Pediat. Res. 7: 616-619 (1973) Adenosine triphosphate pyruvate dehydrogenase Leigh's encephalomyelopathy thiamine mitochondria The Effect of Thiamine Treatment on the Activity of Pyruvate Dehydrogenase: Relation to the Treatment of Leigh's Encephalomyelopathy F. A. HOMMES1191, R. BERGER, AND G. LUIT-DE-HAAN Laboratory of Developmental Biochemistry Department of Pediatrics, University of Groningen School of Medicine, Groningen, The Netherlands Extract Rats received intraperitoneal injection of thiamine (125 mg'/kg body wt) for 4 con- secutive days. There was less inhibition by ATP (9% ± 2%) of the pyruvate dehydro- genase complex of isolated liver mitochondria from treated rats than in liver mitochon- dria from control rats (30% ± 8%). The liver mitochondria of the treated animals contained about 25% more thiamine pyrophosphate than the mitochondria of the controls (0.48 ± 0.03 and 0.36 ± 0.01 nmoles/mg mitochondrial protein, respec- tively) . Roche and Reed have demonstrated inhibition of the phosphorylation of the pyru- vate dehydrogenase complex by thiamine pyrophosphate, thereby maintaining pyru- vate dehydrogenase in the active form. It is suggested that the therapeutic effect of high doses of thiamine given to patients who suffer from Leigh's disease is, at least in part, due to maintainance of the pyruvate dehydrogenase complex in its active form, thus facilitating the oxidation of pyruvate. Speculation Administration of high doses of thiamine to patients suffering from subacute necrotiz- ing encephalomyelopathy seems to have a beneficial effect. Adverse effects, because of interference with a physiologic control mechanism, i.e., inhibition of the phosphoryla- tion of the pyruvate dehydrogenase complex, may, however, interfere with its thera- peutic value. Introduction of thiamine triphosphate, caused by inhibition of thia- Subacute necrotizing encephalomyelopathy or Leigh's mine diphosphate: ATP-phosphotransferase by specific disease [10] is an inborn error of metabolism [9] char- inhibitors circulating in the body fluids of affected pa- acterized by symmetrical lesions of the brainstem. Two tients [3]. theories on the cause of the disease have been pro- Thiamine given in high doses to a patient suffering posed: the first based on the demonstration of a defi- from Leigh's disease does, at least temporarily, improve ciency of pyruvate carboxylase [9] subsequently con- the clinical condition, while the high blood lactic acid firmed by Yoshida et al. [18], Delvin et al. [6], and and pyruvic acid concentrations decrease after admin- Tang et al. [17] and the second based on a deficiency istration of thiamine [5]. Concomitantly, the blood 616 Thiamine treatment in Leigh's disease 617 /3-hydroxybutyrate and acetoacetate concentrations in- Table I. Rates of oxygen uptake by rat liver mitochondria with 1 creased. These data have been interpreted as an acti- pyruvate and malate as substrate vation of the pyruvate dehydrogenase complex by thia- 2 Rate of O2 uptake Treatment of animal and in n at 0/min-mg Inhibition, % mine [5, 8]. addition to basic medium protein Roche and Reed [14] have demonstrated recently that thiamine pyrophosphate inhibits the phosphoryla- No treatment ADP, 5 mM 42.7 22 tion of the pyruvate dehydrogenase, thereby maintain- ATP, 5 mM 33.3 ing the pyruvate dehydrogenase complex in its active form. The question arises, therefore, as to whether ad- Thiamine treated (1) ADP, 5 mM 52.4 ministration of high doses of thiamine can maintain 7 the pyruvate dehydrogenase complex in the nonphos- ATP, 5 mM 49.0 phorylated active form. Rats received intraperitoneal Thiamine treated (2) injections of high doses of thiamine. The activity of ADP, 5 HIM 51.6 9 pyruvate dehydrogenase of liver mitochondria was then ATP, 5 HIM 49.0 determined under phosphorylating and nonphospho- rylating conditions. It was found that thiamine treat- None (2) ADP, 5 mM 61.6 37 ment of the rats resulted in less inhibition by ATP of ATP, 5 mM 38.8 liver pyruvate dehydrogenase as compared with con- trols. Thiamine treated (3) ADP, 5 mM 67.5 12 ATP, 5 HIM 59.5 Materials and Methods 1 The incubation medium, as well as the scheme of injection for Young adult, female rats of the Wistar strain of about thiamine treatment of the rats, is described under Materials and 200 g, body wt, received daily intrapei'itoneal injections Methods. Mean inhibition ± SE for control animals, 30% ± 8%; of 1 ml solution containing 25 mg thiamine dichloride for thiamine-treated animals, 9% ± 2%. 2 Number of animals receiving treatment is indicated in paren- in 0.9% NaCl for 4 consecutive days. The animals were thesis. killed on the 5th day. Liver mitochondria were pre- pared in 0.25 M sucrose as described by Schneider [15]. malate as substrate, in the medium described under The rate of oxygen uptake was measured polarograph- materials and methods in the presence of ADP or ATP. ically [2] in a medium which had the following com- In the presence of ADP and an uncoupler, the pyru- position: KoHPO4, 10 mM; KC1, 15 ITIM; MgCl2, 5 mM; vate dehydrogenase is maximally activated because the EDTA, 2 mM; trishydroxymethylaminomethane hydro- intramitochondrial ATP level is essentially zero [7]. chloride, 50 mM; fiuorocitrate to inhibit further oxida- The same experiment repeated in the presence of ATP tion of citrate, 0.1 mM; carboxyl cyanide ^-trifluoro- and oligomycin to prevent the hydrolysis of ATP dem- methoxyphenyl hydrazone as an uncoupler, 1 fiM; onstrates inhibition of the rate of oxygen uptake. That oligomycin to inhibit the mitochondrial ATPase, 6 the rate of oxygen uptake is directly related to the ac- fig/ml; malate to generate an adequate supply of oxalo- tivity of pyruvate dehydrogenase in this system has acetate, 5 HIM; pyruvate, 0.5 ITIM; ADP or ATP, 5 mM. been demonstrated by showing that the ratio of acetyl- Protein was determined by the biuret method, using CoA over CoA is decreased in the presence of ATP and bovine serum albumin as a standard. that, furthermore, the rate of oxidation of palmitoyl Mitochondrial extracts were prepared with 18% carnithine is higher than that of pyruvate and malate HC1O , followed by neutralization with 30% KHCO . 4 3 [1, 16]. Citrate synthase is, in this system, not rate After standing for 1 hr at 0° the mixture was centri- limiting. Liver mitochondria prepared from thiamine- fuged and the supernatant analyzed for thiamine phos- treated rats show considerably less inhibition of the phates as described by Cooper et al. [3]. pyruvate dehydrogenase by ATP than the nontreated All reagents used were of analytical grade. controls (Table I). Administration of thiamine can ap- parently maintain the pyruvate dehydrogenase in the Results active, nonphosphorylated form, which may be ex- Table I summarizes the results on the rates of oxygen pected from the observations of Roche and Reed [14]. uptake of rat liver mitochondria with pyruvate and Therefore, the mitochondria prepared from thiamine- 618 HOMMES, BERGER, AND LUIT-DE HAAN Table II. Thiamine pyrophosphate content of liver mitochon- Phenomenologically, this could amount to the same dria of rats with and without thiamine treatment1 as postulating an activation of the enzyme complex. If Thiamine pyrophosphate this mechanism is also operative for the effect of thia- Treatment of animal content of liver mitochon- dria in protein, nniol/mg mine in cases of Leigh's disease it is necessary to dem- onstrate that thiamine treatment results in a lower None (1) 0.37 sensitivity to ATP inhibition of the pyruvate dehy- Thiamine treated (1) 0.50 Thiamine treated (2) 0.50 drogenase complex. The data presented in Table I None (2) 0.35 demonstrate that this is indeed the case. Table II dem- Thiamine treated (3) 0.43 onstrates, furthermore, that treatment of the rats with high doses of thiamine results in an increased content 1 Number of animals receiving treatment is indicated in paren- theses. The mean value ± SE for the control animals is 0.36 ± of thiamine pyrophosphate in the mitochondria. It is 0.01 and for the treated animals is 0.48 ± 0.03. therefore highly likely that the mechanism suggested by Reed and Roche [14] is also operative in the case treated rats have to contain more thiamine diphosphate of the thiamine-treated rats and consequently also in than those of the controls. cases of Leigh's disease treated with high doses of thia- Electrophoretic analysis of mitochondrial extracts at mine. pH 5.2 demonstrated that the only fraction which The present findings do not exclude a role of thia- could be made visible with the fluorescing ferricyanide mine triphosphate in the ethiology of the neuropatho- reagent, had the same electrophoretic mobility as thia- logic findings as observed in Leigh's disease [3, 4]. The mine diphosphate. This was observed for control ani- maintainance of the pyruvate dehydrogenase complex mals as well as for thiamine-treated animals. Subse- in its active form, as demonstrated here, is, however, quent analyses of mitochondrial extracts for thiamine an effect of the thiamine administration. How far in- derivatives were therefore carried out on whole ex- terference by the thiamine therapy with a physiologic tracts, without electrophoretic separation. The results control mechanism has any adverse effects remains to are given in Table II. Liver mitochondria prepared be established in future investigations. from thiamine-treated rats contain significantly more thiamine diphosphate than do liver mitochondria pre- Summary pared from control animals. Intraperitoneal injection in rats of high doses of thia- Discussion mine resulted in an increased content of thiamine py- rophosphate of the liver mitochondria. Concomitantly Pincus and associates [12, 13] have advocated the use a decreased sensitivity to inhibition by ATP of liver of thiamine in cases of Leigh's disease as suggested by pyruvate dehydrogenase was found. This result has Lonsdale [11]. That administration of thiamine has a been related to the earlier observed decrease in blood beneficial effect for those patients seems to be well lactate and pyruvate after institution of thiamine ther- documented [5, 8, 11-13].