Introduction
In the preceding article in this series (1), attention was drawn to the activity of
glutathione reductase, superoxide dismutase and catalase in the
liver, heart, kidney, soleus muscle and visceral adipose tissue of
control female rats exposed from the eighth week after birth and
for the ensuing 8 weeks to a diet containing 64% (w/w) starch and
5% (w/w) sunflower oil (Ssun rats) and female rats exposed over the
same period to diets containing 64% D-fructose and 5% sunflower oil
(Fsun rats) or 3.4% sunflower oil mixed with 1.6% salmon oil (Fsal
rats) or safflower oil (Fsaf rats). The activities of these enzymes
were lower in the Fsun rats than in the Ssun rats; these decreases
were attenuated in the Fsaf and Fsal rats, particularly in the
latter. In the light of such findings, the aim of the present study
was to measure in plasma, liver, heart, kidney, soleus muscle and
visceral adipose tissue the concentrations of thiobarbituric acid
reactive substances (TBARS), carbonyl radicals, hydroperoxides and
nitric oxide.
Materials and methods
The four groups of rats (Ssun, Fsun, Fsal and Fsaf)
examined in the present study were the same as those mentioned in
the preceding article in this series (1).
The measurements of TBARS were performed according
to the methods of Salih et al(2) and Bostoglou et al(3), as adapted by Genot (4). The results are expressed as μmol of
malonaldehyde equivalent (5). The
plasma and tissue carbonyl content was measured according to the
method of Levine et al(6),
the results being expressed as μmol per liter plasma or per mg
tissue protein. The hydroperoxides were measured by the method
proposed by Hermes-Lima et al(7), as modified by Eymard and Genot
(8) and using the FOX 2 reagent
(9). The results are again
expressed as μmol by reference to cumene hydroperoxide standards
(Sigma Aldrich, St. Louis, MO, USA). Finally, nitric oxide was
measured by the method of Cortas and Wakid (10), the results being also expressed as
μmol.
All results are presented as mean values (± SEM)
together with the number of individual observations (n) or degree
of freedom (df). The statistical significance of differences
between mean values was assessed by use of the Student’s t-test.
P<0.05 was considered to indicate a statistically significant
result.
Results
TBARS
The plasma concentration and tissue content of TBARS
were invariably higher in the Fsun rats than in the Ssun rats
(Table I). In the fructose-fed
rats, the partial substitution of sunflower oil by either safflower
or salmon oil opposed such an increase. Thus, relative to the
corresponding reference values found in the plasma, liver, heart,
kidney, soleus muscle and visceral adipose tissue of the Ssun rats
(100.0±5.5%; n=36), the measurements made in Fsun, Fsaf and Fsal
rats averaged, respectively, 312.5±15.7 (n=36), 214.2±13.2 (n=36)
and 177.6±11.7% (n=30), all four mean percentages being
significantly different (P<0.05 or less) from one another. The
results recorded in the Fsal rats represented 84.0±4.6% (n=30;
P<0.02) of those recorded in the Fsaf rats (100.0±4.7%;
n=36).
 | Table IPlasma concentration (μM) and tissue
content (μmol/g protein) of thiobarbituric acid reactive
substances. |
Table I
Plasma concentration (μM) and tissue
content (μmol/g protein) of thiobarbituric acid reactive
substances.
| Ssun (n=6) | Fsun (n=6) | Fsal (n=5) | Fsaf (n=6) |
|---|
| Plasma | 24.2±0.9 | 80.1±0.7 | 45.0±1.1 | 53.5±1.2 |
| Liver | 1.5±0.2 | 5.3±0.7 | 2.9±0.6 | 3.4±0.4 |
| Heart | 1.7±0.3 | 2.7±0.1 | 2.3±0.2 | 2.6±0.2 |
| Kidney | 1.4±0.2 | 4.7±0.4 | 3.2±0.6 | 4.0±0.8 |
| Soleus muscle | 1.5±0.2 | 4.7±0.4 | 2.4±0.4 | 2.8±0.4 |
| Visceral adipose
tissue | 0.3±0.1 | 1.1±0.1 | 0.5±0.1 | 0.6±0.1 |
Carbonyl radicals
A situation comparable to that described for TBARS
prevailed in the case of carbonyl radicals (Table II). For instance, in the plasma,
the mean concentrations of carbonyl radicals displayed a Ssun <
Fsal < Fsaf < Fsun hierarchy. Likewise, in the liver, heart,
kidney, soleus muscle and visceral adipose tissue, the carbonyl
content, expressed relative to the corresponding values found in
Ssun rats (100.0±4.0%; n=30) averaged 143.0±5.6 (n=30), 127.2±5.5
(n=30) and 112.3±4.4% (n=20) in the Fsun, Fsaf and Fsal rats,
respectively. These four mean values were close to being
significantly different from one another (P<0.06 or less) and
became so when the two sets of individual data to be compared were
expressed relative to one another. For instance, in the latter
procedure, the difference between Fsun and Fsaf rats yielded a
P-value <0.025, as distinct from a P-value <0.06 when
comparing the data normalized relative to the Ssun reference
measurements.
| Table IIPlasma concentration (μM) and tissue
content (μmol/mg protein) of carbonyl radicals. |
Table II
Plasma concentration (μM) and tissue
content (μmol/mg protein) of carbonyl radicals.
| Ssun (n=6) | Fsun (n=6) | Fsal (n=5) | Fsaf (n=6) |
|---|
| Plasma | 27.09±2.17 | 37.09±0.69 | 32.50±2.48 | 34.18±1.73 |
| Liver | 3.27±0.07 | 4.16±0.06 | 3.45±0.09 | 3.56±0.06 |
| Heart | 3.13±0.20 | 4.34±0.19 | 3.42±0.29 | 3.55±0.14 |
| Kidney | 3.36±0.40 | 5.46±0.60 | 4.05±0.56 | 5.03±0.70 |
| Soleus muscle | 2.04±0.24 | 3.03±0.29 | 2.45±0.11 | 2.70±0.17 |
| Visceral adipose
tissue | 1.16±0.14 | 1.60±0.17 | 1.23±0.13 | 1.53±0.14 |
Hydroperoxides
The results concerning the hydroperoxide content of
the samples yielded similar information to that found in the TBARS
measurements (Table III). Thus,
in all cases (plasma, liver, heart, kidney, soleus muscle and
visceral adipose tissue) the mean values always followed the same
hierarchy, that is, Ssun < Fsal < Fsaf < Fsun. The close
analogy between the results collected in liver, heart, kidney,
soleus muscle and visceral adipose tissue in terms of the
diet-induced changes of TBARS, carbonyl and hydroperoxide content
is shown in Fig. 1.
 | Figure 1Comparison between Ssun (○), Fsun (●),
Fsaf (△) and Fsal (x) rats in terms of tissue content of
thiobarbituric acid reactive substrates (TBARS), carbonyl radicals,
hydroperoxides and nitric oxide. Tissue content of (A) TBARS, (B)
carbonyl radicals, (C) hydroperoxides and (D) nitric oxide. The
results refer to measurements made in liver, heart, kidney, soleus
muscle and visceral adipose tissue, with the exception of nitric
oxide in which case the measurements made in soleus muscle were
ignored. Mean values (± SEM) are expressed relative to the
corresponding values found in Ssun rats and refer to either 25–30
individual measurements (TBARS, carbonyl radicals and
hydroperoxides) or 20–24 individual measurements (nitric
oxide). |
| Table IIIPlasma concentration (μM) and tissue
content (μmol/mg protein) of hydroperoxides. |
Table III
Plasma concentration (μM) and tissue
content (μmol/mg protein) of hydroperoxides.
| Ssun | Fsun | Fsal | Fsaf |
|---|
| Plasma | 34.77±0.82a | 53.26±2.47a | 41.38±0.56b | 45.54±1.40a |
| Liver | 2.74±0.15a | 5.70±0.71a | 3.25±0.34b | 3.75±0.41a |
| Heart | 1.94±0.38b | 3.56±0.21a | 2.82±0.25b | 3.37±0.36b |
| Kidney | 4.86±0.50a | 8.58±1.06a | 5.36±0.88b | 5.56±0.86a |
| Soleus muscle | 3.90±0.07a | 6.48±0.10a | 4.42±0.17b | 4.55±0.06a |
| Visceral adipose
tissue | 3.52±0.53a | 5.37±0.42a | 3.72±1.14b | 3.99±0.97a |
Nitric oxide
The measurements of nitric oxide yielded results
vastly different from those so far considered, in one major respect
(Table IV). In plasma, the nitric
oxide concentration was twice as low (P<0.001) in the Fsun rats
as that in the Ssun rats. However, in muscle, the nitric oxide
content was higher (P<0.02) in the Fsun rats than in the Ssun
rats. The nitric oxide contents of liver, heart, kidney and
visceral adipose tissue were lower in the Fsun rats than in the
Ssun rats, the former averaging 60.9±2.5% (n=24; P<0.001) of the
mean corresponding reference value in the latter (100.0±3.2%;
n=24). The relative magnitude of such a decrease (39.1±4.0%; df=46)
was not significantly different (P>0.39) from that found in the
plasma of the same rats (46.6±3.2%; df=10).
| Table IVPlasma concentration (μM) and tissue
content (μmol/mg protein) of nitric oxide. |
Table IV
Plasma concentration (μM) and tissue
content (μmol/mg protein) of nitric oxide.
| Rats | Ssun (n=6) | Fsun (n=6) | Fsal (n=5) | Fsaf (n=6) |
|---|
| Plasma | 22.17±0.69 | 11.84±0.16 | 18.37±0.64 | 16.66±0.68 |
| Liver | 3.20±0.37 | 1.99±0.06 | 3.46±0.10 | 2.47±0.19 |
| Heart | 2.98±0.09 | 2.11±0.12 | 3.17±0.10 | 2.27±0.16 |
| Kidney | 2.70±0.05 | 1.67±0.12 | 2.42±0.14 | 2.07±0.06 |
| Soleus muscle | 1.84±0.25 | 3.03±0.34 | 2.61±0.43 | 2.69±0.35 |
| Visceral adipose
tissue | 1.41±0.09 | 0.69±0.07 | 1.27±0.11 | 0.86±0.07 |
In analogy, however, with the results recorded for
the other variables (TBARS, carbonyl and hydroperoxides), the
partial substitution of sunflower oil by either safflower or salmon
oil tended, in the fructose-fed rats, to minimize the difference
otherwise found between Ssun and Fsun rats in terms of the nitric
oxide content of plasma, liver, heart, kidney, soleus muscle and
visceral adipose tissue. Relative to this difference (100.0±5.5%;
n=36), that found in the Fsaf rats only represented 67.4±6.4%
(n=36; P<0.001), whilst in the Fsal rats such a difference was
either further decreased (P<0.02) to 37.0±10.4% (n=20) in
plasma, kidney, soleus muscle and visceral adipose tissue or
inverted in algebraic terms with an increment of 21.7±6.7% (n=10;
P<0.02) instead of a decrement of 100.0±7.0% (n=12) when the
respective differences between either Fsal or Fsun and Ssun rats in
liver and heart were compared. The absolute values of the nitric
oxide content of liver, heart, kidney, soleus muscle and visceral
adipose tissue in the Fsal rats averaged 107.2±6.1% (n=25;
P>0.29) of the corresponding values found in the Ssun rats
(100.0±3.6%; n=30).
Fig. 1 shows the
inverse situation found for the nitric oxide tissue content, on one
hand, and the TBARS, carbonyl or hydroperoxide tissue content, on
the other. For reasons already mentioned, the soleus muscle data
for nitric oxide content were omitted from these computations.
Discussion
The measurements made in plasma were generally in
fair agreement with those recorded in the organs considered in the
present study. The data collected in liver, heart, kidney, soleus
muscle and visceral adipose tissue provided coherent results, with
the sole exception of the nitric oxide content of soleus
muscle.
The main findings of the present study are shown in
Fig. 1. It illustrates the
fructose-induced increases in the tissue content of TBARS, carbonyl
radicals and hydroperoxides and the fructose-induced decrease in
the tissue content of nitric oxide. Furthermore, the
fructose-induced changes were reduced in the Fsaf rats and reduced
even further in the Fsal rats. In the latter, the mean values, on
occasion, did not differ significantly from those found in the Ssun
rats.
The accumulation of TBARS, carbonyl radicals and
hydroperoxides in the liver, heart, kidney, soleus muscle and
visceral adipose tissue of fructose-fed rats coincided with a
decreased activity of the enzymes glutathione reductase, superoxide
dismutase and catalase in the same organs of the same rats
(1). Likewise, the response to the
incorporation of either safflower or salmon oil in the diet in
terms of the tissue content of TBARS, carbonyl radicals and
hydroperoxides coincided with the partial correction of the
fructose-induced changes in the activities of these enzymes.
The potential benefit of the latter dietary
manipulations, for example, in terms of either glucose homeostasis
or prevention of arterial hypertension in human subjects, merit
further consideration, as recently reviewed (11).
Acknowledgements
We are grateful to C. Demesmaeker for secretarial
help.
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