Influence of riboflavin supplementation on liver trace element concentration in trained rats fed low-protein diet

Gralak Probl Hig MA Epidemiol i wsp. Influence 2013, 94(3): of riboflavin 527-531 supplementation on liver trace element concentration in trained rats fed... 527 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/ 1/ Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Biological Sciences, Warsaw, Poland 2/ Department of Hygiene and Physiology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland 3/ Department of Endocrinology, Medical University in Lodz, Poland 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 B 2 ), 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 niskobiałkowa 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. Słowa kluczowe: ryboflawina, aktywność fizyczna, niedobór białka, szczur Probl Hig Epidemiol 2013, 94(3): 527-531 www.phie.pl Nadesłano: 19.06.2013 Zakwalifikowano do druku: 13.07.2013 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 the metabolism of trace elements. Aim. The objective was to study the effect of riboflavin (vitamin B 2 ) content, training and protein deficiency on the trace elements concentration in rat liver. Material & methods. The experiment (90 days) was performed on male Wistar rats divided into six groups fed ad libitum with 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 energy from protein and the other four groups were fed the diet containing only 4.5% of energy from protein. Two of the four protein-deficient diets were supplemented with riboflavin in the amount of 75 mg/kg diet as fed. 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) the liver concentrations of iron and lower (p 0.05) of the chromium content were observed. The low-protein diet increased (p 0.05) the liver Cu content in all rats. Conclusions. Riboflavin and the zinc metabolism are correlated. Physical activity increased the iron concentrations and decreased the 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 differences between the studies. Key words: riboflavin, physical activity, protein deficiency, rats 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

528 Probl Hig Epidemiol 2013, 94(3): 527-531 Introduction Riboflavin has been well known for almost 100 years, when it was synthesized. It is absorbed primarily in the proximal small intestine but absorption also occurs in the large intestine. In the mammalian 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 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 of 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 B 12 is also dependent on FAD. Riboflavin has the capacity to form complexes, and the riboflavin supplementation may result in an increased absorption of zinc and iron, thus increasing 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 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 [3-5]. Because exercise stresses metabolic pathways that depend on thiamine, riboflavin, and vitamin B 6, 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 B 6. 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 B 6 status [6, 7]. Aim The objective was to study the effect of riboflavin (vitamin B 2 ) content, protein deficiency and training on the mineral concentration in rat liver. Material and methods The experiment was performed on 41 growing male Wistar rats kept in individual plastic cages in a conditioned room at 24 C and 12-hour light period. The animals were randomly divided into six groups and fed ad libitum with semi-purified diets (Table I). Two diets contained 14.7 MJ gross energy per 1 kg (350 kcal/100 g) and 20% of energy from protein. The 15% of energy were derived from fats including 2% from the essential polyunsaturated fatty acids. The rats of the other four groups were fed an isocaloric diet (14.7 MJ/kg) but with only 4.5% of energy derived from protein (Table I). Two of the four proteinrestricted diets were supplemented with riboflavin (Polfa, Kraków) in the amount of 75 mg/kg diet as fed. It was the 25-fold higher concentration than in the groups without supplementation. Distilled water was freely available to all rats during the experiment. The rats of three groups were trained for one hour daily: one was fed the diet with 20% protein content, the second was fed the diet with 4.5% protein content without the riboflavin addition and the other supplemented with riboflavin. The rats were subjected to the treadmill 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. The liver samples (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 Ethos 900 microwave oven (Milestone). The mineral elements were estimated by the flame (air-acetylene) atomic absorption Table I. Composition of the diets Tabela I. Skład dawek pokarmowych Energy from protein (%) /Energia z białka 20 4.5 Component /Składnik g/kg kcal g/kg kcal Sunflower oil /Olej słonecznikowy 3.6 32.4 5.0 45.0 Lard /Smalec 54.9 492.6 53.6 480.2 Casein /Kazeina 189.7 607.0 45.6 145.9 Egg powder /Proszek jajeczny 16.1 93.3 2.0 11.6 Wheat flour /Mąka pszenna 194.3 676.1 194.3 676.1 Wheat starch /Skrobia pszenna 300 1200 300 1200 Potato starch /Skrobia ziemniaczana 91.4 11.36 Sugar /Cukier 100 399 235.9 941.2 Mineral mix* /Premiks mineralny 40 40 Vitamins** /Premiks witaminowy 10 10 * 1000 g mineral mixture/premiks mineralny: KHPO 4 322.0g, CaCO 3 300.0g, NaCl 167.0g, MgSO 4 102.0g, CaHPO 4 75.0g, FeC 6 P 5 O 7 27.5g, MnSO 4 5.1g, KJ-0.8g, CuSO 4 0.3g, ZnCl 2 0.25g, CoCl 2 0.05g. ** 1000 g vitamin mixture/premiks witaminowy: Vit. D 3 545000 IU, Vit. K 1.0g, Vit. B 12 30μg, Choline chloride 10.0g, Folic acid 1.01g, Biotin 0.03g, Inositol 10.0g, PABA 10.0g, Vit. A 1250000 IU, Vit. B 6 1.5g, Vit. E 2.5g, Vit. B 1 5.0g, Vit. C 25g, Vit. PP 5.0g, Vit. B 2 2.5g, Calcium panthotenate 25.0g.

Gralak MA i wsp. Influence of riboflavin supplementation on liver trace element concentration in trained rats fed... 529 spectrophotometry (Perkin-Elmer 1100B) using hollow cathode lamps with deuterium background correction (except 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 the post-hoc test of 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 principles of animal care were followed. Results and discussion The daily feed intake was similar in all groups. Generally the dietary supplementation of riboflavin (25-fold as compared to un-supplemented groups) did not affect the mineral content in 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 II). The effect was different to the previous study when we did not observe any significant effect of 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 III) 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 I). 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 of 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]. A 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. 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 liver cumulated copper released from other tissues in order to limit the losses of this trace element from the body. Surprisingly, the low-protein diets did not cause an increase of the iron and manganese concentrations 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 the lower magnesium, zinc and copper concentrations 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, but not iron. The present study did not confirm that conclusion. In Table II. Concentration of minerals in the rat liver (x±sd) Tabela II. Stężenie składników mineralnych w wątrobach szczurów (x±sd) Minerals in Dietary protein /Białko w diecie liver (mg/kg) 4.5% energy from protein /4,5% energii /Składniki 20% energy from protein /20% energii z białka Riboflavin suplemented z białka mineralne 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) 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 182±9 175±21 187±26 195±31 166±14 161±19 Cu 4.20±0.16 4.07±0.32 4.49±0.22 4.37±0.12 4.43±0.23 4.37 ±0.14 Mn 0.37±0.09 ab 0.32±0.06 b 0.33±0.06 b 0.40±0.10 ab 0.48±0.08 a 0.35±0.12 ab Cr 4.23±0.40 3.83±0.34 4.34±0.28 4.11±0.21 4.33±0.22 4.34±0.29 a,b Means not sharing the same letter differ at P 0.05 (post-hoc test F Ryan-Einot-Gabriel-Welch) a,b Średnie oznaczone tą samą literą nie różnią się istotnie na poziomie P 0.05

530 Probl Hig Epidemiol 2013, 94(3): 527-531 Table III. Statistical evaluation of experimental factors impact on 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 P = 0.003 NS NS Cu NS NS P < 0.001 Mn NS NS NS Cr NS P = 0.016 NS NS not significant at P 0,05; NS brak istotności na poziomie P 0.05 trained rats as compared to non-trained ones, a higher (p 0.05) liver concentration of iron (35.7±8.3; n=13 v. 30.3±6.3 mg/kg; n=16) was observed. The liver concentration of other trace elements was not significantly affected by the exercise, except for chromium. We stated 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 the exercise-induced hemolysis and partially changes the hematological profile, although without causing 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 the higher iron concentration in the liver of trained rats in our study. However, Liu et al. [18] stated a 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 recirculation from hepatic macrophages [19], which also could 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). Conclusion Riboflavin and the zinc metabolism are correlated. Physical activity increased the iron concentrations and decreased the chromium concentrations in liver, which can be correlated with the increased demands of oxygen and glucose transportation. It is confirmed that protein-deficient diets increase the copper content in liver. Piśmiennictwo / References 1. Agte VV, Paknikar KM, Chiplonkar SA. Effect of riboflavin supplementation on zinc and iron absorption and growth performance in mice. Biol Trace Elem Res 1998, 65(2): 109-115. 2. Chen H, Kimura M, Itokawa Y. Changes in iron, calcium, magnesium, copper, and zinc levels in different tissues of riboflavin-deficient rats. Biol Trace Elem Res 1997, 56(3): 311-319. 3. Gralak MA et al. Effect of restricted feed intake and concentration of iron and manganese in rats. [in:] Metal Ions in Biology and Medicine 8. Cser MA, Sziklai Laszlo I, Etienne J-C, Maymard J, Centeno J, Khassanova L, Collery Ph (eds), John Libbey Eurotext, Paris 2004: 299-302. 4. Gralak MA, et al. Effect of restricted feed intake and concentration of zinc and copper in rats. Biol Trace Elem Res 2004, 98 (1): 85-94. 5. Gralak MA, et al. Effect of restricted feed intake and concentration of calcium and magnesium in rats. Trace Elements and Electrolytes 2004, 21: 89-94. 6. Manore MM. Effect of physical activity on thiamine, riboflavin, and vitamin B-6 requirements. Am J Clin Nutr 2000, 72(2 Suppl): 598S-606S. 7. Woolf K, Manore MM. B-vitamins and exercise: does exercise alter requirements? Int J Sport Nutr Exerc Metab 2006, 16(5): 453-484. 8. Fairweather-Tait S, et al. Riboflavin deficiency and iron absorption in adult Gambian men. Ann Nutr Metab 1992, 36: 34-40. 9. Powers HJ, et al. The effect of riboflavin deficiency in rats on the absorption and distribution of iron. Br J Nutr 1988, 59: 381-387. 10. Powers HJ, et al. Riboflavin deficiency in rat: effects on iron utilization and loss. Br J Nutr 1991, 65: 487-496. 11. Butler BF, Topham RW. Comparison of changes in the uptake and mucosal processing of iron in riboflavin-deficient rats. Biochem Mol Biol Int 1993, 30: 53-61. 12. Adelkan DA, Thurnham DI. The influence of riboflavin deficiency on absorption and liver storage of iron in the growing rat. Br J Nutr 1986, 56: 171-179.

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