ARTICLE
Auteur(s) : Satoko Akiyama1, Mariko
Uehara1, Shin-ichi Katsumata1, Hiroshi
Ihara2, Naotaka Hashizume3, Kazuharu Suzuki1
1Department of Nutritional Science, Faculty
of Applied Bioscience, Tokyo University of Agriculture,
Tokyo
2Department of Laboratory Medicine, Toho University
Ohashi Medical Center, Tokyo
3Department of Health and Nutrition, Wayo
Women’s University, Chiba, Japan
Several researchers have reported that magnesium (Mg) deficiency
increases the iron (Fe) [1, 2], triglyceride (TG) and total
cholesterol (TC) contents in the liver [3] and decreases the Mg
[4-6] and high-density lipoprotein (HDL)-cholesterol (HDL-C) levels
in serum [3]. We previously found that Mg-deficient rats exhibit
increased levels of phosphatidylcholine hydroperoxide (PCOOH), a
primary product of lipid peroxidation in biological membranes, in
plasma and several tissues [7]. Further, Hus et al. [8] reported
that Mg-deficient rats exhibit reduced concentrations of ascorbic
acid (AsA) in the liver. AsA has been known to play important roles
in many biochemical reactions. Antioxidants and antioxidant enzymes
are the primary elements involved in defence responses that protect
organisms from oxidative damage, and AsA is an important biological
antioxidant [9]. AsA and α-tocopherol synergistically react with
organic free radicals; the antioxidant properties of these
compounds are known to be responsible for their biological
activity. These facts suggest that reduced concentrations of AsA
also influence lipid peroxidation in Mg-deficient rats. In this
study, hypothesizing that Mg deficiency may increase the
requirement of AsA, we investigated the effects of dietary AsA
supplementation on lipid peroxidation and the lipid content in the
liver and serum of Mg-deficient rats.
Materials and methods
Eighteen 3-week-old male Sprague-Dawley rats (Clea Japan, Tokyo,
Japan) were housed individually in stainless-steel cages at 22°C,
under a 12-h light/12-h dark cycle. The Tokyo University of
Agriculture Animal Use Committee approved the study, and the
animals were maintained in accordance with the university
guidelines for the care and use of laboratory animals. All the rats
were fed a control diet, formulated on the basis of the AIN-93G
diet [10], over a 2-d acclimatization period. After this period,
the rats were randomly assigned to 3 experimental groups, each
containing 6 rats, and were maintained on one of the following 3
diets: the control diet (C group); a low-Mg diet containing 0.008%
Mg (D group), and a low-Mg diet supplemented with 0.03% AsA (DA
group). The rats in the C and DA groups were pair-fed their
respective diets with the rats in the D group. All the rats were
provided free access to deionized water. Faeces and urine samples
were collected from all the rats for 3 d prior to the analysis of
Mg. The rats were maintained on the different diets for 42 d,
following which they were sacrificed, and blood and liver samples
were collected for analyses. The blood samples were centrifuged,
and the supernatants were used as serum samples. The liver was
perfused with cold 0.9% NaCl solution and resected.
To measure the Mg levels, the urine samples, micropulverized
faecal samples, and liver samples were dried, ashed, and extracted
in 1 mol/L HCl. The Mg concentration was analysed by performing
atomic absorption spectrophotometry (HITACHI A-2000, Tokyo, Japan)
according to the method described by Gimblet et al. [11].
The serum PCOOH fraction was analysed by using the
chemiluminescence high-performance liquid chromatography (CL-HPLC)
method [7, 12].
The serum AsA was assayed by spectrophotometric methods,
according to the procedures described by Ihara et al. [13].
Liver lipids were extracted using a mixture of chloroform and
methanol (in a volume ratio of 2:1), by the method described by
Folch et al. [14]. The TG and TC concentrations in the liver and
serum were measured using enzymatic colorimetric methods, with the
triglyceride E-test and cholesterol C-test Wako kits (Wako Pure
Chemical Industries, Osaka, Japan).
Each result was expressed as the mean ± SE for each group
comprising 6 rats. One-way analysis of variance (ANOVA) was
performed, followed by Fisher’s protected least square difference
(PLSD) test to determine whether the differences among the groups
were significant. The differences were considered significant at p
< 0.05.
Results
The final body weight and weight gain were significantly lower in
the D and DA groups than in the C group but did not differ between
the former 2 groups (table 1). The
apparent absorption, retention, and serum concentrations of Mg were
significantly lower in the D and DA groups than in the C group
(table 2). The serum AsA levels were
significantly lower in the D group than in the C and DA groups but
did not differ between the latter 2 groups (table 3). The serum PCOOH levels were
significantly elevated under conditions of Mg deficiency but were
suppressed by dietary AsA supplementation (table
3). The TG and TC concentrations in the liver were
significantly higher in the D group than in the C and DA groups,
while those in the serum were significantly higher in the D and DA
groups than in the C group but not differ between the former 2
groups (table 3).
Table 1 Changes of body weight and food intake in
control rats (C), Mg-deficient rats (D) and Mg-deficient rats
supplemented with AsA (DA).
|
Group
|
C
|
D
|
DA
|
|
Initial body weight (g)
|
42.07 ± 1.61
|
43.27 ± 1.22
|
44.45 ± 1.18
|
|
Final body weight (g)
|
268.36 ± 3.03a
|
227.78 ± 3.08b
|
228.02 ± 3.39b
|
|
Weight gain (g)
|
226.29 ± 2.28a
|
184.50 ± 2.15b
|
183.57 ± 2.40b
|
|
Food intake (g/day)
|
12.99 ± 0.29
|
13.01 ± 0.19
|
13.05 ± 0.25
|
Table 2 Changes of Mg balance and serum Mg in control
rats (C), Mg-deficient rats (D) and Mg-deficient rats supplemented
with AsA (DA).
|
Group
|
C
|
D
|
DA
|
|
Apparent Mg absorption (mg/day)
|
6.48 ± 0.22a
|
0.78 ± 0.03b
|
0.82 ± 0.04b
|
|
Mg retention (mg/day)
|
6.22 ± 0.184a
|
0.71 ± 0.04b
|
0.78 ± 0.05b
|
|
Serum Mg (mg/dL)
|
1.82 ± 0.05a
|
0.57 ± 0.04b
|
0.43 ± 0.06b
|
Table 3 Changes of serum AsA, PCOOH, TG and TC and
liver TG and TC in control rats (C) Mg-deficient rats (D) and
Mg-deficient rats supplemented with AsA (DA).
|
Group
|
C
|
D
|
DA
|
|
Serum AsA (mg/dL)
|
1.08 ± 0.09a
|
0.78 ± 0.05b
|
1.04 ± 0.08a
|
|
Serum PCOOH (pmol/mL)
|
40.61 ± 3.74b
|
57.94 ± 3.58a
|
45.68 ± 4.36b
|
|
Serum TG (mg/dL)
|
122.16 ± 10.99b
|
145.66 ± 11.77a
|
150.20 ± 15.14a
|
|
Serum TC (mg/dL)
|
93.25 ± 5.43b
|
104.64 ± 8.86a
|
101.07 ± 4.70a
|
|
Liver TG (mg/g)
|
16.30 ± 1.90b
|
21.91 ± 2.29a
|
17.06 ± 0.93b
|
|
Liver TC (mg/g)
|
2.07 ± 0.99b
|
2.51 ± 0.09a
|
2.00 ± 0.12b
|
Discussion
Mg deficiency reduced the final body weights of the animals in the
present study, consistent with previous studies [5, 6, 8]. Dietary
Mg deficiency is reported to affect the growth of rats via a
reduction in the protein utilization [6]. The apparent Mg
absorption and retention and the serum Mg levels decreased in the
Mg-deficient rats in the present study. These results were
consistent with those of previous studies [5, 6]. However, AsA
supplementation did not affect the absorption, retention, and serum
concentrations of Mg in the Mg-deficient rats.
Hsu et al. [8] reported that Mg-deficient rats exhibit a reduced
capacity to utilize glucuronolactone or gulonolactone for the
synthesis of AsA in the liver. In the present study, we observed
reduced AsA levels in the rat sera. It has been suggested that Mg
deficiency interferes with AsA synthesis and increases the AsA
requirement of the body. We have previously reported that
Mg-deficient rats exhibit elevated serum levels of PCOOH [7].
Similar to the results of our previous study, those obtained in the
present study revealed that the serum PCOOH levels were elevated in
Mg-deficient rats. Glutathione catalyzes the conversion of
dehydro-AsA to AsA, and Mg deficiency affects the metabolism of
glutathione [15]. The reduced serum levels of AsA that are induced
by Mg deficiency may in turn increase the PCOOH levels, in
association with an impairment in the glutathione metabolism;
however, we did not analyse the glutathione levels of the rats in
the present study. Nevertheless, dietary AsA supplementation
restored the serum levels of AsA and suppressed the increase in the
PCOOH concentrations induced by Mg deficiency.
It is well known that Mg deficiency is accompanied by an
inflammatory syndrome characterized by release of inflammatory
cytokines and acute phase proteins [16]. This acute phase response
has been shown to be responsible for oxidative stress and lipid
disturbances induced by Mg deficiency. In addition, Ikeda et al.
reported that the serum concentration of IL6, an inflammatory
cytokine that stimulates gene expression of acute phase proteins,
has been shown to be higher in AsA deficient rats, and AsA
deficiency causes changes similar to those that occur in the acute
phase response [17]. Thus the possibility might exist that a less
severe inflammatory response explains the beneficial effect of AsA
supplementation in Mg deficient rats and that the inflammatory
response in Mg deficient rats participates in AsA deficiency.
Mg deficiency increases the very low-density lipoprotein
(VLDL)-cholesterol and LDL-cholesterol levels but decreases the
HDL-cholesterol levels, because this condition decreases the
activity of lecithin-cholesterol acyltransferase (LCAT) [18].
Moreover, Mg deficiency increases the percentage composition of
triglycerides in VLDL, LDL, and HDL, and reduces that of proteins
[19]. AsA plays an important role in cholesterol metabolism.
Compared to normal guinea pigs, guinea pigs deficient in AsA are
reported to exhibit significantly higher levels of cholesterol in
the serum and liver; this is because cholesterol catabolism is
significantly impaired in AsA-deficient animals, owing to a
reduction in the 7α-hydroxylation of cholesterol [20]. Mg
deficiency may secondarily induce a condition similar to that
observed in AsA deficiency and may impair lipid metabolism. In the
present study, we observed elevated serum concentrations of TG and
TC in the Mg-deficient rats; however, dietary AsA supplementation
did not affect these concentrations. Further, we measured the liver
concentrations of TG and TC: these concentrations were elevated in
the Mg-deficient rats but were normalized with AsA
supplementation.
Although many studies have demonstrated that AsA exerts a
cholesterol-lowering effect, its effects on cholesterol metabolism
remain debated [21]. In a previous study on humans, no significant
changes were observed in the plasma cholesterol or TG levels of
hypercholesterolaemic subjects who had been receiving AsA (4 g/d)
orally for 2 months. In our animal experiment, no significant
changes were observed in the serum TC and TG levels after dietary
AsA supplementation although the liver TG and cholesterol levels
were reduced by AsA supplementation in Mg-deficient rats. This
discrepancy may be attributable to differences in the feeding
conditions, such as the dose of AsA, the dietary regimen and the
age of the subjects.
In this study, we investigated the effects of dietary AsA
supplementation on lipid peroxidation and the lipid content in the
liver and serum of Mg-deficient rats. The elevated serum levels of
PCOOH and TG and TC lipids were lowered by AsA supplementation.
These results indicate that Mg deficiency increases the AsA
requirement of the body and that dietary AsA supplementation can
normalize the serum levels of PCOOH and the liver lipid content in
the liver and serum of Mg-deficient rats, without altering the Mg
status.
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