Neurochemistry International 45 (2004) 1021–1028
Tricarboxylic acid cycle enzymes following thiamine deficiency
Parvesh Bubber, Zun-Ji Ke, Gary E. Gibson∗
Department of Neurology and Neuroscience, Burke Medical Research Institute, Weill Medical College,
Cornell University, 785 Mamaroneck Avenue, White Plains, NY 10605, USA
Received 11 February 2004; received in revised form 12 May 2004; accepted 18 May 2004
Available online 10 July 2004
Abstract
Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent
enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never
been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured
in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and
malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the ␣-ketoglutarate dehydrogenase
complex (KGDHC) (−21.5%) and PDHC (−10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase
(STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other
enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes
should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be
reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter
brain function in neurodegenerative disorders.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Mitochondria; Tricarboxylic acid cycle; Thiamine deficiency; Neurodegenerative diseases; Energy metabolism
1. Introduction
Thiamine is a cofactor of key metabolic enzymes and
thiamine deficiency (TD) leads to alterations in brain glucose metabolism (Hakim and Pappius, 1981; Kinnersley
and Peters, 1929). Thiamine pyrophosphate (TPP) is required as a coenzyme for the mitochondrial enzyme complexes ␣-ketoglutarate dehydrogenase complex (KGDHC)
and pyruvate dehydrogenase complex (PDHC). TD diminishes the activities of these enzymes. In general, KGDHC is
Abbreviations: AABS, amino azo benzoic acid; AD, Alzheimer’s disease; BSA, bovine serum albumin; CS, citrate synthase (EC 4.1.3.7);
DTT, dithiothreitol; EGTA, ethylene glycol tetra acetic acid; EDTA, ethylene diamine tetra acetic acid; GTP, guanosine triphosphate; KGDHC,
␣-ketoglutaric acid dehydrogenase complex (KGDHC; EC 1.2.4.2, EC
2.3.1.61, EC 1.6.4.3); ICDH, isocitric acid dehydrogenase (EC 1.1.1.41);
MDH, malate dehydrogenase (EC 1.1.1.37); PDHC, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3); ROS, reactive
oxygen species; STH, succinate thiokinase (EC 6.2.1.4); SDH, succinate
dehydrogenase (EC 1.3.99.1); TCA, tricarboxylic acid; TPP, thiamine pyrophosphate
∗ Corresponding author. Tel.: +1 914 597 2294; fax: +1 914 597 2757.
E-mail address: [email protected] (G.E. Gibson).
0197-0186/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2004.05.007
more severely affected than PDHC (Elnageh and Gaitonde,
1988; Freeman et al., 1987; Sheu et al., 1998). The KGDHC
activity shows a reduction of about 45% in TD mice brains
(Freeman et al., 1987). Studies in rats, show that reduction
can be larger in specific brain regions. For example, TD
leads to a 50 and 57% reduction of KGDHC activity in thalamus and inferior colliculus of rat brain, respectively (Sheu
et al., 1998). Selective decreases in the KGDHC activity (up
to 30%) occur in lateral vestibular nucleus and hypothalamus with chronic thiamine deprivation in rats (Butterworth
et al., 1986). However, enzyme activity decreases to a lesser
extent or is unchanged in other brain structures.
Although TD-induced reductions in thiamine-dependent
enzymes are well documented, few studies have been done
in mice, and the activities of non-TPP-dependent enzymes
of the tricarboxylic acid cycle (TCA) have not been reported. Previous studies that compare enzyme activity to selective neuronal death reveal that diminished mitochondrial
metabolic flux correlates better to selective neuronal death
than the activities of either PDHC or KGDHC (Gibson et al.,
1984). Such findings suggest that factors in addition to the
decreases in the activities of thiamine-dependent enzymes
1022
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
Fig. 1. The TCA cycle.
must underlie selective cell death. This conclusion is supported by immunocytochemical results, which indicate that
neither the immunoreactivities of the thiamine-dependent
enzymes, nor their response to TD, predict which cells will
die in TD (Calingasan et al., 1994).
The thiamine-dependent enzymes PDHC and KGDHC are
key enzymes of mitochondrial metabolism that can be rate
controlling, but all of the enzymes of the TCA cycle may respond to metabolic perturbations as a unit. PDHC catalyses
oxidative decarboxylation of pyruvate, to form acetyl-CoA,
which enters TCA cycle. The TCA cycle is a supramolecular
assembly of eight enzymes including citrate synthase (CS),
aconitase, isocitrate dehydrogenase (ICDH), KGDHC, succinate thiokinase (STH), succinate dehydrogenase (SDH),
fumarase and malate dehydrogenase (MDH) (Fig. 1). Since
all of the enzymes of TCA cycle are responsible for catalyzing sequential reactions, they have been proposed to be
organized in supramolecular complexes termed metabolons
(Lyubarev and Kurganov, 1989). The protein–protein interactions between TPP- and non-TPP-dependent enzymes of
metabolons in the TCA cycle may underlie TD-induced neurodegeneration. To test that possibility, all of the enzymes
of the TCA cycle were measured in TD mouse brain.
Numerous studies suggest that abnormalities in glucose
oxidation and mitochondrial function may be common
causes of neurodegeneration (Beal, 1996; Sheu et al., 1998).
Reductions in the activities of thiamine-dependent enzymes
occur in several age-dependent neurodegenerative disorders including Alzheimer’s disease (Gibson et al., 2000a,b,
1988), Parkinsons’s disease (Gibson et al., 2003; Mizuno
et al., 1994), Progressive Supranuclear Palsy (Park et al.,
2001) and Wernicke–Korsakoff syndrome (Butterworth et
al., 1993). The reductions in KGDHC in AD have been
particularly well-documented and have been reported by
multiple laboratories (for review, see Gibson et al., 1998).
The decline of KGDHC enzyme activity in AD patients
bearing the apolipoprotein E4 allele is highly correlated
(r = 0.7) with the clinical dementia rating score (Gibson
et al., 2000a,b). Abnormalities in the other enzymes of
TCA cycle in neurodegenerative diseases have not been
reported.
The use of a combination of thiamine deficiency and the
inhibitor pyrithiamine to induce experimental TD in the mice
provides a reliable method that produces both the metabolic
and the pathologic characteristics of oxidative stress, inflammation and subsequent neurodegeneration (Gibson et al.,
1984; Butterworth et al., 1985; Calingasan et al., 1998).
Pyrithiamine is an inhibitor of thiamine transport (Nose
et al., 1976) and of thiamine pyrophosphokinase (Johnson
and Gubler, 1965), which synthesizes TPP (Rindi and Perri,
1961). Mice start to lose weight after 8 or 9 days of pyrithiamine treatment. There is little or no neuronal loss on day 8.
This is followed by ataxia, paralysis and extensive neuronal
loss in select brain regions on day 9 or 10.
The current experiments tested whether the enzymes of
the TCA cycle are altered by TD and how early in the course
of the disorder these reductions occur. The results begin to
test the role of TCA cycle enzymes in neurodegeneration.
2. Experimental procedures
2.1. Materials
All chemicals and reagents including enzyme preparations
were purchased from Sigma Chemical Company (St. Louis,
MO, USA).
2.2. Animals
C57Bl/6N mice (Harlan Sprague Dawley Indianpolis, IN,
USA) were used because TD-induced pathology in this strain
has been extensively studied in this strain (Calingasan et al.,
1999). Upon arrival, the animals were housed individually
and were maintained under constant temperature (70 ◦ F), humidity (50%) and 12-h light–dark cycle. The animals were
fed a pelleted diet (ICN Nutritional Biochemicals, Cleveland, OH, USA) and provided distilled water. The animals
were allowed to acclimatize to the environment for 3 days
before the induction of TD. All the procedures were approved by the Institutional Animal Care and Use Committee
of Weill Medical College of Cornell University.
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
2.3. Induction of thiamine deficiency
Mice were divided into three groups [control (n = 12), 8
days of TD (n = 7) and 10 days of TD (n = 12)]. TD was
induced in mice by providing them with a thiamine-deficient
diet (ICN Nutritional Biochemicals, Cleveland, OH, USA)
and giving them daily intraperitoneal injections of the
pyrithiamine (5 ␮g in 0.1 ml of saline/10 g body weight).
The control animals received a normal-thiamine containing diet ad libitum and daily intraperitoneal injections
of saline (0.1 ml/10 g of body weight). The choice of
pyrithiamine-induced TD model and ad libitum-fed animals
as controls has been discussed previously (Calingasan et al.,
1995).
2.4. Preparation of tissue homogenates
Mice were decapitated into liquid nitrogen. The whole
frozen brains (minus olfactory bulbs) were removed and
ground with a pestle under liquid nitrogen. They were stored
at −80 ◦ C. Before running the assays, the brains were homogenized with a teflon glass homogenizer in one of the
following buffers.
1. 50 mM Tris–HCl- AND; 1 mM EDTA; 10% glycerol pH
7.6 (for estimation of SDH, STH, MDH, Fumarase and
CS).
2. 50 mM Tris–HCl; 5 mM sodium citrate; 0.6 mM magnesium chloride pH 7.4; 1 mM DTT; 0.2 mM EGTA; 0.08%
Triton X-100 and 50 ␮M leupeptin (for estimation of
aconitase, ICDH and KGDHC)
3. 50 mM sodium phosphate pH 7.4; 1 mM DTT; 20% Triton X-100 and 50 ␮M leupeptin (for estimation of active
and total PDHC).
One unit of enzyme activity was defined as the amount
of enzyme catalyzing the production of 1 nmole of NADH
or NADPH/min per mg protein.
2.5. Estimation of enzyme activities
2.5.1. Pyruvate dehydrogenase complex (PDHC) [EC
1.2.4.1, EC 2.3.1.12, EC 1.6.4.3]
Active PDHC activity was determined by coupling the
production of acetyl-CoA to the acetylation of aminoazobenzoic acid (AABS) with arylamine acetyltransferase (ArAt).
The reaction mixture (pH 7.8) contained the following (mM
unless indicated otherwise): MgCl2 (1), EDTA (0.5), 0.1%
Triton X-100, DTT (1), 0.05 mg/ml AABS, NAD (2), CoA
(0.1), TPP (0.3), ArAT (100 mU) and sodium pyruvate (5)
in Tris–HCl (50) buffer. The decrease in optical density was
followed at 460 nm at 37 ◦ C for 30 min (Ksiezak-Reding
et al., 1982; McCormack and Denton, 1989) with a spectrophotometer plate reader (Molecular Devices, Sunnyvale,
CA, USA). For the measurement of the total PDHC activity,
the ‘inactive’ (phosphorylated) form of the enzyme com-
1023
plex was converted to the ‘active’ (dephosphorylated) form
by incubating the brain homogenates at 37 ◦ C for 30 min.
Under these conditions, the phosphatase was fully activated
and catalysed the dephosphorylation of PDHC (Hucho et al.,
1972). The rest of the procedure for determining total activity was the same as for the active form.
2.5.2. Citrate synthase (CS) [EC 4.1.3.7]
Estimation of CS activity is based on the chemical coupling of CoASH (released from acetyl-CoA during the
enzymatic synthesis of citrate) to Ellman’s reagent, 5,5
dithiobis-(2-nitrobenzoic acid)[DTNB]. The reaction mixture contained 2 mM oxaloacetic acid, 2 mM acetyl-CoA,
2.5 mM DTNB and 100 mM Tris–HCl, pH 8.0. The increase
in optical density was assessed at 412 nm (Shepherd and
Garland, 1969).
2.5.3. Aconitase [EC 4.2.1.3]
Aconitase in the samples was activated by sodium thiomalate (20 mM) and ferrous ammonium sulfate (4 mM) mixture
for 30 min at 37 ◦ C. The enzyme activity was estimated by
following a linear fluorescence change at 340 nm at 30 ◦ C
in 250 ␮l reaction mixture for 30 min. The reaction mixture
(pH 7.4) contained the following (mM): isocitric dehydrogenase (200 mU), Tris–HCl (50), magnesium chloride (0.6),
sodium citrate (5) buffer and NADP (0.5) (Morton et al.,
1998).
2.5.4. Isocitric acid dehydrogenase (ICDH) [EC 1.1.1.41]
ICDH activity was estimated by following the rate of production of NADPH in 200 ␮l of assay mixture (pH 7.6) for
15 min at 340 nm. The assay mixture contained the following
(mM): magnesium sulfate (20), isocitric acid (8), Tris–HCl
(50) buffer and NADP (0.5) (Bai et al., 1999).
2.5.5. α-Ketoglutaric acid dehydrogenase complex
(KGDHC) [EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3]
KGDHC activity was measured by monitoring the conversion of NAD to NADH with a 96 well fluorometric plate
reader (Molecular Devices; Sunnyvale, CA, USA). The reaction mixture (pH 7.0) contained the following (mM): MgCl2
(1), CaCl2 (1), EDTA (0.5), TPP (0.3), DTT (1), NAD (1),
Coenzyme A (0.163), ␣-ketoglutarate (1) and Tris–HCl (50)
buffer and 0.1% Triton X-100 (Gibson et al., 1988).
2.5.6. Succinate thiokinase (STH) [EC 6.2.1.4]
The STH activity was determined by coupling of GDP
formation to pyruvate kinase [EC 2.7.1.40] and the lactate
dehydrogenase system [EC 1.1.1.27]. The reaction mixture (pH 7.4) contained the following: MgCl2 (10 mM),
CoA (100 ␮M), phosphoenolpyruvate (1.55 mM), GTP
(100 ␮M), NADH (200 ␮M) Tris–succinate (50 mM), pyruvate kinase (830 mU) and lactate dehydrogenase (830 mU).
The rate of disappearance of NADH in the coupled reaction
was determined at 340 nm at 37 ◦ C for 20 min (Sungman,
1969).
1024
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
2.5.7. Succinate dehydrogenase (SDH) [EC 1.3.99.1]
SDH activity was measured spectrophotometrically by
following the rate of reduction of dicholoroindophenol dye
in 200 ␮l reaction mixture (pH 7.6) containing the following
(mM): succinate (40), sodium azide (4), dichloroindophenol
(72 ␮M) in potassium phosphate (100) buffer and EDTA (1)
(Veeger et al., 1969).
2.5.8. Fumarase [EC 4.2.1.2]
Fumarase activity was measured by following the change
in fumarate concentration spectrophotometrically at 250 nm
in 200 ␮l reaction mixture (pH 7.3) containing malate
(50 mM), sodium phosphate (50 mM) buffer, and 0.1%
bovine serum albumin (BSA). The increase in absorbance
was measured for 15 min at 25 ◦ C (Hill and Bradshaw,
1969).
2.5.9. Malic dehydrogenase (MDH) [EC 1.1.1.37]
MDH activity was measured by following the rate of oxidation of NADH in the presence of oxaloacetate. The decrease in the absorbance at 340 nm was determined in 200 ␮l
reaction mixture (pH 7.2) containing the following (mM):
oxaloacetic acid (2) in potassium phosphate (100) buffer and
NADH (1) at 25 ◦ C (Kitto, 1969).
2.5.10. Protein
Protein content was determined with a Bio-Rad 500-006
kit (Bio-Rad Laboratories, Hercules, CA, USA) based on
Bradford dye binding procedure that utilizes the color
change of Coomassie brilliant blue G-250 (Bradford, 1976).
Sigma BSA (A-2153) was used as standard.
2.5.11. Data analysis and statistics
The experiments were repeated two times. All of the measurements of individual samples were carried out in triplicate. The Q-test was used to remove outlying values (Dean
and Dixon, 1951). ANOVA analysis (two-way) was applied
to compare the three groups. Student’s–Newman–Keuls post
hoc test (SPSS Inc., Chicago, IL, USA) was applied after
the ANOVA.
3. Results
Activities of different enzymes in the TCA cycle of mouse
brain varied considerably. The non-thiamine-dependent enzyme MDH had the highest activity of all the enzymes
(Table 1). Its activity was nearly 75 times higher than the
activity of the lowest enzyme and nearly four times higher
than the activity of next closest enzyme. Activities of fumarase, STH and CS enzymes were at the intermediate levels. Thiamine-dependent multi-enzyme complexes such as
PDHC and KGDHC were lower than the other enzymes.
PDHC was twice KGDHC, but only 75% of it was in the active form. Aconitase had the lowest activity of all enzymes
in the TCA cycle.
Table 1
Activities of TCA cycle enzymes in mouse brain
Total PDHC
Active PDHC
PDHC (active/total) × 100
Citrate synthase
Aconitase
Isocitric dehydrogenase
␣-Ketoglutaric dehydrogenase
Succinate thiokinase
Succinate dehydrogenase
Fumarase
Malate dehydrogenase
25.2
18.5
75.4
223.4
12.6
25.9
14.4
44.8
16.0
111.7
896.6
±
±
±
±
±
±
±
±
±
±
±
1.1
1.0
5.1
7.0
0.4
0.8
0.4
1.3
0.7
4.2
29.3
The values represent the mean of 12 independent measurements done
as part of two independent experiments. Each assay of each brain was
done in triplicate. All the values (mean ± S.E.M.) are specific activities
(nmole/min per mg protein).
TD treatment did not change total PDHC activity significantly after 8 or 10 days (Figs. 2 and 3). Total PDHC
activity in mouse brain was measured in the presence of
saturating TPP concentrations. The relative proportion of
the active form of PDHC in the brain homogenate depends
upon the method of killing the animal and the extent of
postmortem ischemia of the brain (Cremer and Teal, 1974).
In the current experiments, the mice brains were frozen
immediately by allowing the head to fall in liquid nitrogen.
Elnageh and Gaitonde (1988) reported that the proportion of
‘active’ form of PDHC in the brain increased with the time
elapsed before freezing the brain in liquid nitrogen (from
79% at 45 s to 101% at 150 s). In the current experiments,
‘active’ PDHC constituted about 75% of the total PDHC
activity. The values are also in broad agreement with those
reported by others (Jope and Blass, 1976). Eight or ten days
of TD treatment did not decrease the active form of PDHC
significantly in comparison to controls (Figs. 2 and 3).
The first three steps of the TCA cycle were not altered by
TD. Citrate synthase catalyses the first step of the TCA cycle.
It is highly exergonic reaction and of central importance
in keeping the entire cycle going in forward direction. No
significant decrease in CS activity was seen after 8 days
or 10 days of TD treatment (Figs. 2 and 3). Similarly, the
activities of next two enzymes of the TCA cycle, ‘aconitase
and ICDH’ did not change after 8 or 10 days of TD treatment
(Figs. 2 and 3).
KGDHC is the next key enzyme of TCA cycle. In the
presence of saturating concentration of TPP, KGDHC enzyme activity diminished significantly [F (2, 28) = 6.9,
P < 0.004] after TD treatment in comparisons to controls. KGDHC decreased after 8 days (P < 0.05) or 10
days (P < 0.01) of TD treatment (Figs. 2 and 3). STH,
a non-thiamine-dependent enzyme, follows KGDHC in the
TCA cycle. It converts succinyl-CoA to succinate using the
thioester bond of succinyl-CoA to drive the synthesis of a
high-energy nucleotide phosphate by substrate level phosphorylation. Its activity changed significantly [F (2, 28)
= 7.1, P < 0.005] with TD. STH activity decreased after 8
1025
MD
H
Fum
ar a
se
SD
H
ST
H
KG
DH
C
0
ICD
H
Ac
oni
tas
To
t al
10
e
PD
HC
Ac
tive
PD
HC
%
Ac
tive
PD
HC
CS
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
% Change
-10
*
-20
*
-30
**
-40
***
% Change
MD
H
Fu
ma
ras
e
SD
H
ST
H
KG
DH
C
ICD
H
Ac
oni
tas
e
PD
HC
CS
Ac
tive
%
PD
HC
Ac
t i ve
0
To
tal
10
PD
HC
Fig. 2. Activities of TCA cycle enzymes following 8 days of TD. Activities were determined in the brains of seven TD mice. The percent change from
the control values in Table 1 was determined for each animal. Values are mean ± S.E.M. of the percent decrease. ANOVA analysis was applied to
compare three groups (Controls, 8 days of TD, 10 days of TD). F-ratio is a measure of how different the means are relative to the variability within each
sample. Student’s–Newman–Keuls post hoc test was applied to determine the significant difference between groups. (∗ ) Denotes significance compared
to control, ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.001.
-10
*
-20
**
-30
****
-40
Fig. 3. Activities of citric acid cycle enzymes following 10 days of TD. Activities were determined in the brains of 12 TD mice. The percent change
from the control values in Table 1 was determined for each animal. Values are mean ± S.E.M. of the percent decrease. ANOVA analysis was applied to
compare three groups (Controls, 8 days of TD, 10 days of TD). F-ratio is a measure of how different the means are relative to the variability within each
sample. Student’s–Newman–Keuls post hoc test was applied to determine the significant difference between groups. (∗ ) Denotes significance compared
to control. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.0001.
days (P < 0.005) of TD treatment in comparison to control
(Fig. 2) but did not vary after 10 days.
SDH catalyses the next sequential enzyme reaction after
STH. It introduces a ‘trans’ double bond in succinate to
form fumarate. TD reduced its activity [F (2, 28) = 15.0,
P < 0.000]. The enzyme activity was significantly decreased after 8 days (P < 0.001) or 10 days (P < 0.0001)
of TD treatment in comparison to controls (Figs. 2 and 3).
Fumarate undergoes stereo specific hydrogenation to form
malate catalysed by fumarase. Its activity did not change
with TD treatment. MDH is the final enzyme of TCA cycle
and catalyses the NAD+ -dependent oxidation of l-malate
to oxaloacetate. Its activity changed significantly [F (2,
28) = 4.64, P < 0.02] in TD-treated mice in comparison
to controls. TD treatment decreased (P < 0.05) enzyme
activity after 8 or 10 days (Figs. 2 and 3).
4. Discussion
The present study is the first to examine the effect of
TD on all of the enzymes of TCA cycle. The reduction in
1026
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
KGDHC activity is one of the earliest biochemical change
observed in TD (Gibson et al., 1984). Reductions are larger
(30–50%) in select brain regions such as vestibular nucleus,
hypothalamus, thalamus and inferior colliculus (Butterworth
et al., 1986; Sheu et al., 1998). The decreased activity in
the presence of optimal TPP is consistent with the concept
that binding of TPP to its target proteins not only enables
enzymatic catalysis, but also helps to stabilize the protein
structure. Previous studies (Sheu et al., 1998) show that
the protein levels of E1k, E2k and E3 (KGDHC subunits)
are unaffected in TD brain despite a marked reduction in
KGDHC activity. Immunocytochemistry reveals that the levels of KGDHC molecules are not significantly reduced-even
at the sub-regional and cellular level. The increase in reactive oxygen species with TD may damage KGDHC, and the
E2k component may be particularly vunerable (Sheu et al.,
1998). Dihydrolipoic acid, part of KGDHC complex, reacts
with hydroxyl radicals (Scott et al., 1995), which inactivates
the enzyme (Sadek et al., 2002).
The combination of previous studies with the current results suggests that the TD-induced reductions in TCA cycle enzymes diminish metabolism. The rate of oxidation of
pyruvate in brain homogenates of pyrithiamine treated rat
decreases to 51–56% (Gubler, 1961) or 76–81% (Bennett
et al., 1966). This reduction occurs even though the current results indicate that PDHC activities are not diminished.
Similarly, our previous studies indicate that metabolic flux
correlates better to selective neuronal death than the activities of either PDHC or KGDHC (Gibson et al., 1984).
Although PDHC and KGDHC are similar in many ways,
activities of KGDHC but not PDHC decline with TD. Both
enzymes were measured in the presence of saturating concentration of TPP. TPP can readily dissociates from PDHC
and TPP restores enzyme activity to normal levels (Gubler,
1961). On the other hand, KGDHC has never been freed of
TPP without inactivating it irreversibly. PDHC contains two
lipoic acid residues covalently bound to each E2 subunit,
which also makes it less susceptible to free radical modification than KGDHC (Humphries and Szweda, 1998). The lack
of change in PDHC activity is in general agreement with the
previous studies (Elnageh and Gaitonde, 1988; Butterworth,
1986). The total activity of enzyme in several regions of the
brain (Butterworth et al., 1985) remained near control levels
in TD rats. The multi-enzyme complex KGDHC is similar
to the PDHC in the intricacy of its protein makeup, cofactors an its mechanism of its action yet it is more responsive to TD than PDHC. This analysis suggests selectivity of
TD-induced oxidative stress to specific enzymes in the TCA
cycle.
The effects of TD on non-TPP-dependent enzymes in the
TCA cycle have never been studied. Although some of them
did not show any effect (fumarase, aconitase and ICDH),
others were affected more than the thiamine-dependent
enzymes (SDH and MDH). Such changes are possibly because of close proximity and orientation of enzymes in
the TCA cycle for maintaining the metabolic flux. Dif-
ferences in the activities of non-TPP-dependent enzymes
with TD treatment may reflect differences in their regulation, orientation or their interaction with TPP-dependent
enzymes in the TCA metabolon. The metabolon concept
of the TCA cycle enzymes supported by various experimental approaches demonstrates specific interactions between five sequential enzymes including fumarase, MDH,
CS, aconitase, and ICDH (Velot et al., 1997). It provides a possibility for enzyme–enzyme interaction of TPPand non-TPP-dependent enzymes thereby influencing the
chemical transformation of metabolic intermediates in the
metabolon microenvironment. SDH is bound to the inner mitochondrial membrane and has FAD bound to one of its subunit. TD significantly diminishes its activity in mice brain.
Administration of the herbicide 2,4-dichlorophenoxyacetic
acid, which reduces thiamine in the organs of mice by
40–50%, reduces liver SDH (Bogdan, 1983), but no measures were made in brain. In addition to its role in the TCA
cycle, SDH is a component of the electron transport chain.
Thus, reduced SDH would also be expected to diminish
electron transport chain activity. It was somewhat surprising
that the loss of a cofactor for some enzymes could influence the activities of other enzymes of the TCA cycle. The
deficiency of these non-TPP-dependent enzymes and the
resulting oxidative deficit could contribute to TD-related
pathology and precipitate the neuronal death.
TD increases oxidative stress, which may inactivate other
metabolic enzymes. Measures of oxidative stress include
elevations of heme oxygenase-1 (HO-1) (Gibson et al.,
1999), ferritin and reactive iron in microglia (Calingasan
et al., 1998). TD increases nitration of proteins and elevates lipid peroxidation in neurons in vulnerable areas
(Calingasan et al., 1998, 1999). Furthermore, TD elevates
the concentration of reactive oxygen species (ROS) in the
thalamus (Langlais and Mair, 1990). The increased reactive oxygen species may diminish the activity of thiamineand non-thiamine-dependent mitochondrial enzymes. For
example, mitochondrial enzymes such as aconitase, CS,
PDHC, SDH and KGDHC are sensitive to oxidative stress
(Nulton-Persson and Szweda, 2001; Navarro et al., 2002;
Melov et al., 1999; Gibson et al., 1998; Bubber and Gibson,
unpublished results). Increased ROS have also been implicated in neurodegeneration including Alzheimer’s disease
(Markesbery, 1997).
The activity measures did not distinguish between brain
regions that are vunerable and non-vunerable to TD. The
changes in activity levels in select regions may be much
greater (Gibson et al., 1989; Butterworth and Giguere, 1984).
Reductions in KGDHC also occur in many regions such as
medial septal nucleus, nucleus of the diagonal band, hippocampus, red nucleus, pontine nuclei and most cranial
nerve nuclei (Calingasan et al., 1995). The effect of TD on
the subcellular and regional distribution of all the TPP- and
non-TPP-dependent enzymes of the TCA cycle will help to
reveal their precise interactions, which lead to selective and
significant cell death in TD.
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
A decline of mitochondrial oxidation and increase in the
oxidative stress are important functional markers of aging
and common risk factors of age related neurodegenerative
disorders like Alzheimer’s and Parkinson’s disease (Beal,
1995; Blass, 1993). TD is a classical model of impaired cerebral oxidative metabolism that leads to selective neuronal
loss and neurological symptoms. These results emphasize
the fact that all enzymes of the TCA cycle are likely to have
an important role in events leading to mitochondrial oxidative stress, tissue damage and neurodegeneration. These
findings may have relevance to human disease in which
thiamine-dependent enzymes are known to be diminished.
Acknowledgements
We acknowledge Burke Medical Research Institute for
the grant support: AG14930, AG 14600 and AG19589.
References
Bai, C., Fernandez, E., Yang, H., Chen, R., 1999. Purification
and stabilization of a monomeric isocitrate dehydrogenase from
Corynebacterium glutamicum. Protein Exp. Purif. 15 (3), 344–348.
Beal, M.F., 1995. Aging, energy, and oxidative stress in neurodegenerative
diseases. Ann. Neurol. 38 (3), 357–366.
Beal, M.F., 1996. Mitochondria, free radicals and neurodegeneration. Curr.
Opin. Neurobiol. 6, 661–666.
Bennett, C.D., Jones, J.H., Nelson, J., 1966. The effects of thiamine
deficiency on the metabolism of the brain. J. Neurochem. 13, 449–459.
Blass, J.P., 1993. Pathophysiology of the Alzheimer’s syndrome.
Neurology 43 (Suppl. 4), S25–S38.
Bogdan, A.S., 1983. Effect of small amounts of 2,4-dichlorophenoxyacetic
acid derivatives on thiamine and riboflavin metabolism in the animal
body. Neurology 2, 59–62.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248–254.
Butterworth, R.F., 1986. Cerebral thiamine-dependent enzyme changes
in experimental Wernicke’s encephalopathy. Metab. Brain Dis. 1 (3),
165–175.
Butterworth, R.F., Giguere, J.F., 1984. Pyruvate dehydrogenase activity in
regions of the rat brain during postnatal development. J. Neurochem.
43 (1), 280–282.
Butterworth, R.F., Giguere, J.F., Besnard, A.M., 1986. Activities of
thiamine-dependent enzymes in two experimental models of thiamine
deficiency encephalopathy 2 alpha-ketoglutarate dehydrogenase.
Neurochem. Res. 11 (4), 567–577.
Butterworth, R.F., Giguere, J.F., Besnard, A.M., 1985. Activities of
thiamine-dependent enzymes in two experimental models of thiamine
deficiency encephalopathy 1. The pyruvate dehydrogenase complex.
Neurochem. Res. 10, 1417–1428.
Butterworth, R.F., Kril, J.J., Harper, C.G., 1993. Thiamine-dependent
enzyme changes in the brains of alcoholics: relationship to the
Wernicke–Korsakoff syndrome. Alcohol Clin. Exp. Res. 17 (5), 1084–
1088.
Calingasan, N.Y., Baker, H., Sheu, K.F., Gibson, G.E., 1994. Distribution
of the ketoglutarate dehydrogenase complex in rat brain. J.
Comp. Neurol. 346, 461–479.
Calingasan, N.Y., Chun, W.J., Park, L.C., Uchida, K., Gibson, G.E., 1999.
Oxidative stress is associated with region-specific neuronal death during
thiamine deficiency. J. Neuropathol. Exp. Neurol. 58 (9), 946–958.
1027
Calingasan, N.Y., Park, L.C., Calo, L.L., Trifiletti, R.R., Gandy, S.E.,
Gibson, G.E., 1998. Induction of nitric oxide synthase and microglial
responses precede selective cell death induced by chronic impairment
of oxidative metabolism. Am. J. Pathol. 153 (2), 599–610.
Calingasan, N.Y., Sheu, K.F., Baker, H., Jung, E.H., Paoletti, F., Gibson,
G.E., 1995. Heterogeneous expression of transketolase in rat brain. J.
Neurochem. 64 (3), 1034–1044.
Cremer, J.E., Teal, H.M., 1974. The activity of pyruvate dehydrogenase
in rat brain during postnatal development. FEBS Lett. 39 (1), 17–20.
Dean, R.B., Dixon, W.J., 1951. Simplified statistics for small numbers of
observations. Anal. Chem. 23 (4), 636–638.
Elnageh, K.M., Gaitonde, M.K., 1988. Effect of a deficiency of thiamine
on brain pyruvate dehydrogenase: enzyme assay by three different
methods. J. Neurochem. 51 (5), 1482–1489.
Freeman, G.B., Nielsen, P.A., Gibson, G.E., 1987. Effect of age
on behavioural and enzymatic changes during thiamine deficiency.
Neurobiol. Aging 8, 429–434.
Gibson, G.E., Haroutunian, V., Zhang, H., Park, L.C., Shi, Q., Lesser,
M., Mohs, R.C., Sheu, R.K., Blass, J.P., 2000a. Mitochondrial damage
in Alzheimer’s disease varies with apolipoprotein E genotype. Ann.
Neurol. 48 (3), 297–303.
Gibson, G.E., Kingsbury, A.E., Xu, H., Lindsay, J.G., Daniel, S., Foster,
O.J.F., Lees, A.J., Blass, J.P., 2003. Deficits in tricarboxylic acid cycle
in brains from patients with Parkinson’s disease. Neurochem. Int. 43,
129–135.
Gibson, G.E., Ksiezak-Reding, H., Sheu, K.F., Mykytyn, V., Blass, J.P.,
1984. Correlation of enzymatic, metabolic, and behavioral deficits in
thiamin deficiency and its reversal. Neurochem. Res. 9 (6), 803–814.
Gibson, G., Nielsen, P., Mykytyn, V., Carlson, K., Blass, J., 1989.
Regionally selective alterations in enzymatic activities and metabolic
fluxes during thiamin deficiency. Neurochem. Res. 14 (1), 17–24.
Gibson, G.E., Park, L.C., Sheu, K.F., Blass, J.P., Calingasan, N.Y., 2000b.
The alpha-ketoglutarate dehydrogenase complex in neurodegeneration.
Neurochem. Int. 36 (2), 97–112.
Gibson, G.E., Park, L.C., Zhang, H., Sorbi, S., Calingasan, N.Y., 1999.
Oxidative stress and a key metabolic enzyme in Alzheimer brains,
cultured cells, and an animal model of chronic oxidative deficits. Ann.
N.Y. Acad. Sci. 893, 79–94.
Gibson, G.E., Sheu, K.F., Blass, J.P., 1998. Abnormalities of mitochondrial
enzymes in Alzheimer’s disease. J. Neural. Transm. 105 (8–9), 855–
870.
Gibson, G.E., Sheu, K.F., Blass, J.P., Baker, A., Carlson, K.C., Harding, B.,
Perrino, P., 1988. Reduced activities of thiamine-dependent enzymes in
the brains and peripheral tissues of patients with Alzheimer’s disease.
Arch. Neurol. 45 (8), 836–840.
Gubler, C.J., 1961. Studies on the physiological functions of thiamine.
I. The effects of thiamine deficiency and thiamine antagonists on the
oxidation of ketoacids by rat tissues. J. Biol. Chem. 236, 3112–3120.
Hakim, A.M., Pappius, H.M., 1981. The effect of thiamine deficiency on
local cerebral glucose utilization. Ann. Neurol. 9 (4), 334–339.
Hill, R.L., Bradshaw, R.A., 1969. Fumarase. In: Lowenstein, J.M. (Ed.),
Methods in Enzymol, vol. 13, pp. 91–92.
Hucho, F., Randall, D.D., Roche, T.E., Burgett, M.W., Pelley, J.W., Reed,
L.J., 1972. -Keto acid dehydrogenase complexes XVII. Kinetic and
regulatory properties of pyruvate dehydrogenase kinase and pyruvate
dehydrogenase phosphatase from bovine kidney and heart. Arch.
Biochem. Biophys. 151 (1), 328–340.
Humphries, K.M., Szweda, L.I., 1998. Selective inactivation of alphaketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of
lipoic acid with 4-hydroxy-2-nonenal. Biochemistry 37 (45), 15835–
15841.
Johnson, I.R., Gubler, C.J., 1965. Studies with thiamine
pyrophosphokinase from rat brain. Fed. Proc. 24, 481.
Jope, R., Blass, J.P., 1976. The regulation of pyruvate dehydrogenase in
brain in vivo. J. Neurochem. 26 (4), 709–714.
Kinnersley, H.W., Peters, R.A., 1929. Observation upon carbohydrate
metabolism in birds I. The relation between lactic acid content of the
1028
P. Bubber et al. / Neurochemistry International 45 (2004) 1021–1028
brain and the symptoms of opisthotonus in rice-fed pigeons. Biochem.
J. 23, 1126–1136.
Kitto. G.B., 1969. Intra and extramitochondrial malate dehydrogenases
from chicken and tuna heart. In: Lowenstein, J.M. (Ed.), Methods in
Enzymol, vol. 13, pp. 106–116.
Ksiezak-Reding, H., Blass, J.P., Gibson, G.E., 1982. Studies on
the pyruvate dehydrogenase complex in brain with the arylamine
acetyltransferase-coupled assay. J. Neurochem. 38 (6), 1627–1636.
Langlais, P.J., Mair, R.G., 1990. Protective effects of the glutamate
antagonist MK-801 on pyrithiamine-induced lesions and amino acid
changes in rat brain. J. Neurosci. 10 (5), 1664–1674.
Lyubarev, A.E., Kurganov, B.I., 1989. Supramolecular organization of
tricarboxylic acid cycle enzymes. Biosystems 22, 91–102.
Markesbery, W.R., 1997. Oxidative stress hypothesis in Alzheimer’s
disease. Free Radic. Biol. Med. 23, 134–147.
McCormack, J.G., Denton, R.M., 1989. Influence of calcium ions on
mammalian intramitochondrial dehydrogenases. Methods Enzymol.
174, 95–118.
Melov, S., Coskun, P., Patel, M., Tuinstra, R., Cottrell, B., Jun, A.S.,
Zastawny, T.H., Dizdaroglu, M., Goodman, S.I., Huang, T.T., Miziorko,
H., Epstein, C.J., Wallace, D.C., 1999. Mitochondrial disease in
superoxide dismutase 2 mutant mice. Proc. Natl. Acad. Sci. USA
96 (3), 846–851.
Mizuno, Y., Matuda, S., Yoshino, H., Mori, H., Hattori, N., Ikebe, S., 1994.
An immunohistochemical study on alpha-ketoglutarate dehydrogenase
complex in Parkinson’s disease. Ann. Neurol. 35 (2), 204–210.
Morton, R.L., Ikle, D., White, C.W., 1998. Loss of lung mitochondrial
aconitase activity because of hyperoxia in bronchopulmonary dysplasia
in primates. Am. J. Physiol. 274 (1 Pt 1), L127–L133.
Navarro, A., Sanchez Del Pino, M.J., Gomez, C., Peralta, J.L., Boveris,
A., 2002. Behavioral dysfunction, brain oxidative stress and impaired
mitochondrial electron transfer in aging mice. Am. J. Physiol.
Regulatory Integrative Comp. Physiol. 282, R985–R992.
Nose, Y., Iwashima A., Nishino H., 1976. Thiamine uptake by rat brain
slices. In: Gubler, C.J., Fujiwara, M., Dreyfus, P.M. (Eds.), Thiamine.
John Wiley and Sons, New York, pp. 157–168.
Nulton-Persson, A.C., Szweda, L.I., 2001. Modulation of mitochondrial
function by hydrogen peroxide. J. Biol. Chem. 276 (26), 23357–
23361.
Park, L.C., Albers, D.S., Xu, H., Lindsay, J.G., Beal, M.F., Gibson, G.E.,
2001. Mitochondrial impairment in the cerebellum of the patients
with progressive supranuclear palsy. J. Neurosci. Res. 66 (5), 1028–
1034.
Rindi, G., Perri, V., 1961. Uptake of pyrithiamine by tissues of rats.
Biochem. J. 80, 329–342.
Sadek, H.A., Humphries, K.M., Szweda, P.A., Szweda, L.I., 2002.
Selective inactivation of redox-sensitive mitochondrial enzymes during
cardiac reperfusion. Arch. Biochem. Biophys. 406 (2), 222–228.
Scott, B.C., Aruoma, O.I., Evans, P.J., O’Neil, C., Vander Vliet, A., Cross,
C.E., Tristchler, H., Halliwell, B., 1995. Liopoic and dihydrolipoic
acids as antioxidant. A critical review. Free Radi. Res. 20, 119–
123.
Sheu, K.F., Calingasan, N.Y., Lindsay, J.G., Gibson, G.E., 1998.
Immunochemical characterization of the deficiency of the alphaketoglutarate dehydrogenase complex in thiamine-deficient rat brain.
J. Neurochem. 70 (3), 1143–1150.
Shepherd, D., Garland, P.B., 1969. Citrate synthase from rat liver. In:
Lowenstein, J.M. (Ed.), Methods in Enzymol, vol. 13, pp. 11–13.
Sungman, Cha., 1969. Succinate thiokinase from pig heart. In: Lowenstein,
J.M. (Ed.), Methods in Enzymol, vol. 13, pp. 62–65.
Veeger, C., DerVartanian, D.V., Zeylemaker, W.P., 1969. Succinate
dehydrogenase. In: Lowenstein, J.M. (Ed.), Methods in Enzymol, vol.
13, pp. 106–116.
Velot, C., Mixon, M.B., Teige, M., Srere, P.A., 1997. Model of a quinary
structure between Krebs cycle enzymes: a model for the metabolon.
Biochemistry 36 (47), 14271–14276.