The investigators hypothesized that acute consumption of Fuji apple juice (AJ) could increase the antioxidant status and/or decrease the oxidative stress (OS) biomarkers, without increasing serum biochemical parameters in patients undergoing maintenance hemodialysis (MHD). In this pre-post pilot feasibility study, patients served as their own controls, received 300 and 150 mL AJ immediately after a dialysis section, on different days, with a 3 week-washout period. Blood was collected at the baseline period, after 30 and 60 min of AJ consumption. OS biomarkers (total antioxidant status (TAS), total oxidant status (TOS), ascorbic acid, catalase (CAT), glutathione peroxidase (Gpx), superoxide dismutase (SOD) and reduced glutathione (GSH) and potassium, phosphorus, uric acid, and glucose concentrations were analyzed.

Subjects and study design

In this pre-post pilot feasibility study, subjects served as their own controls. Participants were recruited from a hemodialysis clinic, located in Florianópolis' metropolitan area, Santa Catarina state (Southern Brazil), from June to August 2015 with the following inclusion criteria: hemodialysis treatment ≥ 3 months, age ≥ 20 years, and body mass index (BMI) ≥ 23 kg/m2. Exclusion criteria were allergy to apple, presence of cancer or acquired immunodeficiency syndrome, kidney transplant less than 6 months before enrolling in the study, taking antioxidant or nutritional supplements during the 30 days before enrollment, having been hospitalized within 6 weeks before the beginning of the study, or suffering from an acute illness.

Subjects were instructed to keep their usual dietary habits for the duration of the study, except for avoiding intake of polyphenol-rich foods (i.e., vegetables, fruits and fruit-containing products, chocolate, tea, coffee, honey, and any alcoholic beverage) within 2 days before and during intervention. On two different days, each volunteer consumed 300 mL Fuji AJ, immediately after a dialysis section. After a washout period of 3 weeks, the volunteers drank 150 mL Fuji AJ in a similar manner as described above. Before and after 30 and 60 min of AJ consumption, blood samples were withdrawn for biochemical analysis. This protocol was chosen taking into account the previous favorable effects of AJ consumption on serum OS biomarkers described for healthy volunteers.

Baseline clinical and biochemical data were obtained from medical records. This study was approved by the ethics committee of human research of the Federal University of Santa Catarina (UFSC) (protocol number 37090614.6.0000.0118) and all participants gave written and informed consent.

Apple juice and analysis

Fuji apples were obtained from the Experimental Seasonal Fields at the Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina' Station in the city of São Joaquin, Santa Catarina, Brazil (latitude 28º 17' 39", longitude 49º 55' 56" and altitude 1.415 m), harvested in their commercial maturation stage during 2015's harvest. After harvest, apples were stored at 4 ± 1°C. To allow for a quick and easy apple intake in large amounts, 300 mL of AJ, equivalent to 3.5 apples, and 150 mL AJ, equivalent to 1.5 apples were used. Before preparing the juice, the fruits were sanitized in sodium hypochlorite. Apples with peel and no seeds were blended in a centrifugal juice extractor without water addition. Each dose of AJ was prepared and consumed immediately.

The contents of dry matter, total soluble solids, potential of hydrogen (pH) and titratable acidity of AJ were measured following official methods. The total phenolic content of the AJ extracts was measured using the Folin-Ciocalteu method colorimetric. The total flavanol content was estimated using p-dimethylaminocinnamaldehyde (DMACA) method. The total antioxidant capacity was determined using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) method. For total monomeric anthocyanin, a spectrophotometric pH differential method was used. Analyses were conducted with each AJ used in the two intervention days, in triplicate.

Blood sampling and laboratory methods

In each experiment day, blood samples from participants were collected in three different moments. Venous blood samples were collected using a vacuum system (Vacutainer Becton Dickinson BD®, São Paulo, Brazil) into heparin, Ethylenediaminetetraacetic acid (EDTA)-containing tubes or additive-free tubes. Plasma and serum were immediately obtained by centrifugation (3,000 revolutions per minute (RPM), 10 min, room temperature) for the measurement of uric acid, phosphorus, glucose, potassium, Total Antioxidant Status (TAS) and Total Oxidant Status( TOS). In order to quantify the endogenous antioxidant enzymes, an aliquot of whole blood was lysed. In order to measure reduced glutathione (GSH), an aliquot of whole blood was preserved in N-ethylmaleimide (NEM). All samples were immediately stored at -80°C for later analysis in duplicate.

Serum uric acid, phosphorus and glucose concentrations were determined using commercially available kits (Labtest Diagnóstica SA, Lagoa Santa, Minas Gerais, Brazil) in an automated multi-biochemical analyzer (Cobas Miras Plus, Roche®, Germany). Serum potassium levels were measured in an automated Dimension Max System (Siemens Healthcare Diagnostics Products®, Germany), according to the manufacturer's instructions.

TAS and TOS assays were measured spectrophotometrically, according to Erel's methods. Ascorbic acid and GSH were determined using high-performance liquid chromatography (HPLC), as previously described. The superoxide dismutase (SOD) activity was evaluated using SOD Assay Kit (Sigma Aldrich®, St. Louis, Missouri, USA) according to the manufacturer's instructions. Catalase (CAT) activity was determined spectrophotometrically according to the previously described method. The glutathione peroxidase (GPx) activity was determined using the NADPH oxidation rate method.

Inclusion Criteria:

• hemodialysis treatment ≥ 3 months, age ≥ 20 years, and body mass index ≥ 23 kg/m2

Exclusion Criteria:

• allergy to apple, presence of cancer or acquired immunodeficiency syndrome, kidney transplant less than 6 months before enrolling in the study, taking antioxidant or nutritional supplements during the 30 days before enrollment, having been hospitalized within 6 weeks before the beginning of the study, or suffering from an acute illness