Effect of Pyridoxamine on Diabetic Retinopathy and Diabetes-Induced Deterioration of

Journal of Applied Sciences Research, 6(9): 1316-1324, 2010
© 2010, INSInet Publication
Effect of Pyridoxamine on Diabetic Retinopathy and Diabetes-Induced Deterioration of
Serum Lipids and Creatinine in Experimental Animals
1
Ghada Ghanem El-Hossary, 1Amany Hassan M. El-Shazly, 2Nadia Samy Ahmed, 3Sahar Mahmoud
Mansour and 3Anisa Saleh Mohamed
1
Pharmacology, 2Nutrition and 3Histology Departments, Research Institute of Ophthalmology,
Giza, Egypt.
Abstract: The present study aimed to investigate the effect of pyridoxine administration on diabetesinduced alterations of serum lipids and creatinine levels and retinal histopathological changes. Forty albino
Wistar rats of both sexes were used and divided into four groups; two groups (I and II) served as controls.
In group III, diabetes was induced by a single intraperitoneal injection of alloxan 100 mg/kg. In group
IV, diabetes was induced and rats received pyridoxine in drinking water (2g/liter) for three months. Serum
glucose, cholesterol, triglyceride and creatinine levels were measured after 2 and 3 months. Then, animals
were sacrificed and subjected to light microscopic examination of the retina. Untreated diabetic animals
exhibited significant deterioration of the measured biochemical parameters in addition to marked
histopathological retinal changes. Treatment with pyridoxine improved significantly the diabetes-induced
deterioration of cholesterol, triglyceride and creatinine in blood at the second and third months as
compared to untreated model. In addition, the retina of these animals appeared fairly normal with minimal
deviation from the control retina suggesting its protective role against diabetic retinopathy.
Key words:
Pyridoxine, experimental diabetes mellitus, glycated end product, diabetic retinopathy,
serum lipids, creatinine.
INTRODUCTION
Diabetic complications appear to be multifactorial
in origin. The biochemical process of advanced
glycation, in particular, has been postulated to play a
central role in these complications. This process is
accelerated in diabetes as a result of chronic
hyperglycemia and increased oxidative stress[1].
Advanced glycated end products (AGEs) are a complex
group of compounds formed via a non enzymatic
reaction between reducing sugars and amine residues
on proteins, lipids or nucleic acids. The major AGEs in
vivo appear to be formed from highly reactive
intermediate carbonyl groups, known as alphadicarbonyls or oxoaldehydes[1,2]. The effects of AGEs
may be receptor-dependent by action on the AGEs
receptor (RAGEs) on the cell surface or receptorindependent intracellular action[3].
Advanced glycation occurs over a prolonged period
affecting long lived proteins. The structural components
of the connective tissue matrix and, in particular,
basement membrane components such as type IV
collagen are prime targets. Other long-lived proteins
can also undergo advanced glycation, including myelin,
tubulin, plasminogen activator 1 and fibrinogen.
Extracellular matrix proteins are susceptible to AGE
modification because of their slow turnover rate. The
formation of intermolecular and intramolecular
crosslinks with collagen as a result of the glycation
process leads to structural alterations leading to
increased stiffness and resistance to proteolytic
digestion[4].
Accumulation of AGEs occurs in most sites of
diabetes complications including the kidney, retina and
atherosclerotic plaques[1]. The kidney is a target for
AGE-mediated damage. It also affects the circulating
AGE concentrations because the kidney is the major
site of clearance of AGEs[5]. Moreover, AGEs have
been localized to retinal blood vessels in patients with
type 2 diabetes and were found to correlate with the
degree of retinopathy[6]. Retinopathy is a major
complication of diabetes in both humans and animal
models. It is due to microvascular lesions together with
dysregulation of an array of biochemical pathways in
the diabetic retina. New therapeutic regimens are
needed to effectively prevent or retard the initiation and
progression of the retinal microvascular cell dysfunction
and death that is characteristic of the vaso-degenerative
stages of diabetic retinopathy[7]. Agents that inhibit
AGE formation were tested extensively such as
aminoguanidine which was one of the first inhibitors of
AGE formation studied[8].
Corresponding Author: Ghada Ghanem El-Hossary, Pharmacology Department, Research Institute of Ophthalmology, Giza,
Egypt. E-mail: [email protected]
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J. Appl. Sci. Res., 6(9): 1316-1324, 2010
Another inhibitor of AGEs formation is
pyridoxamine (pyridoxine) which is a derivative of
vitamin B6. Pyridoxine assists in the balancing of
sodium and potassium as well as promoting red blood
cell production. It is linked to cardiovascular health by
decreasing the formation of homocysteine. Pyridoxine
may help to balance hormonal changes in women and
aid the immune system. Lack of pyridoxine may cause
anemia, nerve damage, seizures, skin problems and
sores in the mouth[9]. It is required for the production
of the monoamine neurotransmitters serotonin,
dopamine, noradrenaline and adrenaline, as it is the
precursor of pyridoxal phosphate: cofactor for the
enzymatic conversion of 5-hydroxytryptophan into
serotonin and levodopa into dopamine, noradrenaline
and adrenaline. As such it has been implicated in the
treatment of depression and anxiety[10]. In murine
models of diabetes, it was reported to reduce
hyperlipidemia and prevent AGE formation[11]. Thomas
et al.[12] have shown that pyridoxamine could
antagonize angiotensin II-induced elevation in serum
and renal AGEs, prevent renal hypertrophy and
decrease salt retention in experimental diabetes[12]. In
addition, it was reported to prevent successfully
diabetes-induced retinal vascular lesions[13].
The aim of the present work is to test the potential
prophylactic effect of pyridoxine oral administration on
diabetic retinal histopathological changes as well as
serum lipids and creatinine in experimentally induced
diabetes in rats.
MATERIALS AND METHODS
Induction of the Animal Model of Diabetes Mellitus:
Wister rats were injected intraperitoneally with a single
dose of 100 mg/kg alloxan (Sigma, Germany). The
blood glucose level was measured after 48 hours then
weekly. Diabetes was defined as a fasting blood
glucose level exceeding 120 mg/dl.
Animal Groups: Forty Albino Wistar rats of both
sexes weighing 140-150 grams were used. They were
housed individually in separate cages under veterinary
supervision. They were used in accordance with
institutional guidelines and with the statement for use
of animals in ophthalmic and vision research. They
were fed with the standard diet and water for three
months and kept in 12 hours dark/light cycles under
controlled temperature and humidity. Animals were
divided into four groups each consisting of ten rats.
A- Control Groups:
Group I: The animals received an equivalent volume
of distilled water once daily by means of a stomach
tube (negative control).
Group II: The animals received pyridoxine (pyridoxine
hydrochloride powder from MP Biomedicals, France)
in their drinking water (2 g/l) for three months
(positive control).
B- Diabetic Model Groups:
Group III: Diabetic model was induced and the
animals were left untreated.
Group IV: Diabetic model was induced and the
animals received pyridoxine in their drinking water (2
grams/liter) for three months starting from the third day
of induction of diabetes.
Biochemical Analysis: Serum glucose, cholesterol,
triglycerides and creatinine levels were measured
enzymatically by colorimetric methods using
commercial kits obtained from Biocon (Germany) for
glucose, Linear Chemicals (Spain) for serum lipids and
Spectrum Diagnostic (Germany) for creatinine.
Histopathological Examination: Histopathological
examination was carried out according to Drury and
Wallington[14]. The eyes were enucleated, dissected and
immediately double fixed in 4% glutraldehyde buffer,
then 1.3% osmium tetraoxide in phosphate buffer (pH
7.3). Retinal specimens were processed and embedded
in araldite Cy 212. Semi-thin sections were stained
with toluidine blue (TB) and examined by light
microscopy.
Statistical Analysis: Values of serum levels of glucose,
cholesterol, triglycerides and creatinine were expressed
as mean±SD. Analysis of variance (ANOVA) and
student t test were performed to compare the values
between groups. A post-hoc test was used to isolate
significant differences if (P < 0.05).
Results:
Biochemical Analysis: The mean glucose, cholesterol,
triglyceride and creatinine serum levels (at the 2nd and
3rd months) are showed in Tables (1, 2, 3 and 4).
Control rats (groups I and II) showed normal values of
the estimated parameters which were not significantly
changed all through the duration of the experiment.
Regarding induction of diabetes model in group III,
rats exhibited a noticeable deterioration in the tested
parameters. The mean blood glucose level increased
significantly to 177.70±19.74 and 170.30±15.08 mg/dl
at the 2nd and 3rd months respectively (Table 1). Also
cholesterol serum level increased to 96.60±7.07 and
99.30±12.17 mg/dl (Table 2) while triglyceride level
reached 166.80±15.43 and 170.30±13.83 mg/dl at the
2nd and 3rd months respectively (Table 3). As for serum
creatinine, it was also elevated to 1.16±0.21 and
1.60±0.17 mg/dl (Table 4). All these values were
significantly higher than the control values.
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J. Appl. Sci. Res., 6(9): 1316-1324, 2010
Table 1: Mean levels (±SD) of serum glucose at the 2nd and 3rd months of the experiment in group I (negative controls), group III (untreated
diabetic model) and group IV (diabetic model treated with pyridoxine in a dose of 2 grams/liter of drinking water for 3 months).
Groups
Group I
Group III
Group IV
Mean ± SD after 2 months
102.00±4.06
177.70±19.74
168.80±22.34
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.733
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Mean ± SD after 3 months
100.00±5.04
170.30±15.08
164.00±18.28
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.922
* Significant difference at p < 0.05; P1 compared to group I; P2 compared to group III.
Table 2: Mean levels (±SD) of serum cholesterol level at the 2nd and 3rd months of the experiment in group I (negative controls), group III
(untreated diabetic model) and group IV (diabetic model treated with pyridoxine in a dose of 2 grams/liter of drinking water for
3 months).
Groups
Group I
Group III
Group IV
Mean ± SD after 2 months
78.90±9.58
96.60±7.07
77.10±8.91
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.003*
0.971
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Mean ± SD after 3 months
79.80±8.56
99.30±12.17
78.60± 1.2
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.999
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.000
*Data are expressed as mean ± SD; n = 10.; * Significant difference at p < 0.05; P1 compared to group I.; P2 compared to group III.
Table 3: Mean levels (±SD) of serum triglyceride level at the 2nd and 3rd months of the experiment in group I (negative controls), group III
(untreated diabetic model) and group IV (diabetic model treated with pyridoxine in a dose of 2 grams/liter of drinking water for
3 months).
Groups
Group I
Group III
Group IV
Mean ± SD after 2 months
113.30±8.62
166.80±15.43
115.00±9.43
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.998
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Mean ± SD after 3 months
115.30±7.58
170.30±13.83
146.00±9.61
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.000
*Data are expressed as mean ± SD; n = 10; *Significant difference at p < 0.05; P1 compared to group I; P2 compared to group III.
Table 4: Mean levels (±SD) of serum creatinine level at the 2nd and 3rd months of the experiment in group I (negative controls), group III
(untreated diabetic model) and group IV (diabetic model treated with pyridoxine in a dose of 2 grams/liter of drinking water for
3 months).
Groups
Group I
Group III
Group IV
Mean ± SD after 2 months
0.62±0.14
1.16±0.21
0.79±0.15
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.068
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Mean ± SD after 3 months
0.58±0.12
1.60±0.17
1.12±0.17
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P1
0.000*
0.000*
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------P2
0.000
*Data are expressed as mean ± SD; n = 10; *Significant difference at p < 0.05; P1 compared to group I. P2 compared to group III.
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After treatment of diabetic rats with pyridoxine
(group IV), it was observed that the serum glucose
levels were still markedly elevated to 168.80±22.34 and
164.00±18.28 mg/dl at the 2nd and 3rd months
respectively. They were still significantly higher than
control group values. Comparing serum glucose levels
of group IV with untreated animals of group III, they
were found to be insignificantly different from diabetic
model values after 2 months and 3 months (Table 1).
On the other hand, serum cholesterol level was
significantly improved in animals treated with
pyridoxine as compared to the untreated model. It
reached 77.10±8.91 and 78.60±11.20 mg/dl at the 2nd
and 3rd months respectively without a significant
difference from control levels. As for serum
triglyceride level, it reached 115.00±9.43 and
146.00±9.61; it was significantly lower than the levels
of the untreated diabetic model after 2 and 3 months,
but still significantly higher than control values at the
3rd month (Table 3). Moreover, group IV showed
markedly improved serum creatinine levels reaching
values that were significantly lower than the untreated
diabetic model values at 2nd and 3rd months. They were
still significantly higher than control group values at 3rd
month. These values were 0.79±0.15 and 1.12±0.17
mg/dl at the 2nd and 3rd months respectively (Table 4).
Histopathological Examination: The histological
examination of the retinas of rats in the negative
controls (group I) showed the normal layers of the
retina (Figs. 1a&b). In addition, the retinae of rats of
group II treated with pyridoxine (positive control)
appeared fairly normal. Light microscopic examination
of the retinae of diabetic rats (group III) revealed clear
spaces in-between the segments of photoreceptors.
Tortuous dilated thick walled capillaries are present
which were abnormally extending from the ganglion
cell layer to the inner nuclear layer. In addition, dark
stained areas near to the wall of dilated capillary may
present leakage from the capillary with evident edema
of the outer and inner nuclear layers were noticed (Fig.
2). Diabetic animals treated with pyridoxine (group IV)
exhibited well protection of the retina against the above
diabetic changes, except for the presence of mild
vacuolations and dark stained bodies in the pigment
epithelium layer and slight disorganization of the outer
photoreceptor layer (Fig. 3).
Discussion: Non-enzymatic chemical modification of
proteins by reducing sugars, known as the Maillard
reaction, is implicated in the development of pathology
during aging and in chronic diseases such as diabetes,
atherosclerosis, and Alzheimers disease[1]. The Maillard
reaction between sugar and protein proceeds through a
labile Schiff base, which isomerizes to a ketoamine
adduct, the Amadori compound. Oxidative
decomposition and further reaction of the Amadori
compound produce advanced glycation end products
(AGEs)[2]. Similarly, advanced lipoxidation end
products (ALEs), such as the malondialdehyde adducts
to lysine, are formed on proteins during lipid
peroxidation reactions through effects on protein
structure, function, and turnover. The accumulation of
AGEs and ALEs in tissue proteins is thought to
contribute to the development of diabetic
complications[15]. Protein-sugar intermediates can also
degrade, largely through sugar autoxidation or through
the degradation of the Schiff base intermediates, to
produce low molecular weight carbonyl compounds.
Reactive carbonyls are also produced during lipid
peroxidation reactions. These electrophilic compounds
can react directly with proteins to form adducts with
lysine or arginine side chains resulting in the induction
of pathogenic protein modifications[16].
In the present study, diabetes was induced in rats
and oral pyridoxine was administered for three months
to ameliorate some diabetic complications on the retina
and serum levels of lipids and creatinine. The retinas
from diabetic untreated animals showed histological
changes reflecting the picture of diabetic retinopathy.
Among the several pathogenic mechanisms that may
contribute to diabetic retinopathy is the formation and
accumulation of AGEs and reactive dicarbonyl
intermediates. Accumulation of AGEs on diabetic
retinal capillary basement membranes contributes to
structural and functional abnormalities on this
specialized extracellular matrix causing intramolecular
and intermolecular cross-link formation rendering it
more resistant to protease modification[17]. During
hyperglycemia, production of reactive oxygen species
by alterations in glucose homeostasis together with
increased AGE formation and polyol pathway activity
may be the common pathways leading to activation of
protein kinase C and downstream signaling events that
lead to development of diabetic complications[18].
In the present work, PM administration protected
the retina from diabetes induced retinopathy in the
treated model group as shown by the histological
results. The results of the present work are in line with
those of Alan Stitt et al[19] who examined the ability of
PM to protect against diabetes-induced retinal vascular
lesions. They observed that diabetes increased the
retinal acellular capillaries more than threefold
accompanied by a significant upregulation of laminin
immunoreactivity in the retinal microvasculature.
Diabetes also increased mRNA expression for the
extracellular matrix proteins fibronectin, collagen IV
and laminin. Treatment with PM protected against
capillary drop-out, limited laminin protein upregulation
and decreased mRNA expression for the three
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J. Appl. Sci. Res., 6(9): 1316-1324, 2010
Fig. 1a: Light micrograph of a semi-thin section of control albino rat retina (group I) showing 1-pigment
epithelium 2- photoreceptors, outer segment (OS), inner segment (IS) 3- outer limiting membrane, 4part of outer nuclear layer (TB X1250).
Fig. 1b: Light micrograph of a semi-thin section of control albino rat retina (group I) showing: 5- outer plexiform
layer, 6- inner nuclear layer. (H) horizontal cell, (B) bipolar cell, (A) amacrine cell, (M) muller cell (TB
X1250).
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J. Appl. Sci. Res., 6(9): 1316-1324, 2010
Fig. 2:
Light micrograph of a semi-thin section of albino diabetic rat retina (group III) showing clear spaces (S)
in-between the segments of photoreceptors. A tortuous dilated thick walled capillary (C) is present and
abnormally extending from the ganglion cell layer to the inner nuclear layer. The dark stained areas
(arrows) near to the wall of dilated capillary may present leakage from the capillary. Edema of the outer
and inner nuclear layers is evident. (H) horizontal cell, (B) bipolar cell, (A) amacrine cell, (M) muller
cell (TB X1250).
basement membrane components in the retinal
microvasculature. They explained that PM may act to
limit AGE modifications that initiate inappropriate
cross-linking of extracellular matrix proteins[19]. It can
also prevent protein modification by ALEs which may
be an important consideration for neural tissues such as
the retina that possess high levels of polyunsaturated
fatty acids, which are particularly susceptible to
peroxidation reactions[16].
The vaso-degenerative stage of diabetic retinopathy
is characterized by progressive vascular dysfunction
and loss of retinal capillary viability. Hence, there is an
increase in the number of acellular capillaries in the
retinal microvasculature[20]. It was demonstrated that
PM could inhibit the formation of acellular strands in
the retinas of diabetic rats, maintaining microvascular
cellularity at normal (nondiabetic) levels and protecting
against premature pericyte cell death[19,20] which is also
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J. Appl. Sci. Res., 6(9): 1316-1324, 2010
Fig. 3:
Light micrograph of a semi-thin section of albino diabetic rat retina treated with pyridoxine for three
months (group IV). It shows the presence of mild vacuole (V) in pigment epithelium layer. Notice the
increased dark stained bodies in the pigment epithelium and slight disorganization of the outer
photoreceptor layer (Ph) (TB X1250).
in accordance with the results of the present study.
These results indicate the potential role of the
AGE/ALE inhibitor PM to protect against a range of
pathological changes in the diabetic retina and may be
useful for treating diabetic retinopathy.
In the present work, the serum levels of
cholesterol, triglyceride and glucose were determined
and were markedly elevated in the untreated diabetic
model. After 2 and 3 months of pyridoxine intake, the
cholesterol and triglyceride levels improved
significantly compared to untreated diabetic group and
were insignificantly different from controls except for
the triglyceride level after 3 months as it was still
significantly higher than controls. As for glucose levels,
they were found to be insignificantly different from
diabetic model values after 2 and 3 months.
Additionally, kidney function was assessed by
measuring creatinine serum level which was
deteriorated significantly in the untreated diabetic
model group. It showed a marked significant
improvement after 2 months of pyridoxine
administration, whereas after 3 months, serum
creatinine increased due to sustained diabetic effect; but
still it was significantly improved as compared to
untreated diabetic model.
In agreement with the results of the present
investigation, Alderson et al.[22] demonstrated that PM
could reduce plasma lipids in diabetic rats. In addition,
Mets et al.[16] supported a role for lipoxidation reactions
in the chemical modification of proteins and
development of complications in diabetic and
prediabetic hyperlipidemic states. They demonstrated
that the protective effects of PM are consistent with its
role in reducing plasma lipids and trapping
intermediates in lipoxidation reactions[16].
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J. Appl. Sci. Res., 6(9): 1316-1324, 2010
Regarding the kidney, Nakamura et al.[23] have
reported that PM can retarded the development of
nephropathy in streptozotocin-diabetic rats and Zucker
(obese, hyperlipidemic) rats. They explained that this
effect is due to the inhibitory action of PM on the
formation of AGEs and ALEs in vivo. They suggested
that the presence of severe hyperlipidemia and the
reno-protective effects of PM in both the diabetic and
the Zucker rats indicate that lipids rather than
carbohydrates might be the primary source of chemical
modification of proteins in diabetes[23]. In the renal
tissue, PM appears to inhibit the principal steps that
lead to chemical modification of proteins by low
molecular weight carbonyl compounds derived from
either sugars or lipids, in addition to its inhibition of
the formation of AGEs derived from Amadori
adducts[24]. These carbonyl compounds, also called
reactive carbonyl species such as methylglyoxal, were
found to be elevated in diabetes. They modify critical
arginine and lysine residues in matrix proteins and
interference with renal cell-matrix interactions.
Pyridoxamine was also found to inhibit the pathogenic
effects of elevated methylglyoxal levels leading to renal
protection[25]. It has to be considered that the use of
pyridoxamine must be carried out cautiously because
excess ingestion of pyridoxine may itself cause
peripheral sensory neuropathy[26].
In conclusion, pyridoxamine is a promising drug
candidate for treatment of diabetic nephropathy,
retinopathy and hyprelipidemia. However, the
mechanism of PM therapeutic action is poorly
understood. Further studies are recommended to
investigate more protective roles, proper safe dosing
and mechanisms of action.
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