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Effects of Various Concentrations of Quail Egg Solution on Glycemia and Antioxidant Parameters of Alloxan-induced Diabetic Rats

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Journal of Advances in Medical and Pharmaceutical
5(4): 1-7, 2016, Article no.JAMPS.22723
ISSN: 2394-1111
SCIENCEDOMAIN international
Effects of Various Concentrations of Quail Egg
Solution on Glycemia and Antioxidant Parameters of
Alloxan-induced Diabetic Rats
Patrick Emeka Aba
, Domnic Chibuike Igwebuike
and Jonas Anayo Onah
Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Nigeria.
Department of Biochemistry, University of Nigeria, Nsukka, Nigeria.
Department of Veterinary Surgery, University of Abuja, Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Author PEA designed the study and
wrote the protocol. Author DCI managed the animals, collected all data, performed the statistical
analysis, and wrote the first draft of the manuscript. Author JAO did the literature search and also
wrote part of the manuscript. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/JAMPS/2016/22723
(1) Hamdy A. Sliem, Internal Medicine, Suez Canal University, Egypt and College of Dentistry, Qassim University and EL-Jouf
University, Saudi Arabia.
Sema Kalkan Uçar, Ege University, Turkey.
Li Yao, Zhejiang Chinese Medical University, China.
Complete Peer review History:
Received 22
October 2015
Accepted 5
November 2015
Published 20
November 2015
This study evaluated the effects of different concentrations of quail egg solution on glycemia and
some antioxidant markers in alloxan-induced diabetic rats. Thirty adult male albino rats were
assigned to 5 groups of 6 rats per group. Groups 2, 3, 4 and 5 were made diabetic by single
intraperitoneal injection of 160 mg/kg alloxan monohydrate. Upon establishment of diabetes (fasting
blood glucose levels above 126 mg/dl), the rats in groups 2, 3 and 4 were respectively administered
with 30, 15 and 7.5 mg/ml orally daily for 21 days. Groups 1 and 5 were administered with distillled
water. Fasting blood glucose (FBG) levels of the rats were assessed 1 h, 6 h, 24 h and 21 days
post treatment. On the 21
day, blood samples were collected for malondialdehyde (MDA),
superoxide dismutase (SOD), and reduced glutathione (RSH) assays. Results indicate that
administration of quail egg solution at the concentration of 30 mg/ml to the diabetic rats significantly
(p< 0.05) reduced the FBG from 343.80 to 87.20 mg/dl on day 21 post treatment. There was
significant (p< 0.05) reduction in the mean MDA values of the 30 mg/ml-treated groups compared to
Original Research Article
Aba et al.; JAMPS, 5(4): 1-7, 2016; Article no.JAMPS.22723
the negative control group. The SOD activity and glutathione levels of 30 mg/ml-treated diabetic rats
were significantly (p< 0.05) higher compared to that of the diabetic untreated group. It was
concluded that administration of quail egg solution especially at the concentration of 30 mg/ml to the
diabetic rats resulted in hypoglycemia and improvement in the levels and activities of in vivo
antioxidant parameters.
Keywords: Quail egg; glycemia; antioxidant; diabetic rats.
Diabetes mellitus is a chronic metabolic disorder
that is fast becoming a global problem with huge
social, health and economic consequences. It is
estimated that in 2010, there were about 285
million people (approximately 6.4% of the adult
population) suffering from diabetes. This number
is estimated to increase to 430 million in near
future in the absence of better control or cure [1].
Data from World Health Organization (WHO),
suggest that Nigeria has the greatest number of
people living with diabetes in Africa [2].
Traditionally, types 1 and 2 diabetes mellitus are
recognized by WHO, although gestational
diabetes mellitus and other types of diabetes
have been described. Basically, the clinical signs
of diabetes mellitus include polyuria, polydipsia,
polyphagia, weakness, asthenia blurred vision
and impaired wound healing. Diagnosis involves
assay of fasting blood glucose levels,
measurement of glycosylated haemoglobin,
assay of urinary sugar and plasma insulin values
while treatment tailored towards reduction in the
fasting blood sugar levels are achieved by
dietary control, exercise and use of oral
hypoglycemics [3].
Oxidative stress associated with diabetes
mellitus has been widely reported. Diabetic
patients present with increased levels of
malondialdehyde consequent upon exuberant
cellular lipid peroxidation. Increased lipid
peroxidation presents a close relationship with
the high glycemic levels and oxidative stress in
diabetes mellitus [4]. Superoxide dismutase and
glutathione are antioxidants that protect against
lipid peroxidation. Assay of MDA, SOD and gluta-
thione are important in assessment of severity
and amelioration of diabetes mellitus [5,6].
The synthetic drugs in use for treatment of
diabetes mellitus are not only expensive but have
complicated mode of intake and have several
side effects. Therefore the search for alternative
therapy has been advocated for. Numerous
nutritional and therapeutic values of quail egg
have been reported. The quail eggs are rich
sources of antioxidants, minerals and vitamins
[7]. Experts in natural medicine believe that quail
egg has positive effects on people with
hypertension, liver problem, hyperlipidemia and
anaemia [8].
This study evaluated the effects of quail egg
solution on glycemia and in vivo antioxidant
parameters of alloxan-induced diabetic rats.
2.1 Animals
Adult male Wistar albino rats of 10 to 16 weeks
and average weight of 160±15 g were obtained
from the Animal House of the Faculty of
Biological Sciences, University of Nigeria,
Nsukka, Enugu state, Nigeria. The animals were
acclimatized for the duration of 7 days under
standard environmental conditions with a 12 h
light/dark cycle maintained on a regular feed
(vital feed) and water ad libitum.
2.2 Quail Egg
Quail eggs used were obtained from the Faculty
of Veterinary Medicine, University of Nigeria,
Nsukka, Enugu state, Nigeria Farm. The freshly
laid eggs weighed between 10-15 g.
2.3 Experimental Design
Thirty adult male albino wistar rats were
assigned into 5 groups of 6 rats per group.
Following establishment of diabetes mellitus on
the 2
day post induction, the rats were treated
with different concentrations of quail egg solution
as follows:
Group Treatment
One Non diabetic rats administered 10 ml/kg distilled water (positive control)
Two Diabetic rats administered 30 mg/ml quail egg solution (highest concentration)
Three Diabetic rats administered 15 mg/ml quail egg solution (medium concentration)
Four Diabetic rats administered 7.5 mg/ml quail egg solution (lowest concentration)
Five Diabetic rats administered 10 ml/kg distilled water (Negative control)
Aba et al.; JAMPS, 5(4): 1-7, 2016; Article no.JAMPS.22723
Upon establishment of diabetes, the quail egg
solution was administered daily through the oral
route for 21 days. The FBG levels were
assessed 1 h, 6 h 24 h and 21 days post
treatment while blood for assay of oxidative
stress markers was collected on the 21
day post
2.4 Preparation of Quail Egg
An empty beaker was weighed (A g). The shells
of the quail eggs were broken with spatula and
the contents emptied into the beaker. The weight
of the beaker and the contents were recorded as
B g. The weight of the contents of the egg alone
was obtained by subtracting the weight of the
beaker alone from the weight of the beaker and
its contents. Thus the weight of the egg yolk and
albumen, C was expressed mathematically thus:
C (g) = B (g)-A (g)
C (g) was solubilized in a calculated quantity of
distilled water to make a desired concentration
of quail egg solution and thereafter, serial
dilutions of the stock solution were made for
the different groups.
2.5 Induction of Experimental Diabetes
Diabetes was induced in rats using the method
described by [9]. The rats were fasted for 16 h
prior to induction of diabetes. Diabetes was
induced by single intraperitoneal injection of
alloxan monohydrate at the dose of 160 mg/kg.
Diabetes was established on day 2 post
induction on confirmation of FBG levels above
7 mmol/l or 126 mg/dl.
2.6 Blood Collection
Blood samples were collected from the animals
into EDTA bottle using orbital techniques for the
biochemical determinations after plasma harvest.
Blood samples were collected from the
retrobulbar plexus of the median canthus of the
eye of the rats [10].
2.7 Estimation of Superoxide Dismutase
Superoxide dismutase activity was assayed by
the method of [11]. Plasma (0.5) ml was diluted
to 1.0 ml with ice cold water, followed by 2.5 ml
ethanol and 1.5 ml chloroform (chilled reagent).
The mixture was shaken for 60 seconds at C
and then centrifuged. The enzyme activity in the
supernatant was determined as follows. The
assay mixture contained 1.2 ml of sodium
pyrophosphate buffer, 0.1 ml of PMS and 0.3 ml
of NBT and approximately diluted enzyme
preparation in a total volume of 3 ml. The
reaction was started by the addition of 0.2 ml
NADH. After incubation at 30°C for 90 seconds,
the reaction was stopped by the addition of 1 ml
glacial acetic acid. The reaction mixture was
stirred vigorously and shaken with 4 ml n-
butanol. The mixture was allowed to stand for 10
minutes, centrifuged and butanol layer was
separated. The colour intensity of the chromogen
in the butanol layer was measured in a
spectrophotometer at 520 nm. A system devoid
of enzyme served as control. One unit of enzyme
activity is defined as the enzyme concentration,
which gives 50% inhibition of NBT reduction in
one minute under assay conditions. SOD activity
was expressed as U/ml of plasma.
2.8 Estimation of Lipid Peroxidation
Lipid peroxidation was estimated by measuring
spectrophotometrically, the level of the lipid
peroxidation product, malondialdehyde (MDA) as
described by [12]. A volume, 0.1 ml of the serum
was mixed with 0.9 ml of H
O in a test tube. A
volume, 0.5 ml of 25% TCA (trichloroacetic acid)
and 0.5 ml of 1% TBA (thiobarbituric acid) in
0.3% NaOH were also added to the mixture. The
mixture was boiled for 40 minutes in water-bath
and then cooled in cold water. Then 0.1 ml of
20% sodium dodecyl sulfate (SDS) was added to
the cooled solution and mixed properly. The
absorbance was taken at wavelength 532 nm
and 600 nm against a blank.
- A
x 100 (mg/dl)
2.9 Estimation of Reduced Glutathione
The reduced glutathione level was determined by
the method of [13]. This method was based on
the development of yellow colour when 5,5’-
dithio-bis-2-nitrobenzoic (DTNB) is added to
compound containing sulphydryl groups. The
colour developed was read at 412 nm in
2.10 Statistical Analyses
Data obtained were analyzed using One-way
Analysis of Variance (ANOVA). Variant means
were separated using Duncans Multiple range
Aba et al.; JAMPS, 5(4): 1-7, 2016; Article no.JAMPS.22723
Pos hoc Test. Results were presented as Mean ±
Standard Error of the Mean (Mean ±SEM).
There was a significant (p<0.05) decrease in the
FBG levels of the groups treated with quail egg
solution 24 h post treatment when compared to
that of the diabetic untreated group. The group
treated with 30 mg/ml of quail egg (group 2)
showed the most profound decrease in FBG
levels when compared with the other treated
groups. The FBG levels of 30 mg/ml-treated rats
was comparable to that of the normal control
groups 21 days post treatment.
The plasma malondialdehyde values of rats in
group 2 were significantly (p<0.05) reduced
compared to that of the negative control group
but was statistically comparable (p>0.05) to
those of the normal control group.
There was significant (p<0.05) increase in the
mean activity of SOD for the group treated with
30 mg/ml of quail egg solution when compared
with the diabetic untreated group. There was no
significant (p>0.05) difference in the mean
activity of SOD of the 15 and 7.5 mg/kg quail
egg-treated rats when compared to the diabetic
untreated group.
There was a significant (p<0.05) increase in
the reduced glutathione concentration in
the groups treated with quail egg solution
when compared to the diabetic untreated group.
There was no significant (p>0.05) difference
in the reduced glutathione levels between the
15 and 7.5 mg/dl quail egg solution-treated
Table 1. Effect of quail egg solution on the fasting blood glucose levels of alloxan-induced
diabetic rats
induction (PI)
1 h PI
6 h PI
24 h PI
21 Days PI
1 84.80±4.39
2 92.80±1.98
3 91.40±2.24
4 96.80±1.11
5 92.00±1.47
Different superscript along the same column indicate significant difference at p<0.05
Fig. 1. Effect of subchronic administration of quail egg solution on malondialdehyde values of
alloxan-induced diabetic rats
Aba et al.; JAMPS, 5(4): 1-7, 2016; Article no.JAMPS.22723
Fig. 2. Plasma superoxide dismutase activity of alloxan-induced diabetic rats treated with quail
egg solution
Fig. 3. Reduced glutathione values of alloxanized rats treated with varying concentrations of
quail egg solution
The elevated glucose levels observed in groups
2-5 rats compared to the group 1 rats (Table 1)
was attributed to the effect of alloxan
monohydrate administered to the rats in groups
2-5. Alloxan monohydrate, a glucose analogue
has been shown to produce hyperglycemia
through either partial or complete destruction of
beta cells of the islets of langerhans which
Aba et al.; JAMPS, 5(4): 1-7, 2016; Article no.JAMPS.22723
secrets insulin [14]. Insulin deficiency leads to
increased blood glucose levels [15]. The
elevated glucose level was significantly reduced
in all the treated groups especially in the 30
mg/ml-treated diabetic rats which decreased
from 343.80±6.00 mg/dl to 87.20±3.22 mg/dl 21
days post-treatment. The reduction was credited
to the effect of quail egg solution. Quail egg
contains important nutrients such as amino acids
(leucine, valine and alanine), vitamins (vitamin E)
and minerals such as zinc [16]. Leucine has
been reported to lower blood glucose levels in
type 2 diabetic patients [17]. Leucine is one of
the most potent insulin secretagogues among the
branched chain amino acids that facilitate
glucose induced insulin release from pancreatic
beta cells [17]. Alanine equally plays a key role in
maintaining glucose levels in the body by helping
to convert glucose to energy [18]. Marvin et al,
[19] reported that supplementation of vitamin E
might alter insulin receptors in muscle or adipose
tissue by increasing membrane motility.
The results obtained for MDA showed that the
diabetic rats treated with the highest
concentration of quail egg solution had
significantly lower levels of MDA compared to
other groups (Fig. 1). This indicates that at very
high concentration, quail egg may mitigate lipid
peroxidation. This observation may be attributed
to antioxidant content of quail egg such as
Vitamin E. Antioxidants are known to protect
against lipid peroxidation [20].
Similarly, the diabetic rats treated with quail egg
solutions demonstrated significant increases in
the SOD activities compared to the negative
control group (Fig. 2). This may be related to the
Zinc content of quail egg [21]. Zinc is a co-factor
for the enzyme SOD thus facilitates the activity of
SOD [22].
The depleted reduced glutathione in the diabetic
untreated rats compared to the treated group
(Fig. 3) is a consequence of diabetes. Reduced
glutathione level is usually depleted in diabetic
conditions due to hyperglycemia which channels
glucose to the polyol pathway depleting NADPH
required for glutathione regeneration [23].
Mitigation of hyperglycemia may therefore
improve the levels of reduced glutathione. It is
also possible that the zinc content of quail egg
may inhibit glutathione depletion [24].
Administration of quail egg solution especially at
the concentration of 30 mg/ml to diabetic rats
resulted in hypoglycaemia and improvement in
the in vivo antioxidant marker parameters.
It is not applicable.
All authors hereby declare that “principles of
laboratory animal care” (NIH publication No 85-
23, revised 1985) were followed, as well as
specific national laws. All experimemts have
been examined and approved by the appropriate
ethics committee.
Authors have declared that no competing
interests exist.
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© 2016 Aba et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Peer-review history:
The peer review history for this paper can be accessed here:
... Whole eggs have predominantly been criticized for their associated risk of developing chronic diseases [24], yet the benefits of WE consumption have also been reported [13]. For instance, several groups have suggested that WE provide antioxidant properties [13,25], either through antioxidant peptides in the egg yolk [11] or other reactive oxygen species-reducing nutrients [26]. Other studies examining the role of quail egg consumption in rat models of T2DM have demonstrated upregulation of glutathione metabolism in alloxan-induced T2DM in Wistar rats [25] and improved oxidative stress profiles in streptozotocin-injected rats [27]. ...
... For instance, several groups have suggested that WE provide antioxidant properties [13,25], either through antioxidant peptides in the egg yolk [11] or other reactive oxygen species-reducing nutrients [26]. Other studies examining the role of quail egg consumption in rat models of T2DM have demonstrated upregulation of glutathione metabolism in alloxan-induced T2DM in Wistar rats [25] and improved oxidative stress profiles in streptozotocin-injected rats [27]. Raza and colleagues [27] identified that in diabetic rat liver glutathione content and glutathione S-transferase (GST) activity were decreased 65% while also observing that brain glutathione and GST activity were increased two-fold as a result of a T2DM phenotype. ...
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Nutrigenomic evidence supports the idea that Type 2 Diabetes Mellitus (T2DM) arises due to the interactions between the transcriptome, individual genetic profiles, lifestyle, and diet. Since eggs are a nutrient dense food containing bioactive ingredients that modify gene expression, our goal was to examine the role of whole egg consumption on the transcrip-tome during T2DM. We analyzed whether whole egg consumption in Zucker Diabetic Fatty (ZDF) rats alters microRNA and mRNA expression across the adipose, liver, kidney, and prefrontal cortex tissue. Male ZDF (fa/fa) rats (n = 12) and their lean controls (fa/+) (n = 12) were obtained at 6 wk of age. Rats had ad libitum access to water and were randomly assigned to a modified semi-purified AIN93G casein-based diet or a whole egg-based diet, both providing 20% protein (w/w). TotalRNA libraries were prepared using QuantSeq 3' mRNA-Seq and Lexogen smallRNA library prep kits and were further sequenced on an Illu-mina HighSeq3000. Differential gene expression was conducted using DESeq2 in R and Benjamini-Hochberg adjusted P-values controlling for false discovery rate at 5%. We identified 9 microRNAs and 583 genes that were differentially expressed in response to 8 wk of consuming whole egg-based diets. Kyto Encyclopedia of Genes and Genomes/Gene ontol-ogy pathway analyses demonstrated that 12 genes in the glutathione metabolism pathway were upregulated in the liver and kidney of ZDF rats fed whole egg. Whole egg consumption primarily altered glutathione pathways such as conjugation, methylation, glucuronidation, and detoxification of reactive oxygen species. These pathways are often negatively affected during T2DM, therefore this data provides unique insight into the nutrigenomic response of dietary whole egg consumption during the progression of T2DM.
... [6][7][8] These results are also similar to reports in animals on the blood glucose lowering effects of eggs. [33][34][35][36][37] On the other hand a study has reported that administration of quail eggs in comparison to chicken eggs in diabetic rats did not reduce the blood glucose levels. [19] The beneficial effect of eggs on reducing high blood glucose may be related to the levels of unsaturated fatty acid content. ...
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... This implies that the 30 mg/ml quail egg pretreatment prevented excessive leakage of ALT into the circulation probably by stabilizing hepatocyte membranes or by mopping the acetaminophen metabolites. The antioxidant properties of quail egg have been widely reported (Aba et al., 2015). Presence of Vitamins B2 and C in quail eggs has also been associated with hepatoprotection (Takate et. ...
... Antioxidants are known to mop up free radicals (Aba et al., 2015). It is also possible that the extract may have stimulated the regeneration of the islet cells. ...
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Collection of blood from small laboratory animals is necessary for a wide range of scientific research and there are a number of efficient methods available for that. It is important that blood sample collection from experimental animals should be least stressful because stress will affect the outcome of the study. Various regulatory agencies and guidelines have restricted the use of animals and the techniques used for blood collection in laboratory animals. This article deals with the approved blood collection techniques for laboratory animals like rodents, lagomorphs and nonrodents. Permission of the Institute Animal Ethics Committee has been obtained for the use of animals for demonstrating the techniques.
Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status a cellular adaptive response occurs requiring functional chaperones, antioxidant production and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring plasma reduced and oxidized glutathione, as well as pentosidine, protein carbonyls lipid oxidation products 4-hydroxy-2-nonenal and F2-isoprostanes in plasma, and lymphocytes, whereas the lymphocyte levels of the heat shock proteins (HSP) HO-1, Hsp72, Sirtuin-1, Sirtuin-2 and thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. Plasma GSH/GSSG showed a significant decrease in type 2 diabetes as compared to control group, associated with increased pentosidine, F2-isoprostanes, carbonyls and HNE levels. In addition, lymphocyte levels of HO-1, Hsp70, Trx and TrxR-1 (P<0.05 and P<0.01) in diabetic patients were higher than in normal subjects, while sirtuin-1 and sirtuin-2 protein was significantly decreased (p<0.05). In conclusion, patients affected by type 2 diabetes are under condition of systemic oxidative stress and, although the relevance of downregulation in sirtuin signal has to be fully understood, however induction of HSPs and thioredoxin protein system represent a maintained response in counteracting systemic pro-oxidant status. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.
The reaction of human 5-aminolaevulinate dehydratase with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.
Dietary zinc deficiency can cause increased lipid peroxidation while zinc supplementation inhibited this process. The aim of this study was to investigate the effect of dietary zinc on malondialdehyde (MDA) product as an index of endogenous lipid peroxidation, glutathione (GSH) and protein thiols (PrSHs) as well as superoxide dismutase (SOD) activity in rat blood, liver and pancreas. Young male rats were fed a zinc deficient (ZD) basal diet containing congruent to 0.5 ppm zinc or were fed ad libitum (AL) a zinc adequate diet (30 ppm zinc) for 3 weeks. The ZD rats were then fed the basal diet supplemented with either 100 ppm zinc or 1000 ppm zinc for another 3 weeks. The zinc concentration of the investigated tissues reflected the dietary zinc content. Plasma, liver and pancreas MDA measurements from ZD rats revealed significant increases (P < 0.05, < 0.001) as compared to AL control values, the highest increase was in pancreas. ZD rats also displayed significant decreases in their blood and liver GSH content (P < 0.001, < 0.05) and SOD activity (P < 0.001) as well as serum PrSHs (P < 0.001) as compared to AL control values. However, these measurements in pancreas were insignificantly changed except GSH content was significantly increased (P < 0.05). Feeding ZD rats a diet containing 100 ppm or 1000 ppm zinc resulted in a significant reduction of the endogenous MDA formation (P < 0.05, < 0.001) in their tissues with the reversal of changes in the other parameters, so that their levels were nearly restored to AL control values especially in response to 1000 ppm zinc.(ABSTRACT TRUNCATED AT 250 WORDS)
Transition metals catalyze free radical-mediated oxidation of lipids and lipoproteins. This process is currently studied because of its potential relevance to pathological processes like atherosclerosis. Formation of thiobarbituric acid-reacting substances from polyenoic fatty acids is frequently used to follow oxidation of lipids and plasma lipoproteins. We describe here how Cu(II)- and Fe(III)-catalyzed oxidation of human low density lipoprotein or soy bean phospholipids and the photometric evaluation of the thiobarbituric acid-reaching substances formed can be conducted in the same 96-well microtiter plate. The procedure showed a correlation of 0.98 with conventional two-stage fluorimetric and spectrophotometric methods and also showed better reproducibility. The plate method can handle up to one plate per hour with considerably less labor than the test tube assays. The plate procedure required small volumes of diluted samples of lipoproteins lipids and reagents. The method was suitable for testing the concentration-dependent antioxidant potency of substances like probucol, butylated hydroxytoluene, and alpha-tocopherol. The method can also be used to follow the kinetics of oxidation of lipoproteins.