effect of DCA on the urinary excretion of lactate and pyruvate has not been investigated. We describe the in vivo effects of DCA on urinary lactate and pyruvate excretion in a patient with congenital lactic acidosis and in healthy human controls, and the contribution of urinary lactate and pyruvate excretion to the decrease of these plasma compounds.
* Present address: Department of Pediatrics. School of Medicine, Tokyo University. Address for correspondence: Hiroko Kodama, Department of Pediatrics, Jichi Medical mikawachi-machi. Kawachi-gun, Tochigi-ken 329-04, Japan.
0009-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
School,
Mina-
266
Materials and methods
Patient
The patient is a 2-yr-old boy who was well until 5 mth of age when he showed hypotonia and growth retardation. Subsequently, he developed spastic paraplegia, strabismus, nystagnus, epileptic seizure and mental retardation. Laboratory findings confirmed metabolic acidosis (pH 7.25, B.E. - 13) and lactic acidemia (blood lactate, 99 mg/dl; blood pyruvate, 4.5 mg/dl). Neither protein nor glucose were detected in his urine, and renal function was normal. The activities of the pyruvate dehydrogenase complex, pyruvate carboxylase, cytochrome c oxidase and phosphoenol-pyruvate carboxylase in fibroblasts were normal [lo]. The enzymatic defect thus remained unknown.
r30 P
200-
0
3 hr
Fig. 1. Effect of DCA on plasma lactate and pyruvate (A) and urinary lactate and pyruvate (B.C) in the patient with congenital lactic acidosis. Closed circle is lactate and open circle is pyruvate. C lactate/C creatinine expresses urinary lactate (mg/dl) x serum creatinine (mg/dl)/plasma lactate (mg/dl) X urinary creatinine (mg/dl) x 100%.
267
Method After an overnight fast, 50 mg/kg and 30 mg/kg doses of DCA were administered orally to the patient and normal adult controls respectively who had given their informed consent. Blood and urine samples were collected from the patient every 3 h for 6 and 3 h, respectively. Control blood samples were obtained every 1.5 h for 4.5 h, and beginning 1.5 h before the administration of DCA, control urine samples were collected at 1.5 h intervals for a total of 6 h. Food and drink were not given to the patient and controls during the loading test. Blood and urine samples were deproteinized as soon as possible and kept frozen at -20°C until use. The urinary volumes of the control nos. 1, 2 and 3 were measured and recorded. Analyses Lactate and [11,12]. Before Creatinine in [13,14]. Urinary of Tanaka and
pyruvate levels in the plasma and urine were measured enzymatically the analysis, urine was decolorized by activated charcoal treatment. the plasma and urine was determined by the method of Foiin-Wu organic acids, extracted and oxime-trimethylsilylated by the method Hine [15] with some modification, were analysed by gas chromatog-
A g 1.4. 0 1.2.c s l.OI ; 0.8: 0.60) ; 0.4:
0.2-
._ ;
; 0
1.5
3
4.5 hr
0
1.5
Fig. 2. Effect of DCA on plasma lactate (A) and urinary lactate (B,C,D) Control 1. closed circle and closed bar; control 2, open circle and open oblique line bar. C lactate/C creatinine is the same as that in Fig. 1.
3
4.5
hr
in 4 healthy bar; control
adult controls. 3, triangle and
268
raphy-mass spectrometry (Shimadzu GC-MS 9020-DF). Urinary amino acids were analyzed by an ammo acid autoanalyzer. Results Lactate and pyruvate in the plasma and urine
Figure 1 shows lactate and pyruvate levels in the plasma and urine of the patient with congenital lactic acidosis during DCA treatment. Urinary excretion of lactate and pyruvate increased, while plasma concentrations decreased. Therefore, lactate and pyruvate clearances per creatinine clearance (urinary lactate, mg/dl x plasma creatinine, mg/dl/urinary creatinine, mg/dl X plasma lactate, mg/dl x 100%) also increased. Figures 2 and 3 show the changes of lactate and pyruvate in plasma and urine of control individuals. A temporary decrease of the plasma lactate and pyruvate was observed in controls 1, 2 and 3. Urinary lactate and pyruvate per excreted creatinine increased in all controls under the ad~nistration of DCA. The same was true for the ratio of lactate and pyruvate clearance to creatinine clearance and for the total excreted lactate and pyruvate. We concluded that lactate and pyruvate excretion was increased by DCA.
g
1.04A
IO.*aI
1Td
5 0.6; e 100.4E B ii: 0.2-
b
0 0
0
1.5
0
3
4.5 hr
0
1.5
3
4.5 ht
0
15
3
45
B
0
0
/
Q
a
0
200 t : 100 ._ 3
1.5
3
4 5 hr
hr
Fig, 3. Effect of DCA on plasma pyruvate (A) and urinary pyruvate (B.C.Df in healthy adult controls. Symbols are the same as those in Fig. 2.
269
CC-MS analysis of urinary organic acids Figure 4 shows urinary organic acids in the lactic acidosis patient. The levels of lactate, pyruvate, 3-hydroxybutyrate and fumarate in his urine without DCA treatment were higher than those in age-matched controls. Those compounds increased more under DCA treatment. Urinary glycolate, which is a metabolite of DCA [16], also increased during treatment. Other organic acids such as citrate, isocitrate, aconitate, a-ketoglutarate, succinate, malate and adipate were unchanged under DCA. In the controls, the urinary concentrations of lactate, pyruvate, 3-hydroxybutyrate and glycolate increased slightly, while the concentrations of the other organic acids did not change under DCA. a
Fig. 4. Chromatqrams of urinary organic acids from the patient with congenital lactic acidosis before (a) and after (b) the administration of DCA. A 3% OV-17 capillary column was used for analyses. Column temperature was 90 to 280°C. Peaks: 1= lactate; 2 = glycolate; 3 = 3-hydroxybutyrate; 4 = pyruvate; 5 = urea; 6 = fumarale; 7 = succinate; 8 = a-ketoglutarate; 9 = aconitate: 10 = citrate; 11= &citrate: 12 = p-hydroxyphenyllactic acid; 13 = hippurate; 14 = heptadecanoic acid (internal standard). The urine corresponding to 20 pg of creatinine added to 30 pg of heptadecanoate as an internal standard was applied to a column.
270
Amino acids in the urine Under DCA, excreted amino acids did not change either in the patient acidosis nor in the controls.
with lactic
Discussion Our in vivo observations on plasma are consistent with previous reports by others [l-6]. Ulhich et al [8,17] reported that D-lactate is transported by the Na’-dependent transport system in the brush border in the rat kidney and that the transport is inhibited by the administration of short fatty acids or those Cl or OH substituted analogs to the luninal perfusate. These findings along with ours indicate that DCA inhibits the specific tubular reabsorption of lactate and pyruvate and increases urinary excretion of these compounds. However, these urinary increments were small. Therefore the main factor which lowers plasma lactate and pyruvate appears be the activation of PDH but not the increments of urinary lactate and pyruvate. Acknowledgements Supported by a grant (06-07) from National Center for Nervous, Muscular Disorders of the Ministry of Health and Welfare of Japan. thank T. Fukunaga and M. Nakano for technical assistance.
Mental and The authors
References 1 Coude FX, Saudubray JM. DeMaugre F. Marsac C, Leroux JP. Dichloroacetate as treatment for congenital lactic acidosis. N Engl J Med 1978; 299: 1365-1366. 2 Evans OB, Stacpoole PW. Brief treatment with dichloroacetate does not modify suspected subacute necrotizing encephalomyelitis. Ann Neurol 1980; 8: 647-648. 3 McKhann G, Francois B. Evrard P. Long term use of low doses of sodium dichloroacetate in a child with congenital lactic acidosis. Pediatr Res 1980; 14: 167. 4 Green AA, Weindling AM, Soltesz G. Ross B, Jenkins PA. Dichloroacetate in the patient of congenital lactic acidosis. J Inher Metab Dis 1984; 7: 26. 5 Stacpoole PW. Harman EM, Cury SH. Baumgartner TG, Pharm D. Misbin RI. Treatment of lactic acidosis with dichloroacetate. N Engl J Med 1983; 309: 390-396. 6 Evans OB. Stacpoole PW. Prolonged hypolactatemia and increased total pyruvate dehydrogenase activity by dichloroacetate. Biochem Pharmacol 1982; 31: 1295-1300. 7 Kuroda Y, Toshima K. Watanabe T, Kobashi H, Ito M. Takeda E, Miyao M. Effects of dichloroacetate on pyruvate metabolism in rat brain in vivo. Pediatr Res 1984; 18: 936-938. 8 Ulhich KJ, Rumrich G, Klass S. Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney II. Specificity for aliphatic compounds. Pfltigers Arch 1982: 395: 220-226. 9 Ullrich KJ, Rumrich G, Klass S, Fasold H. Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney III. Specificity for aromatic compounds. Pfltigers Arch 1982; 395: 227-231. 10 Okabe I, Kodama H, Shimoizumi H, Kamoshita S, Miyabayashi S. Treatment of lactic acidosis: effects of dichloracetate on lactate and pyruvate levels in the cerebrospinal fluid. Europ J Pediatr 1986: 145: 159. 11 Hohorst HJ. L-(+)-Lactate. Determination with lactate dehydrogenase and DPN. in: Bergmeyer HU. ed. Methods of enzymatic analysis. New York: Academic Press, 1963: 266-271. 12 Bucher T, Czak R, Lamprecht W, Latzko E. Pyruvate. In: Bergmeyer HU. ed. Methods of enzymatic analysis. New York: Academic Press, 1963: 253-259.
271 13 Chasson AL. Determination of creatinine by means of automatic chemical analysis. Am J Clin Path01 1961; 35: 83. 14 Bonsnes RW, Tausskey HH. On the calorimetric determination of creatinine by the Jaffe reaction. J Biol Chem 1945; 158: 581-591. 15 Tanaka K, Hine DG. Compilation of gas chromatographic retention indices of 163 metabolically important organic acids, and their use in detection of patients with organic acidurias. J Chromatogr 1982; 239: 301-322. 16 Crabb DW, Harris RA. Mechanism responsible for the hypoglycemic actions of dichloroacetate and 2chloropropionate. Arch Biochem Biophys 1979; 198: 145-152. 17 Ullrich KJ, Rumrich G, Klbss S. Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney. I. Transport kinetics of D-lactate, Na+-dependence and effect of inhibitors. Pfliigers Arch 1982; 395: 212-219.
Clinrco Chimicu Acto, 160 (1986) 26.5-211 Elsevier
CCA 03609
Renal effects of dichloroacetate Hiroko
Kodama
and (’Department
in vivo
a, Seiji Yamaguchi “, Ichiro Okabe a, Masaaki Tadao Orii b, Shigehiko Kamoshita a.* u Depurtment of Pediatrics, Jichi Medical School,
Kodama
‘,
h Depurtment of Pediatrics, School of Medicine, Gifu Universit, of Marine Biochemistry, School of Fisheries Sciences, Kitasato University (Jupun) (Received
14 April 1986; revision
Key words: Dichloroucetute;
Luck
23 July 1986)
acidosis: Urinuv
lactate; Urinq
pyruvnte
We have investigated the effect of dichloroacetate (DCA) on the urinary excretion of lactate and pyruvate in a patient with congenital lactic acidosis and in healthy human controls. DCA administered orally in doses of 50 mg/kg and 30 mg/kg decreased plasma lactate and pyruvate of both the patient and the controls, while the urinary excretion of lactate and pyruvate was increased. However, these urinary increments were too small to contribute to the decrease of plasma lactate and pyruvate.
Introduction Sodium dichloroacetate (DCA) has been used to treat lactic acidosis as a congenital condition, in hepatic disease, in renal disease and in diabetes mellitus [l-5], because it reduces blood lactate and pyruvate. This effect is thought to be due to activation of the pyruvate dehydrogenase (PDH) complex which in turn is initiated by inhibition of PDH kinase [6,7]. On the other hand, aliphatic and aromatic compounds inhibit reabsorption of monocarboxylic acid in the proximal renal tubule [8,9]. However, the effect of DCA on the urinary excretion of lactate and pyruvate has not been investigated. We describe the in vivo effects of DCA on urinary lactate and pyruvate excretion in a patient with congenital lactic acidosis and in healthy human controls, and the contribution of urinary lactate and pyruvate excretion to the decrease of these plasma compounds.
* Present address: Department of Pediatrics. School of Medicine, Tokyo University. Address for correspondence: Hiroko Kodama, Department of Pediatrics, Jichi Medical mikawachi-machi. Kawachi-gun, Tochigi-ken 329-04, Japan.
0009-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
School,
Mina-
266
Materials and methods
Patient
The patient is a 2-yr-old boy who was well until 5 mth of age when he showed hypotonia and growth retardation. Subsequently, he developed spastic paraplegia, strabismus, nystagnus, epileptic seizure and mental retardation. Laboratory findings confirmed metabolic acidosis (pH 7.25, B.E. - 13) and lactic acidemia (blood lactate, 99 mg/dl; blood pyruvate, 4.5 mg/dl). Neither protein nor glucose were detected in his urine, and renal function was normal. The activities of the pyruvate dehydrogenase complex, pyruvate carboxylase, cytochrome c oxidase and phosphoenol-pyruvate carboxylase in fibroblasts were normal [lo]. The enzymatic defect thus remained unknown.
r30 P
200-
0
3 hr
Fig. 1. Effect of DCA on plasma lactate and pyruvate (A) and urinary lactate and pyruvate (B.C) in the patient with congenital lactic acidosis. Closed circle is lactate and open circle is pyruvate. C lactate/C creatinine expresses urinary lactate (mg/dl) x serum creatinine (mg/dl)/plasma lactate (mg/dl) X urinary creatinine (mg/dl) x 100%.
267
Method After an overnight fast, 50 mg/kg and 30 mg/kg doses of DCA were administered orally to the patient and normal adult controls respectively who had given their informed consent. Blood and urine samples were collected from the patient every 3 h for 6 and 3 h, respectively. Control blood samples were obtained every 1.5 h for 4.5 h, and beginning 1.5 h before the administration of DCA, control urine samples were collected at 1.5 h intervals for a total of 6 h. Food and drink were not given to the patient and controls during the loading test. Blood and urine samples were deproteinized as soon as possible and kept frozen at -20°C until use. The urinary volumes of the control nos. 1, 2 and 3 were measured and recorded. Analyses Lactate and [11,12]. Before Creatinine in [13,14]. Urinary of Tanaka and
pyruvate levels in the plasma and urine were measured enzymatically the analysis, urine was decolorized by activated charcoal treatment. the plasma and urine was determined by the method of Foiin-Wu organic acids, extracted and oxime-trimethylsilylated by the method Hine [15] with some modification, were analysed by gas chromatog-
A g 1.4. 0 1.2.c s l.OI ; 0.8: 0.60) ; 0.4:
0.2-
._ ;
; 0
1.5
3
4.5 hr
0
1.5
Fig. 2. Effect of DCA on plasma lactate (A) and urinary lactate (B,C,D) Control 1. closed circle and closed bar; control 2, open circle and open oblique line bar. C lactate/C creatinine is the same as that in Fig. 1.
3
4.5
hr
in 4 healthy bar; control
adult controls. 3, triangle and
268
raphy-mass spectrometry (Shimadzu GC-MS 9020-DF). Urinary amino acids were analyzed by an ammo acid autoanalyzer. Results Lactate and pyruvate in the plasma and urine
Figure 1 shows lactate and pyruvate levels in the plasma and urine of the patient with congenital lactic acidosis during DCA treatment. Urinary excretion of lactate and pyruvate increased, while plasma concentrations decreased. Therefore, lactate and pyruvate clearances per creatinine clearance (urinary lactate, mg/dl x plasma creatinine, mg/dl/urinary creatinine, mg/dl X plasma lactate, mg/dl x 100%) also increased. Figures 2 and 3 show the changes of lactate and pyruvate in plasma and urine of control individuals. A temporary decrease of the plasma lactate and pyruvate was observed in controls 1, 2 and 3. Urinary lactate and pyruvate per excreted creatinine increased in all controls under the ad~nistration of DCA. The same was true for the ratio of lactate and pyruvate clearance to creatinine clearance and for the total excreted lactate and pyruvate. We concluded that lactate and pyruvate excretion was increased by DCA.
g
1.04A
IO.*aI
1Td
5 0.6; e 100.4E B ii: 0.2-
b
0 0
0
1.5
0
3
4.5 hr
0
1.5
3
4.5 ht
0
15
3
45
B
0
0
/
Q
a
0
200 t : 100 ._ 3
1.5
3
4 5 hr
hr
Fig, 3. Effect of DCA on plasma pyruvate (A) and urinary pyruvate (B.C.Df in healthy adult controls. Symbols are the same as those in Fig. 2.
269
CC-MS analysis of urinary organic acids Figure 4 shows urinary organic acids in the lactic acidosis patient. The levels of lactate, pyruvate, 3-hydroxybutyrate and fumarate in his urine without DCA treatment were higher than those in age-matched controls. Those compounds increased more under DCA treatment. Urinary glycolate, which is a metabolite of DCA [16], also increased during treatment. Other organic acids such as citrate, isocitrate, aconitate, a-ketoglutarate, succinate, malate and adipate were unchanged under DCA. In the controls, the urinary concentrations of lactate, pyruvate, 3-hydroxybutyrate and glycolate increased slightly, while the concentrations of the other organic acids did not change under DCA. a
Fig. 4. Chromatqrams of urinary organic acids from the patient with congenital lactic acidosis before (a) and after (b) the administration of DCA. A 3% OV-17 capillary column was used for analyses. Column temperature was 90 to 280°C. Peaks: 1= lactate; 2 = glycolate; 3 = 3-hydroxybutyrate; 4 = pyruvate; 5 = urea; 6 = fumarale; 7 = succinate; 8 = a-ketoglutarate; 9 = aconitate: 10 = citrate; 11= &citrate: 12 = p-hydroxyphenyllactic acid; 13 = hippurate; 14 = heptadecanoic acid (internal standard). The urine corresponding to 20 pg of creatinine added to 30 pg of heptadecanoate as an internal standard was applied to a column.
270
Amino acids in the urine Under DCA, excreted amino acids did not change either in the patient acidosis nor in the controls.
with lactic
Discussion Our in vivo observations on plasma are consistent with previous reports by others [l-6]. Ulhich et al [8,17] reported that D-lactate is transported by the Na’-dependent transport system in the brush border in the rat kidney and that the transport is inhibited by the administration of short fatty acids or those Cl or OH substituted analogs to the luninal perfusate. These findings along with ours indicate that DCA inhibits the specific tubular reabsorption of lactate and pyruvate and increases urinary excretion of these compounds. However, these urinary increments were small. Therefore the main factor which lowers plasma lactate and pyruvate appears be the activation of PDH but not the increments of urinary lactate and pyruvate. Acknowledgements Supported by a grant (06-07) from National Center for Nervous, Muscular Disorders of the Ministry of Health and Welfare of Japan. thank T. Fukunaga and M. Nakano for technical assistance.
Mental and The authors
References 1 Coude FX, Saudubray JM. DeMaugre F. Marsac C, Leroux JP. Dichloroacetate as treatment for congenital lactic acidosis. N Engl J Med 1978; 299: 1365-1366. 2 Evans OB, Stacpoole PW. Brief treatment with dichloroacetate does not modify suspected subacute necrotizing encephalomyelitis. Ann Neurol 1980; 8: 647-648. 3 McKhann G, Francois B. Evrard P. Long term use of low doses of sodium dichloroacetate in a child with congenital lactic acidosis. Pediatr Res 1980; 14: 167. 4 Green AA, Weindling AM, Soltesz G. Ross B, Jenkins PA. Dichloroacetate in the patient of congenital lactic acidosis. J Inher Metab Dis 1984; 7: 26. 5 Stacpoole PW. Harman EM, Cury SH. Baumgartner TG, Pharm D. Misbin RI. Treatment of lactic acidosis with dichloroacetate. N Engl J Med 1983; 309: 390-396. 6 Evans OB. Stacpoole PW. Prolonged hypolactatemia and increased total pyruvate dehydrogenase activity by dichloroacetate. Biochem Pharmacol 1982; 31: 1295-1300. 7 Kuroda Y, Toshima K. Watanabe T, Kobashi H, Ito M. Takeda E, Miyao M. Effects of dichloroacetate on pyruvate metabolism in rat brain in vivo. Pediatr Res 1984; 18: 936-938. 8 Ulhich KJ, Rumrich G, Klass S. Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney II. Specificity for aliphatic compounds. Pfltigers Arch 1982: 395: 220-226. 9 Ullrich KJ, Rumrich G, Klass S, Fasold H. Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney III. Specificity for aromatic compounds. Pfltigers Arch 1982; 395: 227-231. 10 Okabe I, Kodama H, Shimoizumi H, Kamoshita S, Miyabayashi S. Treatment of lactic acidosis: effects of dichloracetate on lactate and pyruvate levels in the cerebrospinal fluid. Europ J Pediatr 1986: 145: 159. 11 Hohorst HJ. L-(+)-Lactate. Determination with lactate dehydrogenase and DPN. in: Bergmeyer HU. ed. Methods of enzymatic analysis. New York: Academic Press, 1963: 266-271. 12 Bucher T, Czak R, Lamprecht W, Latzko E. Pyruvate. In: Bergmeyer HU. ed. Methods of enzymatic analysis. New York: Academic Press, 1963: 253-259.
271 13 Chasson AL. Determination of creatinine by means of automatic chemical analysis. Am J Clin Path01 1961; 35: 83. 14 Bonsnes RW, Tausskey HH. On the calorimetric determination of creatinine by the Jaffe reaction. J Biol Chem 1945; 158: 581-591. 15 Tanaka K, Hine DG. Compilation of gas chromatographic retention indices of 163 metabolically important organic acids, and their use in detection of patients with organic acidurias. J Chromatogr 1982; 239: 301-322. 16 Crabb DW, Harris RA. Mechanism responsible for the hypoglycemic actions of dichloroacetate and 2chloropropionate. Arch Biochem Biophys 1979; 198: 145-152. 17 Ullrich KJ, Rumrich G, Klbss S. Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney. I. Transport kinetics of D-lactate, Na+-dependence and effect of inhibitors. Pfliigers Arch 1982; 395: 212-219.