Neurochemical research, 2003 - KD, GPx

8/7/2019 Neurochemical research, 2003 - KD, GPx

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Ketogenic Diet Increases Glutathione Peroxidase Activity

in Rat Hippocampus

Denize R. Ziegler,1 Leticia C. Ribeiro,2 Martine Hagenn,3 lonara R. Siqueira,2

Emeli Arajo,2 Iracy L. S. Torres,2 Carmem Gottfried,1,2 Carlos Alexandre Netto,2

and Carlos-Alberto Gonalves2

(Accepted April 23, 2003)

Ketogenic diets have been used in the treatment of refractory childhood epilepsy for almost

80 years; however, we know little about the underlying biochemical basis of their action. In

this study, we evaluate oxidative stress in different brain regions from Wistar rats fed a ketogenic

diet. Cerebral cortex appears to have not been affected by this diet, and cerebellum presented

a decrease in antioxidant capacity measured by a luminol oxidation assay without changes in

antioxidant enzyme activitiesglutathione peroxidase, catalase, and superoxide dismutase. In

the hippocampus, however, we observed an increase in antioxidant activity accompanied by an

increase of glutathione peroxidase (about 4 times) and no changes in lipoperoxidation levels.

We suggest that the higher activity of this enzyme induced by ketogenic diet in hippocam-

pus might contribute to protect this structure from neurodegenerative sequelae of convulsive

disorders.

KEY WORDS: Ketogenic diet; oxidative stress; glutathione peroxidase; lipoperoxidation; hippocampus.

INTRODUCTION

Brain tissue is particularly vulnerable to oxidative

damage, possibly because of its high consumption of

oxygen and the consequent generation of high quantities

of reactive oxygen species (ROS) during oxidative phos-

phorylation (1). Moreover, several enzymes expressed in

brain, including monoamine oxidase, tyrosine hydroxy-

lase, and L-amino acid oxidase, lead to hydrogen peroxide

formation as a normal by-product of their activity. Sev-

eral regions of the brain are particularly rich in iron,

which promotes the production of damaging oxygen

free radical species. Furthermore, the brain is relatively

poorly endowed with protective antioxidant enzymes or

antioxidant compounds (2,3).

ROS formation has been implicated in damage to

cerebral tissue in several nervous pathologies, such as

ischemia-reperfusion injury, Parkinsons disease, and

epilepsy (4). Active oxygen species concentrations are

often increased during seizure activity, and both initia-tion and propagation of lipid peroxidation have been sug-

gested to play a role in epileptogenesis (57).

The ketogenic diet (KD) has been used in the treat-

ment of refractory childhood epilepsy since the early

1920s and reemerged as an important alternative clini-

cal approach in the 1990s. The efficacy of the diet as a

treatment for human epilepsy has been suggested by

clinical evidence (8,9). The KD is designed to stimulate

0364-3190/03/12001793/0 2003 Plenum Publishing Corporation

Neurochemical Research, Vol. 28, No. 12, December 2003 ( 2003), pp. 17931797

1 Centro de Cincias da Sade, Universidade do Vale do Rio dos Sinos,

So Leopoldo, RS, Brazil.2 Departamento de Bioqumica, ICBS, Universidade Federal do Rio

Grande do Sul, Porto Alegre, RS, Brazil.3 Departamento de Fisiologia, ICBS, Universidade Federal do Rio Grande

do Sul, Porto Alegre, RS, Brazil.4 Address reprint requests to: Denize Righetto Ziegler, Centro de

Cincias da Sade, Unisinos, Av. Unisinos, 950Caixa postal: 275,

93022-000, So Leopoldo RS, Brazil. Fax: 55-51-590 8122; E-mail:

[email protected]


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1794 Ziegler et al.

the biochemical effects of fasting by maintaining a state

of ketosis, the resulting condition provides for much of

the cerebral energy requirements in the form of ketone

bodies. Despite the clinical success, there have been

remarkably few studies pointing to the possible mechan-

isms of that diet, as well as to their effects on distinct

aspects regarding central nervous system metabolism(9,10).

It should be interesting to investigate whether KD

changes brain antioxidant activities. In this study we

evaluate oxidative stress in different brain regions (hip-

pocampus, cerebral cortex, and cerebellum) from rats fed

a KD. We measured lipid peroxidation assayed by levels

of thiobarbituric acid reactive substances (TBARS), total

antioxidant reactivity (TAR) estimated through measure-

ments of luminol oxidation assay, and antioxidant enzyme

activitiescatalase, superoxide dismutase and glutathione

peroxidase.

EXPERIMENTAL PROCEDURE

Reagents and Equipment. All chemicals were purchased from

Sigma (St. Louis, MO, USA) except for the RANSOD kit, which was

purchased from RANDOX. Total antioxidant capacity was assayed using

a beta liquid scintillation spectrometer (Wallac model 1409), and the

enzyme activities were measured with a double-beam spectrophotome-

ter with temperature control (Hitachi U-2001).

Animals and Diet. Male 30-day-old Wistar rats came from the

local breeding colony (ICBS-UFRGS). Animals were weight matched

and divided into two groups: control rats that received regular labo-

ratory chow (Nuvilab-CR1, Nuvital, Brazil) and treated rats that

received a KD (Table I) for 8 weeks (10). They were maintained ina ventilated room at 21C, with free access to food and water on a

12-h light/dark cycle. All animal procedures were in accordance with

the NIH guidelines for the care and use of laboratory animals and

were approved by the local authorities.

Tissue Preparation. Animals were killed by decapitation, and the

brain regions were dissected on ice. Brain tissue was homogenized in

the incubation medium used for each technique and centrifuged at

1000 g for 10 min at 4C, and the supernatant was immediately

used for the lipid peroxidation and antioxidant reactivity measure-

ments. For the enzyme activity determinations, the brain tissue was

kept frozen at 70C for up to 1 week.

Thiobarbituric Acid Reactive Substances (TBARS) Assay. TBARS

were determined immediately after tissue homogenization by a fluores-

cence method (11). After extraction with n-buthanol, fluorescence was

measured at 515 nm excitation and 555 nm emission. Values wereexpressed as nM TBARS/g tissue, using malondialdehyde standards pre-

pared from 1,1,3,3-tetramethoxypropane.

Total Antioxidant Reactivity (TAR) Assay. The method was

based on Lissi et al. (12) and Desmarchelier et al. (13). The reac-

tion mixture contains the free radical source 2 mM 2,2 azobis

(2-amidopropane (ABAP) and 6 mM luminol in glycine buffer

(0.1 M, pH 8.6). Incubation of this mixture at 20C generates an

almost constant light intensity that was measured in a scintillation

counter (Beckman) working in the out of coincidence mode. The

TAR values were determined by measuring the initial decrease of

luminol luminescence, calculated as the ratio lo/l, where lo is the

luminescence intensities in the absence of additives and l

is the luminescence intensity after addition of a small aliquot of

the sample. A comparison of the ratio lo/l of Trolox (20 nM) and

the samples allows obtaining TAR values as equivalents of Trolox

concentration.

Catalase (CAT) Assay. CAT activity was assayed by the method

of Aebi (14), which is based on the disappearance of H2O2 at 240 nm.

Brain tissue was homogenized 1:10 (w/v) in 10 mM potassium phos-

phate buffer, pH 7.6. One unit is defined as one M of hydrogen

peroxide consumed per minute, and the specific activity is reported

as units per milligram of protein.Superoxide Dismutase (SOD) Assay. The assay for SOD activ-

ity was carried out with the RANSOD kit (Randox, USA). Cerebral

tissue was homogenized 1:10 (w/v) in 10 mM potassium phosphate

buffer, pH 7.4. This method is based on the formation of red

formazan from the reaction of 2-(4-iodophenyl)-3-(4-nitrophenol)-

5-phenyltetrazolium chloride (INT) and superoxide radical (pro-

duced in the incubation medium from xanthine oxidase reaction),

which is assayed in a spectrophotometer at 505 nm. The inhibition

of the produced chromogen is proportional to the activity of the

SOD present in the sample. A 50% inhibition is defined as 1 unit

of SOD, and specific activity is expressed as units per milligram of

protein.

Glutathione Peroxidase (GPx) Assay. GPx activity was measured

by the method of Wendel (15), except for the concentration of NADPH,

which was adjusted to 0.1 mM. Tissue was homogenized 1:10 (w/v) in10 mM potassium phosphate buffer, pH 7.6. Tert-butyl-hydroperoxide

was used as substrate. NADPH disappearance was monitored with a

spectrophotometer at 340 nm. One GPx unit is defined as 1 Mol of

NADPH consumed per minute, and specific activity is reported as units

per milligram of protein.

Protein Determination. Protein was measured by the method of

Lowry et al. (16) using bovine serum albumin as standard.

Statistical analysis. Results are expressed as mean values SEM.

Students t test was used to analyze the significance of differences

between control and experimental groups.

Table I. Composition of the Control and Ketogenic Diets

Control diet* g/100 g Ketogenic diet g/100 g

Total fat 11 Lard 69Sunflower oil 0.5

Protein 22 Protein 24

Fiber 3 Fiber 1Ash 6 Ash 4Vitamin 2 Vitamin 1.5Carbohydrates 52 Carbohydrates 0

*Commercial nonpurified diet, Nuvilab-CR1 (Curitiba, Brazil).Casein, purity 87% (from Herzog, Porto Alegre, Brazil) supplementedwith 0.15% L-Methionine (from Merck, Rio de Janeiro, Brazil).Mineral mixture (from Roche, So Paulo, Brazil), mg/100 g of ration:NaCl, 557; Kl, 3.2; KH2PO4, 1556; MgSO4, 229; CaCO3, 1526;FeSO4.7H2O, 108; MnSO4.H2O, 16; ZnSO4.7H2O, 2.2; CuSO4.5H2O,1.9; CoCl2.6H2O, 0.09.Vitamin mixture (from Roche, So Paulo, Brazil), mg/100 g of ration:vitamin A, 4; vitamin D, 0.5; vitamin E, 10; menadione, 0.5; choline,200; PABA 10; inositol 10 mg; niacin, 4; pantothenic acid, 4; riboflavin,0.8; thiamine, 0.5; pyridoxine, 0.5; folic acid, 0.2; biotin, 0.04; vitaminB12, 0.003.


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Fig. 2. Effects of KD on TAR assay in rat hippocampus (Hc),cerebral cortex (Cx) and cerebellum (Cb). TAR was measured bydecrease of luminol luminescence. Results are expressed aspercentage of control. Columns represent mean SEM of eightindependent experiments performed in duplicate. The mean TARvalues (expressed as pM eq. Trolox/mg protein) from control group

were 57.37 9.8 (Hc), 70.42 1.3 (Cx), and 74.97 1.2 (Cb).*Values significantly different from control group, as determined byStudents t test (P 0.05).

RESULTS

Thirty-day-old rats fed a KD for 8 weeks gained

weight similarly to controls. We used a semiquantitative

method for evaluate ketonemia at sacrifice day. Ketonemia

in control rats was less than 0.4 mM and in ketogenic rats

was about 2.4 mM.Brain regions examined showed distinct behavior

in regard to TBARS levels (Fig. 1). In cerebral cortex

and hippocampus, TBARS levels were similar between

KD-treated animals and controls, suggesting that lipo-

peroxidation within those structures was not altered.

However, TBARS levels were significantly increased

in cerebellum of KD-treated rats, indicating an increase

in lipoperoxidation resulting from the change in the diet

composition.

The analysis of total antioxidant capacity (TAR)

in cerebral cortex revealed no significant differences

between the two groups. However, rats treated with a KDhad a significant increase in their antioxidant capacity in

hippocampus and a significant decrease in the cerebellum

(Fig. 2).

We found changes of TBARS and TAR in hippo-

campus and cerebellum, and therefore we decided to inves-

tigate antioxidant enzyme activities in these regions. There

were no significant differences between experimental

groups regarding the three enzymes activitiessuperoxide

dismutase (SOD), catalase (CAT), and glutathione peroxi-

dase (GPx) in cerebellum. However, in hippocampus, CAT

activity decreased around 50% and GPx was much more

active, around 400%, in KD-fed animals, although SOD

activity did not vary between groups (Table II).

DISCUSSION

The understanding of possible mechanisms that

underlie the therapeutic effects of KD in epileptic dis-orders is very important. It has been widely suggested

that nutritive dietary constituents can promote or hinder

the development of several chronic diseases (17), mainly

because of an increased susceptibility to or protection

against free radicals, respectively. This is, to our know-

ledge, the first study to focus on the relationship between

KD and oxidative stress in CNS.

Oxidative stress has been defined as the increase in

steady-state concentrations of active oxygen species,

either resulting from an overproduction of radical species

and/or as a consequence of antioxidant defenses deple-

tion. It has been widely recognized that the susceptibility

to oxidative stress differs according to specific brainregion (1820). According to this characteristic, oxida-

tive stress has distinct effects in the brain structures

studied. Cerebral cortex seems to have not been

affected by KD, maintaining the lipoperoxidation level

or the total antioxidant capacity. However, in KD rat

cerebellum, there was a decrease in antioxidant capa-

city not resulting from a drop in antioxidant enzyme

activities. Increased lipoperoxidation may be due to a

Fig. 1. Effects of KD on TBARS levels in rat hippocampus (Hc),cerebral cortex (Cx), and cerebellum (Cb). Wistar rats were fed withcontrol or ketogenic diet (KD). TBARS levels were measured at515-nm excitation and 555-nm emission wavelengths. Columnsrepresent mean SEM of six independent experiments performed induplicate. Students t test was used to evaluate the significance ofdifferences between paired group means.*Values significantly different from control diet group (P 0.01).


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1796 Ziegler et al.

Table II. Effects of Treatment with KD on Enzyme Activities in Hippocampus and CerebellumHomogenates from Rats

Enzymes activities (units/mg protein)

Groups CAT SOD GPx

Hippocampus control 0.120 0.016 56.84 3.14 3.39 0.41Hippocampus KD 0.068 0.005* 51.31 2.56 13.60 1.40*Cerebellum control 1.720 0.190 71.40 6.42 9.52 0.85Cerebellum KD 1.720 0.240 66.60 3.03 11.02 1.65

Note: Results are mean SEM of eight independent experiments performed in duplicate. One CAT unit isdefined as 1 M of H2O2 consumed per minute. One SOD unit is defined as 50% inhibition of red for-mazan formation. One GPx unit is defined as 1 M of NADPH consumed per minute.*Values significantly different from diet control group as determined by Students t test (P 0.05).

higher susceptibility of this structure to the effects of

ROS caused by the change in diet composition. The

hippocampus, however, showed an opposite profile. Wehave observed an increase in antioxidant defense capa-

city, and, probably associated to that fact, there was no

change in lipoperoxidation. This increase was due, at

least partially, to an increase in the activity of the antioxi-

dant GPx enzyme, that might have been stimulated by

several factors, among them an increase in ROS pro-

duction itself. The increase in GPx activity was so impor-

tant as to guarantee protection, even though CAT activity

decreased. GPx appears to play a major role in metaboliz-

ing hydrogen peroxide in neural tissue (21,22). We do

not know whether these effects are caused by high circu-

lating levels of ketone bodies or by the lipid components

of a KD.

Ketone bodies are able to affect oxidative stress in

nonneural cells. Cultures of polymorphonuclear leukocytes

and red blood cells from healthy subjects exposed to ketone

bodies presented a reduced production of superoxide (23)

and accumulation of oxidized glutathione, respectively

(24). Acetoacetate, but not beta-hydroxybutyrate, increased

lipid peroxidation in cultured human umbilical vein

endothelial cells (25). Further studies in cultured neural

cells from different brain regions will be useful to detail

our results and to characterize the possible direct effect of

ketone bodies.

Another possibility to explain the differences that wefound would be conceiving that the lipid component of a

KD could change lipid composition of the membranes

and/or cellular antioxidant activity. For example, changes

in fatty acid unsaturation of mitochondria membranes are

accompanied by changes in the susceptibility and gener-

ation of reactive oxygen species (26). Moreover, polyun-

saturated fatty acids could play a role by direct control of

gene expression in many neurological diseases involving

oxidative stress (27). However, at this moment there is no

evidence relating degree of fatty acid unsaturation in the

several KD formulas and efficacy of this diet in epilepticdisorders.

CONCLUSION

There are many hypotheses about how a KD can

affect epileptic diseases. The relationship between

free radical and scavenger enzymes with epilepsy has

been found, and ROS have been implicated in seizure-

induced neurodegeneration (see Schwartzkroin [28] for

a review).

Our data suggest that ketogenic diet maybe protec-tive in epileptic disorders by affecting antioxidant activ-

ity, particularly that of GPx. Supporting that, a reduced

intracellular GPx activity in children resistant to con-

ventional pharmacological therapy, the main indication

of KD, has been reported (29). Additionally, in the rat

model of epilepsy induced by pilocarpine, an increase of

hippocampal GPx during the first hour of status epilep-

tic was observed (30). A high activity of GPx induced

by KD in hippocampus might contribute to protect this

structure from the neurodegenerative sequelae of epilep-

tic disorders.

ACKNOWLEDGEMENTS

Supported by Brazilian funds from Conselho Nacional de Desen-

volvimento Cientfico e Tecnolgico (CNPq), PRONEX (66.136/

1996-0) and Fundao de Amparo a Pesquisa do Rio Grande do

Sul (FAPERGS). The authors are very grateful to Dr. Adriana Bello

Klein and Dr. Suzana Lores Arnaiz for their comments on the

manuscript.


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REFERENCES

1. Castagne, V., Gautschi, M., Lefevre, K., Posada, A., and Clarke,P. G. H. 1999. Relationships between neuronal death and the cel-lular redox status: Focus on the developing nervous system. Prog.Neurobiol. 59:397423.

2. Coyle, J. T. and Puttfarcken, P. 1993. Oxidative stress, glutamate,

and neurodegenerative disorders. Science 262:689695.3. Halliwell, B. and Gutteridge, J. M. C. 1999. Free radicals in biologyand medicine. Pages 721731, in Halliwell, B. and Gutteridge,J. M. C. (eds.), Oxidative stress and disorders of the nervous sys-tem: General principles, Oxford University Press, New York.

4. Pazdernik, T. L., Layton, M., Nelson, S. R., and Samson, F. E.1992. The osmotic/calcium stress theory of brain damage: Arefree radicals involved? Neurochem. Res. 17:1121.

5. Willmore, L. J. 1990. Post-traumatic epilepsy: Cellular mechanismsand implications for treatment. Epilepsia 31:S67S73.

6. Brigelius-Flohe, R. 1999. Tissue-specific functions of individualglutathione peroxidase, Free Radic. Biol. Med. 27:951965.

7. Dringen, R. 2000. Metabolism and functions of glutathione inbrain. Prog. Neurobiol, 62:649671.

8. Swink, T. D., Vining, E. P. G., and Freeman, J. M. 1997. Theketogenic diet. Adv. Pediat. 44:297329.

9. Vining, E. P. G. 1999. Clinical efficacy of the ketogenic diet.

Epilepsy Res. 37:181190.10. Ziegler, D. R., Arajo, E., Rotta, L., Perry, L. M., and Gonalves, C. A.

2002. ketogenic diet increases protein phosphorylation in brain slicesof rats. J. Nutr. 132:483487.

11. Fraga, C. G., Leibovitz, B. E., and Tappel, A. L. 1988. Lipid per-oxidation measured as thiobarbituric acid-reactive substances intissue slice: Characterization and comparison with homogenatesand microsomes. Free Radic. Biol. Med. 4:155161.

12. Lissi, E., Salim-hanna, M., Pascual, C., and del Castilho, M. D.1995. Evaluation of total antioxidant potential (TRAP) and totalantioxidant reactivity (TAR) from luminol-enhanced chemilumin-escence measurements. Free Radic. Biol. Med. 18:153158.

13. Desmarchelier, C., Barros, S., Repetto, M., Latorre, L. R., Kato, M.,Coussio, J., and Ciccia, G. 1997. 4-Nerolidylcatechol from Potho-morphe spp. scavenges peroxyl radicals and inhibits Fe(II)-dependent DNA damage. Planta Med. 63:561563.

14. Aebi, H. 1984. Catalase in vitro. Methods Enzymol. 105:121126.15. Wendel, A. 1981. Glutathione peroxidase. Methods Enzymol.77:325332.

16. Lowry, O H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J.1951. Protein measurement with the Folin phenol reagent. J. Biol.Chem. 193:265275.

17. Kim, S. K. 1999. National Institutes of Health workshop: Roleof nutrient regulation of signal transduction in metabolic diseases.Am. J. Clin. Nutr. 70:544571.

18. Arnaiz, S. L., Travacio, M., Llesuy, S., and Arnaiz, G. R. L. 1998.Regional vulnerability to oxidative stress in a model of experi-mental epilepsy. Neurochem. Res. 23:14771483.

19. Candelario-Jalil, E., Al-Dalain, S. M., Castillo, R., Martinez, G.,

and Fernandez, O. S. 2001. Selective vulnerability to kainate-induced oxidative damage in different rat brain regions. J. Appl.Toxicol. 21:403407.

20. Homi, H. M., Freitas, J. J., Curi, R., Velasco, I. T., and Junior, B. A.2002. Changes in superoxide dismutase and catalase activities of ratbrain regions during early global transient ischemia/reperfusion.Neurosci. Lett. 333:3740.

21. Choi, B. H. 1993. Oxygen, antioxidants and brain dysfunction.Yonsei Med. J. 34:110.

22. Sinet, P. M., Heikkila, R. E., and Cohen, G. 1980. Hydrogenperoxide production in rat brain in vivo. J. Neurochem. 34:14211428.

23. Sato, N., Shimizu, H., Shimomura, Y., Suwa, K., and Kobayashi,I. 1992. Mechanism of inhibitory action of ketone bodies on theproduction of reactive oxygen intermediates (ROIS) by polymor-phonuclear leukocytes. Life Sci. 51:113118.

24. Jain, S. K. and McVie, R. 1999. Hyperketonemia can increase lipid

peroxidation and lower glutathione levels in human erythrocytes invitro and in type 1 diabetic patients. Diabetes 48:18501855.

25. Jain, S. K., Kannan, K., and Lim, G. 1998. Ketosis (acetoacetate)can generate oxygen radicals and cause increased lipid peroxida-tion and growth inhibition in human endothelial cells. Free Radic.Biol. Med. 25:10831088.

26. Herrero, A., Portero-Otin, M., Bellmunt, M. J., Pamplona, R., andBarja, G. 2001. Effect of the degree of fatty acid unsaturation ofrat heart mitochondria on their rates of H 2O2 production and lipidand protein oxidative damage. Mech. Ageing Dev. 122:421443.

27. Ntambi, J. M. and Bene, H. 2001. Polyunsaturated fatty acid reg-ulation of gene expression. J. Mol. Neurosci. 16:2738001.

28. Schwartzkroin, P. 1999. Mechanisms underlying the anti-epilepticefficacy of the ketogenic diet. Epilepsy Res. 37:171180.

29. Weber, G. F., Maertens, P., Meng, X. Z., and Pippenger, C. E.1991. Glutathione peroxidase deficiency and childhood seizures

Comment. Lancet15:14431444.30. Bellissimo, M. I., Amado, D., Abdalla, D. S., Ferreira, E. C.,Cavalheiro, E. A., and Naffah-Mazzacoratti, M. G. 2001. Superox-ide dismutase, glutathione peroxidase activities and the hydroper-oxide concentration are modified in the hippocampus of epilepticrats. Epilepsy Res. 46:121128.

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Neurochemical research, 2003 - KD, GPx