effects of liver failure on branched-chain α-keto acid dehydrogenase complex in rat liver and...
TRANSCRIPT
Effects of liver failure on branched-chain a-keto acid dehydrogenasecomplex in rat liver and muscle: comparison between acute
and chronic liver failure
Takashi Honda1, Yoshihide Fukuda1, Isao Nakano1, Yoshiaki Katano1, Hidemi Goto1,Masaru Nagasaki2, Yuzo Sato2, Taro Murakami3, Yoshiharu Shimomura3,*
1Therapeutic Medicine, School of Medicine, Nagoya University, Nagoya 466-8550, Japan2Research Center of Health, Physical Fitness and Sports, Nagoya University, Nagoya 464-8601, Japan
3Department of Materials Science and Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan
Background/Aims: Branched-chain a-keto acid dehydrogenase (BCKDH) complex catalyses the committed step in
the branched-chain amino acid (BCAA) catabolic pathway. In many cases of liver failure, the serum BCAAs/aromatic
amino acids ratio (Fisher’s ratio) decreases, and BCAAs have been administered to patients with liver failure to correct
this ratio. We conducted an animal study to examine whether the effects on hepatic BCKDH complex differ between
acute liver failure (ALF) and chronic liver failure (CLF).Methods: ALF and CLF was induced in rats by a single high-dose injection and 21 weeks of repeated low-dose
injections of carbon tetrachloride, respectively. Plasma BCAA and branched-chain a-keto acid (BCKA) levels, and
activities and protein amounts of hepatic BCKDH complex and kinase were measured.
Results: ALF was characterized by elevated plasma BCAA and BCKA levels and decreased hepatic BCKDH activity.
CLF was characterized by decreased plasma BCAA and BCKA levels and increased hepatic BCKDH activity. This
increase in BCKDH activity in CLF was associated with the decreased BCKDH kinase, which is responsible for the
BCKDH inactivation.
Conclusions: The results obtained in the present study suggest that BCAA catabolism is suppressed in ALF andincreased in CLF.
q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Branched-chain amino acid; Branched-chain a-keto acid; Branched-chain a-keto acid dehydrogenase;Carbon tetrachloride; Liver cirrhosis; Muscle
1. Introduction
Branched-chain amino acids (BCAAs) are essential
amino acids in animals and comprise approximately
35% of the indispensable amino acids in muscle
proteins and 40% of the preformed amino acids required
by mammals [1]. Mammals have a catabolic system for
BCAAs. The first step in this catabolic pathway is
reversible transamination to form branched-chain a-keto
acids (BCKAs), which is catalyzed by branched-chain
aminotransferase (BCAT). The second step is irrevers-
ible oxidative decarboxylation of the BCKAs, which is
catalyzed by branched-chain a-keto acid dehydrogenase
(BCKDH) complex. This is the rate-limiting step in the
catabolic pathway, regulating the BCAA catabolism [1,
2]. The complex is regulated by covalent modification;
BCKDH kinase phosphorylates and inactivates the
complex, and BCKDH phosphatase dephosphorylates
and activates the complex [3]. Phosphorylation regulates
0168-8278/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jhep.2003.11.003
Journal of Hepatology 40 (2004) 439–445
www.elsevier.com/locate/jhep
Received 23 March 2003; received in revised form 4 October 2003;
accepted 3 November 2003* Corresponding author. Address: Department of Bioscience, Nagoya
Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
Tel.: þ81-52-735-5198; fax: þ81-52-735-5198.
E-mail address: [email protected] (Y. Shimomura).
Abbreviations: AAA, aromatic amino acid; ALP, alkaline phosphatase;
ALT, alanine aminotransferase; AST, aspartate aminotransferase; BCAA,
branched-chain amino acid; BCAT, branched-chain aminotransferase;
BCKA, branched-chain a-keto acid; BCKDH, branched-chain a-keto
acid dehydrogenase; ECL, enhanced chemiluminescence; PVDF,
polyvinylidene difluoride.
the enzyme activity in response to alterations in
physiological conditions.
Most previous studies have focused on the rat liver
because BCKDH activity is markedly higher in liver in
comparison to other organs [4]. The liver lacks significant
BCAT activity [5] Therefore, this tissue has almost no
ability to catabolize BCAAs directly, resulting in significant
contribution toward disposal of BCKAs produced by other
organs.
During liver failure in humans, the molar ratio of
BCAAs to aromatic amino acids (AAAs), called Fisher’s
ratio, can be an important marker of liver failure.
However, the dynamics of serum levels of BCAAs and
AAAs appear to differ between acute liver failure (ALF)
and chronic liver failure (CLF). Reported concentrations
of serum BCAAs during ALF in humans have not been
consistent: decreased [6], similar [7,8] and increased [9]
serum BCAAs concentrations in comparison to normal
concentrations have been reported, although markedly
increased concentrations of serum AAAs have been
obtained. In contrast, decreased serum BCAA and slightly
increased serum AAA concentrations have been reported
consistently in CLF [7–9]. Whether both ALF and CLF
can result in a decreased Fisher’s ratio, justifying BCAA
therapy to correct it remains unclear. Oral BCAA
supplementation has improved production of albumin in
the liver of patients with liver cirrhosis (LC) [10],
increased the cumulative survival of patients with
decompensated cirrhosis [11], and improved the mental
state of cirrhotic patients with chronic encephalopathy
[12,13]. However, administration of BCAA to patients
with ALF can cause nitrogen overload and accelerate
hyperammonemia and hepatic encephalopathy. Therefore,
with respect to BCAA metabolism, the two liver failure
conditions may differ.
Although BCKDH complex plays an important role in
BCAA catabolism, little is known about its activity in
relation to either ALF or CLF. In the present animal study,
we examined hepatic and muscular BCKDH activities in
both ALF and CLF.
2. Materials and methods
2.1. Animals
The experimental protocol was reviewed and approved by the AnimalCare Committee of Nagoya University School of Medicine. Male Sprague–Dawley rats (CLEA Japan, Tokyo, Japan) were housed in the conventionalanimal room and had free access to water and a controlled diet based onAIN76 [14] with slight modifications according to AIN93 [15].
2.1.1. Induction of ALFALF was induced in 8-week-old rats weighing 250–260 g ðn ¼ 7Þ by
intraperitoneal injection of 2.0 ml/kg body weight of carbon tetrachloride(CCl4; Wako Pure Chemical Industries, Osaka, Japan) as a 20% mixturewith olive oil (Katayama Chemical, Osaka, Japan) (ALF group). Ratsinjected with olive oil alone served as controls ðn ¼ 6Þ (control group).Rats were sacrificed under pentobarbital anesthesia 24 h after injection
between 18:00 and 20:00, and all rats were deprived of food forapproximately 10 h and weighed prior to being sacrificed. Blood wastaken by syringe from the inferior vena cava and the liver andgastrocnemius muscle were removed rapidly, freeze-clamped at liquidnitrogen temperature, and stored at 280 8C until analyses. All liverswere examined histologically and ALF was confirmed in all rats treatedwith CCl4.
2.1.2. Induction of CLF (LC)LC was induced in 7-week-old rats weighing 160–190 g ðn ¼ 7Þ by
repeated injections of CCl4. Briefly, CCl4 mixed with an equal volume ofolive oil was injected subcutaneously twice per week at 0.5 ml/kg bodyweight, and 0.05% sodium phenobarbital (Katayama Chemical, Osaka,Japan) was given orally in drinking water to promote LC, as reportedpreviously (LC group) [16,17]. Rats injected with olive oil alone served ascontrols ðn ¼ 7Þ (control group). After 21weeks of the experimental period,all rats were treated in the same manner as described in the ALF section.Ascites was detected in all rats treated with CCl4. Histological examinationconfirmed the development of LC in the rats treated with CCl4. Sodiumphenobarbital was given only in rats treated with CCl4. Therefore weexamined the effect of sodium phenobarbital on the hepatic BCKDHcomplex activity in normal rats and confirmed that 0.05% sodiumphenobarbital did not significantly affect the enzyme activity (data notshown).
2.2. Liver and muscle specimens
Frozen liver and skeletal muscle samples were weighed quickly andhomogenized as described previously [18] and were used for analyses.
2.3. Materials
2-Keto[1-14C]isocaproate was purchased from Amersham Japan(Tokyo, Japan). Lambda protein phosphatase was obtained from NewEngland BioLabs (Beverly, MA, USA). Broad-specificity protein phos-phatase [19] and antisera against the E1 and E2 components of BCKDHcomplex [20] were prepared as previously described. Monoclonalantibodies against BCKDH kinase were kindly provided from Dr TomohiroTamura of National Institute of Advanced Industrial Science andTechnology (AIST; Sapporo, Japan). All other reagents were ofbiochemical grade.
2.3.1. Enzyme assaysThe activities of the active/dephosphorylated form (actual activity) and
totally dephosphorylated form (total activity) of hepatic BCKDH complexwere determined separately by spectrophotometric methods based onmeasurement of the NADH production rate [21]. BCKDH kinase wasassessed as previously described [20] with a minor modification: 50 mMa-chloroisocaproate (kinase inhibitor) was added to the BCKDH assaybuffer. Actual and total activities of BCKDH complex in the skeletalmuscle were measured radiochemically with a-keto[1-14C]isocaproate assubstrate [18].
Citrate synthase activity was measured according to the method ofShepherd and Garland [22]. Citrate synthase is a marker enzyme ofmitochondria and it was used to assess the mitochondrial damage caused byCCl4 injection.
2.3.2. Immunoprecipitation of BCKDH complex in liver
extractsImmunoprecipitation of BCKDH complex in liver extracts was
performed as previously reported [23,24]. The preparation obtained wasused in the Western blotting analyses for determination of BCKDH kinasebound to the complex.
2.3.3. Western blottingSamples containing 3.0 mg total protein or immunoprecipitates were
separated by 12.5% polyacrylamide gel electrophoresis in the presence ofSDS (SDS-PAGE) and electrotransferred to polyvinylidene difluoride(PVDF) membranes as previously reported [23,24]. Immunoblotting wasperformed using appropriate primary antibodies (polyclonal antiserumagainst the BCKDH E1 or E2 component, or monoclonal antibodies against
T. Honda et al. / Journal of Hepatology 40 (2004) 439–445440
BCKDH kinase) and secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (H þ L) antibodies orhorseradish peroxidase-conjugated anti-mouse immunoglobulin G anti-bodies). Protein bands on the membrane were visualized using an enhancedchemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Uppsala,Sweden). The E1a and E2 components were easily visualized. However,the signal for E1b was too weak for accurate quantification [25,26].Immunoreactive bands were quantified with the use of NIH Image (Version1.61) software.
2.3.4. Northern blottingTotal cellular RNA was extracted from freeze-clamped liver with
ISOGEN (Wako, Tokyo, Japan) following the instructions of themanufacturer. The RNA concentrations were determined spectro-photometrically and then the samples (20 mg) were electrophoreticallyran in 1.2% agarose-formaldehyde gels [27]. Northern blotting wasperformed as previously described [28]. 32P-labeled cDNAs for ratBCKDH E1a and E2 were used as probes for the hybridization [26].Sample integrity and equal loading of RNA were monitored by stainingwith ethidium bromide after electrophoresis. The abundance ofBCKDH E1a and BCKDH E2 mRNA was expressed relative to theexpression levels in the control rats. The radioactivity of blottedmembranes was determined using a laser imaging analyzer BAS1000(Fuji Film, Tokyo, Japan).
2.3.5. Analyses of blood samplesPeripheral blood components (Table 1) were determined by routine
laboratory methods. Concentrations of plasma BCAAs and BCKAs wereanalyzed with a fluorometric method [29] and spectrophotometric endpointassay [30], respectively.
2.4. Statistical analysis
All values are expressed as mean ^ standard error (SE). Differencesbetween the experimental rats and control rats were analyzedstatistically by unpaired Student’s t-test. P values ,0.05 wereconsidered significant.
3. Results
3.1. Characteristics of control and experimental rats
(Table 1)
Rats with ALF and CLF weighed significantly less than
the control rats. Blood chemistry findings were typical for
ALF and LC in the respective groups of rats.
3.2. Plasma BCAA and BCKA concentrations
The concentration of plasma BCAAs in the ALF group
was significantly greater than that in the control group
(Fig. 1A), whereas that in the cirrhosis group was slightly,
but significantly, lower than that in the control group
(Fig. 1B). The results for plasma BCKAs were similar to
those of plasma BCAAs (Fig. 1C and D).
3.3. Hepatic BCKDH complex, BCKDH kinase, and citrate
synthase activities
Total hepatic BCKDH complex activity in the ALF
group was less than half of that in the control group
(P , 0:001; Fig. 2A). Moreover, the actual activity of
hepatic BCKDH complex in the ALF group was markedly
lower than that in the control group (P , 0:01; Fig. 2C). In
contrast, the actual activity of hepatic BCKDH complex in
LC group was more than twice that in the control group
(P , 0:01; Fig. 2D).
Kinase activities in the cirrhosis and the corresponding
control groups were 0.49 ^ 0.08 and 0.87 ^ 0.08 min21,
respectively, and the former was significantly lower than the
latter ðP , 0:05Þ: On the other hand, the kinase activities in
the ALF and the corresponding control groups were
0.34 ^ 0.07 and 0.37 ^ 0.06 min21, respectively, and did
not differ significantly. It appears that the activities for the
ALF and the control groups, which were measured under
the same conditions, were underestimated, because
the activity in the control group was too low compared to
that of the control group corresponding to the cirrhosis
group. It may result from requirement of almost twice tissue
amounts in the kinase assay due to so low total activity of
the BCKDH complex in the ALF group. In this case, the
protein concentration in the assay mixture was about twice
that for the regular assay. This consideration is supported by
our previous report [20].
Hepatic citrate synthase activity in both the ALF
group and the LC group did not differ significantly
Table 1
Characteristics of acute liver failure model and liver cirrhosis model
Acute liver failure model Liver cirrhosis model
Control ðn ¼ 6Þ Acute liver failure ðn ¼ 7Þ Control ðn ¼ 7Þ Liver cirrhosis ðn ¼ 7Þ
Body weight (g) 253.3 ^ 3.3 241.4 ^ 1.4* 595.7 ^ 8.4 428.6 ^ 24.3***
Liver weight (g) 7.1 ^ 0.3 9.8 ^ 0.3*** 17.4 ^ 0.3 16.1 ^ 1.8
Liver weight/body weight (%) 2.8 ^ 0.1 4.0 ^ 0.1*** 2.9 ^ 0.1 3.7 ^ 0.3*
AST (IU/l) 83.0 ^ 2.7 4046.0 ^ 433.6*** 66.0 ^ 7.8 235.1 ^ 37.2***
ALT (IU/l) 27.0 ^ 2.4 2223.1 ^ 313.2*** 36.0 ^ 8.4 108.6 ^ 8.1***
Alkaline phosphatase (IU/l) 1015.0 ^ 55.2 1637.0 ^ 137.3** 369.4 ^ 27.0 971.7 ^ 202.3*
Total bilirubin (mg/dl) ND 0.8 ^ 0.2** ND 0.2 ^ 0.1*
Albumin (g/dl) 3.4 ^ 0.0 3.2 ^ 0.1 4.2 ^ 0.1 2.6 ^ 0.2***
NH3 (mg/dl) 97.2 ^ 9.4 139.1 ^ 21.5 97.0 ^ 18.0 130.5 ^ 14.3
Platelet ( £ 103/ml) 85.1 ^ 2.8 81.7 ^ 4.6 75.7 ^ 3.8 50.7 ^ 7.9*
Values represent means ^ SE. Significantly different from control in each group (*P , 0:05; **P , 0:01; ***P , 0:001). Significance of statistical
differences was assessed by unpaired Student’s t-test. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ND, not detected.
T. Honda et al. / Journal of Hepatology 40 (2004) 439–445 441
from activity in the corresponding control groups
(Fig. 2E and F).
3.4. Levels of hepatic BCKDH complex subunits
and BCKDH kinase
The relationship between the amount of proteins loaded
on the gel and the intensities of signals obtained after blots
of BCKDH E2 on the membrane showed a good
proportionality (Fig. 3). We confirmed that other proteins
also showed the same proportionality.
The level of the E1a subunit in ALF group was 39% of
that in the control group (P , 0:01; Fig. 4A). This value was
similar to the total activity of the enzyme complex described
above. The level of the E1a subunit in LC rats was slightly
higher than that in the control rats (Fig. 4B). Amount of the
E2 subunit in ALF group was slightly lower and that in
cirrhosis rats was slightly higher than the level in respective
control groups, but the differences were not statistically
significant (Fig. 4C and D).
The level of the BCKDH kinase protein in the ALF
group was slightly, but not significantly, lower than that in
the control rats (Fig. 4E). The level of the kinase protein in
LC rats was significantly lower than that in the correspond-
ing control rats (P , 0:01; Fig. 4F).
3.5. Abundance of BCKDH E1a and BCKDH E2 mRNA
in liver
Northern blot analysis showed that the abundance of
BCKDH E1a mRNA was significantly lower in the ALF
group than that in the control rats (P , 0:01; Fig. 5A),
whereas the abundance did not differ between the
cirrhosis and the corresponding control groups (Fig. 5B).
The abundance of BCKDH E2 mRNA in either the ALF
group or the cirrhosis group did not differ significantly
from those in the corresponding control groups (Fig. 5C
and D).
3.6. BCKDH complex activity in muscle
Total muscle BCKDH complex activity in both liver
failure groups was similar to activity in the corresponding
control groups (Fig. 6A and B). However, the actual activity
of muscle BCKDH complex in the ALF group was twice
that of the control group (P , 0:05; Fig. 6C), whereas the
Fig. 1. Plasma BCAA and BCKA concentrations in acute liver failure
(ALF) and liver cirrhosis (LC). Values are mean 6 SE. Asterisks
indicate significant differences from the respective control group
(**P < 0.01, ***P < 0.001).
Fig. 2. Total and actual hepatic BCKDH activities and hepatic citrate
synthase activity in acute liver failure (ALF) and liver cirrhosis (LC).
Values are mean 6 SE. Asterisks indicate significant differences from
the respective control group (**P < 0.01, ***P < 0.001).
Fig. 3. Relationship between the amounts of proteins (0.5–5.0 mg of
liver extracts) loaded on the gel and the intensity of signals on the
membrane. Blots on the membrane were stained with antiserum
against BCKDH E2.
T. Honda et al. / Journal of Hepatology 40 (2004) 439–445442
actual activity in the cirrhosis group did not differ from that
of the control group (Fig. 6D).
4. Discussion
In the present study, we observed different responses
of the BCKDH complex to ALF and CLF. In the
presence of ALF, hepatic BCKDH complex activity was
markedly decreased, and concentrations of plasma
BCAAs and BCKAs were increased, suggesting that
hepatic BCKA catabolism was suppressed. On the other
hand, in the presence of CLF, enzyme activity was not
suppressed; rather the actual activity was increased, and
the concentrations of both plasma BCKAs and BCAAs
were decreased, suggesting BCKA catabolism may be
enhanced in the cirrhotic liver in comparison to that of
normal liver.
When the proportion of the active, non-phosphorylated
form of the enzyme in the total population of the molecule
was designated as the ‘activity state’, the activity state of
BCKDH complex was correlated inversely with BCKDH
kinase activity under various physiological conditions
[31–35]. In the present study, the activity and the protein
amount of BCKDH kinase were significantly decreased, and
inversely, the activity state of the BCKDH complex was
significantly increased in CLF. Considering that BCKDH
kinase regulates the BCKDH complex activity, the BCKDH
kinase may be damaged in CLF, resulting in increased
BCKDH complex activity and low plasma concentrations
of BCKAs and BCAAs. It was reported that serum levels of
Fig. 4. Protein levels for hepatic BCKDH complex subunits (E1a and
E2) and BCKDH kinase in acute liver failure (ALF) and liver cirrhosis
(LC). Liver extracts (3 mg protein/lane) or immunoprecipitates
(10 ml/lane) were applied on the gel for electrophoresis as described
in Section 2. ALF and LC data are expressed as percentages of
respective controls. Typical images of the Western blots are showed
above each bar. Values are mean 6 SE. Asterisks indicate significant
differences from the respective controls (**P < 0.01).
Fig. 5. mRNA for hepatic BCKDH subunits (E1a and E2) in acute liver
failure (ALF) and liver cirrhosis (LC). Each sample (20 mg RNA/lane)
was applied on the gel for electrophoresis as described in Section 2.
ALF and LC data are expressed as percentages of respective controls.
Values are mean 6 SE. Asterisks indicate significant differences from
the respective controls (**P < 0.01).
Fig. 6. Total and actual muscle BCKDH activities in acute liver failure
(ALF) and liver cirrhosis (LC). Values are mean 6 SE. Asterisks
indicate significant differences from the respective control group
(*P < 0.05).
T. Honda et al. / Journal of Hepatology 40 (2004) 439–445 443
cytokines were elevated in CLF [36–38] and that cytokines
increased the rate of the oxidation of the amino acids [39].
Furthermore, it was demonstrated that administration of
cytokines activated BCKDH complex in rat muscle [40].
These findings suggest that there may be a role for
proinflammatory cytokines in increased hepatic BCKDH
complex activity in LC rats. Further investigations are
warranted to clarify the mechanisms responsible for effects
of CLF on the BCKDH activation. On the other hand, the
activity and the protein amount of the kinase did not appear
to be affected by ALF. It is compatible that the activity state
of BCKDH complex was not altered by ALF.
In contrast to the distinct features of BCAA catabolism
between these two liver failure models, the activity of
hepatic citrate synthase (mitochondrial marker enzyme) was
similar in both ALF and CLF. This indicates that the distinct
hepatic BCKDH complex activities in the two liver failure
models are not due to CCl4-induced mitochondrial dis-
orders. According to Western blotting analysis, the level of
the E1a subunit of BCKDH complex showed a trend similar
to that of the total activity of the complex under both liver
failure conditions, whereas the level of the E2 subunit was
not altered. These results suggest that the reduced BCKDH
activity is due to decreased levels of the E1 subunit. This
may be related to findings that dietary protein deficiency
reduces levels of E1a protein relative to those of E2 protein
[26] and that E1a is susceptible to enzyme inactivation [41].
These findings suggest that the integrity of the BCKDH
complex may be regulated by the amount of E1 subunit [26].
The abundance of BCKDH E1a mRNA was also decreased
by ALF, suggesting that the gene expression of the subunit
may be impaired by the liver failure.
In the present study, the activity state of liver BCKDH
complex in two control rat groups were 41% (in the control
rats for ALF rats) and 20% (in the control rats for the
cirrhosis rats). In normal rats, a wide range of the activity
state of the liver complex was reported [42], because the
activity state is greatly affected by many factors such as a
protein content in the diet, the time of the day for liver
sampling from rats, and gender of rats [32–34]. The reasons
for the difference in the activity states between two control
groups in the present study was not elucidated, but the age
of rats on the final day of the experiment was different,
probably resulting in the different activity state. In a
preliminary experiment, normal rats fed a laboratory chow
(CE-2 from CLEA Japan, Tokyo) at 8 weeks of age had 85%
of the activity state, when rats were sacrificed at the same
conditions as ALF rats.
BCAAs can be oxidized by skeletal muscle to produce
energy. In the present study, muscular BCKDH complex
activity was increased significantly to approximately twice
that observed in ALF. However, the BCKDH complex
activity was not changed in CLF, suggesting that muscular
BCAA catabolism is increased only in ALF. Such activation
of the muscular BCKDH complex with high serum BCAA
concentrations was also observed in rats stressed with
exercise [18] and with diabetes [25,33]. Although muscular
BCKA catabolism was promoted, it did not appear to be
sufficient to compensate for the decreased hepatic BCKA
oxidation observed in the rats.
In conclusion, the results in the present study suggest that
ALF suppresses and that CLF promotes BCAA catabolism.
It has been reported that BCAA administration improves
protein turnover in rats and humans with LC [43–45],
suggesting that BCAA administered to patients with
cirrhosis is appropriate. Furthermore, BCAA may be a
preferable substrate to fulfill the energy requirements of
patients with cirrhosis [46], because energy efficacy of
BCAAs is higher than that of other substrates such as
glucose and fatty acids and glucose tolerance is deteriorated
in 70 – 80% of LC patients [47]. However, BCAA
administration in patients with ALF, especially those with
high serum BCAA concentrations, may not be appropriate
because of the potential for BCAA overload. The findings
obtained in the present study support that BCAA adminis-
tration therapy is suitable for patients with LC but not those
with ALF.
Acknowledgements
This work was supported in part by a grant-in-aid for
scientific research (14370022 to Y.S.) from the Ministry of
Education, Science, Sports and Culture, Japan.
References
[1] Harper AE, Miller RH, Block KP. Branched-chain amino acid
metabolism. Annu Rev Nutr 1984;4:409–454.
[2] Harris RA, Popov KM, Zhao Y, Shimomura Y. Regulation of
branched-chain amino acid catabolism. J Nutr 1994;124:
1499S–1502S.
[3] Damuni Z, Reed LJ. Purification and properties of the catalytic
subunit of the branched-chain alpha-keto acid dehydrogenase
phosphatase from bovine kidney mitochondria. J Biol Chem 1987;
262:5129–5132.
[4] Harris RA, Zhang B, Goodwin GW, Kuntz MJ, Shimomura Y,
Rougraff P, et al. Regulation of the branched-chain alpha-ketoacid
dehydrogenase and elucidation of a molecular basis for maple syrup
urine disease. Adv Enzyme Regul 1990;30:245–263.
[5] Hutson SM. Subcellular distribution of branched-chain aminotrans-
ferase activity in rat tissues. J Nutr 1988;118:1475–1481.
[6] Clemmesen JO, Kondrup J, Ott P. Splanchnic and leg exchange of
amino acids and ammonia in acute liver failure. Gastroenterology
2000;118:1131–1139.
[7] Fischer JE, Rosen HM, Ebeid AM, James JH, Keane JM, Soeters PB.
The effect of normalization of plasma amino acids on hepatic
encephalopathy in man. Surgery 1976;80:77–91.
[8] Rosen HM, Yoshimura N, Hodgman JM, Fischer JE. Plasma amino
acid patterns in hepatic encephalopathy of differing etiology.
Gastroenterology 1977;72:483–487.
[9] Watanabe A, Hayashi S, Higashi T, Obata T, Sakata T, Takei N,
et al. Characteristics change in serum amino acid levels in
different types of hepatic encephalopathy. Gastroenterol Jpn 1982;
17:218–223.
[10] Kato M, Yoshida T, Moriwaki H, Muto Y. Effect of branched-chain
amino acid (BCAA) enriched-nutrient mixture on albumin meta-
bolism in cirrhotic patients. Acta Hepatol Jpn 1991;32:692–699.
T. Honda et al. / Journal of Hepatology 40 (2004) 439–445444
[11] Yoshida T, Muto Y, Moriwaki H, Yamato M. Effect of long-term oral
supplementation with branched-chain amino acid granules on the
prognosis of liver cirrhosis. Gastroenterol Jpn 1989;24:692–698.
[12] Marchesini G, Dioguardi FS, Bianchi GP, Zoli M, Bellati G, Roffi L,
et al. Long-term oral branched-chain amino acid treatment in chronic
hepatic encephalopathy. A randomized double-blind casein-con-
trolled trial. The Italian Multicenter Study Group. J Hepatol 1990;
11:92–101.
[13] Plauth M, Egberts EH, Hamster W, Torok M, Muller PH, Brand O,
et al. Long-term treatment of latent portosystemic encephalopathy
with branched-chain amino acids. A double-blind placebo-controlled
crossover study. J Hepatol 1993;17:308–314.
[14] Committee on Standards for Nutritional Studies, Report of the
American Institute of Nutrition ad hoc committee on standards for
nutritional studies. J Nutr 1977;107:1340–1348.
[15] Reeves PG, Nielsen FH, Fahey Jr. GC. AIN-93 purified diets for
laboratory rodents: final report of the American Institute of Nutrition
ad hoc writing committee on the reformulation of the AIN-76A rodent
diet. J Nutr 1993;123:1939–1951.
[16] Proctor E, Chatamra K. High yield micronodular cirrhosis in the rat.
Gastroenterology 1982;83:1183–1190.
[17] Kajiwara K, Okuno M, Kobayashi T, Honma N, Maki T, Kato M, et al.
Oral supplementation with branched-chain amino acids improves
survival rate of rats with carbon tetrachloride-induced liver cirrhosis.
Dig Dis Sci 1998;43:1572–1579.
[18] Shimomura Y, Suzuki T, Saitoh S, Tasaki Y, Harris RA, Suzuki M.
Activation of branched-chain alpha-keto acid dehydrogenase complex
by exercise: effect of high-fat diet intake. J Appl Physiol 1990;68:
161–165.
[19] Harris RA, Paxton R, Parker RA. Activation of the branched-chain
alpha-ketoacid dehydrogenase complex by a broad specificity
protein phosphatase. Biochem Biophys Res Commun 1982;107:
1497–1503.
[20] Shimomura Y, Nanaumi N, Suzuki M, Popov KM, Harris RA.
Purification and partial characterization of branched-chain alpha-
ketoacid dehydrogenase kinase from rat liver and rat heart. Arch
Biochem Biophys 1990;283:293–299.
[21] Nakai N, Kobayashi R, Popov KM, Harris RA, Shimomura Y.
Determination of branched-chain alpha-keto acid dehydrogenase
activity state and branched-chain alpha-keto acid dehydrogenase
kinase activity and protein in mammalian tissues. Methods Enzymol
2000;324:48–62.
[22] Shepherd D, Garland PB. Citrate synthase from rat liver. Methods
Enzymol 1969;13:11–16.
[23] Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 1970;227:680–685.
[24] Obayashi M, Sato Y, Harris RA, Shimomura Y. Regulation of the
activity of branched-chain 2-oxo acid dehydrogenase (BCODH)
complex by binding BCODH kinase. Fed Eur Biochem Soc Lett 2001;
491:50–54.
[25] Li Z, Murakami T, Nakai N, Nagasaki M, Obayashi M, Xu M, et al.
Modification by exercise training of activity and enzyme expression
of hepatic branched-chain alpha-ketoacid dehydrogenase complex in
streptozotocin-induced diabetic rats. J Nutr Sci Vitaminol (Tokyo)
2001;47:345–350.
[26] Zhao Y, Popov KM, Shimomura Y, Kedishvili NY, Jaskiewicz J,
Kuntz MJ, et al. Effect of dietary protein on the liver content and
subunit composition of the branched-chain alpha-ketoacid dehydro-
genase complex. Arch Biochem Biophys 1994;308:446–453.
[27] Sambrook J, Fritsch EF, Maniatis T. Electrophoresis of RNA through
gels containing formaldehyde, 2nd ed. Molecular cloning: a
laboratory manual, Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press; 1989. pp. 7.43–7.45.
[28] Murakami T, Shimomura Y, Fujitsuka N, Nakai N, Sugiyama S,
Ozawa T, et al. Enzymatic and genetic adaptation of soleus muscle
mitochondria to physical training in rats. Am J Physiol 1994;267:
E388–E395.
[29] Gleeson M, Maughan RJ. A simple enzymatic fluorimetric method for
the determination of branched-chain L-amino acids in microlitre
volumes of plasma. Clin Chim Acta 1987;166:163–169.
[30] Goodwin GW, Kuntz MJ, Paxton R, Harris RA. Enzymatic
determination of the branched-chain alpha-keto acids. Anal Biochem
1987;162:536–539.
[31] Wagenmakers AJ, Schepens JT, Veerkamp JH. Effect of starvation
and exercise on actual and total activity of the branched-chain 2-oxo
acid dehydrogenase complex in rat tissues. Biochem J 1984;223:
815–821.
[32] Kobayashi R, Shimomura Y, Murakami T, Nakai N, Fujitsuka N,
Otsuka M, et al. Gender difference in regulation of branched-chain
amino acid catabolism. Biochem J 1997;327:449–453.
[33] Aftring RP, Miller WJ, Buse MG. Effects of diabetes and starvation on
skeletal muscle branched-chain alpha-keto acid dehydrogenase
activity. Am J Physiol 1988;254:E292–E300.
[34] Popov KM, Zhao Y, Shimomura Y, Jaskiewicz J, Kedishvili NY,
Irwin J, et al. Dietary control and tissue specific expression of
branched-chain alpha-ketoacid dehydrogenase kinase. Arch Biochem
Biophys 1995;316:148–154.
[35] Huang YS, Chuang DT. Down-regulation of rat mitochondrial
branched-chain 2-oxoacid dehydrogenase kinase gene expression by
glucocorticoids. Biochem J 1999;339:503–510.
[36] Khoruts A, Stahnke L, McClain CJ, Logan G, Allen JI.
Circulating tumor necrosis factor, interleukin-1 and interleukin-6
concentrations in chronic alcoholic patients. Hepatology 1991;13:
267–276.
[37] Tilg H, Wilmer A, Vogel W, Herold M, Nolchen B, Judmaier G, et al.
Serum levels of cytokines in chronic liver diseases. Gastroenterology
1992;103:264–274.
[38] Ludwiczek O, Kaser A, Novick D, Dinarello CA, Rubinstein M,
Vogel W, et al. Plasma levels of interleukin-18 and interleukin-18
binding protein are elevated in patients with chronic liver disease.
J Clin Immunol 2002;22:331–337.
[39] Garcia-Martinez C, Llovera M, Lopez-Soriano FJ, del Santo B,
Argiles JM. Lipopolysaccharide (LPS) increases the in vivo oxidation
of branched-chain amino acids in the rat: a cytokine-mediated effect.
Mol Cell Biochem 1995;148:9–15.
[40] Nawabi MD, Block KP, Chakrabarti MC, Buse MG. Administration
of endotoxin, tumor necrosis factor, or interleukin 1 to rats activates
skeletal muscle branched-chain alpha-keto acid dehydrogenase. J Clin
Invest 1990;85:256–263.
[41] Shimomura Y, Kuntz MJ, Suzuki M, Ozawa T, Harris RA.
Monovalent cations and inorganic phosphate alter branched-chain
alpha-ketoacid dehydrogenase-kinase activity and inhibitor sensi-
tivity. Arch Biochem Biophys 1988;266:210–218.
[42] Block KP, Aftring RP, Buse MG, Harper AE. Estimation of branched-
chain alpha-keto acid dehydrogenase activation mammalian tissues.
Methods Enzymol 1988;166:201–213.
[43] Usui T, Moriwaki H, Hatakeyama H, Kasai T, Kato M, Seishima M,
et al. Oral supplementation with branched-chain amino acids
improves transthyretin turnover in rats with carbon tetrachloride-
induced liver cirrhosis. J Nutr 1996;126:1412–1420.
[44] Okuno M, Moriwaki H, Kato M, Muto Y, Kojima S. Changes in the
ratio of branched-chain to aromatic amino acids affect the secretion of
albumin in cultured rat hepatocytes. Biochem Biophys Res Commun
1995;214:1045–1050.
[45] Marchesini G, Bianchi G, Merli M, Amodio P, Panella C, Loguercio
C, et al. Nutritional supplementation with branched-chain amino acids
in advanced cirrhosis: a double-blind, randomized trial. Gastroenter-
ology 2003;124:1792–1801.
[46] Kato M, Miwa Y, Tajika M, Hiraoka T, Muto Y, Moriwaki H.
Preferential use of branched-chain amino acids as an energy substrate
in patients with liver cirrhosis. Intern Med 1998;37:429–434.
[47] Megyesi C, Samols E, Marks V. Glucose tolerance and diabetes in
chronic liver disease. Lancet 1967;2:1051–1056.
T. Honda et al. / Journal of Hepatology 40 (2004) 439–445 445