key: cord-310883-t5r4xqj3
authors: Grundler, Franziska; Mesnage, Robin; Goutzourelas, Nikolaos; Tekos, Fotios; Makri, Sotiria; Brack, Michel; Kouretas, Demetrios; Wilhelmi de Toledo, Françoise
title: Interplay between oxidative damage, the redox status, and metabolic biomarkers during long-term fasting
date: 2020-08-25
journal: Food Chem Toxicol
DOI: 10.1016/j.fct.2020.111701
sha: 
doc_id: 310883
cord_uid: t5r4xqj3

Obesity and its related metabolic disorders, as well as infectious diseases like covid-19, are important health risks nowadays. We recently documented that long-term fasting improves metabolic health and enhanced the total antioxidant capacity. The present study investigated the influence of a 10-day fasting on markers of the redox status in 109 subjects. Reducing power, ABTS radical scavenging capacity, and hydroxyl radical scavenging capacity increased significantly, and indicated an increase of circulating antioxidant levels. No differences were detected in superoxide scavenging capacity, protein carbonyls, and superoxide dismutase when measured at baseline and after 10 days of fasting. These findings were concomitant to a decrease in blood glucose, insulin, HbA1c, total cholesterol, LDL and triglycerides as well as an increase in total cholesterol/HDL ratio. In addition, the well-being index as well as the subjective energy levels increased, documenting a good tolerability. We documented an interplay between redox and metabolic parameters, as lipid peroxidation baseline levels (TBARS) affected the ability of long-term fasting to normalize lipid levels. A machine learning model showed that a combination of antioxidant parameters measured at baseline predicted the efficiency of the fasting regimen to decrease LDL levels. In conclusion, we demonstrated that long-term fasting enhanced the endogenous production of antioxidant molecules, that act protectively against free radicals, and in parallel improved the metabolic health status. Our results suggest that the outcome of long-term fasting strategies could be depending on the baseline values of the antioxidative and metabolic status of subjects.

achieve these objectives could be long-term fasting, that is defined as voluntary food 1 abstinence from 2 days to several weeks (Wilhelmi de Toledo et al., 2020b). The study of 2 various forms of fasting on metabolic health has been gaining ground in recent years with 3 more and more studies showing beneficial effects such as inactivating of the mechanistic 4 target of rapamycin (mTOR) signaling pathways as well as several others resulting in 5 metabolic normalization, enhanced autophagy and apoptosis followed by cell regeneration, 6 increased brain-derived neurotrophic factor (BDNF) levels in brain leading to enhanced 7 cognition, mood as well as increased neuronal plasticity and regeneration. Fasting also 8 enhanced the transcription of cytoprotective enzymes, mitochondrial biogenesis all of these 9 effects leading to restoration and conservation of functional integrity of cells and tissues (de even the occurrence of heart attacks and stroke (Knott et al., 2020) . Earlier studies also 18 suggest that this can be applicable to understand redox systems as a recent study 19

successfully developed a neural network to predict oxidative damage using measurements of patients. The BDNF, which has its production stimulated by ketosis during fasting (Mattson 25 et al., 2018) , is known to control the nuclear translocation of the transcription factor Nrf2 to 26 J o u r n a l P r e -p r o o f activate antioxidant defenses (Bouvier et al., 2017) . Furthermore, the mild oxidative stress 1 caused by the reduction in adenosine triphosphate (ATP)/adenosine monophosphate (AMP) 2 ratio has a similar effect to activate transcription of cytoprotective enzymes sulfiredoxin 1, 3 thioredoxin reductase 1, heme oxygenase-1, as well as several others conjugation and 4 elimination enzymes (Burton et al., 2016) . 5

This article is a continuation of our previous study on the effect of a 10-day fasting on 6

indicators of redox status of humans which showed that while the total antioxidant capacity 7 (TAC) was enhanced, TBARS, an important indicator of lipid peroxidation, were reduced 8 (Wilhelmi de Toledo et al., 2020a). The aim of the present article was to analyze additional 9 redox parameters and to correlate the redox status with markers of glucose and lipid status, 10 the improvement of which leads to a better metabolic health and a reduced risk of metabolic 11

diseases. It was also evaluated if the baseline levels in markers of the redox status can 12 predict the success of the fasting regimen using a machine learning approach. 

The 109 study participants were recruited out of a total of 182 subjects who were admitted 11 to the Buchinger Wilhelmi Clinic (BWC) and fulfilled the following criteria: Subjects were 12 aged between 18 and 70 years and underwent at least 7 to maximum 13 (10 ± 3 days) days 13 of fasting. Two blood samplings at the beginning and at the end of the experiment were 14 conducted. One week prior as well as during the fasting period the intake of micronutrient 15 supplements was advised to be stopped, except for magnesium supplementation. Clinical 16 experience showed that magnesium intake during the fasting course seems to protect 17 against muscle cramps that possibly could be provoked by the recommendation to drink 18 sufficient liquids during fasting. Subjects were excluded when they had a predefined 19 contraindication to fasting as described in the guidelines of fasting therapy (Wilhelmi de 20

Toledo et al., 2013) like cachexia, anorexia nervosa, advanced kidney, liver or 21 cerebrovascular insufficiency, dementia or other chronic psychiatric diseases, as well as 22 pregnant or lactating women. Furthermore, subjects who could not follow the study 23 procedure due to an inability to speak German, English or French, or subjects that were 24 participating in another study, were also excluded. Altogether, 37 subjects did not meet the 25 J o u r n a l P r e -p r o o f inclusion criteria, and 35 subjects declined to participate. One subject terminated the study 1 earlier as defined in the protocol due to low hemoglobin and sodium levels. a 600 kcal diet of either rice and vegetables or fruits. To initiate the fasting period, a laxative 7 (20-40 g Na 2 SO 4 in 500 ml water) was administered. During fasting, an enema was applied 8 every other day to remove intestinal remnants and desquamated mucosal cells. A calorie 9

intake of ~250 kcal/day was ensured by the daily intake of 250 ml freshly squeezed organic 10 juice at midday, and 250 ml of vegetable soup in the evening, as well as 20 g honey per day. 11

The subjects were advised to drink daily at least 2-3 l of water or non-caloric herbal teas. A 12 stepwise reintroduction of food with an ovo-lacto-vegetarian organic diet from 800 to 1600 13 kcal/day followed the fasting period. was done at the 10 ± 3 fasting day (time-point #2, post). Subjects' height was measured 20 with seca 285 (Seca, Hamburg, Germany) and the waist circumference was assessed with a 21 measuring tape, placed halfway between the lowest rib and the iliac crest. Body weight was 22 measured daily (Seca 704/635, Seca, Hamburg, Germany) between 7:00 am and 9:00 am by 23 trained nurses, while subjects were lightly dressed. Additionally, blood pressure and heart 24 rate were measured on the non-dominant arm after a rest, while subjects were seated (boso 25 Carat professional; BOSCH +SOHN GmbH u. Co. KG). 26

Subjects self-reported their energy level on a numeric rating scale from 0 (weak) to 10 1 (powerful) before and after fasting. Furthermore, the well-being index (WHO-5) was self-2 assed by answering five statements scored from 0 (at no time) to 5 (all of the time). After 3 building the sum of the five scores and multiplying it with 4 the WHO-5 was given as a 4

percentage between 0 % and 100 % (Bech, 2004) . Possible adverse events were 5 documented in a report form by the medical staff. Germany) (in 2.5 N HCl) for the sample, or 500 μl of 2.5 N HCl for the blank, were added to 9 the pellet. The samples were incubated in the dark at room temperature for 1 h with 10 intermittent vortexing every 15 min and were centrifuged at 15,000 x g for 5 min at 4˚C. 11

The supernatant was discarded and 1 ml of 10% TCA was added, vortexed and centrifuged 12 at 15,000 x g for 5 min at 4˚C. The supernatant was discarded and 1 ml of ethanol-ethyl 13 acetate (1:1 v/v) was added, vortexed and centrifuged at 15,000 x g for 5 min at 4˚C. This 14 washing step was repeated twice. The supernatant was discarded and 1 ml of 5 M urea (pH 15 = 2.3) was added, vortexed and incubated at 37˚C for 15 min. The samples were 16 centrifuged at 15,000 x g for 3 min at 4˚C and the absorbance was read at 375 nm. The 17 calculation of the protein carbonyls concentration was based on the molar extinction 18 coefficient of DNPH. Total plasma protein was assayed using the Bradford protein assay. 19

The superoxide anion radical-scavenging ability of plasma was measured using a slightly 20 modified protocol of Ak and Gülçin, (Ak and Gülçin, 2008) . In this method, superoxide anion 21 In the reducing power assay, a plasma sample was dissolved in phosphate buffer (0.2 M, pH 4 = 6.6) at different concentrations. An aliquot (10 μl) of the sample solution was added to 5 490 μl of 1% potassium ferricyanide and incubated at 50° C for 20 min. The samples were 6 cooled on ice for 5 min. Then, 250 μl of 10% TCA was added and the samples were 7 centrifuged (1700g, 10 min, and 25° C). Subsequently, 250 μl of dH 2 O and 50 μl of 0.1% 8 ferric chloride were added to the supernatant and the samples were incubated at RT for 10 9 min. The absorbance was monitored at 700 nm (Yen and Duh, 1994) . incubated at 95° C for 10 min. Then, the samples were cooled on ice for 5 min, centrifuged 16 (1700g, 10 min, and 25° C), and the absorbance was monitored at 520 nm. In each 17 experiment, the sample without H2O2 was considered as blank and the sample without The free radical-scavenging activity of the samples was also determined by ABTS radical 20 cation (ABTS•+) decolorization assay as previously described by Cano et al. (Cano et al., 21 2000) , with some modifications. In brief, ABTS•+ radical was produced by mixing 2 mM 22 ABTS with 30 μM H 2 O 2 and 6 µM horseradish peroxidase (HRP) enzyme in 50 mM PBS (pH = 23 7.5). Immediately, following the addition of the HRP enzyme, the contents were vigorously 24 mixed, incubated at room temperature in the dark and the reaction was monitored at 730 25 nm until stable absorbance was obtained. Subsequently, 10 µl of plasma were added in the 26 reaction mixture and the decrease in absorbance at 730 nm was measured. In each 1 experiment, the tested sample alone containing 1 mM ABTS and 30 µM H 2 O 2 in 50 mM PBS 2 (pH = 7.5) was used as a blank, while the formed ABTS•+ radical solution alone with 10 µl 3 H2O was used as a control. 4

The determination of SOD activity in RBCL was based on the method of nitroblue tetrazolium 5 salt (NBT) as described in the study by Oberley and Spitz (Oberley and Spitz, 1984) . More 6 specifically, this assay included a negative control which was prepared by mixing 800 µl of 7 

The aim of this study was to understand the interplay between markers of the redox status 3 and the metabolic changes observed during a long-term fasting in 109 individuals. For this 4 purpose, 12 markers of the redox status were evaluated (Figure 1 ). This included markers of 5 oxidative stress/damage (TBARS, carbonyls), as well as markers of the antioxidant capacity 6 (GSH, TAC, superoxide scavenging capacity, reducing power, hydroxyl radical scavenging 7 capacity, ABTS•+ radical scavenging capacity, as well as GPx, SOD and GR activities). The 8 mean age of the study population was 57 years and 62 % of the participants were female 9

( The heatmap displays the correlations between the redox parameters (rows) and the 2 metabolic parameters (columns). Dendrograms shows the relationships between the 3 different parameters evaluated using the hierarchical clustering of Euclidean distance. The 4 colour scale shows the coefficient of correlations. Statistical significance was tested (* 5 p<0.05 ; ** p< 0.01 ; *** p< 0.001). 2019), and show that these metabolic improvements are reproducible. In addition, well-10 being improvements were confirmed with new methods (i.e. energy levels, Figure 3H ; 11 WHO-5 index, Figure 3I ). Next, the changes in 12 biomarkers of antioxidant systems caused 12 by long-term fasting were studied ( Figure 3J to 3U). Gender differences were limited to 13 TBARS levels which were higher at baselines in males. Gender did not have a substantial 14 effect on the effects of fasting on redox parameters. Glutathione levels ( Figure 3J ) and its 15 regulation through GR ( Figure 3M ) and GPx activities ( Figure 3N ) were unchanged. However, 16 the antioxidant capacity reflected by the TAC ( Figure 3L ) and the reducing power ( Figure 3U ) 17 were increased. This ultimately increased ROS scavenging potential as showed by an 18 increase hydroxyl radical scavenging activity ( Figure 3R ) and an increased ABTS scavenging 19 ( Figure 3S ). This improvement of the antioxidant capacity was concomitant to a decrease in 20 markers of oxidative stress/damage TBARS ( Figure 3T ). The model was validated on an independent test set consisting of 44 patients (40% of the 2 data), with a statistically significant correlation between the predicted LDL decrease and the 3 actual LDL decrease (p= 0.016). It was also evaluated if metabolic parameters at baseline 4 (described in Figure 2 ) could predict the changes in lipid peroxidation during fasting but 5 failed to establish a model with good predictive abilities. It was demonstrated that long-term fasting can increase the endogenous production of a 25 number of antioxidant molecules that act protectively against free radicals. These findings 26 are in contrast to the preconceived idea that the antioxidant reserves would decrease during 1 fasting due to a lack of absorbed micronutrients with known antioxidative roles (e.g. vitamin 2 E and B, zinc, selenium) after the cessation of food intake. When exogenous antioxidants are 3 missing, endogenous antioxidants are sufficient to maintain homeostasis. This includes uric 4 acid and bilirubin, two important endogenous antioxidants (Ames et al., 1981; Sedlak et al., 5 2009 ). Although bilirubin was not measured in this study, we measured the increase in uric 6 acid and its association with TAC, that was discussed in detail in the previous article 7 Besides showing that long-term fasting improves the redox status and metabolic health 1 indicators, an interplay between these parameters was described. The strongest correlation 2 observed was between uric acid levels and the TAC, which was expected, since much of the 3 TAC is due to the circulating uric acid (Janaszewska and Bartosz, 2002) . However, the most 4 important biological correlation was observed between TBARS and lipid metabolism. In 5 general, TBARS decreased significantly after long-term fasting, as did total cholesterol, LDL 6 and triglyceride levels, with a parallel decrease in waist circumference and BMI. Higher lipid 7 peroxidation levels at baseline, reduced the ability of long-term fasting to normalize 8

Our machine learning algorithm confirmed that the levels of TBARS is a reliable predictor of 10 how well a person will respond to long-term fasting. This model was trained and evaluated number of studies are showing that response to diet is personal and that these differences 20 can affect disease susceptibility (Zeevi et al., 2015) . This can be due to differences in 21 lifestyle, genetics, or even gut microbiome composition (Berry et al., 2020) . We suggest in 22 this study that the response to fasting can also be individual and showed that it can depend 23 on the baseline antioxidative status. Factors driving these differences would have to be 24 evaluated in further studies including measurement of gut microbiome composition, as it was 25 repeatedly showed to be an important factor driving personal susceptibility to disease 26 (Tierney et al., 2020) . In addition, it was also shown that the gut microbiome dramatically 27 changed during fasting, and were correlated to changes in energy metabolism ( Since lipids are used as energy substrates during fasting, it is reasonable to assume that the 3 decrease in lipid levels, could partly lead to decreased lipid peroxidation. We documented in 4 an unpublished study that the more atherogenic, small dense LDL particles diminished 5 significantly after 14 fasting days. Since they are the most oxidizable, it seems logical that 6 the lipid peroxidation levels decrease (Chaudhary et al., 2017) . Furthermore, HDL is the 7 greatest antagonist of lipid oxidation and it was documented that total cholesterol/HDL 8 increased significantly during long-term fasting (Brites et al., 2017) . 9 10

The results of our study conclude that fasting improves oxidative stress indicators by 12

increasing the antioxidant capacity of the blood plasma through the increase in TAC, 13 reducing power, ABTS radical scavenging capacity and hydroxyl radical scavenging capacity. 14 At the same time, fasting reduces lipid peroxidation and improves various metabolic 15 indicators, especially lipids. Furthermore, total cholesterol/HDL improved significantly. 16

Although the prognostic ability of the redox status to predict metabolic changes caused by 17 long-term fasting will have to be confirmed on a larger cohort, the remarkable performance 18 of this model considering the relatively small number of patients suggest that the antioxidant 19 status is a crucial determinant of the normalization of lipid levels during long-term fasting. 20

Altogether, our results show that the effects of long-term fasting on lipid metabolism are 21 influenced by the redox status, and that these effects can be forecasted based on the levels 22

of redox parameters before fasting through the use of machine learning approaches. We 23 recommend that long-term fasting strategies should be personalized and adjusted to the 24 metabolic and antioxidative baseline status of the subjects. 

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• Long-term fasting increases the antioxidant capacity and decreases oxidative damage • It improves the metabolic health status • High TBARS levels at baseline limit the LDL reduction during long-term fasting • The antioxidant status is related with the lipid lowering effect of long-term fasting

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work

☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data