ALINE LUIZA FUHR

Título da Dissertação: MACRONUTRIENTES E ÁCIDOS GRAXOS DE DIETAS ENTERAIS: UMA COMPARAÇÃO ENTRE RÓTULOS E ACHADOS ANALÍTICOS

Orientadora: Profa. Dra. Grasiele Scaramal Madrona

Data da Defesa: 26/06/2020

INTRODUCTION
In recent decades, at the level of public health, there has been an increase in factors that influence clinical changes inherent to the need for alternative ways of feeding, such as enteral nutrition. Whether in the hospital or at home, enteral nutrition is indicated for individuals in whom the oral route is insufficient to meet nutritional needs or is impossible. The supply of nutrients occurs through liquid diets, whether these are manipulated (prepared manually with food) or industrialized enteral formulations (liquids or powders to be diluted). The scenario of comorbidities points to the notoriety of nutritional care for the prevention and treatment of pathologies. All this notoriety incites the possibility of adulteration, as has already been done in other food compounds with nutritional appeal. Only a few studies have sought to assess the accuracy of the composition of enteral diets and nutritional supplements with respect to macronutrients and fatty acids. In this sense, there are still gaps regarding composition and quality analysis of the lipids used in enteral nutrition formulas.
AIMS
Analyze and compare to the nutritional information on the label of industrialized enteral formulas marketed in Brazil, the following aspects: macronutrient content, caloric density, nutritional quality and fatty acid profile.
MATERIAL AND METHODS
Ten liquid enteral nutritional formulas were purchased on the local market in the cities of Maringa and Foz do Iguaçu (Parana State, Brazil), as well as on online shopping sites. The samples choice was due to the commercial availability, but also intended the insertion of formulas with varied therapeutic indications (children's diet therapy, for diabetics, for healing, standard diet, with protein appeal and with the addition of fibers). The lyophilized samples were kept at -20 ºC until analysis. The reagents used were all of analytical grade (Merck) and the chromatographic standards used were from Sigma. The macronutrients determination was carried out in triplicate, according to AOAC (2000). The protein content was assessed by the Kjeldahl method (method nº 920.87 AOAC, 1995). The determination of carbohydrates was carried out according to the phenol-sulfuric method described by Dubois (1956). The lipid content was obtained from the methodology established by Parnell (1899) and Bligh & Dyer (1959), with adaptations. The contents of protein, carbohydrates and total lipids were converted to 100mL, considering the density of the enteral diets. The total calories per mL of each formulation (caloric density) was estimated from the sum of the macronutrients found analytically (Heimburger and Weinsier, 1985). In addition, from the extracted lipid content, fatty acids were derivatized into fatty acid methyl esters (FAME) according to the method described by Santos et al. (2014). Chromatographic analyzes were carried out in a gas chromatograph coupled to the Thermo Scientific flame detector (GC-FID) TR-1310 model, equipped with a TR-FAME column (120 m long, 0.25 mm internal diameter and 0 mm film), 25 μm) and flame ionization detector, operating in split mode with a flow of 120 mL min-1. The injector temperature was of 230ºC and 225ºC for the detector. The injections were made in triplicate with an injection volume of 1 μl, from the organic phase that was collected and maintained at -20 °C after derivatization. FAMEs were identified by comparing the retention times between the constituents of the samples in relation to the standard fatty acid methyl esters (Supelco® 37 Component FAME Mix, Sigma Aldrich, St. Louis, EUA). The peak area was determined using the Chromeleon 7 software and results were expressed as percentages of the relative area (%). The nutritional quality of the lipid fraction was determined from three indices: a) Atherogenicity Index (AI); b) Thrombogenicity Index (TI); c) Hypocholesterolemic/Hypercholesterolemic (H/H) fatty acids ratio; d) P/S [ΣPUFA / ΣMUFA] ratio (Fehily et al. 1992; Santos-Silva, Bessa, and Santos-Silva 2002). Finally, the data were analyzed using Microsoft Excel® 2013 software. For the comparison between the values obtained and the nutritional contents described on the labels, a tolerance of ± 20% (80-120% adequacy) was considered in relation to the values of nutrients declared by the manufacturers, as defined by Resolution nº 360/2003 of the National Health Surveillance Agency (ANVISA) (Brazil 2003), as well as by the Food Drug Administration (FDA).
RESULTS AND DISCUSSION
Of the formulas evaluated, only one was produced in Brazil, the others were imported from China (1), Holland (2), Germany (5) and Argentina (1). Three formulations (A4, A9 and A10) presented levels of lipids below the tolerability established by ANVISA, varying from 1.15-1.85-fold the labeled contents. Of these, the difference was significant only for formulation A4 (p<0.05). The levels obtained for total proteins were significantly different (p<0.05) to the labels for formulations A1 and A6, in which there was variation of 2.76 to 2.41-fold lower than those values reported by the manufacturer, respectively. In this regard, it should be noted that, in the case of enteral formulations, the protein stands out for its fundamentality to healthy individuals but especially for with specific pathologies. The non-equivalence of the protein content in relation to the label is worrying when one considers that the nutritional prescription for the critical patient is based on evidence, and for this reason, it is expected that the prescribed is the one offered. The amount of protein administered and the patient's clinical moment may be relevant to optimize ICU mortality results and in the long term after discharge from intensive care. Among the ten formulations evaluated in this study, the differences in carbohydrate content were significant (p <0.05) for seven (A1, A2, A4, A5, A6, A9, A10), with the label values being higher than the ones evidenced by analyzes. Four samples showed discrepancies as to the tolerability of ± 20% (A1, A2, A6 and A7) with variations between 1.28 to 1.77-fold higher for glycids, noting that one of these formulations (A6) had a claim for glycemic control. The administration of carbohydrate values above the recommended is associated with problems such as hyperglycemia, increased CO2 production, lipogenesis and increased insulin. In addition, samples A1 and A2 coincidentally showed contents of 1.34 and 1.46-fold higher than labeled total carbohydrate values, as well as lower protein levels, reaching 2.76 and 2.41-fold lower than labeling, respectively. Furthermore, the caloric density values were similar to those presented on the label by the manufacturers for all formulations. In this regard, it is noted that the results for the health and mortality of patients will also depend on the proper prescription and the accuracy of the offer of what was prescribed. The administration of enteral nutrition with total security of what is being prescribed is this process basis, considering different external factors that can still interfere in this scope. Regarding the fatty acid profile found analytically, it should be noted that 20 fatty acids were quantified in the lipid materials of the enteral formulations, of which 17 were identified. Of these, eight were saturated fatty acids, three monounsaturated and six polyunsaturated. Notably, five samples (A2, A3, A6, A8 and A9) showed saturated fatty acids levels above the labeling, exceeding the tolerability of 20% with a variation of 2.65 (formulation A8) up to 10.36-fold (formulation A3) higher; however, only in two of them the difference between what was found and the label was not significant (p> 0.05). Moreover, in formulations A2, A8 and A9 the lowest percentages of monounsaturated fatty acids were identified, corresponding to 5.11 ± 2.84%, 14.52 ± 0.71%, and 13.67 ± 3.43%, respectively. The results obtained were significantly different for six (A1, A4, A5, A6, A8, A9) of the ten formulations with respect to MUFA lower than the 20% variation for A4 (59.99 ± 0.20%), A5 (75.22 ± 0.57%), A6 (48.49 ± 2.18%) and A9 (13.67 ± 3.43%), reaching up to 2.29-fold below the labeled value for A4. For polyunsaturated fat, samples A3, A5 and A1 showed, respectively, the lowest proportions in relation to the total fatty acids identified (A3: 5.25 ± 0.45%; A5: 14.46 ± 0.31%; A1 : 16.93 ± 0.32%). In five formulations (A3, A4, A7, A9, A10) EPA and DHA were not detected, despite the fact that in all of their labels there was a claim of the addition of fish oil and existence of these fatty acids in the composition. It was found that the sample A8 was the one that presented the best proportion of EPA + DHA (0.47 ± 0.19%), which had the highest amount of fish oil claimed on the label. On the other hand, the analytical results showed that the relationship between PUFA/SFA (PS) found in samples A4, A5 and A9 were lower than expected. The ratios between hypocholesterolemic and hypercholesterolemic (HH) fatty acids in samples A3 (0.98) and A8 (1.92) were lower in relation to the other formulations, suggesting hypercholesterolemic potential Also, formulations A3 (0.30), A8 (0.29) and A9 (0.25) had higher thrombogenic indices (TI). Considering the atherogenicity index (AI), A6 (0.15) and A9 (0.15), followed by A2 (0.10) and A3 (0.10), showed the higher values. On the other hand, an anti-inflammatory fatty acid profile is recommended for enteral diets, with caution in the supply of saturated fats and even in terms of the n-6: n3 ratio, since such markers are reflected in the clinical outcomes of enteral nutrition therapy.
CONCLUSIONS
In this work, we found discrepancies between values shown on the labels and those obtained for some enteral formulations, in the case of macronutrients such as protein and carbohydrate, and regarding the composition of fatty acids. Of the formulations, 30% presented all macronutrient parameters in line with the label. Two samples showed discrepant values between that described on the label regarding protein; four samples showed values between 1.28 to 1.77-fold those described on the label for carbohydrates; in three formulations, the lipid content exceeded the tolerability limit. Nevertheless, the caloric density values did not show any significant difference in relation to the values labeled by the manufacturers. Still, for five formulations, the content of saturated fatty acids exceeded those shown on the label, whereas for three of them the levels of monounsaturated were lower than the alleged contents. Fatty acids from fish oil were not found in five samples that claimed this ingredient. Thus, the discrepancy between the values warns of the need for supervision as to the actual composition of enteral formulations, which are administered to individuals with specific nutritional needs, who may have their clinical outcomes compromised, whether in the ICU, in the outpatient clinic or at home level. As already clearly established, not only precise levels of calories, but also macronutrients can have repercussions on the outcomes of patients in critical health condition, as well as those who need enteral nutrition at home, which justifies ensuring the right nutrition in the right time.
Key words: Formulated foods. Enteral nutrition. Food analysis. Centesimal composition. Nutritional therapy. Quality control

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