The increase in TAG after fat intake is defined as postprandial hyperlipidaemia. Clinical studies have shown that increased postprandial concentrations of TAG are associated with coronary and arterial disease(Reference Karpe, Steiner, Uffelman, Olivecrona and Hamsten1, Reference Patsch, Miesenbock, Hopferwieser, Muhlberger, Knapp, Dunn, Gotto and Patsch2) and with insulin resistance(Reference Cardona and Tinahones3). Studies in healthy volunteers have shown that postprandial hyperlipidaemia causes a marked increase in oxidative stress and a worsening in endothelial function(Reference Bae, Bassenge, Kim, Kim, Kim, Lee, Park and Schwemmer4). Postprandial lipoproteins, especially chylomicrons and VLDL, appear to generate oxygen radicals on the endothelial surface that react with nitric oxide and reduce its availability(Reference De Koning and Rabelink5).
Our group has previously detected a close association between TAG and uric acid concentrations. Hypertriacylglycerolaemia due to increased levels of VLDL is associated with hyperuricaemia with reduced renal excretion of urates(Reference Tinahones, Collantes, C-Soriguer, Gonzalez-Ruiz, Pineda, Anon and Sánchez Guijo6, Reference Tinahones, Perez-Lindon, C-Soriguer, Pareja, Sanchez-Guijo and Collantes7) and the connection between hyperuricaemia and hypertriacylglycerolaemia has a genetic association(Reference Cardona, Tinahones, Collantes, Escudero, Garcia-Fuentes and Soriguer8, Reference Cardona, Tinahones, Collantes, Escudero, Garcia-Fuentes and Soriguer9).
Many questions remain concerning hyperuricaemia. Current debate is centred on whether high concentrations of uric acid represent a marker of cardiovascular risk or a cardiovascular risk factor itself. This debate is fuelled by the paradoxical effect of purine metabolism on oxidative stress; on one hand the action of xanthine oxidase on xanthine produces uric acid and superoxide radicals belonging to the reactive oxygen species(Reference Berry and Hare10), and on the other, uric acid is an abundant aqueous antioxidant that accounts for almost two-thirds of all free-radical scavenging activity in human serum(Reference Maxwell, Thomason, Sandler, Leguen, Baxter, Thorpe, Jones and Barnett11). Allantoin, formed from the non-enzymatic oxidation of urate, is a marker of the antioxidative effect of uric acid(Reference Benzie, Chung and Tomlinson12).
In short, we know that a high-fat meal is accompanied by an increase in oxidative stress but we do not know the clinical importance of the concentration of uric acid or whether its antioxidative ability plays any part in the oxidative damage resulting from a high-fat meal. Accordingly, the main aim of the present study was to determine the association between uric acid and the increase in oxidative stress following a high-fat meal in healthy persons.
Material and methods
We selected twenty-five healthy persons who were accompanying patients on primary care visits, thirteen men and twelve women. These persons, who all had a normal oral glucose tolerance test and had experienced no change in weight over the previous 3 months, consumed a 60 g high-fat meal with a commercial liquid preparation (Supracal®; SHS International, Liverpool, UK). Only water was permitted during the process, and no physical exercise was undertaken. The commercial preparation of 125 ml contains 60 g fat, of which 12 g are saturated, 35·35 g are monounsaturated, and 12·75 g are polyunsaturated. Each 100 ml contains < 1 g lauric acid, < 1 g myristic acid, 4·8 g palmitic acid, 1·4 stearic acid, 27·7 g oleic acid, 9·6 g linoleic acid, 1·4 behenic acid, and 0·5 g lignoceric acid.
Blood samples were obtained from the antecubital vein and placed in chilled vacutainer tubes (BD vacutainer™, London, UK). The serum was immediately separated by centrifugation for 30 min at 956 rpm and frozen at − 80°C until analysis. The study protocol was approved by the local Bioethics Committee. All subjects gave informed consent to the study, which was approved by the Ethics Committee of Virgen de la Victoria Clinical University Hospital.
Measurements were made in all subjects of age (in years), weight, height (to calculate the BMI, calculated as the weight (kg) divided by the height squared (m2)), and waist and hip circumferences (to calculate the waist:hip ratio, calculated as the waist circumference divided by the hip circumference)(Reference Lohmann, Roche and Martorell13). Measurements and corresponding calculations were made in serum samples of glucose, total protein, uric acid, cholesterol, TAG, HDL-cholesterol, apoAI, apoB, creatinine, C-reactive protein, as well as 24 h uric acid urinary excretion. Additionally, at 3 h, measurements were made of cholesterol, TAG, apoB, uric acid, and pro-oxidant and antioxidant variables. The measurements were made with a Dimension autoanalyser from Dade Behring Inc. (Deerfield, IL, USA). Additionally, insulin and dehydroepiandrosterone sulphate levels were quantified by RIA provided by BioSource S.A. (Nivelles, Belgium) and Beckman Coulter Inc. (Fullerton, CA, USA), respectively.
Lipid peroxidation products (malondialdehyde+4-hydroxyalkenals) and glutathione reductase (GSH-Rd) activity were estimated using reagents purchased from Oxis International (Portland, OR, USA), i.e. LPO-586 and GR-340 kits, respectively. Additionally, glutathione-S-transferase activity was evaluated using reagents purchased from BioVision Inc. (Mountain View, CA, USA), i.e. GST Colorimetric Activity Assay kit.
The protein carbonyl content was evaluated using the method of Levine et al. (Reference Levine, Garland, Oliver, Amici, Climent, Lenz, Ahn, Shaltiel and Stadtman14). Total nitrites (nitrites and nitrates; NOx) was used as a marker of nitric oxide (NO) levels and assayed following the Griess method(Reference Ricart-Jané, Llobera and López-Tejero15) in serum. Glutathione peroxidase (GSH-Px) activity was evaluated by the Flohé and Gunzler method(Reference Flohé and Gunzler16), whereas catalase and total superoxide dismutase activities were assayed following the Aebi technique(Reference Aebi17) and Marklund method(Reference Marklund18), respectively. The CV of the techniques were less than 10 %.
Pearson correlations were made between study variables in order to evaluate their association. Comparisons between experimental conditions (baseline versus after high-fat meal) were determined using a paired-sample test (Wilcoxon). A multiple regression analysis was done to study the associations between the variables that correlated. In all cases the rejection level for a null hypothesis was a P = 0·05 for two tails. Calculations were performed with SPSS software (version 11.0; Statistical Package for the SPSS Iberica, Madrid, Spain).
Table 1 shows the characteristics of the study subjects; none of them fulfilled the Adult Treatment Panel III criteria for the metabolic syndrome.
* Calculated as: (urinary uric acid × serum creatinine)/(serum uric acid × urinary creatinine) × 100.
Table 2 shows the associations between the oxidative stress measurements and the levels of uric acid and TAG prior to the high-fat meal. The baseline levels of plasma TAG were inversely correlated with the antioxidant variables: GSH-Rd, glutathione-S-transferase and superoxide dismutase (r − 0·57, r − 0·60, r − 0·64, respectively), and directly with the oxidant variables: NOx, lipid peroxidation and carbonylated proteins (r 0·74, r 0·49, r 0·55, respectively). Moreover, as well as a strong inverse association between NOx and GSH-Rd (r − 0·76), strong correlations were found between oxidant variables and antioxidant variables (Table 2).
Correlations were significant: *P < 0·05.
†Antioxidant defence variables: CAT, catalase; GSH-Px, glutathione peroxidase; GSH-Rd, glutathione reductase; GST, glutathione-S-transferase; SOD, superoxide dismutase. Marker of vasodilatations: NOx, total nitrites (nitrites and nitrates). Pro-oxidant variables: LPO, lipid peroxidation; ProCar, protein carbonyl.
Table 3 shows the comparison of the means of those variables studied both before and after the high-fat meal. There was a significant increase in the values of TAG, lipid peroxidation, carbonylated proteins, and a significant reduction in GSH-Px, GSH-Rd, NOx and plasma uric acid after the high-fat meal.
Median values were significantly different from those of the baseline (Wilcoxon paired-sample test): *P < 0·05.
† Antioxidant defence variables: CAT, catalase; GSH-Px, glutathione peroxidase; GSH-Rd, glutathione reductase; GST, glutathione-S-transferase; SOD, superoxide dismutase. Marker of vasodilatations: NOx, total nitrites (nitrites and nitrates). Pro-oxidant variables: LPO, lipid peroxidation; ProCar, protein carbonyl.
Table 4 shows the correlations of the differences in the variables before and after the high-fat meal. There was a negative association between the difference in the plasma levels of uric acid and the glutathione-S-transferase and a positive correlation with the carbonylated proteins (r − 0·59, r 0·74, respectively).
TAG post, postprandial TAG; Δ, difference between baseline (before) and after fat overload (increase).
Correlations were significant: *P < 0·05.
† See Table 3 for abbreviations of variables.
Finally, Table 5 shows a multiple regression analysis of the difference in the plasma uric acid levels according to the other variables studied. The increase in the levels of carbonylated proteins after the high-fat meal was the only variable that predicted significantly the variance of this difference in uric acid levels.
ΔProCar, protein carbonyl difference between baseline (before) and after fat overload (increase).
* Variables not entering the model: ΔGSH-Rd, ΔCAT, ΔTAG post, ΔGST, ΔLPO, sex and BMI (see Table 3 for abbreviations of variables).
The results of the present study show that a high-fat meal in healthy persons produces an increase in the plasma levels of postprandial TAG and generates strong oxidative stress, because circulating antioxidant defences were decreased.
The association between plasma TAG levels and oxidative stress variables is known, as an accumulation of TAG and an intracellular increase in fatty acids produces an increase in cell catabolism and a release of electrons in the mitochondria, with the resulting increase in free radicals(Reference Manuel-y-Keenoy, Van Campenhout, Aerts, Vertommen, Abrams, Van Gaal, Van Gils and De Leeuw19). This increase in free radicals provokes a rise in oxidation and activates the antioxidant mechanisms of the organism. The increased oxidative variables after a high-fat meal and the inability of the antioxidant defences of the organism to detain this increase is known as postprandial oxidative stress(Reference Ursini and Sevanian20, Reference Bowen and Borthakur21). Oxidative stress has been involved in the pathogenesis of CVD and in the genesis of the metabolic syndrome(Reference Furukawa, Fujita, Shimabukuro, Iwaki, Yamada, Nakajima, Nakayama, Makishima, Matsuda and Shimomura22). Moreover, oxidative stress that accompanies postprandial lipidaemia can modify the endothelial function(Reference Bae, Bassenge, Kim, Kim, Kim, Lee, Park and Schwemmer4). In the present study we found an inverse association between the antioxidant defences of the organism and the plasma levels of TAG, even under fasting conditions, and that a high-fat meal results in an important increase in lipid peroxidation and carbonylated proteins and a decrease in both GSH-Px and GSH-Rd activity. Porter et al. (Reference Porter, Pearson, Suarez-Mendez and Blann23) found that a low plasma concentration of GSH-Px constituted a risk factor for coronary disease, and low GSH-Px activity was even detected in atherosclerotic carotid plaques(Reference Lapenna, de Gioia, Ciofani, Mezzetti, Ucchino, Calafiore, Napolitano, Di Ilio and Cuccurullo24). Patients undergoing haemodialysis were found to have a reduced plasma level of GSH-Px, partially explaining the presence of atherogenesis in chronic kidney disease(Reference Roxborough, Mercer, McMaster, Maxwell and Young25). Endogenous GSH-Px is an important antioxidant, protecting against oxidative stress in the atherogenic process; a reduction in GSH-Px levels produced by a high intake of fat could contribute to the onset of atherosclerosis associated with postprandial hypertriacylglycerolaemia.
The most relevant finding of the present study, though, is that a high-fat meal that increases oxidative stress produces a decrease in circulating antioxidant defences and in uric acid levels, and that this reduction has a very close association with the increase in oxidative stress.
Uric acid is the final product of purine metabolism in man. In many species, uric acid is metabolized to alantoin by urate oxidase, an enzyme activity that has been lost in man by a defect in gene transcription(Reference Glantzounis, Tsimoyiannis, Kappas and Galaris26).
In the 1980s, Ames et al. proposed that uric acid may have important biological effects as antioxidants, and they showed with in vitro experiments that it is a powerful scavenger of reactive oxygen species(Reference Ames, Cathcart, Schwiers and Hochstein27). Apart from its action as a scavenger of radicals, uric acid also exerts its action chelating metals like iron or copper, converting them into poor reactive species unable to catalyse reactions(Reference Davies, Sevanian, Muakkassah-Kelly and Hochstein28).
Consistent with the antioxidant role of uric acid in vivo is the hypothesis that the loss of urate oxidase in man (and the accompanying rise in serum levels of uric acid) improves antioxidant defence. Watanabe et al. (Reference Watanabe, Kang, Feng, Nakagawa, Kanellis, Lan, Mazzali and Johnson29) proposed that high levels of uric acid in man provide an evolutionary advantage as the hyperuricaemia maintains blood pressure better in conditions of low dietary salt intake.
A double-blind study evaluated the effects of the systematic administration of 100 mg uric acid compared with 1000 mg vitamin C(Reference Waring, Webb and Maxwell30). The authors found a significant increase in the antioxidant capacity of the serum after administration of uric acid and vitamin C, but the effect of the uric acid was substantantially greater. Another clinical study suggested an inverse relation between the concentration of uric acid and oxidative stress during aerobic exercise(Reference Mikami, Yoshino and Ito31). High concentrations of circulating uric acid may prevent oxidative stress in vivo during intense physical activity and protect against oxidative stress in other situations(Reference Waring, Convery, Mishra, Shenkin, Webb and Maxwell32). The present findings are related with this hypothesis to the extent that we show that uric acid is reduced after acute stress (after a high-fat meal) and that this reduction is supposedly because it exerts its antioxidant effect and is converted into allantoin and other products of oxidation(Reference Kaur and Halliwell33); chronic stress would probably cause an increase in uric acid in order to raise the antioxidant capacity of the plasma. Numerous studies have related uric acid with atherosclerosis(Reference Franse, Pahor, Di Bari, Shorr, Wan, Somes and Applegate34), but some authors suggest that the development of atherosclerosis is accompanied by a compensatory increase in antioxidant capacity, due mainly to the increased serum levels of uric acid(Reference Nieto, Iribarren, Gross, Comstock and Cutler35). Experimental findings in animal models found that early elevation of uric acid, during or shortly after acute ischaemic stroke, could confer significant protection against neurological deficit. This is in agreement with the protective effect of uric acid seen in other models of brain disease mediated by free radicals(Reference Yu, Bruce-Keller, Goodman and Mattson36).
The close association in the present study between the reduction in uric acid after a high-fat meal and the increase in carbonylated proteins may be explained by the role of uric acid, as it can repair tryptophan indolyl and tyrosine phenoxyl radicals(Reference Hoey and Butler37), and it is an oxidizable substrate for the haem protein–hydrogen peroxide system, able to protect against oxidative damage, acting as an electron donor(Reference Halliwell, Gutteridge, Halliwell and Gutteridge38).
The present findings reinforce the direct association between uric acid and hypertriacylglycerolaemia, where oxidative stress can be the binding factor. We have previously reported the direct relation between TAG levels and uric acid figures(Reference Tinahones, Collantes, C-Soriguer, Gonzalez-Ruiz, Pineda, Anon and Sánchez Guijo6, Reference Tinahones, Perez-Lindon, C-Soriguer, Pareja, Sanchez-Guijo and Collantes7). The present study, and others, have shown that TAG and postprandial hyperlipidaemia are directly associated with oxidative stress. Uric acid is a powerful antioxidant, and the data from the present study suggest that the reduction seen after a high-fat meal is because it exerts its antioxidant action acutely. The hypothesis may also be completed arguing that the chronic increase in oxidative stress caused by hypertriacylglycerolaemia may produce a chronic increase in uric acid and antioxidant defences which reflects the increase in oxidative stress, thereby raising the antioxidant capacity of the plasma. If this is confirmed, it would reinforce the theory that uric acid is only a marker of risk, and that prudency would be advisable when using uric acid-lowering drugs in patients with hyperuricaemia.
The authors wish to thank all the subjects for their collaboration, and IMABIS. We also gratefully acknowledge the help of Ian Johnstone for his expertise in preparing this manuscript. There are no conflicts of interest. F. C. and F. J. T. worked as investigators, conducted the data management and wrote the manuscript; E. C. worked as a clinical investigator and helped make the database; I. Tunez, I. Tasset and L. G.-S. worked as advice investigators and assisted with the laboratory work. The research group belongs to the ‘Centros de Investigación En Red’ (CIBER, CB06/03/0018) of the Instituto de Salud Carlos III, SAF 2006/12 894 of the MCYT, Madrid, Spain.