Influence of riboflavin supplementation on liver trace element

GralakHig
Probl
MA,
Epidemiol
et al. Influence
2013, 94(4):
of riboflavin
839-842
supplementation on liver trace element concentration in trained rats ...
839
Influence of riboflavin supplementation on liver trace
element concentration in trained rats fed low-protein diet
Wpływ dodatku ryboflawiny na stężenie pierwiastków śladowych w wątrobie trenowanych
szczurów karmionych dietą niskobiałkową
Mikołaj A. Gralak 1/, Bogdan Dębski 1/, Aneta Lewicka 2/, Jerzy Bertrandt 2/, Anna Kłos 2/,
Agnieszka W. Piastowska-Ciesielska 3/, Anna B. Stryczek 1/, Agata Morka 1/
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Biological Sciences, Warsaw, Poland
Department of Hygiene and Physiology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland
3/
Department of Endocrinology, Medical University in Łodź, Poland
1/
2/
Wprowadzenie. Wysiłek wydaje się obniżać stan odżywienia u osób
trenujących, szczególnie w przypadku marginalnych niedoborów
lub minimalnych depozytów składników odżywczych w organizmie.
Współczesne badania sugerują, że wysiłek może zwiększać zapotrzebowanie
na ryboflawinę i ma wpływ na metabolizm pierwiastków śladowych.
Cel pracy. Zbadanie wpływy dodatku ryboflawiny (witaminy B2), wysiłku
i zawartości białka w diecie na stężenie pierwiastków śladowych w wątrobie
szczurów.
Materiał i metody. Doświadczenie (90 dni) przeprowadzono na samcach
Wistar podzielonych na sześć grup. Szczury były karmione ad libitum
dawkami półsyntetycznymi. Obie dawki pokarmowe zawierały 14,7 MJ
energii brutto/kg (350 kcal/100 g). Dwie grupy otrzymywały dietę
zawierającą 20% energii z białka a pozostałe cztery grupy dietę zawierającą
tylko 4,5% energii z białka. Dwie z grup niedoborowych w białko
otrzymywały dodatkowo 75 mg ryboflawiny/kg dawki. Szczury z trzech grup
były poddawane wysiłkowi przez godzinę dziennie (bieżnia).
Wyniki. Stężenie cynku w wątrobie było istotnie niższe u zwierząt
suplementowanych ryboflawiną niż u zwierząt nie suplementowanych.
U szczurów poddanych treningowi w porównaniu do nie trenowanych
stwierdzono istotnie wyższe stężenie żelaza i istotnie niższe stężenie chromu
w wątrobie. Dieta nisko-białkowa spowodowała istotny wzrost stężenia
miedzi w wątrobie wszystkich szczurów.
Wnioski. Ryboflawina i metabolizm cynku są ze sobą powiązane. Wysiłek
zwiększa stężenie żelaza i zmniejsza stężenie chromu w wątrobie. Dawka
pokarmowa niedoborowa w białko zwiększa stężenie miedzi w wątrobie.
Potrzebne są dalsze badania, które pozwolą wyjaśnić różnice między
doświadczeniami.
Introduction. Exercise appears to decrease nutrient status in active
individuals with preexisting marginal vitamin intakes or marginal body
stores. Current research suggests that exercise may increase the requirements
for riboflavin and influence metabolism of trace elements.
Aim. The objective was to study the effect of riboflavin (vitamin B2)
content, training and protein deficiency on trace element concentration
in rat liver.
Material & methods. The experiment (90 days) was performed on male
Wistar rats divided into six groups and fed ad libitum semi-purified diets.
All diets contained 14.7 MJ gross energy/kg (350 kcal/100 g). Two groups
were fed the diet containing 20% of the energy from protein and another
four groups were fed diet containing only 4.5% of the energy from protein.
Two of four protein-deficient diets were supplemented with riboflavin in
amount of 75 mg/kg diet as fed. The rats of three groups were trained
for one hour daily.
Results. The liver Zn concentration was significantly lower in animals
supplemented with riboflavin than in rats fed the diet with normal riboflavin
content. In trained rats as compared to non-trained ones, higher (p≤0.05)
liver concentration of iron and lower (p≤0.05) of chromium content were
observed. The low-protein diet increased (p≤0.05) the liver Cu content
in all rats.
Conclusions. Riboflavin and zinc metabolism are related. Physical activity
increased iron concentrations and decreased chromium concentrations in
liver. It is confirmed that protein-deficient diets increase the liver copper
content in all rats. Further studies are necessary to explain the differences
between studies.
Key words: riboflavin, physical activity, protein deficiency, rats
Słowa kluczowe: ryboflawina, aktywność fizyczna, niedobór białka, szczur
© Probl Hig Epidemiol 2013, 94(4): 839-842
www.phie.pl
Nadesłano: 26.07.2013
Zakwalifikowano do druku: 25.11.2013
Introduction
Riboflavin has been well known for almost 100
years, when it was synthesized. It is absorbed primarily in the proximal small intestine but the absorption
also occurs in the large intestine. In the mammalian
Adres do korespondencji / Address for correspondence
Prof. dr hab. Mikołaj A. Gralak
Katedra Nauk Fizjologicznych, Wydział Medycyny Weterynaryjnej SGGW
ul. Nowoursynowska 166, 02-787 Warszawa, Poland
tel. +48 22 593 62 45, e-mail: [email protected]
organism, the active forms of riboflavin are synthesized
in the mitochondria: flavin mononucleotide (FMN)
which can be further converted to flavin adenine dinucleotide (FAD). Both contribute to cellular growth
mainly through energy production. They are prostetic
840
groups of flavin enzymes bearing proton in intermediary metabolism of amino acids and fatty acids. Several
vitamins are dependent on ribofavin for synthesis and
homeostasis. The conversion of folate to its active form
5-methyl tetrahydrofolate (5-MTHF), is dependent on
FAD. The activation of pyridoxine to pyridoxal 5’-phosphate is dependent on FMN. Moreover the microbial
synthesis of vitamin B12 is also dependent on FAD.
Riboflavin has the capacity to form complexes,
and supplementation with riboflavin may result in an
increased absorption of zinc and iron, thus increasing the cellular transport. Therefore, riboflavin may
have direct as well as indirect effects on growth [1].
The iron concentration in the heart, liver, and spleen
was decreased in the riboflavin-deficient group as
compared with the control group. The calcium and
magnesium concentrations in tibia were also decreased
in the riboflavin-deficient group. However, the copper
concentration was increased in the heart and liver and
the zinc concentration in tibia was also increased [2].
Our earlier studies have shown that vitamins B can
partially prevent changes in mineral element metabolism in rats restricted in feed intake [3, 4, 5].
Because exercise stresses metabolic pathways that
depend on thiamine, riboflavin, and vitamin B6, the
requirements for these vitamins may be increased in
athletes and active individuals. Exercise appears to decrease nutrient status even further in active individuals
with preexisting marginal vitamin intakes or marginal
body stores. Current research suggests that exercise may
increase the requirements for riboflavin and vitamin B6.
Active individuals who have poor diets, especially those
restricting energy intakes or eliminating food groups
from the diet are at greatest risk for poor thiamine,
riboflavin, and vitamin B6 status [6, 7].
Aim
The objective was to study the effect of riboflavin
(vitamin B2) content, protein deficiency and training
on mineral concentration in rat liver.
Material and methods
The experiment was performed on 41 growing
male Wistar rats kept in individual plastic cages in
conditioned room at 24°C and 12-hour light period.
The animals were randomly divided into six groups
and fed ad libitum semipurified diets (Table 1). Two
diets contained 14.7 MJ gross energy per 1 kg (350
kcal/100 g) and 20% of energy originated from protein. 15% of energy was derived from fats including
2% from the essential polyunsaturated fatty acids. The
rats of the other four groups were fed the isocaloric diet
(14.7 MJ/kg) but with only 4.5% of the energy from
protein (Table 1). Two of the four protein-restricted
diets were supplemented with riboflavin (Polfa,
Kraków) in the amount of 75 mg/kg diet as fed. It
Probl Hig Epidemiol 2013, 94(4): 839-842
Table I. Composition of the diets
Tabela I. Skład dawek pokarmowych
Energy from protein (%)
/Energia z białka
Component /Składnik
20
g/kg
Sunflower oil /Olej słonecznikowy
Lard /Smalec
Casein /Kazeina
Egg powder /Proszek jajeczny
Wheat flour /Mąka pszenna
Wheat starch /Skrobia pszenna
Potato starch /Skrobia ziemniaczana
Sugar /Cukier
Mineral mix* /Premiks mineralny
Vitamins** /Premiks witaminowy
4.5
kcal
g/kg
kcal
3.6
32.4
5.0
45.0
54.9
189.7
16.1
194.3
300
91.4
100
40
10
492.6
607.0
93.3
676.1
1200
–
399
–
–
53.6
45.6
2.0
194.3
300
11.36
235.9
40
10
480.2
145.9
11.6
676.1
1200
–
941.2
–
–
* 1000 g mineral mixture/premiks mineralny: KHPO4 – 322.0 g, CaCO3
– 300.0 g, NaCl – 167.0 g, MgSO4 – 102.0 g, CaHPO4 – 75.0 g, FeC6P5O7
– 27.5 g, MnSO4 – 5.1g, KJ – 0.8 g, CuSO4 – 0.3 g, ZnCl2 – 0.25 g, CoCl2
– 0.05 g
** 1000 g vitamin mixture/premiks witaminowy: Vit. D3 – 545000 IU, Vit.
K – 1.0 g, Vit. B12 – 30 μg, Choline chloride – 10.0 g, Folic acid – 1.01 g,
Biotin – 0.03 g, Inositol 10.0 g, PABA – 10.0 g, Vit. A – 1250000 IU, Vit. B6
– 1.5 g, Vit. E – 2.5 g, Vit. B1 – 5.0 g, Vit. C – 25 g, Vit. PP – 5.0 g, Vit. B2
– 2.5 g, Calcium panthotenate – 25.0 g
was the 25-fold higher concentration than in groups
without the supplementation. Distilled water was
freely available to all rats during the experiment. The
rats of three groups were trained for one hour daily:
one fed a diet with 20% protein content, the second
fed a diet with 4.5% protein content without addition
of riboflavin and the other the diet supplemented
with riboflavin. The rats were subjected to tread-mill
exercise 5 days a week. The speed of running track was
20 m/min.
The experiment lasted 90 days and then the
animals were killed in ethyl ether narcosis by cervical
displacement. The livers were removed and kept until
analysis at -18°C. Samples of the livers (0.5 g) were
mineralized in the mixture of 5 ml HNO3 (Merck
1.00441) and 1 ml H2O2 (Merck 107298) in hermetic
high-pressure vessels in microwave oven Ethos 900
(Milestone). Mineral elements were estimated by the
flame (air-acetylene) atomic absorption spectrophotometry (Perkin-Elmer 1100B) using hollow cathode
lamps with deuterium background correction (except
for the copper analysis). The external standards were
prepared on the base of Titrisol Standards (Merck) of
the particular elements.
For the statistical evaluation a one-way (group)
and a multi-way analysis of variance (addition of riboflavin “dietary protein” exercise activity) were applied.
For the evaluation of significance among the groups
post-hoc test F Ryan-Einot-Gabriel-Welsch was used
(p≤0.05) (SPSS 12.0 pl).
The experiment had an approval of the Local
Commission of Animal Welfare and the principles of
animal care were followed.
Gralak MA, et al. Influence of riboflavin supplementation on liver trace element concentration in trained rats ...
Results and discussion
The daily feed intake was similar in all groups.
Generally dietary supplementation of riboflavin
(25-fold as compared to unsupplemented groups)
did not affect mineral content in the rat liver, except
for the zinc level. The Hepatic Zn concentration was
significantly lower (15%) in animals supplemented
with riboflavin (163±16 mg/kg; n=12) than in rats
fed the diet (4.5% GE from protein) with the normal
riboflavin content (192 ±28 mg/kg; n=14) (Table 2).
The effect was different to the previous study when we
had not observed any significant effect of the riboflavin supplementation in the diets containing 20% GE
from protein. However in that study the feed intake
was restricted to 50% and 30% of the consumption
registered in the un-supplemented group fed ad libitum
[4]. The supplementation of vitamin B2 may result in
an increased absorption of zinc because it can form
complexes with metals [1]. So it is possible that zinc
had been bound with riboflavin and stored in other
tissues than liver (e.g. in bones). However, the negative
effect of the riboflavin overdose cannot be excluded.
The supplementation of riboflavin significantly increased (Table 3) the liver manganese concentration in
rats fed the low-protein diet, from 0.33±0.06 (n=6)
to 0.48±0.08 mg/kg (n=5) (Table 1). The significant
interactions between riboflavin, protein level and
exercise do not allow for a harmonized conclusion.
Based on the available literature one might expect
a higher iron concentration in the liver of rats supplemented with riboflavin. It was shown that riboflavin had a significant influence on iron utilization
in riboflavin-deficient men [8] and rats [9, 10]. The
riboflavin deficiency impairs iron absorption [10]
resulting mainly from a reduced uptake of iron into
enterocytes [11]. Smaller percentage of the absorbed
59 Fe dose was present in the livers of riboflavin-deficient animals [9]. It was probably related to the lower
liver concentration of ferritin, iron binding protein,
stated in the riboflavin-deficient rats [12]. As in our
study no diets were deficient in riboflavin it can be
concluded that an overdose of dietary riboflavin did
not improve iron absorption/utilization.
841
The low-protein diet increased (p≤0.05) the liver
Cu content from 4.15±0.22 (n=15) to 4.42±0.17
mg/kg (n=14). This change confirms our earlier studies with rats fed a diet containing 4.5% GE from protein [4]. Then we observed even a greater increase of
the copper concentration (x±SEM) from 3.63±0.31
to 6.35±0.59 mg/kg. Probably the liver accumulated
copper released from other tissues in order to limit the
its losses from the body. Surprisingly, the low-protein
diets did not cause an increase in the concentration of
iron and manganese which was observed in previous
studies [3]. There was no literature available which
could help to explain these differences.
Exercise appears to increase the plasma level of
free radicals. This phenomenon is accompanied by
a lower magnesium, zinc and copper concentration
in plasma and an increase of magnesium, iron, copper and selenium in red blood cells [13]. It has been
also stated that after the exercise the excretion of zinc,
copper and chromium increases in urine [14]. In the
former study in rats [15], we concluded that training
(one-hour running) increased the liver accumulation
of magnesium, zinc and copper, and not iron. The
present study did not confirm that conclusion. In
trained rats as compared to non-trained ones, higher
(p≤0.05) liver concentrations of iron (35.7±8.3;
n=13 v. 30.3±6.3 mg/kg; n=16) were observed. The
liver concentration of other trace elements was not
significantly affected by the exercise, except for chromium. We noted that the liver chromium content was
decreased (p ≤ 0.05) in trained animals (4.00±0.29;
n=13 v. 4.27±0.35 mg/kg; n=16). The lower chromium level was probably related to its high urinary
excretion in trained individuals [14].
High-intensity exercise leads to exercise-induced
hemolysis and partially changes the hematological
profile, although not causing the iron deficiency or
iron-deficiency anemia even in the presence of low
iron intake [16]. The serum iron levels might significantly increase (1.3 times) immediately after running
[17]. The iron released from red blood cells might be
captured by hepatocytes, and this could explain higher
iron concentrations in livers of trained rats in our
Table II. Concentration of minerals in the rat liver (x±SD)
Tabela II. Stężenie składników mineralnych w wątrobach szczurów (x±SD)
Dietary protein /Białko w diecie
Minerals in liver
(mg/kg)
20% energy from protein
4.5% energy from protein
/Składniki mineral/20% energii z białka
/4,5% energii z białka
ne w wątrobie
Sedentary rats (n=10) Exercised rats (n=6) Sedentary rats (n=8) Exercised rats (n=6) Sedentary rats (n=6) Exercised rats (n=5)
a,b
a,b
Fe
30.5±6.4
40.9±11.6
30.8±3.6
32.4±3.3
30.3±3.3
34.5±4.3
Zn
Cu
Mn
Cr
182±9
4.20±0.16
0.37±0.09ab
4.23±0.40
175±21
4.07±0.32
0.32±0.06b
3.83±0.34
187±26
4.49±0.22
0.33±0.06b
4.34±0.28
195±31
4.37±0.12
0.40±0.10ab
4.11±0.21
166±14
4.43±0.23
0.48±0.08a
4.33±0.22
161±19
4.37±0.14
0.35±0.12ab
4.34±0.29
– Means not sharing the same letter differ at p≤0.05 (post-hoc test F Ryan-Einot-Gabriel-Welch)
– Średnie oznaczone tą samą literą nie różnią się istotnie na poziomie p≤0,05
842
Probl Hig Epidemiol 2013, 94(4): 839-842
Table III. Statistical evaluation of experimental factors influencing concentration of minerals in the liver of rats
Tabela III. Ocena statystyczna wpływu czynników doświadczalnych na
stężenie minerałów w wątrobie szczurów
Minerals in liver
/Składniki mineralne w wątrobie
Experimental factors /Warunki doświadczenia
Riboflavin
/Ryboflawina
Exercise
/Wysiłek
Protein
/Białko
Fe
NS
p=0.024
NS
Zn
Cu
Mn
Cr
p=0.003
NS
NS
NS
NS
NS
NS
p=0.016
NS
p<0.001
NS
NS
recirculation from hepatic macrophages [19], which
could also increase the hepatic iron concentration. The
synthesis of hepcidin is up-regulated by pro-inflammatory cytokines Il-1β and Il-6 [19]. In women, acute
exercise increased the hepcidin production, which was
preceded by a significant increase in IL-6 immediately
post-exercise and followed by a significant decrease in
the serum iron level nine hours post-exercise [20].
Nevertheless, additional studies are needed to determine the changes in iron metabolism after the exercise
of different characteristics (time/endurance).
NS – not significant at p≤0.05; NS – brak istotności na poziomie p≤0,05
Conclusion
study. However, Liu et al. [18] stated lower expression
of divalent metal transporter 1 (DMT1), ferroportin1
(FPN1), and heme-carrier protein 1 (HCP1) of duodenum epithelium in strenuously exercised rats. The
authors also indicated that inflammation induced by
strenuous exercise increased the transcriptional level
of the hepatic hepcidin gene. Hepcidin inhibits both
the iron absorption from the small intestine and its
Riboflavin and zinc metabolism are related.
Physical activity increased the iron concentrations
and decreased the chromium concentrations in liver,
which can be correlated with increased demands of
oxygen and glucose transportation. It is confirmed
that protein-deficient diets increase the also copper
content in liver.
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