Indian Journal of Experimental Biology Vol. 40, October 2002, pp. 11 83- 11 86

Protective influences of a-ketoglutarate on lipid peroxidation and antioxidant status in ammonium acetate treated rats

S Velvizhi , K B Oakshayani & P Subramanian*

Department of Bioche mistry, Faculty o f Science, Annamalai University, Annamalainagar 608002, India

Received 14 March 2002; revised 31 July 2002

The effects of a-ketoglutarate on ammonium acetate induced hyperammonemia were studied biochemicall y in experi­mental rats. The levels of c ircul atory, non-protein nitrogen, serum transaminases and thiobarbituric ac id reactive substances were significantly increased in ammonium acetate treated rats . These levels were significantly decreased in a-ketoglutarate and ammonium acetate treated rats. Similar patterns of alterations were observed in the levels of free fatty ac ids, tri glyc­erides, phopholipids and cholesterol inbetween various groups. Further non-enzy matic (vitamins C and E) and enzymatic (superox ide dismutase and cata lase) antioxidants were significantly decreased in ammonium acetate treated rats; and were significantl y increased in a-ketoglutarate and ammonium ace tate treated rats . The biochemical alterati ons during a­ketoglutarate treatment could be due to (i) the detoxification of excess ammonia, (ii ) by participating in the non-enzymatic oxidative decarboxy lat ion in the hydrogen perox ide decompositio n process and (iii) by enhancing the proper metaboli sm of fats which could suppress oxygen radicals generati on and thus prevent the lipid peroxidat ive damages in rats.

Ammonia is formed in mammals and humans as a product of catabolism of proteins and other nitroge­nous compounds. At high levels, ammon ia is neuro­toxic, leading to functional disturbances of the central nervous system, which can lead to coma and death '. Ammonia intoxication impairs mitochondrial func­tion2 which could lead to decreased A TP synthesis and also to increased formation of free radical s3

. The major toxic effects of ammonia probably involve changes in cellular pH and the depletion of certain citric acid cycle intermediates, particularly a-ketoglu­tarate2

. Impairment in the formation of urea (in urea cycle) can occur either as a consequence of primary genetic defect or through secondary suppress ion of enzyme activities of urea cycle. Either of the process results in hyperammonemia2.3. Sustained hyperam­monemia in mice leads to increased lipid peroxidation in liver and brain, reflecting an oxidative stress condi­tion 4 .

a-ketoglutarate (a-KG) is an intermed iate of citric acid cycle and is the natural ubiquitous co llector of amino groups in body ti ssues. Further, an important function of a-KG occurs in the formation of car­nitine5

. Because of its chemical structure, a- KG is a potent natural detoxifying agent. In some applica-

·Correspondent author: Phone: 04 144-20346 Fax: 04144-38080 E-mail : psub @rcdiffmail.com

tions, it is far more powerful than vitamin C. One such application is as an antidote to cyanide poisoning where it binds to the cyanide and prevents the circula­

tion of free cyanide6. a-KG was found to offer protec­

tion in heart surgery and is known to decrease the lev­els of ischaemic markers , creatin kinase and troponin T7. Conditions of hyperammonemi a can be treated

with a-ketoglutaric acid8.

Systematic investigations on the levels of lipid per­oxidation products and the levels of non-enzymatic and enzymatic antioxidants under the conditions of hyperammonemia are lacking. The present study deals with the systematic investigation on the levels of products of lipid peroxidation (thiobarbituric acid re­active substances- TBARS) and the levels of vitamins C and E (non-enzymatic antioxidants) and superoxide dismutase and catalase (enzymic antioxidants) under the conditions of hyperammonemi a and during the

treatment of a-KG in rats. Further, the levels of urea, non-protein nitrogen (NPN), serum transaminases and lipid profile variables (free fatty acids, triglycerides, phospholipids and cholesterol) have also been inves­tigated .

Materials and Methods Adult male Wistar rats (24; six months o ld) ob­

tained from Central Animal HOllse, Faculty of Medi­cine, Annamalai University were kept at room tem­perature (30° ± 2°C). Animals were randomized and

1184 INDIAN J EXP BIOL, OCTOBER 2002

separated into four groups (Group I - contro l, Group­II - ammonium acetate treated , Group 111 - ammonium

acetate and a-KG treated and Group IV - a -KG treated). Animals were handled , ethically treated and sacri ficed as per the rules and instructions of Ethical Committee of An imal Care of Annamalai Uni versity in accordance with the Ind ian National Law on animal care and use. Food pellets (Karnataka State Agro Corporation Ltd, Bangalore, Indi a) and water were available ad libitum to the animals.

a -ketoglutarate (di sodium salt) was purchased from Sigma Chemicals Co., USA. Ammonium acetate and other chemi cals used in this study were of analytical grade.

Group I (control) animals were inj ected intraperi­toneall y with phys io logical saline throughout the ex­perimental period (4 weeks) . Group II animals were treated with ammonium acetate (100 mg/kg body weight) intraperitoneall / everyday th roughout the experimental period. Group III animals were treated with ammonium acetate at the same dose as group II animals followed by the administrati on of a ­ketoglutarate solution (2 g/kg body weight) orall /o for 4 weeks. Group IV animals were treated with (J.­

KG solution at the same dose as group III animals for 4 weeks.

Biochemical determinations-At the end of the experimental peri od blood samples were collected in heparini zed tubes with great care by sino-occular puncture and pl asma samples were separated by cen­trifugation. Pl asma vitamin C II , vitamin E1 2, TBARS I" al anine and aspartate transaminases l4

,

I d .. 15 bl d 16 ·d b 00 non-protem nitrogen 00 urea , superoxi e dismutase 17

, catalase in hemolysate l8, plasma free

fatty ac ids 19, triglycerides2o, phospholipids21 and cho­Iesterof2 were estimated.

Statistical analysis-Mean ± SO values of the bio­chemical variables were calculated. Analysis o f variance

fo llowed by 'Least Significant Di ffe rence ' (LSD) was carried out to detec t the signi ficant differences be­tween control and experimental groups.

Results

Body weight changes--Ammonium acetate treated rats (Group II) showed increases in body weight compared with contro ls (Table I) . Ammonium

acetate and a -KG treated rats (Group III ) and Group

IV (a-KG treated rats) showed less significant differ­ences in body weights when compared with contro l rats.

Biochemical analyses-The concentration of TBARS (Table 2) in pl asma was increased signi fi­cantly in ammonium acetate treated rats (Group II ).

Ammonium acetate and a -KG tre8. ted group showed significantly low levels of TB A RS when compared with the corresponding ammonium acetate group

(Table 2). a -KG treated rats (Group IV) showed sig­nificantly low levels of TB ARS whe n compared to control rats. Similar patterns were observed on the levels of urea and non-protein nitrogen in between groups. Acti vities of li ver marker enzy mes, alanine transaminase (ALT) and aspartate transUl'1 inase (AST) were also altered in a similar manner in be­tween g roups.

Administration of ammonium acetate caused a sig­nificant decrease in the levels of vitamins C and E in pl asma when compared to contro ls. A mmonium ace­

tate and a -KG treated groups showed signifi cantl y increased levels of vitamins C and E in plasma when co mpared with co rresponding ammonium acetate

treated group. a -KG treated rats showed signi ficant increases in the levels of antiox idant vitamins (C and E) when compared with controls. Similar alterations were observed on the activiti es of enzymatic antioxi­dants (catalase and superoxide dismutase) in between groups.

Tab le 1- Body weight (g) changes in eac h group

IV alues arc mean±SD from 6 animals in each group]

Groups Cont ro l

Initial body weight 200.1 ±4.8 Final body weight 257.5 ±2.7 Net gain in weight 57.3 ± 4.2

ANOVA fo llowed by Least Significanl Di fference (LSD). " Significant (P < 0.05) when compared with cont ro l.

Ammonium acetate

205.0± 6.3

295.8±3.7

89. 1 ±3.7"

b Signi fica nt (P < 0.05) when compared with ammonium acetale treated.

Ammonium a- KG acetate + a-KG

196.3± 3.5 199.0± 2.5

264.5 ± 8.8 263.8 ± 6.3

68. 1±6.7 b 64.8±6.6 1

VELVIZH I et al., (X-KETOGLUTARAT E & ANTIOXIDANTS 11 85

The levels of free fa tty ac ids, trig lycerides, phos­pholipids and cholesterol were significantly increased in ammonium acetate treated rats (Table 3). The lev­

els were decreased in ammonium acetate and a -KG treated rats and were insigni ficantly diffe rent in a -KG treated group when compared with control s.

Discussion The present results revealed that ammonium ace­

tate treated rats gained significantl y higher body weights compared to contro l rats. It may be due to increased levels of lipids during hyperammonemi a as observed in the present study. Group III (a- KG with ammonium acetate) rats gained body weights simil ar to control rats. Thi s could be due to detox ifying effects of a _KG23

.

It is a well establi shed fac t that ammoni a intoxica­tion enhances lipid perox idation and generates free radical s3

. Thi s could lead to increased levels of

T BARS and decreased leve ls of enzymatic and non­enzymatic antioxidants in group II rats. Elevated lev­els of a-KG (in group III ) could offer protecti on against ox idative damages by participating in the non­enzy matic oxidati ve decarboxylation in the hydrogen peroxide decomposition process 24 .

Serum transaminases (ALT and AST) are sensiti ve indicators of liver cell injur/ 5

. Elevated levels of AL T and AST in ammonium acetate treated rats might be related to damage and destruction of the li ver ti ssue26

.27

. By detoxifying ammoni a, and also by elevating glutamate, g lutamine, and other amino ac ids levels a-KG could be involved in wound repair of liver and could decrease the acti vities of transami­nases.

Increased levels of urea and non-protein nit rogen may indicate the elevated levels of ammonia in am­monium acetate treated rats in the present study. a­KG collects amino (-NH2) groups and traps ammoni a

Table 2-Changes in the biochemical vari Jb les in four groups

[Values are mean ± SD from 6 animab in each group]

Variable Control

TBARS (n moles/ml ) 2.4±0.26

Vitamin C (mg/dl ) 1.7±0.10

Vitamin E (mg/dl) 1.1 6 ±O. IO

Catalase (Ilmoles/min/mgHb) 56.2±6. 1

SOD (50% inhibiti on of NBT reac- 2.3±0.1 5 tion/mgHb)

Urea (mg/dl ) 10.8± 1.2

Non-prote in nit rogen (mgldl) 24.1 ±2.0

ALT (lUlL) 30.4±2.62

AST (lUlL) 106.3±8.06

ANOVA followed by Least Significant Di fference (LSD). ' Sign ificant (P<0.05 ) when compared with contro l.

Ammoni um ace tate

3.8±O.24 "

0.68±0.09"

0.53±0.05"

33.8±4.36"

0.75 ±0.05"

23 .5 ±2.2 1"

55.6±6.8 "

86.0±5.73"

18 1.6± 13.3"

bSigni ficant (P<0.05) when compared with ammonium acetate treated . CSignificant (P<0.05 ) when compared with contro l.

Ammonium ace tate + ex-KG

2.6±0. 13 b

1.25±0. IS b

0.98±0.Q3 b

48.9±4.0 1

1.8 1 ±O. IOb

13.3 ± 1.2 b

34.9± 1.7l b

48.5±2.9 1 b

142 .0±6. 1 b

Table 3-Changes in the lipid profi le levels in four groups

[Values are mean ± SD from 6 animals in each group)

ex- KG F-value

1.9±O. 16 C 84.64

2.4±0. 12c 173.8

2.4 ± 0.02 C 273.0

75.6±9.2 c 46.0 1

3. 1 ±0.04 c 597.2

10.08 ± 1.24 c 94.36

19.4 ± 1.83 c 108.44

28. 1 ±2.1 326.5

97.6±4.83 115.7

Va ri a bl e Contro l Ammonium Ammonium ex- KG F - values acetate

Free fa lly ac ids (mg/dl ) 65.8±3.0 a 11 4.3±6.0

Triglycerides (mg/dl) 85 .1 ±7.2 . 144.8± 7.4

Phospholipids (mg/dl ) 85.0 iE I . 232.5 ±8.2

a Cholesterol (mg/dl) 108.0±4.38 204.0±4.38

ANOVA followed by Least Significant Di ffe rence (LSD). "Significant (P<0.05 ) when compared w;th cont ro l. bSignificalll (P<0.05) when compared v. ith ammonium acetate treated.

ace tate + ex- KG

b 68.9 ±4.6 165.87 83.8± 1.67 b

86.4 ±6.5 82.9 I09.4± 8.6 b

8 1.6 ± 2.6 709.55 11 0.O±7.7 b

I06.6± 4.1 550.19 129.3± 6.02

1186 INDIAN J EXP BIOL, OCTOBER 2002

in blood and in body ti ssues forming glutamate, glutamine and other amino acids by performing deamination and transami nation reactions5 thus de­toxifying ammonia.

Ammonium acetate could deplete the levels of a­KG and other Krebs cycle intermediatesX and thus elevate the levels of acety l CoA. The elevated levels of acetyl CoA could lead to increased levels of lipid profile (free fatty acids, triglycerides, phospholipids and cholesterol) as observed in the present study. Fur­ther, another important function of a-KG occurs in the formation of carnitine5

.28

. Carnitine acts as a car­rier of fatty acids into ce ll mitochondri a so that proper cataboli sm of fats can proceed29

. The decreased a-KG levels in ammonium acetate treated rats could lead to accumulation of fatty ac ids which may be reversed during the treatment of a-KG.

In conclusion, exogenously administered a-KG could cause the biochemical alterations by (i) detoxi­fying excess ammon ia, (ii) particirating in the non­enzymatic oxidative decarboxylation in the hydrogen peroxide decomposition process and (iii) er1lancing the proper metabolism of fat s.

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