Clinica Chhnica Acta, 205 (1992) 75-85 © 1992 ElsevierSciencePublishers B.V. All rights reserved 0009-8981/92/$05.00
75
CCA 05204
Selenium and glutathione peroxidase variations induced by polyunsaturated fatty acids oral supplementation in humans Giuseppe Bellisola I, Silvio Galassini 2'4, Giuliano Moschini 3'4, Giovanni Poli I, Giuseppe Perona 5 and Giancesare Guidi I ILaboratory of Clinical Clwmistry, C.O.C Valeggio s. M. (VR). Institute of Clhtical Chemistry, and 2Institute of Biological Chemistry. University of Verona, 3Ph),~ics Department. University of Padua, 41NFN Laboratori Na:ionali di Legnaro ( PD), Padua attd Shlstitute of Medical Pathology. Division of ltaematolog),. Universio, of Verona (Italy)
(Received 21 June 1991; revision received4 November 1991;accepted 8 November 1991) Key wordy: Blood platelets; Erythrocytes;Glutathione peroxidase;Selenium;Serum selenium; Fish oil
Summary Serum and erythrocyte selenium, erythrocyte and platelet glutathione-peroxidase (GSH-Px) activities, and erythrocyte reduced glutathione (GSH) content were measured in 25 healthy adult individuals before and after daily supplementation with 20 ml offish oil for 10 weeks. Serum-Se decreased from 0.83 ± 0.01 #mol/i to 0.75 .4- 0.02 #mol/i (mean ± S.E.M.) (P < 0.01); erythrocyte-Se decreased from 4.39 ± 0.17 nmol/g hemoglobin (Hb) to 2.83 4- 0.15 nmol/g (P < 0.001). GSH-Px activities increased both in erythrocytes (6.93 4- 0.24 iu/g vs 8.18 4- 0.27 iu/g Hb, P < 0.01) and in platelets (69.2 4- 2.8 iu/g vs 90.9 4- 3.6 iu/g protein, P < 0.001). The concentration of GSH in erythrocytes fell from 9.56 4- 0.29 #mol/g Hb to 5.90 4- 0.30 #mol/g Hb (P < 0.001). The effects on plasma iipids were evident only for triglycerides (before 1.96 4- 0.16 mmol/l, after 1.75 4- 0.14 mmol/l, P < 0.001). We hypothesise the enrichment o f erythrocyte and platelet membranes with polyunsaturated fatty acids (PUFAs), following fish oil intake, can generate increased amounts of lipid peroxides and thus allosterically activate GSH-Px: with time this is harmful for the integrity of the enzyme molecule and Se release may result. We suggest that the Se status o f individuals given PUFAs is assessed before and during intake; Se supplements should only be given when serum and/or erythrocyte Se are reduced.
Correspondence to: G.C. Guidi, Clin. Chem. Laboratory, C.O.C., 1-37067Valeggios.M., Italy.
76 Introduction
Some beneficial effects have been observed in individuals taking supplements of polyunsaturated fatty acids (PUFAs). A recent review [1] reports that the principal effects are: diminution of serum lipid content [2], reduction of platelet aggregation [3], increased levels of plasminogen activator and concurrent reduction of inhibitors of plasminogen activator [4], decreased erythrocyte viscosity, reflecting increased cell deformability [5]. In spite of these apparently favourable effects of PUFAs, assumed mainly as fish oil, agreement about its use as a nutritional supplement has not been reached mainly because of lack of knowledge about possible side effects. As fish oil contains highly unsaturated fatty acids, namely omega-3 fatty acids whose double bonds are readily susceptible to autoxidation, there is an increased generation of peroxyl radicals during fish oil supplementation [6,7] and their reduction (both enzymatically or non-enzymatically) to PUFA hydroperoxides is dependent on the efficiency of hydrogen donors. Therefore the capacity of cell anti-6xidant systems is crucial in this regard, mainly in connection with the role played by some known vitamins [8,9], enzymes [10-12] and trace elements [13]. To evaluate the influence of oral supplementation with PUFAs on some antioxidant mechanisms, we have measured serum and erythrocyte selenium contents, platelet and erythrocyte glutathione peroxidase enzyme activities (GSH-Px; E.C. 1. I 1.1.9) and erythrocyte reduced glutathione (GSH) concentration before and after supplementation with fish oil. The results are reported here. Materials and Methods
Individuals attd PUF.4s reghnen Twenty-five adult healthy individuals (16 males and 9 females; aged 30-54, median 48) were invited to add to thcir usual diet a tablespoon (about 20 ml) of fish oil every day for 10 weeks. Informed consent was obtained from all the individuals. A commercial fish oil preparation (Chimifarm-Verona, Italy) from the same stock, containing 9.9% eicosapentaenoic acid and 13.1% docosahexaenoic acid was employed as the source of PUFAs: thus each individual was taking about 4.6 g of omega-3 fatty acids daily.
Biological spechnens Blood samples were collected in the morning after an overnight fast; 9-ml portions of the blood were anti-coagulated with 1 ml trisodium citrate (Carlo Erba-Milan, Italy) 0.113 mol/i and platelet rich plasmas (PRPs) were Separated from erythrocytes (RBC) by centrifugation at 250 × g for 10 min at room temperature as previously described [!4]. Serum samples (approx. 4 ml) were obtained by centrifugation of clotted whole blood at 1800 × g for 10 rain and portions were stored at -20°C in polypropylene tubes (previously checked for contaminant Se), for Se determination within 2 months.
77
Serum seleniun~ ( serwn-Se ) A l-ml quantity of each serum sample was pre-concentrated on a 3.0-#m pore size, 25-ram diameter filter (Millipore-Bedford, U.S.A.) after organic matrix digestion, using tellurium dioxide (Merck-Darmstadt, Germany) as co-precipitant and as an internal standard [15]. Materials and chemicals employed in this step had been previously checked for contaminant Se. Se was measured by proton induced X energy emission (P.I.X.E.) with an AN 2000 van de Graaff accelerator (INFN-Legnaro, Italy). Limits of detection were 0.025 #moi/.l and linearity was maintained as low as 0.063 #mol/l.
Seleniunl in erythrocytes A l-ml quantity of packed erythrocytes (RBC), previously washed with 0.15 mol/i NaCI solution, was subjected to the same procedure as described for serum. Se was expressed as nmol/g Hb.
Glutathione peroxidase activity ht erythrocytes (RBC-GSH-Px) Enzyme activities were measured as described [16] in 50 #1 of the lysate from R BC and in a final volume of 2.5 ml using tert-butyl-hydroperoxide (Merck-Darmstad, Germany) as starter (final 0.2 mmol/l) at 25°C and were expressed as iu/g Hb.
Ghttathione peroxidase activity & platelets (PLT-GSII-Px) After centrifugation of 3-4 ml of PRP at 1,500 x g supernatant plasma was tak.~n off. Sedimented platelets (PLT) were resuspended and washed twice by centrifugation in cold 5-mmol/l Tris HCl-buffered 0.15 mol/I NaCI (Carlo Erba-Milan, Italy) solution (pH 7.4), containing 1 mmol/l ¢-aminocaproic acid (¢-ACA) (Sigma-St. Louis, U.S.A.) and 1 mmol/I Na2EDTA (Carlo Erba-Milan, Italy); washed and centrifuged PLT were then lysed for 1 h at + 4°C with 0.2-0.3 ml of cold digitoninsaturated medium containing 10 mmoi/I e-ACA and 2 mmol/i Na2EDTA in 50 mmol/l Tris-HCl buffer (pH 7.0) [14]; after centrifugation at 10,000 x g for 5 rain at + 4°C, PLT-GSH-Px activities were measured in 25 #1 of the supernatant PLT lysates using the previ0usl), described procedure and were expressed as iu/g pretein; proteins were measured using the method of Lowry et al. [17].
Ghttathione content of erythrocytes ( RBC-GSH) A 1.8-ml quantity of each 1/10 RBC lysate (with water), was treated with 3 ml precipitating solution (0.21 mol/i glacial metaphosphoric acid, 6 retool/1 Na2EDTA, 8.2 mol/l HCI; Carlo Erba-Milan, Italy). After filtration, i ml ofeach eluate was added to a cuvette containing 4"ml of 0.3 mol/i (pH 8.9), Na2HPO 4 (Carlo Erba-Milan, Italy) and 0.25 ml of 0.5 mmol/l di-thiobis-nitrobenzoic acid (Sigma-St. Louis, U.S.A.). Readings were made at 412 nm against a blank as described [18], and expressed as #mol/g Hb.
78
Statistical analysis The homogeneity of variances was chec!:ed for all the variables and a significafice level well above 5% was obtained for each one. The significance of tile differences between means observed before and after the experiment were caiculatedusing the two-tailed Student t test for paired samples. Correlation coefficients were obtained by linear regression. Results During our study toxic reactions or side effects were not observed and throughout the biochemical tests of li~/er, kidney and hematologic function were normal.
Selenium The mean value of serum Se before supplementation was 0.83.4- 0.01 t~mol/l. This was significantly higher than the mean value observed after supplementation (0.75 ± 0.02 /xmol/l; P < 0.01). Also Se in erythrocytes before fish oil showed a TABLE ! Mean values and S.E.M. ofSe. GSH-Px activity. GSH and serum lipids before and after 10 weeks offish oil supplementation Before mean .4- S.E.M. Serum-Se umol/I (min.-max.) RBCs-Se nmol/g lib (min.-max.) RBCs-GSH-Px iu/g Hb (min.-max.) RBCs-GSH p.mol/g Hb (min.-max.) PLTs-GSH-Px iu/g prot. (min.-max.) Cholesterol mmol/I (min.-max.) Triglycerides mmol/l (min.-max.) HDL chol. retool/1 (min.-max.)
0.83
0.01
After mean .4- S.E.M. 0.75
P
0.02 < 0.01
0.66-0.98 4.39 0.17
0.59-0.94 2.83 0.15
1.98-4.72 6.93 0.24
I. 12-3.38 8.18 0.27
4.90-9.50 9.56 0.29
5.30-10.40 5.9~ 0.30
5.90-12.40 69.15 2.75
3.40-9. I0 90.90 3.57
52.40 - 100.40 5.98 0.19
59. IO- 124.60. 5.90 0.19
4.28-7.99 1.96 0.16
4.12-7.84 1.75 0.14
1.01-4.57 1.15 0.05
0.88-4.19 1.14 0.10
0.77-1.86
0.69- i .70
< 0.001
< 0.01
< 0.001
< 0.001
n.s.
< 0.001
n.s.
79 significantly higher mean value (4.39 ± 0.17 nmol/g Hb) than that measured after supplementation (2.83 ± 0.15 nmol/g Hb; P < 0.001) (Table I). Moreover a positive correlation was observed between serum-Se and RBC-Se (r = 0.60; P < 0.01) only before treatment (Fig. IA); after treatment r = 0.04, P = n.s. (Fig. IB).
Glutathione-peroxidase The mean RBC-GSH-Px activity was 6.9 ± 0.2 iu/g Hb before oil supplementation and significantly increased after treatment (8.2 4- 0.3 iu/g Hb; P < 0.01); the mean basal GSH-Px of platelets increased concomitantly from 69.2 .4- 2,Siu/g protein up to 90.9 ± 3.6 iu/g protein at the end of supplementation (P < 0.001) (Table I). RBC-GSH-Px activities showed positive correlation both with serum-Se (r = 0.51, P < 0.01) and with RBC-Se (r = 0.61, P < 0.01) (Fig. IC, Fig. 2A) only
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Fig. 1. Linearregressionplots of erythrocytevs serumseleniumconce,trations(top) and of crythrocyte GSH-Px activities vs serum seleniumconcentrations(bottom) before (A. C) and after (B, D) fish oil supplement.
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Fig. 2. Linear regression plots of erythrocyte GSH-Px activities vs selenium concentrations (top) and of platelet GSH-Px activities vs serum selenium concentrations (bo'ttom) before (A, C) and after (B. D) fish oil supplement. before PUFAs; after, r = 0.22, P = n.s. and r = 0.32, P = n.s., respectively (Fig. ID, Fig. 2B), On the other hand P L T - G S H - P x activities displayed positive correlation with serum-Se both before (r = 0.72; P < 0.001) and after (r = 0.46; P < 0.05) treatment (Fig. 2C,D).
Erythrocyte ghaathione Erythrocyte G S H showed a mean value o f 9.56 ±' 0.29 t~mol/g H b before oil supplement, but fell down to a mean value o f 5.90 -,- 0.30 #mol/g Hb (P < 0.001) at the end o f treatment.
Plasma triglycerides and choh,sterol After fish oil a significant decrease o f the mean concentration o f triglycerides in
81 plasma was observed in respect of the basal mean value (before: 1.96 ± 0.16 mmol/l; after: 1.75 ~ 0.14 mmol/i; P < 0.001); on the contrary the mean values of cholesterol and HDL cholesterol remained unchanged throughout the experiment (Table I). Discussion
Our results support the hypothesis of an involvement of some anti-oxidant systems during oral supplementation with PUFAs. A relationship between atocopherol and PUFAs content in pho,~pholipids is reported [19], but little is at present known about the role played in this context by other radical scavengers such as Se, GSH-Px and GSH. Selenium
Basal serum-Se content in our individuals showed a mean value close to that observed in a previous study (about 0.8 #moi/l) where it doubled after supplementation with selenite [20]. Others reported values significantly higher than ours in normal individuals living in different countries [21]. Serum-Se is considered a good indicator of body Se status, since it reflects alimentary intake [22]; the wide variability of serum-Se observed in people from different geographical areas is explained by different Se intakes [23]. The basal serum-Se levels shown by our individuals is in support of a relatively low alimentary availability as already described for some Italian sub-populations [24,25]; nevertheless neither Italy can be considered a country poor in rutritional Se, nor clinical or epider,niological reports are known in this regard. Following PUFAs a significant reduction of mean serum-Se concentration was observed and some individual data approached the lowest values measured in individuals living in countries known to be poor in Se [26]. Such an observation suggests that PUFAs intake could imbalance Se ,aatus, mostly when the nutritional Se availability is already inadequate. Moreover while serum-Se represents a short term indicator of Se status, RBC-Se is a long term indicator [27]. Positive correlation between serum-Se and RBC-Se can reasonably be expected in steady-state nutritional conditions, as actually we have observed before PUFAs. On the contrary the reduction of RBC-Se and the lack of correlation with serum-Se after PUFAs further support an impairment of Se status. Besides, as Se represents the prosthetic group of GSH-Px [28], a decrease of GSH-Px activity ought to have been the consequence of RBC-Se reduction. GSH-Px activities
After PUPAs, GSH-Px activity increased in both erythrocytes and platelets. As regards erythrocytes, this fact seemed paradoxical because: (a) RBC-GSH-Px activity is usually reduced when RBC-Se is low [29] and on the contrary when RBC-Se is high [30]; (b) erythrocytes do not synthesise proteins and it seems that GSH-Px molecule is unable to incorporate Se after translation [311; (c) actually we found lowered RBC-Se concentration after PUFAs. To explain the increased RBC-GSH-Px activity after PUFAs, the enzymatic ac-
82 tivation through the allosteric mechanism must be taken into account [32]. Erythrocytes are particularly susceptible to augmenting the degree of unsaturation of membrane lipids following a diet rich in fish oil [5]. The resulting tendency of the membrane to autoxidize and consequently tO generate lipid hydroperoxides [6,7] is counteracted by GSH-Px, the main erythrocyte enzyme involved in protecting cell membrane from peroxidative damages [33]. Cells can better face the peroxide injury either by synthesising new enzyme molecules at the erythroblaste stage, a measure of delayed efficacy [34] and requiring additional Se availability, or through allosteric activation for immediate needs [32]. In short term the latter mechanism is efficient for immediate reduction of lipid hydroperoxides; nevertheless in the long term, by exposing the active enzyme site to increased oxidation, it can be harmful for the molecule function and integrity. In fact, upon oxidation with substrate peroxides, the Se moiety shuttles between its reduced selenol acid form and either selenenic or the more oxidized seleninic acid form, depending on the concentration of peroxides [35]. In physiological conditions, provided that the donor hydrogen substrate GSH is adequately concentrated, selenenic acid represents the main reversible oxidation form of the Se moiety [36]. When either peroxide formation is raised or GSH is decreased or both, seleninic acid becomes the plevailing oxidation form of Se [35] and Se may eventually be released from the molecule [37,38]. Acttially the RBC of our subjects showed a fall in GSH COntent, which is most likely to be connected with the enhanced enzyme rate. These fads support the hypothesis that the reduction of RBC-Se after PUFAs may be a cons'equence of the release of Se from both enzyme molecules and erythroeytes. As regards PLT, an increased GSH-Px activity was previously described following Se as well as fish oil supplementation, even though at different rates, the former rate being higher [20,39]. Platelets are non-nucleated cells containing some mRNA, thus we cannot completely ignore the possibility that the increased enzyme activity may result from the de novo translation of mRNA coding for GSH-Px. But we report reduction of serum-Se after PUFAs, and a direct relationship has already been documented between the depression of serum-Se due to reduced intake and low PLT-GSH-Px activities [30,40] in individuals not supplemented with PUFAs. This makes the translation hypothesis less likely. On the other hand the different fatty acid composition of the membrane after PUFAs in platelets can generate lipid hydroperoxides [7] effective as activators, and a trend for PLT-GSH-Px to respond allosterically, like the erythrocyte enzyme, can be suggested. Nevertheless since we did not measure platelet Se nor GSH, our data are in this regard less conclusive than for erythrocytes. Others [38,41] have reported increased GSH-Px activities both in RBC and PLT following PUFAs administration and a reduced mean serum-Se concentration was described in psoriatic patients both before and after PUFAs [42], thus suggesting some involvement of Se in this condition and further emphasizing the occurrence of a peroxide-induced activation of GSH-Px. The reduction of serum triglycerides concentration after fish oil can be regarded as a beneficial outcome of supplementation, as observed by others [43]. The effects we have observed in the course of the experiment support the hypothesis that PUFAs administration, at least as fish oil, may impair some key mechanism of defence against peroxidative damage in blood cells. We conclude that similar ef-
83
fects could occur in other body cells or districts as reported by others for t~tocopherol [19,44]; moreover the observed reduction of serum-Se should be regarded as an undesired side effect, mainly in view of some described relationships between low serum-Se and increased risk of pathological events [45-49]. We suggest that the Se status of individuals administered with PUFAs is assessed before and during long term intake regimens. Moreover Se supplements should be provided when reduced serum and/or RBC-Se are observed following PUFAs intake.
Acknowledgements This work was supported in part by a grant from Regione Veneto.
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43 44 45 46 47
48 49
by platelel..glutathione peroxidase activity and other blood parameters. Am J Clin Nutr 1983;37:887-897. Olivieri O, Negri M, De Gironcoli M, el. al. Effects ofdietary fish oil on malondialdehyde production and glutathione peroxidase activity in hypcrlipidacmic patients. Scand J Clin Lab Invest 1988;48:659-665. Corrochcr R, FerrarJ S, De Gironcoli M, et al. Effect offish oil supplementation on erythrocyle lipid pattern, malondialdchyde production and iglutathionc-peroxidaso activity in psoriasis. Clin Chim Acta 1989,179:121-132. Harris WS. Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J Lipid Res 1989;30:785-807. Chautan M, Calaf R, L~onardi Jet al. Inverse modifications of heart and liver (x-tocopherol status by various dietary •-6/n-3 polyunsaturated fatty acid ratios. J Lipid Res 1990;31:2201-2208. Schrauzer GN. So, cancer and New Zealand. Am J Clin Nutr 1980:33:1982-1984. Wang YX, Bocher K, Router H, et al. Selenium and myocardial infarction: glutathione peroxidase in platelets. Klin Wochenschr 1981:59:817-818. Salonen TJ, Alfthan G, Huttunen JC, Pikkaraincn J, Puska P. Association between cardiovascular deatll and myocardial infarction and serum-Se in a matched-paired lo]~gitudinal study. Lancet 1982;ii:175-179. Moore JA, Noiva R, Wells [C. Selenium concentration in plasma ofpatients with arteriographically defined coronary atherosclerosis. Clin Chem 1984;30:117l-I 173. Jackson ML. So: geochemical distribution and association with human heart and cancer death rat~s and longevity in China and the United States. Biol Trace Elem Res 1988:15:13-21.
75
CCA 05204
Selenium and glutathione peroxidase variations induced by polyunsaturated fatty acids oral supplementation in humans Giuseppe Bellisola I, Silvio Galassini 2'4, Giuliano Moschini 3'4, Giovanni Poli I, Giuseppe Perona 5 and Giancesare Guidi I ILaboratory of Clinical Clwmistry, C.O.C Valeggio s. M. (VR). Institute of Clhtical Chemistry, and 2Institute of Biological Chemistry. University of Verona, 3Ph),~ics Department. University of Padua, 41NFN Laboratori Na:ionali di Legnaro ( PD), Padua attd Shlstitute of Medical Pathology. Division of ltaematolog),. Universio, of Verona (Italy)
(Received 21 June 1991; revision received4 November 1991;accepted 8 November 1991) Key wordy: Blood platelets; Erythrocytes;Glutathione peroxidase;Selenium;Serum selenium; Fish oil
Summary Serum and erythrocyte selenium, erythrocyte and platelet glutathione-peroxidase (GSH-Px) activities, and erythrocyte reduced glutathione (GSH) content were measured in 25 healthy adult individuals before and after daily supplementation with 20 ml offish oil for 10 weeks. Serum-Se decreased from 0.83 ± 0.01 #mol/i to 0.75 .4- 0.02 #mol/i (mean ± S.E.M.) (P < 0.01); erythrocyte-Se decreased from 4.39 ± 0.17 nmol/g hemoglobin (Hb) to 2.83 4- 0.15 nmol/g (P < 0.001). GSH-Px activities increased both in erythrocytes (6.93 4- 0.24 iu/g vs 8.18 4- 0.27 iu/g Hb, P < 0.01) and in platelets (69.2 4- 2.8 iu/g vs 90.9 4- 3.6 iu/g protein, P < 0.001). The concentration of GSH in erythrocytes fell from 9.56 4- 0.29 #mol/g Hb to 5.90 4- 0.30 #mol/g Hb (P < 0.001). The effects on plasma iipids were evident only for triglycerides (before 1.96 4- 0.16 mmol/l, after 1.75 4- 0.14 mmol/l, P < 0.001). We hypothesise the enrichment o f erythrocyte and platelet membranes with polyunsaturated fatty acids (PUFAs), following fish oil intake, can generate increased amounts of lipid peroxides and thus allosterically activate GSH-Px: with time this is harmful for the integrity of the enzyme molecule and Se release may result. We suggest that the Se status o f individuals given PUFAs is assessed before and during intake; Se supplements should only be given when serum and/or erythrocyte Se are reduced.
Correspondence to: G.C. Guidi, Clin. Chem. Laboratory, C.O.C., 1-37067Valeggios.M., Italy.
76 Introduction
Some beneficial effects have been observed in individuals taking supplements of polyunsaturated fatty acids (PUFAs). A recent review [1] reports that the principal effects are: diminution of serum lipid content [2], reduction of platelet aggregation [3], increased levels of plasminogen activator and concurrent reduction of inhibitors of plasminogen activator [4], decreased erythrocyte viscosity, reflecting increased cell deformability [5]. In spite of these apparently favourable effects of PUFAs, assumed mainly as fish oil, agreement about its use as a nutritional supplement has not been reached mainly because of lack of knowledge about possible side effects. As fish oil contains highly unsaturated fatty acids, namely omega-3 fatty acids whose double bonds are readily susceptible to autoxidation, there is an increased generation of peroxyl radicals during fish oil supplementation [6,7] and their reduction (both enzymatically or non-enzymatically) to PUFA hydroperoxides is dependent on the efficiency of hydrogen donors. Therefore the capacity of cell anti-6xidant systems is crucial in this regard, mainly in connection with the role played by some known vitamins [8,9], enzymes [10-12] and trace elements [13]. To evaluate the influence of oral supplementation with PUFAs on some antioxidant mechanisms, we have measured serum and erythrocyte selenium contents, platelet and erythrocyte glutathione peroxidase enzyme activities (GSH-Px; E.C. 1. I 1.1.9) and erythrocyte reduced glutathione (GSH) concentration before and after supplementation with fish oil. The results are reported here. Materials and Methods
Individuals attd PUF.4s reghnen Twenty-five adult healthy individuals (16 males and 9 females; aged 30-54, median 48) were invited to add to thcir usual diet a tablespoon (about 20 ml) of fish oil every day for 10 weeks. Informed consent was obtained from all the individuals. A commercial fish oil preparation (Chimifarm-Verona, Italy) from the same stock, containing 9.9% eicosapentaenoic acid and 13.1% docosahexaenoic acid was employed as the source of PUFAs: thus each individual was taking about 4.6 g of omega-3 fatty acids daily.
Biological spechnens Blood samples were collected in the morning after an overnight fast; 9-ml portions of the blood were anti-coagulated with 1 ml trisodium citrate (Carlo Erba-Milan, Italy) 0.113 mol/i and platelet rich plasmas (PRPs) were Separated from erythrocytes (RBC) by centrifugation at 250 × g for 10 min at room temperature as previously described [!4]. Serum samples (approx. 4 ml) were obtained by centrifugation of clotted whole blood at 1800 × g for 10 rain and portions were stored at -20°C in polypropylene tubes (previously checked for contaminant Se), for Se determination within 2 months.
77
Serum seleniun~ ( serwn-Se ) A l-ml quantity of each serum sample was pre-concentrated on a 3.0-#m pore size, 25-ram diameter filter (Millipore-Bedford, U.S.A.) after organic matrix digestion, using tellurium dioxide (Merck-Darmstadt, Germany) as co-precipitant and as an internal standard [15]. Materials and chemicals employed in this step had been previously checked for contaminant Se. Se was measured by proton induced X energy emission (P.I.X.E.) with an AN 2000 van de Graaff accelerator (INFN-Legnaro, Italy). Limits of detection were 0.025 #moi/.l and linearity was maintained as low as 0.063 #mol/l.
Seleniunl in erythrocytes A l-ml quantity of packed erythrocytes (RBC), previously washed with 0.15 mol/i NaCI solution, was subjected to the same procedure as described for serum. Se was expressed as nmol/g Hb.
Glutathione peroxidase activity ht erythrocytes (RBC-GSH-Px) Enzyme activities were measured as described [16] in 50 #1 of the lysate from R BC and in a final volume of 2.5 ml using tert-butyl-hydroperoxide (Merck-Darmstad, Germany) as starter (final 0.2 mmol/l) at 25°C and were expressed as iu/g Hb.
Ghttathione peroxidase activity & platelets (PLT-GSII-Px) After centrifugation of 3-4 ml of PRP at 1,500 x g supernatant plasma was tak.~n off. Sedimented platelets (PLT) were resuspended and washed twice by centrifugation in cold 5-mmol/l Tris HCl-buffered 0.15 mol/I NaCI (Carlo Erba-Milan, Italy) solution (pH 7.4), containing 1 mmol/l ¢-aminocaproic acid (¢-ACA) (Sigma-St. Louis, U.S.A.) and 1 mmol/I Na2EDTA (Carlo Erba-Milan, Italy); washed and centrifuged PLT were then lysed for 1 h at + 4°C with 0.2-0.3 ml of cold digitoninsaturated medium containing 10 mmoi/I e-ACA and 2 mmol/i Na2EDTA in 50 mmol/l Tris-HCl buffer (pH 7.0) [14]; after centrifugation at 10,000 x g for 5 rain at + 4°C, PLT-GSH-Px activities were measured in 25 #1 of the supernatant PLT lysates using the previ0usl), described procedure and were expressed as iu/g pretein; proteins were measured using the method of Lowry et al. [17].
Ghttathione content of erythrocytes ( RBC-GSH) A 1.8-ml quantity of each 1/10 RBC lysate (with water), was treated with 3 ml precipitating solution (0.21 mol/i glacial metaphosphoric acid, 6 retool/1 Na2EDTA, 8.2 mol/l HCI; Carlo Erba-Milan, Italy). After filtration, i ml ofeach eluate was added to a cuvette containing 4"ml of 0.3 mol/i (pH 8.9), Na2HPO 4 (Carlo Erba-Milan, Italy) and 0.25 ml of 0.5 mmol/l di-thiobis-nitrobenzoic acid (Sigma-St. Louis, U.S.A.). Readings were made at 412 nm against a blank as described [18], and expressed as #mol/g Hb.
78
Statistical analysis The homogeneity of variances was chec!:ed for all the variables and a significafice level well above 5% was obtained for each one. The significance of tile differences between means observed before and after the experiment were caiculatedusing the two-tailed Student t test for paired samples. Correlation coefficients were obtained by linear regression. Results During our study toxic reactions or side effects were not observed and throughout the biochemical tests of li~/er, kidney and hematologic function were normal.
Selenium The mean value of serum Se before supplementation was 0.83.4- 0.01 t~mol/l. This was significantly higher than the mean value observed after supplementation (0.75 ± 0.02 /xmol/l; P < 0.01). Also Se in erythrocytes before fish oil showed a TABLE ! Mean values and S.E.M. ofSe. GSH-Px activity. GSH and serum lipids before and after 10 weeks offish oil supplementation Before mean .4- S.E.M. Serum-Se umol/I (min.-max.) RBCs-Se nmol/g lib (min.-max.) RBCs-GSH-Px iu/g Hb (min.-max.) RBCs-GSH p.mol/g Hb (min.-max.) PLTs-GSH-Px iu/g prot. (min.-max.) Cholesterol mmol/I (min.-max.) Triglycerides mmol/l (min.-max.) HDL chol. retool/1 (min.-max.)
0.83
0.01
After mean .4- S.E.M. 0.75
P
0.02 < 0.01
0.66-0.98 4.39 0.17
0.59-0.94 2.83 0.15
1.98-4.72 6.93 0.24
I. 12-3.38 8.18 0.27
4.90-9.50 9.56 0.29
5.30-10.40 5.9~ 0.30
5.90-12.40 69.15 2.75
3.40-9. I0 90.90 3.57
52.40 - 100.40 5.98 0.19
59. IO- 124.60. 5.90 0.19
4.28-7.99 1.96 0.16
4.12-7.84 1.75 0.14
1.01-4.57 1.15 0.05
0.88-4.19 1.14 0.10
0.77-1.86
0.69- i .70
< 0.001
< 0.01
< 0.001
< 0.001
n.s.
< 0.001
n.s.
79 significantly higher mean value (4.39 ± 0.17 nmol/g Hb) than that measured after supplementation (2.83 ± 0.15 nmol/g Hb; P < 0.001) (Table I). Moreover a positive correlation was observed between serum-Se and RBC-Se (r = 0.60; P < 0.01) only before treatment (Fig. IA); after treatment r = 0.04, P = n.s. (Fig. IB).
Glutathione-peroxidase The mean RBC-GSH-Px activity was 6.9 ± 0.2 iu/g Hb before oil supplementation and significantly increased after treatment (8.2 4- 0.3 iu/g Hb; P < 0.01); the mean basal GSH-Px of platelets increased concomitantly from 69.2 .4- 2,Siu/g protein up to 90.9 ± 3.6 iu/g protein at the end of supplementation (P < 0.001) (Table I). RBC-GSH-Px activities showed positive correlation both with serum-Se (r = 0.51, P < 0.01) and with RBC-Se (r = 0.61, P < 0.01) (Fig. IC, Fig. 2A) only
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Fig. 1. Linearregressionplots of erythrocytevs serumseleniumconce,trations(top) and of crythrocyte GSH-Px activities vs serum seleniumconcentrations(bottom) before (A. C) and after (B, D) fish oil supplement.
80
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Fig. 2. Linear regression plots of erythrocyte GSH-Px activities vs selenium concentrations (top) and of platelet GSH-Px activities vs serum selenium concentrations (bo'ttom) before (A, C) and after (B. D) fish oil supplement. before PUFAs; after, r = 0.22, P = n.s. and r = 0.32, P = n.s., respectively (Fig. ID, Fig. 2B), On the other hand P L T - G S H - P x activities displayed positive correlation with serum-Se both before (r = 0.72; P < 0.001) and after (r = 0.46; P < 0.05) treatment (Fig. 2C,D).
Erythrocyte ghaathione Erythrocyte G S H showed a mean value o f 9.56 ±' 0.29 t~mol/g H b before oil supplement, but fell down to a mean value o f 5.90 -,- 0.30 #mol/g Hb (P < 0.001) at the end o f treatment.
Plasma triglycerides and choh,sterol After fish oil a significant decrease o f the mean concentration o f triglycerides in
81 plasma was observed in respect of the basal mean value (before: 1.96 ± 0.16 mmol/l; after: 1.75 ~ 0.14 mmol/i; P < 0.001); on the contrary the mean values of cholesterol and HDL cholesterol remained unchanged throughout the experiment (Table I). Discussion
Our results support the hypothesis of an involvement of some anti-oxidant systems during oral supplementation with PUFAs. A relationship between atocopherol and PUFAs content in pho,~pholipids is reported [19], but little is at present known about the role played in this context by other radical scavengers such as Se, GSH-Px and GSH. Selenium
Basal serum-Se content in our individuals showed a mean value close to that observed in a previous study (about 0.8 #moi/l) where it doubled after supplementation with selenite [20]. Others reported values significantly higher than ours in normal individuals living in different countries [21]. Serum-Se is considered a good indicator of body Se status, since it reflects alimentary intake [22]; the wide variability of serum-Se observed in people from different geographical areas is explained by different Se intakes [23]. The basal serum-Se levels shown by our individuals is in support of a relatively low alimentary availability as already described for some Italian sub-populations [24,25]; nevertheless neither Italy can be considered a country poor in rutritional Se, nor clinical or epider,niological reports are known in this regard. Following PUFAs a significant reduction of mean serum-Se concentration was observed and some individual data approached the lowest values measured in individuals living in countries known to be poor in Se [26]. Such an observation suggests that PUFAs intake could imbalance Se ,aatus, mostly when the nutritional Se availability is already inadequate. Moreover while serum-Se represents a short term indicator of Se status, RBC-Se is a long term indicator [27]. Positive correlation between serum-Se and RBC-Se can reasonably be expected in steady-state nutritional conditions, as actually we have observed before PUFAs. On the contrary the reduction of RBC-Se and the lack of correlation with serum-Se after PUFAs further support an impairment of Se status. Besides, as Se represents the prosthetic group of GSH-Px [28], a decrease of GSH-Px activity ought to have been the consequence of RBC-Se reduction. GSH-Px activities
After PUPAs, GSH-Px activity increased in both erythrocytes and platelets. As regards erythrocytes, this fact seemed paradoxical because: (a) RBC-GSH-Px activity is usually reduced when RBC-Se is low [29] and on the contrary when RBC-Se is high [30]; (b) erythrocytes do not synthesise proteins and it seems that GSH-Px molecule is unable to incorporate Se after translation [311; (c) actually we found lowered RBC-Se concentration after PUFAs. To explain the increased RBC-GSH-Px activity after PUFAs, the enzymatic ac-
82 tivation through the allosteric mechanism must be taken into account [32]. Erythrocytes are particularly susceptible to augmenting the degree of unsaturation of membrane lipids following a diet rich in fish oil [5]. The resulting tendency of the membrane to autoxidize and consequently tO generate lipid hydroperoxides [6,7] is counteracted by GSH-Px, the main erythrocyte enzyme involved in protecting cell membrane from peroxidative damages [33]. Cells can better face the peroxide injury either by synthesising new enzyme molecules at the erythroblaste stage, a measure of delayed efficacy [34] and requiring additional Se availability, or through allosteric activation for immediate needs [32]. In short term the latter mechanism is efficient for immediate reduction of lipid hydroperoxides; nevertheless in the long term, by exposing the active enzyme site to increased oxidation, it can be harmful for the molecule function and integrity. In fact, upon oxidation with substrate peroxides, the Se moiety shuttles between its reduced selenol acid form and either selenenic or the more oxidized seleninic acid form, depending on the concentration of peroxides [35]. In physiological conditions, provided that the donor hydrogen substrate GSH is adequately concentrated, selenenic acid represents the main reversible oxidation form of the Se moiety [36]. When either peroxide formation is raised or GSH is decreased or both, seleninic acid becomes the plevailing oxidation form of Se [35] and Se may eventually be released from the molecule [37,38]. Acttially the RBC of our subjects showed a fall in GSH COntent, which is most likely to be connected with the enhanced enzyme rate. These fads support the hypothesis that the reduction of RBC-Se after PUFAs may be a cons'equence of the release of Se from both enzyme molecules and erythroeytes. As regards PLT, an increased GSH-Px activity was previously described following Se as well as fish oil supplementation, even though at different rates, the former rate being higher [20,39]. Platelets are non-nucleated cells containing some mRNA, thus we cannot completely ignore the possibility that the increased enzyme activity may result from the de novo translation of mRNA coding for GSH-Px. But we report reduction of serum-Se after PUFAs, and a direct relationship has already been documented between the depression of serum-Se due to reduced intake and low PLT-GSH-Px activities [30,40] in individuals not supplemented with PUFAs. This makes the translation hypothesis less likely. On the other hand the different fatty acid composition of the membrane after PUFAs in platelets can generate lipid hydroperoxides [7] effective as activators, and a trend for PLT-GSH-Px to respond allosterically, like the erythrocyte enzyme, can be suggested. Nevertheless since we did not measure platelet Se nor GSH, our data are in this regard less conclusive than for erythrocytes. Others [38,41] have reported increased GSH-Px activities both in RBC and PLT following PUFAs administration and a reduced mean serum-Se concentration was described in psoriatic patients both before and after PUFAs [42], thus suggesting some involvement of Se in this condition and further emphasizing the occurrence of a peroxide-induced activation of GSH-Px. The reduction of serum triglycerides concentration after fish oil can be regarded as a beneficial outcome of supplementation, as observed by others [43]. The effects we have observed in the course of the experiment support the hypothesis that PUFAs administration, at least as fish oil, may impair some key mechanism of defence against peroxidative damage in blood cells. We conclude that similar ef-
83
fects could occur in other body cells or districts as reported by others for t~tocopherol [19,44]; moreover the observed reduction of serum-Se should be regarded as an undesired side effect, mainly in view of some described relationships between low serum-Se and increased risk of pathological events [45-49]. We suggest that the Se status of individuals administered with PUFAs is assessed before and during long term intake regimens. Moreover Se supplements should be provided when reduced serum and/or RBC-Se are observed following PUFAs intake.
Acknowledgements This work was supported in part by a grant from Regione Veneto.
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9 10 11 12
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