Alternative green solvents associated with ultrasound-assisted extraction: A green chemistry approach for the extraction of carotenoids and chlorophylls from microalgae.

This study proposes a method for the ultrasonic extraction of carotenoids and chlorophyll from Scenedesmus obliquus and Arthrospira platensis microalgae with green solvents. Ethanol and ethanolic solutions of ionic liquids were tested with a variety of extraction parameters, including number of extractions, time of extraction, and solid-liquid ratio R(S/L), to determine the optimal conditions. After selecting the most effective green solvent (ethanol), the process conditions were established: R(S/L) of 1:10, three extraction cycles at 3 min each), giving an extraction yield of 2602.36 and 764.21 µgcarotenoids.gdried biomass-1; and 22.01 and 5.81 mgchlorophyll.gdried biomass-1 in S. obliquus and A. platensis, respectively. The carotenoid and chlorophyll extracts obtained using ethanol were shown to be potent scavengers of peroxyl radical, being 5.94 to 26.08 times more potent α-tocopherol. These findings pave the way for a green strategy for valorizing microalgal biocompounds through efficient and environmentally friendly technological processes.

α-tocopherol as a selective modulator of toxicogenic damage induced by antineoplastic agents cyclophosphamide and doxorubicin.

The aim of this study was to determine the oxidative/antioxidative effects, modulatory and selective potential of α-tocopherol (vitamin E) on antineoplastic drug-induced toxicogenetic damage. The toxicity, cytotoxicity and genotoxicity induced by antineoplastic agents cyclophosphamide (CPA) and doxorubicin (DOX) was examined utilizing as models Saccharomyces cerevisiae, Allium cepa, Artemia salina and human peripheral blood mononuclear cells (PBMCs) in the presence of α-tocopherol. For these tests, concentrations of α- tocopherol 100 IU/ml (67mg/ml), CPA 20 µg/ml, DOX 2 µg/ml were used. The selectivity of α-tocopherol was assessed by the MTT test using human mammary gland non-tumor (MCF10A) and tumor (MCF-7) cell lines. Data showed cytoplasmic and mitochondrial oxidative damage induced by CPA or DOX was significantly diminished by α-tocopherol in S. cerevisiae. In addition, the toxic effects on A. salina and cytotoxic and mutagenic effects on A. cepa were significantly reduced by α-tocopherol. In PBMCs, α-tocopherol alone did not markedly affect these cells, and when treated in conjunction with CPA or DOX, α-tocopherol reduced the toxicogenetic effects noted after antineoplastic drug administration as evidenced by decreased chromosomal alterations and lowered cell death rate. In human mammary gland non-tumor and tumor cell lines, α-tocopherol produced selective cytotoxicity with 2-fold higher effect in tumor cells. Evidence indicates that vitamin E (1) produced anti-cytotoxic and anti-mutagenic effects against CPA and DOX (2) increased higher selectivity toward tumor cells, and (3) presented chemoprotective activity in PBMCs.

Guidance for formulating ingredients/products from Chlorella vulgaris and Arthrospira platensis considering carotenoid and chlorophyll bioaccessibility and cellular uptake.

This study aimed to investigate the impact of different microalgal matrices on the bioaccessibility and uptake by Caco-2 cells of carotenoids and chlorophylls. In this way, the microalgal ingredients/products (whole dry biomass [WDB], whole ultrasonicated paste [WUP], and liposoluble pigment emulsion [LPE]) obtained from Chlorella vulgaris and Arthrospira platensis were submitted to in vitro simulated digestion. Apical uptake of pigments in micelles generated during the simulated digestion by Caco-2 human intestinal cells was determined. The influence of simulated digestion on carotenoid and chlorophyll stability and bioaccessibility was assessed by HPLC-PDA-MS/MS and the carotenoids and chlorophylls' bioaccessibility and cellular uptake were shown to be boosted according to the matrix (LPE > WUP > WDB). Our findings showed that Chlorella vulgaris and Arthrospira platensis could be considered in formulations when carotenoids and chlorophylls are the target molecules in the ingredients/products.

Insights on the Bioaccessibility of Natural Pigments from Diatom Chaetoceros calcitrans.

This study aimed to investigate the bioaccessibility of carotenoids and chlorophylls from the biomass of microalgae Chaetoceros calcitrans. The samples were submitted to an in vitro digestion protocol, and the compounds were determined by HPLC-PDA-MS/MS. A total of 13 compounds were identified in all tests. After in vitro digestion, the relative bioaccessibility of carotenoids and chlorophylls ranged from 4 to 58%. The qualitative profile of carotenoids reflected the initial sample, with all-E-zeaxanthin (57.2%) being the most bioaccessible compound, followed by all-E-neochrome (31.26%), the latter being reported for the first time in the micellar fraction. On the other hand, among the chlorophylls only pheophytin a (15.01%) was bioaccessible. Furthermore, a chlorophyll derivative (Hydroxypheophytin a') was formed after in vitro digestion. Considering all compounds, xanthophylls (12.03%) and chlorophylls (12.22%) were significantly (p < 0.05) more bioaccessible than carotenes (11.22%). Finally, the considerable individual bioaccessibilities found, especially for zeaxanthin, demonstrate the bioactive potential of this bioresource. However, the large reduction in the totality of compounds after in vitro digestion suggests that additional technological strategies should be explored in the future to increase the efficiency of micellarization and enhance its bioactive effects.

Bioaccessibility of microalgae-based carotenoids and their association with the lipid matrix.

The composition of microalgae can contribute to nutritious and functional diets. Among the functional compounds, carotenoids are in focus since positive effects on human health have been established, which are in turn related to their bioaccessibility. In addition to essential nutrients, our hypothesis was that microalgae biomasses could be used as sources of bioaccessible carotenoids. Thus, this study determined for the first time the bioaccessibility of carotenoids from biomass of Scenedesmus bijuga and Chlorella sorokiniana and their possible relationship with the lipid composition of the matrix. The samples were submitted to in vitro digestion protocol, and carotenoids were determined by HPLC-PDA-MS/MS. Individual bioaccessibility of carotenoids was ≥ 3.25%. In general, compounds in their cis conformation were more bioaccessible than trans; and total carotenes more than total xanthophylls. Twelve compounds were bioaccessible from the biomass of S. bijuga, and eight in C. sorokiniana. In S. bijuga, the bioaccessibility of total carotenoids was 7.30%, and the major bioaccessible carotenoids were 9-cis-ß-carotene (43.78%), 9-cis-zeaxanthin (42.30%) followed by 9-cis-lutein (26.73%); while in C. sorokiniana, the total bioaccessibility was 8.03%, and 9-cis-ß-carotene (26.18%), all-trans-ß-carotene (13.56%), followed by 13-cis-lutein (10.71%) were the major compounds. Overall, the total content of lipids does not influence the bioaccessibility of total carotenoids. Still, the lipid composition, including structural characteristics such as degree of saturation and chain length of the fatty acid, impacts the promotion of individual bioaccessibility of carotenes and xanthophylls of microalgae. Finally, the results of this study can assist the development of microalgae-based functional food ingredients and products.

Phytochemical analysis and evaluation of the antioxidant and antiproliferative effects of Tucumã oil nanocapsules in breast adenocarcinoma cells (MCF-7).

In this work was to develop an inedited nanocapsule with tucumã oil (Astrocaryum vulgare). The oil presents of phytosterols (squalene and ß-sitosterol), all-trans-beta-carotene, acids oleic and palmitic. Antioxidant activity showed a good performance in DPPH and ABTS assays. The nanocapsules were prepared and demonstrated in their characterization particle size (206 ± 0.69 nm). The cytogenotoxicity evaluation was performed using the MTT, dichlorofluorescein, nitric oxide and dsDNA PicoGreen® assays. Antitumor efficacy assays in MCF-7 cells demonstrated that free oil and tucumã nanocapsules had IC50 of 130 and 50 µg/mL, respectively. Thus, previous studies of toxicity are relevant, as they generate future subsidies, aiming at the potential application of nanostructures and in addition, the promising effect of NCs of tucumã oil on the antiproliferative effect in breast adenocarcinoma cells was evidenced.

Insights on the intestinal absorption of chlorophyll series from microalgae.

The bioaccessibility and subsequent uptake by Caco-2 human intestinal cells of chlorophyll pigments from Scenedesmus obliquus were determined for the first time. In order to evaluate the impact of different types of the matrix on bioaccessibility of chlorophyll from microalgae, three different products were evaluated: isolated chlorophyll extract (ICE); wet ultrasonicated biomass (WUB); and whole dried biomass (WDB). The samples were submitted to in vitro digestion model according to the INFOGEST protocol, and Caco-2 cells determined the intestinal uptake. Chlorophyll pigments were determined by HPLC-PDA-MS/MS. A total of ten chlorophyll pigments (8,318.48 µg g-1) were separated in S. obliquus biomass, with chlorophyll a (3,507.76 µg g-1) and pheophytin a' (1,598.09 µg g-1) the major ones. After in vitro digestion, all tested products showed bioaccessible chlorophylls. However, the total bioaccessibility results were as follows: ICE (33.45%), WUB (2.65%), WDB (0.33%). Five compounds were bioaccessible in ICE, three in WUB, and one in WDB. The hydroxypheophytin a showed the highest bioaccessibility (212%) in ICE, while pheophytin a' in WUB (11%) and WDB (2%). As a result, bioavailability estimates of ICE using the Caco-2 cell showed hydroxypheophytin a (102.53%), followed by pheophytin a' (64.69%) as the chlorophyll pigments most abundant in intestinal cells. In summary, from a nutritional perspective, these three types of the matrix (WDB, WUB, and ICE) influence the promotion of chlorophyll bioaccessibility. In this way, the data suggest that chlorophylls bioaccessibility from ICE is greater than that in WDB and WUB. Therefore, ICE should be considered a product that provides bioavailable chlorophyll and could be the best choice, such as ingredients in the development of functional foods chlorophyll-based.

HPLC-PDA-MS/MS as a strategy to characterize and quantify natural pigments from microalgae.

Interest in pigment composition of microalgae species is growing as new natural pigments sources are being sought. However, we still have a limited number of species of microalgae exploited to obtain these compounds. Considering these facts, the detailed composition of carotenoids and chlorophylls of two species of green microalgae (Chlorella sorokiniana and Scenedesmus bijuga) were determined for the first time by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-PDA-MS/MS). A total of 17 different carotenoids were separated in all the extracts. Most of the carotenoids present in the two microalgae species are xanthophylls. C. sorokiniana presented 11 carotenoids (1408.46 µg g-1), and S. bijuga showed 16 carotenoids (1195.75 µg g-1). The main carotenoids detected in the two microalgae were all-trans-lutein and all-trans-ß-carotene. All-trans-lutein was substantially higher in C. sorokiniana (59.01%), whereas all-trans-ß-carotene was detected in higher quantitative values in S. bijuga (13.88%). Seven chlorophyll compounds were identified in both strains with different proportions in each species. Concentrations of chlorophyll representing 7.6% and 10.2% of the composition of the compounds present in the biomass of C. sorokiniana and S. bijuga, respectively. Relevant chlorophyll compounds are reported for the first time in these strains. The data obtained provide significant insights for microalgae pigment composition databases.

Dietary intake of tyrosine and phenylalanine, and p-cresyl sulfate plasma levels in non-dialyzed patients with chronic kidney disease / Ingestão dietética de tirosina e fenilalanina e níveis plasmáticos de p-cresil sulfato em pacientes com doença renal crônica não dialisados

ABSTRACT Background: Patients with chronic kidney disease (CKD) present an imbalance of the gut microbiota composition, leading to increased production of uremic toxins like p-cresyl sulfate (PCS), product from bacterial fermentation of the amino acids tyrosine (Tyr) and phenylalanine (Phe) from the diet. Thus, diet may be a determinant in the uremic toxins levels produced by the gut microbiota. The aim of this study was to evaluate the possible relationship between Tyr and Phe intake and PCS plasma levels in non-dialysis CKD patients. Methods: Twenty-seven non-dialysis CKD patients (stages 3 and 4) without previous nutritional intervention were evaluated. The dietary intake was evaluated using a 24-hour recall, 3-day food record and protein intake was also estimated by Protein Nitrogen Appearance (PNA). The plasma levels of PCS were measured using reverse phase high performance liquid chromatography. Results: The evaluated patients (GRF, 34.8 ± 12.4 mL/min, 54.2 ± 14.3 years, BMI, 29.3 ± 6.1 kg/m2) presented mean protein intake of 1.1 ± 0.5 g/kg/day), Tyr of 4.5 ± 2.4 g/day and Phe of 4.6 ± 2.5 g/day. PCS plasma levels (20.4 ± 15.5 mg/L) were elevated and positively associated with both, Tyr (r = 0.58, p = 0.002) and Phe intake (r = 0.53, p = 0.005), even after adjustments for eGFR and age. Conclusion: This study suggests that the diet is an important modulator of the uremic toxins plasma levels produced by the gut microbiota, in non-dialysis CKD patients.

RESUMO Introdução: Pacientes com doença renal crônica (DRC) apresentam desequilíbrio na composição da microbiota intestinal, gerando toxinas urêmicas, como o p-cresil sulfato (PCS), pela fermentação bacteriana dos aminoácidos tirosina (Tyr) e fenilalanina (Phe) da dieta. Assim, a dieta pode ser determinante nos níveis de toxinas urêmicas produzidos pela microbiota intestinal. O objetivo deste estudo foi avaliar a possível relação entre a ingestão de Tyr e Phe e os níveis plasmáticos de PCS em pacientes com DRC não dialisados. Métodos: Foram avaliados 27 pacientes com DRC em tratamento conservador (estágios 3 e 4), sem intervenção nutricional prévia. A ingestão alimentar foi avaliada pelo recordatório alimentar de 24h (R-24h) de 3 dias, e a ingestão proteica também foi verificada através do Protein Nitrogen Appearance (PNA). Os níveis plasmáticos de PCS foram determinados por cromatografia líquida de fase reversa. Resultados: Os pacientes avaliados (TFG, 34,8 ± 12,4 mL/min, 54,2 ± 14,3 anos, IMC 29,3 ± 6,1 kg/m2) apresentaram ingestão média de proteína de 1,1 ± 0,5 g/kg/dia, Tyr de 4,5 ± 2,4 g/dia e Phe de 4,6 ± 2,5 g/dia. Os níveis plasmáticos de PCS (20,4 ± 15,5 mg/L) foram elevados e positivamente associados à ingestão de Tyr (r = 0,58, p = 0,002) e Phe (r = 0,53, p = 0,005), mesmo após ajustes pela TFG e idade. Conclusão: Este estudo sugere que a dieta é um importante modulador dos níveis plasmáticos de toxinas urêmicas produzidas pela microbiota intestinal em pacientes com DRC não dialisados.

Dietary intake of tyrosine and phenylalanine, and p-cresyl sulfate plasma levels in non-dialyzed patients with chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) present an imbalance of the gut microbiota composition, leading to increased production of uremic toxins like p-cresyl sulfate (PCS), product from bacterial fermentation of the amino acids tyrosine (Tyr) and phenylalanine (Phe) from the diet. Thus, diet may be a determinant in the uremic toxins levels produced by the gut microbiota. The aim of this study was to evaluate the possible relationship between Tyr and Phe intake and PCS plasma levels in non-dialysis CKD patients. METHODS: Twenty-seven non-dialysis CKD patients (stages 3 and 4) without previous nutritional intervention were evaluated. The dietary intake was evaluated using a 24-hour recall, 3-day food record and protein intake was also estimated by Protein Nitrogen Appearance (PNA). The plasma levels of PCS were measured using reverse phase high performance liquid chromatography. RESULTS: The evaluated patients (GRF, 34.8 ± 12.4 mL/min, 54.2 ± 14.3 years, BMI, 29.3 ± 6.1 kg/m2) presented mean protein intake of 1.1 ± 0.5 g/kg/day), Tyr of 4.5 ± 2.4 g/day and Phe of 4.6 ± 2.5 g/day. PCS plasma levels (20.4 ± 15.5 mg/L) were elevated and positively associated with both, Tyr (r = 0.58, p = 0.002) and Phe intake (r = 0.53, p = 0.005), even after adjustments for eGFR and age. CONCLUSION: This study suggests that the diet is an important modulator of the uremic toxins plasma levels produced by the gut microbiota, in non-dialysis CKD patients.

Exploratory data of the microalgae compounds for food purposes.

This brief data article refers to the previous exploration of Scenedesmus obliquus and Phormidium autumnale biomass about the possibility of using these microalgae species as an unconventional functional food. Data on chemical composition, fatty acids, volatile compounds, and carotenoid profiles were determined. In parallel, are provided the antioxidant capacity (reducing capacity - RC and reactive oxygen species deactivation - ORAC) of aqueous, lipophilic, and carotenoid extracts isolated from microalgae biomass. Both species have similar compounds in their biomass. However, S. obliquus was statistically different with a lower saturated fatty acid (STF) followed by higher mono (MUFA) and polyunsaturated (PUFA) content, also showed higher antioxidant potential for acetone extract and isolated carotenoids. On the other hand, P. autumnale aqueous extract showed high RC and ORAC. The significance of the experimental data was determined using the t-test (p < 0.05) based on the Statistica 7.0 software. These findings led us to explore the microalgae S. obliquus in an in vivo experimental model.

Principais causas da rejeição de rim em pacientes transplantados / Main causes of rejection of rim in transplanted patients

Objetivo: descrever as principais causas da rejeição de rim em pacientes transplantados apontadas pela literatura. Metodologia: trata-se de um estudo de revisão. A coleta de dados foi realizada na Literatura Latino-Americana e do Caribe em Ciências da Saúde e na Scientific Electronic Library Online. Para a seleção dos estudos, adotaram-se os seguintes critérios de elegibilidade: artigos no formato texto completo e publicado em língua portuguesa. Foram excluídos os estudos que abordavam a rejeição em outros tipos de transplantes. Resultados: a partir da análise dos estudos evidencia-se que as complicações cardiovasculares, digestivas, tumorais e infecciosas estão com maior incidência entre os pacientes transplantados e que a causa relaciona-se com a terapia imunossupressora. Conclusão: acredita-se que o estudo possa contribuir para as boas práticas assistenciais a tal clientela, e que assim se possam implementar medidas preventivas embasadas nas melhores evidências científicas.

Objective: to describe the main causes of kidney rejection in transplanted patients mentioned at the literature. Methodology: this is a review study. Data collection was carried out in the Latin American and Caribbean Literature in Health Sciences and the Scientific Electronic Library Online. In order to select the studies, the following eligibility criteria were adopted: articles in full text format and published in Portuguese. Studies addressing rejection in other types of transplants were excluded. Results: from the analysis of the studies it is evident that cardiovascular, digestive, tumor and infectious complications are more frequent among transplanted patients and that the cause is related to immunosuppressive therapy. Conclusion: it is believed that the study can contribute to the good practices of assistance to such clientele, and so that preventive measures based on the best scientific evidence can be implemented.

Experimental exposure to gasohol impairs sperm quality with recognition of the classification pattern of exposure groups by machine learning algorithms.

Contamination caused by leakage at gas stations leads to possible exposure of the general population when in contact with contaminated water and soil. The present study aimed to evaluate the reproductive effects of exposure of adult male rats to gasohol and evaluate the performance of machine learning (ML) algorithms for pattern recognition and classification of the exposure groups. Rats were orally exposed to 0 (control), 16 (EA), 160 (EB), or 800 mg kg-1 bw day-1 of gasohol (EC), for 30 consecutive days. Sperm quality of the groups exposed to two higher doses was reduced in comparison to the control group. The sperm parameters decreased were: daily sperm production, sperm number in the caput/corpus epididymis, progressive motility, mitochondrial activity, and acrosomal membrane integrity. Sperm transit time in the epididymis cauda and sperm isolated head were increased in EB and EC. Sertoli cells number was decreased in these groups, but their support capacity was maintained. ML methods were used to identify patterns between samples of control and exposure groups. The results obtained by ML methods were very promising, obtaining about 90% of accuracy. It was concluded that the exposure of rats to different doses of gasohol impair spermatogenesis and sperm quality, with a recognizable classification pattern of exposure groups at ML.

Identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale using ultrasound-assisted extraction.

The identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale was determined. The ultrasound-assisted extraction was utilized for obtaining the chlorophyll compounds from biomass. A total of eleven molecules were separated in microalgae chlorophyll extract, with pheophytin a' (371µg·g-1) and chlorophyll a (159.3µg·g-1) as the major ones. The chlorophyll extract was shown to be a potent scavenger of peroxyl radical, being almost 200 times more potent than α-tocopherol. These facts suggest the microalgae Phormidium autumnale as potential source of bioactive tetrapyrrole compounds.

Carotenoid profile of three microalgae/cyanobacteria species with peroxyl radical scavenger capacity.

Carotenoids from cyanobacteria Aphanothece microscopica Nageli and green microalgae Chlorella vulgaris and Scenedesmus obliquus were identified. The total carotenoid content, based on dry weight of biomass, of A. microscopica Nägeli, C. vulgaris and S. obliquus were 1398.88µg/g, 1977.02µg/g and 2650.70µg/g, respectively. A total of 23 different carotenoids were separated in all the extracts, the major ones being all-trans-ß-carotene (29.3%) and all-trans-lutein (28.1%) in Scenedesmus; all-trans-echinenone (22.8%) and all-trans-ß-carotene (17.7%) in Chlorella; all-trans-echinenone (28.3%) and all-trans-ß-carotene (26.2%) in Aphanothece. The carotenoid extracts were shown to be a potent scavenger of peroxyl radical, with values of 31.1 (Chlorella), 14.0 (Scenedesmus) and 7.3 (Aphanothece) times more potent than α-tocopherol.

Treino em auto-observação e adesão à dieta em adulto com diabetes tipo 2 / Training on self-observation and adherence to diet in an adult with type 2-diabetes

Este estudo verificou os efeitos do treino de auto-observação sobre a adesão à dieta em um adulto com diabetes Tipo 2. A coleta de dados ocorreu por meio de entrevistas no domicílio do participante. Após levantamento da linha de base, iniciou-se o treino no uso de protocolos para registro de automonitoramento (Passo 1), seguido de treino do relato verbal do paciente sobre a sua alimentação no dia anterior (Passo 2) e de treino no planejamento da adesão à dieta (Passo 3). O cálculo do Índice de Adesão à Dieta (IAD) do paciente e seus relatos verbais nortearam a análise dos resultados, que indicaram aumento na frequência de respostas de auto-observação do comportamento alimentar e no IAD no Passo 1. Esse ganho foi mantido no Passo 2 e maximizado com o treino no planejamento da adesão à dieta no Passo 3.(AU)

The present study verified the effects of self-observation over the adherence to a prescribed diet for an adult with Type 2-diabetes. The data collection was brought up by means of home interviews. Following the baseline determination, the following steps were performed: a training on the use of protocols for self-monitoring registration (Step 1), a training on the patient's verbal report over his feeding on the former day (Step 2), and a training on how to plan the adherence to the diet (Step 3). The calculation of the patient's Adherence Index to the Diet (AID) along with his verbal reports guided the data analysis, which revealed an increase in the frequency of self-observation of feeding behavior and in the AID on Step 1. This gain was maintained on Step 2 and further maximized by the training on planning the adherence to the diet on Step 3.(AU)

Treino em auto-observação e adesão à dieta em adulto com diabetes tipo 2 / Training on self-observation and adherence to diet in an adult with type 2-diabetes

Este estudo verificou os efeitos do treino de auto-observação sobre a adesão à dieta em um adulto com diabetes Tipo 2. A coleta de dados ocorreu por meio de entrevistas no domicílio do participante. Após levantamento da linha de base, iniciou-se o treino no uso de protocolos para registro de automonitoramento (Passo 1), seguido de treino do relato verbal do paciente sobre a sua alimentação no dia anterior (Passo 2) e de treino no planejamento da adesão à dieta (Passo 3). O cálculo do Índice de Adesão à Dieta (IAD) do paciente e seus relatos verbais nortearam a análise dos resultados, que indicaram aumento na frequência de respostas de auto-observação do comportamento alimentar e no IAD no Passo 1. Esse ganho foi mantido no Passo 2 e maximizado com o treino no planejamento da adesão à dieta no Passo 3.

The present study verified the effects of self-observation over the adherence to a prescribed diet for an adult with Type 2-diabetes. The data collection was brought up by means of home interviews. Following the baseline determination, the following steps were performed: a training on the use of protocols for self-monitoring registration (Step 1), a training on the patient's verbal report over his feeding on the former day (Step 2), and a training on how to plan the adherence to the diet (Step 3). The calculation of the patient's Adherence Index to the Diet (AID) along with his verbal reports guided the data analysis, which revealed an increase in the frequency of self-observation of feeding behavior and in the AID on Step 1. This gain was maintained on Step 2 and further maximized by the training on planning the adherence to the diet on Step 3.