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Research Article
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Influence of L-arginine on the Heart Histology and Function Markers of Metabolic Syndrome in Female Wistar Albino Rats |
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Anthony C.C. Egbuonu,
Ifeoma I. Ijeh,
Lawrence U.S. Ezeanyika
and
Onyechi O. Obidoa
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ABSTRACT
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High blood pressure (a condition associated with vascular constriction) is a major feature of metabolic syndrome (MES). MES, a constellation of metabolic disorders, is prevalently higher in females and was associated with a reduced concentration of a vasodilator molecule, Nitric Oxide (NO). L-arginine (ARG), a precursor of NO may improve MES, warranting this study. Two groups (n = 8) of female Wistar albino rats were (per orally for twenty eight days) exposed to a single dose of 60 mg kg-1 b.wt. of ARG and 3 mL kg-1 b.wt. of distilled water, DW, respectively as treated and control groups. Significant differences in means were separated by students t-test (p<0.05; p<0.01) and results expressed as Mean±Standard deviation. ARG exposure caused a significant reduction (p<0.01) in sodium ion (Na+) concentration (136.42±1.66 mmol L-1; 6.54%), but a non-significant decrease (p>0.05) in potassium ion (K+) concentration (4.54±0.66 mmol L-1; 14.01%) in the rats serum, suggesting improved/reduced blood pressure. ARG treatment in the rats had a significant increase (p<0.01) in Alkaline Phosphatase (ALP) activity (8.30±0.23 IU L-1; 196.43%) in the rats serum, indicating adverse influence on high metabolic organs, including the brain. Sodium ion had a significant negative correlation (r = 0.01) with potassium ion, whereas the heart histomorphology revealed degenerations in the ARG-fed rats, apparently confirming the observations and suggestions thereto. Thus, ARG may improve blood pressure in the rats, perhaps at the expense of compromised heart function and histology of the rats. These may be pointing to a new arginine phenomenon, hence warrant follow up.
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Received: January 10, 2013;
Accepted: February 23, 2013;
Published: May 21, 2013
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INTRODUCTION
High blood pressure (a condition associated with vascular constriction) is
a major feature of metabolic syndrome (MES). It was associated with a reduced
concentration of nitric oxide, NO (a vasodilator molecule) and characterized
by a cluster of cardiovascular risk factors, including high blood pressure (Deedwania
and Gupta, 2006; Gallagher et al., 2010)
and increased health challenges (Pelucchi et al.,
2010; Siddiqui, 2011). Metabolic syndrome scourge
is pandemic (Gotto et al., 2006; Grundy,
2008) and is prevalent in children (Pedrosa et al.,
2011). The syndrome afflicts about 20-30% of the adult population the world
over (Grundy, 2008; Chaabo et
al., 2010), including Nigeria (30.7%) as reported by Ijeh
et al. (2010). The increasing prevalence of MES (Bakoma
et al., 2011) may be higher among the female gender, with huge implications.
Garlichs et al. (2000) had associated reduced
NO concentration with the pathophysiology of MES. NO regulated cardiovascular
function (McGrowder and Brown, 2007) and at abnormal
concentration, had elicited pathological condition (Lokhande
et al., 2006) in animals. A reduction in the concentration of ARG
affected the biological activity of NO (Subratty et al.,
2007; Harisa, 2011), suggesting exogenous supply
of ARG my influence the activity of NO in animals. Indeed, possible role of
ARG in metabolic processes that could improve MES features in animals was suggested
(Sepehri et al., 2006; Van
Waardenburg et al., 2007; Ezeanyika and Egbuonu,
2011; Harisa, 2011) and appear to be supported by
recent studies (Egbuonu and Ezeanyika, 2012a, b;
Egbuonu and Ezeanyika, 2013).
Since high blood pressure is a major feature of MES (Expert
Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults,
2001 (NCEP) (USA); Gallagher et al., 2010),
this study aimed to ascertain the effect of ARG on the heart histology and function
markers of MES in female rats. Objectives set to achieve the stated aim include
the study of the effect of ARG on serum alkaline phosphatase activity, sodium
ion and potassium ion concentrations as well as on the heart histology of female
Wistar albino rats. In particular, potassium ion contributes to the optimal
functioning of the cells and the organs and its deficiency (hypokalaemia) was
associated with cardiac dysfunction (Bush, 1991) and high
blood pressure (Siani et al., 1987; Strazzullo
et al., 1990). On the other hand, elevated sodium ion concentration
(hypernatraemia) indicated high blood pressure (Jaitovich
and Bertorello, 2010).
MATERIALS AND METHODS Chemicals: The chemicals (analytical grade) used in this study were products of reputable companies based in Europe and America.
Concentration determination/justification: Based on the WHO reported
daily ARG oral intake (Marshal, 1994) and the concentration
used in earlier studies (Alexander et al., 2004;
Egbuonu et al., 2010a-c),
the concentration of ARG used in this study was calculated and adjusted to 60
mg kg-1 b.wt.
Animals and treatment: The female Wistar rats (60-80 g) used in this
study were procured from the animal house of the Faculty of Biological Sciences
University of Nigeria. The animal study was according to International guidelines
for the care and use of laboratory animals in Biomedical Research (CCAC,
1985; WMA/APS, 2002). The animal study was conducted
between August and September, 2010.
The rats were acclimatized for a week and randomized into two groups (sample size of eight rats each) based on their body weight. Group B rats were exposed to ARG (60 mg kg-1 b.wt.) whereas Group A rats were given Distilled Water (DW) (3 mL kg-1 b.wt.). The rats were exposed orally for 28 consecutive days. The rats were kept in a well-ventilated stainless steel cages at room temperature (28±2°C) and tropical humid condition. They were maintained under twelve hours of light alternating with twelve hours of darkness. The rats were allowed free access to tap water and standard rat chow (Grand Cereals and Oil Mills Limited, Jos, Nigeria) throughout the experimental period. Sample collection and preparation: After 28 days oral intubation, the animals were fasted overnight and sacrificed on day 29. The respective blood sample of the animals was collected by ophthalmic venous plexus or retro orbital sinus venipuncture, using sterile capillary tube to direct blood into clean non-anticoagulated glass tubes. Clotted blood in each tube was centrifuged (at 3000 rpm for 10 min) to yield the serum. The respective serum was aspirated separately into stoppered polystyrene tube and stored in a deep freezer for subsequent use in determining the biochemical markers of MES related to heart function. Organ specimen (heart) excised from the sacrificed rats for histology were fixed in 10% formaldehyde buffered saline (formal saline) until used.
Our choice for using female rats in this study was based on the recent listing
of female gender as an independent risk factor for the development of MES (Ravikiran
et al., 2010) and reports that MES was prevalently higher in females
(Mangat et al., 2010; Kilic
et al., 2010).
Parameters determined
Serum alkaline phosphatase (ALP) activity: The serum alkaline phosphatase
(ALP) activity was assayed by the method of Walter and Schutt
(1974). This is based on the principle that alkaline phosphatase could hydrolyze
colorless phosphate esters of various alcohols and phenols yielding p-nitrophenol
as the yellow nitrophenolate ion in alkaline solution that was measured colorimetrically
at 405 nm.
Photometric estimation of sodium (Na+) and potassium (K+) ions concentrations: The estimation of sodium and potassium ions in serum was with flame emission photometer. This is based on the principle that passing a liquid sample through a nebuliser (atomizer) could excite the atoms which on falling back to the ground state, emit light of characteristic wavelength, color and intensity. On passing through a suitable filter, a photosensitive detector measures the emitted light as the amount (concentration) of the atom (metallic ion) present.
Organ histology: Organ specimen (heart) for histological examination
was promptly excised from the sacrificed rats and fixed in 10% formaldehyde
buffered saline (formal saline) as reported by Egbuonu et
al. (2010c). Briefly, after dehydration (in graded levels (70-100%)
of alcohol), clearing (in xylene impregnated with paraffin wax) and sectioning
(at 5 microns thickness using rotary microtome) the sections were floated on
a water bath maintained at a temperature of 2-3°C below melting point of
the paraffin wax. The sections were dried on a hot plate maintained at a temperature
of 2-3°C above the melting point of the paraffin and stained. Then, the
sections were mounted, using haematoxylin and eosin.
Statistical analysis: Data were analyzed by Students t-test to
determine the significant differences in means, using the Statistical Package
for the Social Sciences (SPSS) for Windows version 16.0 (SPSS Inc., Chicago,
IL., USA). Results were expressed as mean and standard deviation (Mean±SD)
of eight rats per group at significance levels of p<0.05 and p<0.01. Furthermore,
correlation of the results for possible association among the studied parameters
was by Pearsons bivarate methods (r = 0.01).
RESULTS Serum alkaline phosphatase (ALP) activity: Results of this study reveal that rats exposed to ARG had a significant (p<0.01) increase in their serum ALP activity (8.30±0.23 IU L-1) compared with those exposed to DW (2.80±0.10 IU L-1). This is an increase of over one-fold (196.43%) in ARG-fed group relative to the control (Fig. 1). Serum sodium ion (Na+) concentration: As depicted in Fig. 2, the sodium ion concentration in serum decreased in the ARG-treated rats (136.42±1.66 mmol L-1) relative to control (145.97±1.22 mmol L-1). The observation, representing a decrease of 6.54% was statistically significant (p<0.01). Serum potassium ion (K+) concentration: The results of this study as presented in Fig. 3 show that ingesting ARG by rats non-siginificantly decreased (p>0.05) the serum K+ ion concentration (4.54±0.66 mmol L-1) in comparison with the control (5.28±0.44 mmol L-1). This observation represents a decrease of 14.01% relative to the control value.
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Fig. 1: |
Effect of DW and ARG on serum ALP activity of rats |
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Fig. 2: |
Effect of DW and ARG on serum sodium ion concentration of
rats |
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Fig. 3: |
Effect of DW and ARG on potassium ion concentration in serum
of rats |
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Fig. 4: |
Heart section of untreated, control (Group A) rats showing
normal papillary muscles (arrow heads). H and E stains, x400 |
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Fig. 5: |
Heart section of rats treated with high ARG (Group B) showing
degeneration of the papillary muscles (arrow heads). H and E stains x400 |
Histomorphology of the heart: Heart sections of the control (Group A)
showed typical histology with normal papillary muscles (Fig. 4).
Table 1: |
Correlations output of serum potassium ion and sodium ion |
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**Correlation is significant at the 0.01 level (2-tailed) |
Sections collected from rats treated with ARG (Group B) showed moderate degeneration
of the papillary muscles (Fig. 5).
Correlation of results: The results of Pearsons correlation analysis showed that sodium ion concentration had a significant negative correlation (r = 0.01) with potassium (Table 1). DISCUSSION
The female gender is an independent risk factor for the development of metabolic
syndrome, MES (Ravikiran et al., 2010). MES,
a constellation of metabolic disorders, is prevalently higher in the females.
It was associated with a reduced concentration of NO (a vasodilator molecule)
and characterized by a cluster of cardiovascular risk factors, including hypertension
(high blood pressure) (Deedwania and Gupta, 2006; Gallagher
et al., 2010). Possible role of ARG in metabolic processes that could
improve MES in animals was suggested (Van Waardenburg et
al., 2007; Ezeanyika and Egbuonu, 2011) and
appear to be supported by recent studies (Egbuonu and Ezeanyika,
2012a, b; Egbuonu and Ezeanyika,
2013). High blood pressure (a condition associated with vascular constriction
and heart function) is a major feature of MES. These therefore warranted this
study.
ARG ingestion by rats elicited a significant increase (p<0.01) in ALP activity
in the rats serum, indicating alterations of pancreatic function (Fraulob
et al., 2010) and other high metabolic organs, possibly the heart
and brain. The ALP enzyme is present in various high metabolic organs hence
is not a specific marker of toxic effect. In similar studies, we demonstrated
that L-arginine exposure improved the kidney/renal function (Egbuonu
and Ezeanyika, 2013) and liver function (Egbuonu et
al., 2013), but damaged the brain function (unpublished).
Sodium ion accumulation caused high blood pressure in animals (Jaitovich
and Bertorello, 2010). ARG ingestion to the female rats decreased (p<0.01)
the sodium ion (Na+) concentration, implying lowered blood pressure
and possible benefit on MES in the rats. This is consistent with earlier reports
of ARG-induced vasodilation (Rang et al., 2003)
and reduction of hypertension or high blood pressure (Alexander
et al., 2004) in animals. The possible vasodilatory activity of ARG
via its metabolite, NO, could enhance the increase in the wall of the blood
vessels resulting in lowered blood pressure. On the other hand, ARG treated
rats had a decrease in serum potassium ion (K+) concentration. Decreased
potassium ion (hypokalaemia) resulted to neurological and cardiac dysfunctions
in animals (Bush, 1991), but the observation was not statistically
significant (p>0.05) hence may not be treatment related.
Furthermore, correlation of the results showed that sodium ion had a significant
negative correlation (r = 0.01) with potassium ion, suggesting overriding effect
of ARG on sodium ion over potassium ion in relation to blood pressure reduction
in this study. Generally, degenerations observed in the heart sections of the
rats appear to indicate ARG-induced adverse influence on their heart histomorphology,
apparently confirming the observation on the serum ALP activity and the suggestion
thereto. Agent-induced histomorphologic alterations in organs was reported by
Egbuonu et al. (2010c). It appears that the possible
blood pressure lowering potential of ARG elicited adverse influence on the heart
histology. We did neither know nor study how this could have occurred but L-arginine-induced
complex effect had been reported in animals and attributed to the multiple catabolic
pathways of ARG (Schriek et al., 2007). Arginine
paradox (a phenomenon referring to the dependence of cellular NO production
on exogenous L-arginine concentration despite the theoretical saturation of
nitric oxide synthase enzymes with intracellular L-arginine) may be fundamental
to this complexity.
CONCLUSION In conclusion, this study suggests that exposure to ARG may improve blood pressure (a major feature of MES) in the female rats, perhaps at the expense of compromised heart histology and function of the rats. This may be pointing to a an entirely new arginine phenomenon that warrants further investigation and caution in the use of ARG against MES associated with heart function in female rats.
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