Human & Experimental Toxicology (2002) 21, 153 ± 158

www.hetjournal.com

Mitigation of free radical toxicity in

hyperoxaluric condition by a novel
derivative eicosapentaenoate-lipoate
M Lenin, LM Latha, M Nagaraj and P Varalakshmi*
Department of Medical Biochemistry, PGIBMS, University of Madras, Taramani Campus, Chennai-600 113, India

Lipoic acid (LA) and eicosapentaenoic acid (EPA) have been The three drugs, namely LA, EPA and EPA-LA had reversed
shown to ameliorate the changes associated with hyper- the above changes, but the effect was more pronounced in
oxaluria. This prompted us to study the effect of EPA-LA, a EPA-LA-treated stone formers. These features highlight the
new derivative, in experimental urolithiatic condition. beneficial effect of EPA-LA wherein the potency of two
Foreign body implantation method followed by supple- drugs has been combined. The practical outcome of these
mentation of ammonium oxalate was adopted to induce findings is that the cellular antioxidant defence can be
stone formation in the bladder. Significant depletion in the increased by the supplementation of lipoate and its
antioxidant status was observed in the kidney and bladder derivative EPA-LA. Human & Experimental Toxicology
of stone-forming animals, associated with increased lipid (2002) 21, 153 ± 158.
peroxidation. The present observations provide supporting
evidence to the hypothesis that free radicals might be Key words: antioxidants; eicosapentaenoic acid; hyperoxaluria;
involved in causing toxicity in hyperoxaluric condition. lipoic acid

Introduction
Calcium oxalate calculi of the kidney is a common poisoning, radiation damage, diabetes, neurodege-
clinical problem estimated to occur in approximately nerative diseases and AIDS. 10 Our laboratory has
12% of the population, with a recurrence rate of 70 ± established the anti-urolithiatic property of lipoic acid
80% in males and 47± 60% in females.1 The biochem- and its protective role exhibited against free radical
ical mechanism of stone formation is still unknown. toxicity during experimental hyperoxaluria. 7,8
However, free radicals are intricately involved in the Eicosapentaenoic acid (EPA), a polyunsaturated
process of crystal deposition in the renal tissues.2 fatty acid (PUFA) found in fish oil belongs to the
Several studies indicating the role of free radical n-3 polyunsaturated ¬-linolenic acid family.11 It is
reactions in the causation of renal stone formation an important component of practically all cell
have been recently published. 3 ± 6 Earlier studies con- membranes and it is essential for normal human
ducted in our laboratory demonstrated an increase in growth and development. 12 Chemically, EPA is
lipid peroxidation and a decrease in the levels of cis-5,8,11,14,17-eicosapentaenoic acid, and thera-
antioxidants in kidney tissue rats fed with calculi- peutically it has been categorized as a hypolipidemic,
producing diet.7,8 cardioprotectant and antiplatelet agent.13 Buck et al.
¬-Lipoic acid and its reduced form, dihydrolipoic have reported that fish oil supplementation preven-
acid, have been referred to as universal antioxidants ted nephrocalcinosis and hypercalciuric symptoms in
that function in both aqueous and membrane phases.9 experimental animals.14
A number of experimental as well as clinical studies The above reports prompted us to investigate the
point to the usefulness of ¬-lipoate as a therapeutic pharmacological efficacy of the derivative EPA-LA on
agent for such diverse conditions as myocardial and free radical damage manifested during bladder-stone
cerebral ischemia ± reperfusion injury, heavy metal formation. LA and EPA-ethyl ester were used for
comparison purpose.

*Correspondence: P Varalakshmi, Department of Medical Bio-

chemistry PGIBMS, University of Madras, Taramani Campus, Materials and methods
Chennai-600 113, India.
E - mail: [email protected] Drug and chemicals
Received 6 August 2001; revised 1 February 2002; accepted 14 EPA-LA the derivative, EPA-ethyl ester and LA were
February 2002 gifted by Scotia Pharmaceuticals, Carlisle, UK,
© Arnold 2002 10.1191/0960327102ht231oa
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 Mitigation of oxalate toxicity by lipoate derivative
M Lenin et al.

154

Laxdale (Stirling, UK) and Astra Medica (Frankfurt, implanted+AO-administered+EPA for 15 days;
Germany) respectively. Thiobarbituric acid and re- group VII Ð zinc-implanted+AOx-administered+LA
duced glutathione were purchased from Sisco for 15 days; group VIII Ð zinc-implanted+AOx-
Research Laboratories, Mumbai, India. Dithionitro- administered+EPA-LA for 15 days. Corn oil was used
benzoic acid was obtained from BDH Chemicals, as vehicle.
Poole, England, UK. All other chemicals and solvents After the experimental period the animals were
were of analytical grade. sacrificed by cervical decapitation and kidney and
bladder tissues were excised. The excised tissues
Induction of bladder stone were processed as whole tissue and washed in cold
Male albino rats of Wistar strain (150 ± 180 g) were 0.15 M KCl. They were then homogenized in
acclimatized to laboratory conditions for a week. Tris ± HCl buffer (0.01 M, pH 7.4) to give a 10%
Calcium oxalate crystallization was induced by for- homogenate. Aliquots were used for further assay.
eign body implantation technique.15 In brief, the Protein was estimated by the method of Lowry
animals were anaesthetized with ether and sub- et al.16 Lipid peroxidation (LPO) was assayed by the
jected to suprapubic cystomy. A preweighed sterile method of Devasagayam.17 Superoxide dismutase
zinc piece (15‹2 mg) was implanted in the bladder. (SOD) activity was assayed according to the method
The bladder was closed by a single suture and the of Marklund and Marklund.18 The method of Rotruck
lower abdomen was sutured in layers. The animals et al. was adopted to assay glutathione peroxidase
were allowed to recover for 3 days. Experimental (GPx).19 Catalase (CAT) activity was assayed by the
animals were used after obtaining prior permission method of Sinha.20
and handled according to the University and local Glutathione (GSH) content was determined based
legislation. on its reaction with 5,5 0-dithiobis(2-nitro-benzoic
acid), according to the method of Moron et al.21
Experimental set-up Ascorbic acid (Vit C) level was estimated by the
The animals were divided into eight groups with six method of Omaye et al. 22 and vitamin E (Vit E) was
rats in each group: group I Ð sham-operated control determined by the method of Baker and Frank.23
animals, wherein the bladder was cut open and All data were expressed as mean‹SD. Statistical
sutured without implantation to nullify the changes significance between the data of different groups
due to trauma of surgery; group II Ð zinc-implanted were evaluated by LSD. p values less than 0.05 were
animals administered 2% ammonium oxalate (AOx) considered as significantly altered.
for 15 days (1 mL/rat/day p.o.); group III Ð sham-
operated animals administered EPA-ethyl ester (EPA)
(oral gavage, 26 mg/kg body weight/day p.o.) for 15 Results
days; group IV Ð sham-operated animals adminis-
tered LA (oral gavage, 20 mg/kg body weight/day p.o.) Table 1 depicts the levels of malondialdehyde
for 15 days; group V Ð sham-operated animals released indicating the extent of lipid peroxidation
administered EPA-LA (oral gavage, 50 mg/kg body in the kidney and bladder tissues. Significant
weight/day p.o.) for 15 days; group VI Ð zinc- increase ( p<0.05) in the basal and induced levels of

Table 1 Effect of EPA, LA and EPA-LA derivative on tissue lipid peroxidation in control and experimental animals (values are expressed as
mean‹SD for six animals)
Lipid peroxidation Group I Group II Group III Group IV Group V Group VI Group VII Group VIII
(nmol MDA formed/
min/mg protein)
Kidney
Basal 2.5‹0.31 4.1‹0.3 a 2.7‹0.11 2.5‹0.25 2.6‹0.16 3.6‹0.32 ab 2.7‹0.16 bc 2.6‹0.18 bc
Ascorbate-induced 5.4‹0.42 8.2‹0.67 a 5.7‹0.47 5.5‹0.43 5.4‹0.51 7.7‹0.49 a 6.1‹0.25 abc 5.6‹0.26 bc
FeSO4-induced 15.1‹1.2 18.8‹1.4 a 16.0‹1.09 15.1‹1.12 15.2‹1.13 17.9‹1.32 a 16.8‹1.08 ab 15.3‹0.93 bc

Bladder
Basal 1.36‹0.1 2.9‹0.23 a 1.7‹0.12 1.4‹0.11 1.4‹0.16 2.5‹0.26 ab 2.2‹0.13 abc 1.9‹0.125 abcd
Ascorbate-induced 4.8‹0.32 6.9‹0.54 4.9‹0.36 4.7‹0.33 4.8‹0.51 6.4‹0.33 a 5.4‹0.27 abc 4.9‹0.23 bcd
FeSO4-induced 7.1‹0.84 10.9‹0.63a 7.7‹0.59 7.3‹0.83 7.3‹0.68 9.2‹0.72 ab 8.1‹0.53 abc 7.41‹0.51 bc
Treatment of groups: group I Ð sham-operated control+corn oil; group II Ð implanted+2% AOx (1 mL/day p.o.); group III Ð sham+EPA (26
mg/kg body weight/day p.o.); group IV Ð sham+LA (20 mg/kg body weight/day p.o.); group V Ð sham+EPA Ð LA (50 mg/kg body weight/
day p.o.); group VI Ð implanted+AOx+EPA; group VII Ð implanted+AOx+LA; group VIII Ð implanted+AOx+EPA-LA. Drugs were
administered by oral gavage for a period of 15 days and the vehicle used was corn oil. Comparisons are made as follows: a Ð with group I; b
Ð with group II; c Ð with group VI; d Ð with group VII. The letters a, b, c, d represent statistical significance at p<0.05.

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 Mitigation of oxalate toxicity by lipoate derivative
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155
Table 2 Effect of EPA, LA and EPA-LA derivative on tissue enzymic antioxidants in control and experimental animals (values are expressed
as mean‹SD for six animals)
Enzymes (U/min/mg protein) Group I Group II Group III Group IV Group V Group VI Group VII Group VIII
Kidney
SOD 8.1‹0.18 4.3‹0.32 a 8.2‹0.71 8.2‹0.65 8.2‹0.81 6.5‹0.47 ab 6.3‹0.57 ab 7.7‹0.62 bcd
GPx 8.4‹0.75 4.3‹0.39 a 8.3‹0.76 8.4‹0.65 8.5‹0.83 5.7‹0.72 ab 6.2‹0.73 ab 7.3‹0.51 abcd
CAT 188‹13.2 102‹9.36 a 154‹13.7 156‹12.2 159‹11.9 116.0‹7.7 a 124.0‹9.6 ab 132‹10.8 abc

Bladder
SOD 6.5‹0.56 3.3‹0.27 a 6.5‹0.6 6.5‹0.74 6.5‹0.06 4.2‹0.16 ab 5.1‹0.42 abc 5.4‹0.41 abc
GPx 6.9‹0.59 2.4‹0.35 a 6.8‹0.44 6.95‹0.68 6.9‹0.4 3.2‹0.52 ab 4.4‹0.42 abc 6.1‹0.5 abcd
CAT 123‹11.1 74‹6.8 a 120‹10.1 119‹10.3 125‹9.8 94.0‹10.2 ab 37.0‹9.4 ab 105‹11.2 abcd
Enzyme units are expressed as: SOD, the amount of enzymes required to bring about 50% inhibition of auto oxidation of pyrogallol; GPx,
micromoles of GSH consumed and CAT, micromoles of H2O2 consumed. Treatment of groups: see Table 1. Comparisons are made as in
Table 1.

lipid peroxidation was observed in the stone formers Discussion

(group II) compared to that of the control animals
(group I). In the present study, the foreign body introduced into
Oxalate- and crystal-mediated peroxidative the bladder acts as a nidus, enabling deposition of
changes of the kidney and bladder tissues were calcium oxalate over it. In our recent publication, we
restricted to a major extent in group VIII rats treated have reported that calcium oxalate deposition was
with EPA-LA and group VII rats treated with LA. EPA- maximum around the zinc implant, within the blad-
treated group VI animals did not demonstrate any der of group II animals.24 Significant reductions in
significant reduction in LPO levels, which may be stone weights were observed with the treatment
due to its polyunsaturated nature. procedures, which was maximum with EPA-LA
Table 2 delineates the changes in the activities of (90%) followed by LA (56%) and minimal with EPA
antioxidative enzymes (SOD, GPx, CAT) in kidney (38%).
and bladder. These tissue endogenous antioxidant Oxalate, an essential stone-forming constituent has
defence capabilities were found to be inhibited in been shown to induce lipid peroxidation. 25 Peroxide
the lithogenic group (group II) compared to that of formation in vivo may produce serious consequen-
control rats (group I). ces in the tissues. 26 The increase in LPO might
Changes in the kidney and bladder tissue levels of be attributed to the supplementation of AOx. The
non-enzymic antioxidants GSH, Vit C and Vit E are peroxidative changes induced in the cell are encoun-
given in Table 3. Stone formers (group II) demonstra- tered by the elaborate defence mechanism including
ted a decrease in the levels of GSH and both the enzymic and non-enzymic antioxidants.27 These cel-
vitamins in kidney. Depletion of these components lular antioxidants are armoured with the capacity to
were also observed in the bladder of lithogenic rats deal with the free radical species produced by normal
(group II). metabolic process, but is limited.28
Replenishing activity was found to be most Thiol compounds are well known for the free
pronounced in groups VII and VIII, which might radical scavenging property. Dihydrolipoic acid has
be attributed to the antioxidative nature of lipoic been reported to be cytoprotective against mucosal
acid. damage produced by different antirheumatic drugs.29

Table 3 Effect of EPA, LA and EPA-LA derivative on tissue non-enzymic antioxidants in control and experimental animals (values are
expressed as mean‹SD for six animals)
Parameters (·g/mg protein) Group I Group II Group III Group IV Group V Group VI Group VII Group VIII
Kidney
GSH 8.2‹0.87 4.1‹0.61 a 8.2‹0.63 8.4‹0.65 8.3‹0.61 5.3‹0.42 ab 6.8‹0.49 abc 6.4‹0.54 ab
Vit C 2.2‹0.05 1.0‹0.08 a 2.1‹0.2 2.2‹0.20 2.2‹0.2 1.1‹0.09 a 1.9‹0.17 abc 1.97‹0.16 abc
Vit E 1.4‹0.04 0.47‹0.2 a 1.3‹0.11 1.5‹0.10 1.4‹0.12 0.6‹0.36 a 1.1‹0.09 abc 1.1‹0.07 abc

Bladder
GSH 4.7‹0.38 1.9‹0.2a 4.7‹0.38 4.9‹0.43 4.8‹0.42 2.1‹0.18 a 3.6‹0.35 abc 4.2‹0.38 abcd
Vit C 0.9‹0.08 0.3‹0.03 a 0.87‹0.08 1.03‹0.1 0.98‹0.08 0.35‹0.04 a 0.75‹0.06 abc 0.89‹0.06 bcd
Vit E 1.3‹0.11 0.5‹0.03 a 1.2‹0.09 1.29‹0.12 1.3‹0.11 0.6‹0.05 a 1.04‹0.08 abc 1.2‹0.09 bcd
Treatment of groups: see Table 1. Comparisons are made as in Table 1.

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156

Lipoate, labeled as a potent free radical scavenger,30 an increase in the susceptibility of membranes to
administered to group VII rats caused a significant peroxidation. 40,41 Hogberg et al. reported that GSH
fall in the peroxidation level. depletion induced peroxidation and cell lysis.42 Exog-
EPA-treated rats (group VI) did not show a marked enous oxidant injury of renal tubular epithelia was
decrease in LPO level. Being a PUFA, EPA is afforded by GSH deficiency.43,44 Increased oxidative
prone to peroxidation. However, as an inducer of stress increases the formation and efflux of oxidized
the expression of antioxidant enzymes SOD, GPx glutathione.45 Hiramatsu et al. 46 have reported an
and CAT the peroxidative level is maintained inverse correlation between the reduced content of
lower than that of the stone formers. 31 The GSH and increased levels of peroxidation. Protection
nephroprotective action of n-3 PUFA has been is conferred to the tissues and membranes by the
reported in animal models of adriamycin- and synergistic action of Vit C and E, through interactions
gentamicin-induced renal toxicities. 32,33 EPA has between water- and lipid-soluble substances.46 The
potentiating effects on the drug administered along levels of GSH, Vit C and Vit E were restored to near-
with it.34 normal levels with lipoate and EPA-LA treatments.
In our present study, EPA supplemented as a Lipoate, a dithiol compound, is reported to afford
derivative of lipoate protects the former from suscept- protection to the cell membranes by the possible
ibility to peroxidation. When administered with the interaction with the above antioxidants via the Vit E
antioxidant Vit E, EPA has been reported to attenuate cycle. 47 Schlemmer et al. reported the prevention of
peroxidation. 35,36 Thus, the potent antioxidant and experimentally induced calcinosis with EPA-LA treat-
cytoprotective property of LA gets added upon with ment to rats.48
the adjuvant effect and antioxidant expression prop- From the present study it is evident that both LA
erty of EPA, enabling the derivative to suppress and EPA-LA offer remarkable protection against
peroxidation to a greater extent than that of lipoate peroxidation, which impairs cellular stability.
and eicosapentaenoate. EPA-LA seems to be superior to EPA and LA in
Ammonium-oxalate-supplemented stone-forming effectively enhancing the activities and levels of
rats showed a decrease in SOD activity responsible enzymic and non-enzymic antioxidants. This ven-
for dismutation. This may be due to the oxidative ture highlights further the antioxidant nature of
assault rendered by the oxalate content in the lipoate and the therapeutic effectiveness of combin-
tissues. 37 The inhibition of GPx activity in both ing the properties of EPA and lipoate in curtailing
kidney and bladder of calculogenic rats (group II) the free-radical-mediated toxicity arising during
occurs in concurrence with the depletion in GSH stone formation.
level. This observation is supported by an earlier
report. 38 Rister and Bachner speculated that cata-
lase activity decreases during oxidative stress.39 Acknowledgements
Oxalate has been reported to induce peroxidation,
which indirectly inhibits catalase in the calculo- One of the authors, M. Lenin, acknowledges the
genic rats. Council of Scientific and Industrial Research, New
Antioxidant defences, particularly those of the Delhi, India for financial assistance in the form of
glutathione redox system, may be induced, despite Senior Research Fellowship.

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