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Because the latter passage describes a classical fast to coincide with a period of mourning, a modern day Daniel Fast is most commonly followed for 21 days [ 41 ].

The purpose of the present study was to investigate the effects of a 21 day Daniel Fast on biomarkers of oxidative stress, antioxidant status, and nitric oxide in human subjects. To our knowledge, this was the first scientific investigation of the Daniel Fast in relation to markers of oxidative stress.

Six female subjects were vegetarians. One female subject had a diagnosis of well-controlled type II diabetes and one male subject had a history of coronary artery bypass graft surgery.

Subject characteristics are presented in Table 1 and medication use of subjects is presented in Table 2. No restrictions were placed on subjects regarding body mass necessary for enrollment. Hence, the BMI of subjects ranged from All subjects were nonsmokers. Thirty-four subjects were classified as exercise-trained, performing 1.

Based on the above information and aside from the overweight or obese status of 22 subjects, most were considered "relatively" healthy and active.

Medical and activity data were obtained via physical assessment and questionnaires. Each subject was informed of all procedures, potential risks, and benefits associated with the study through both verbal and written form in accordance with the approved procedures of the University Institutional Review Board for Human Subjects Research H All subjects signed an informed consent form prior to participating.

During the initial visit to the laboratory, subjects completed all paperwork and had hemodynamic and anthropometric variables measured. Subjects were provided detailed instructions for the fast and were given food logs for dietary recording.

Subjects were provided an outline of foods that are allowed on the fast as well as commonly consumed foods that are disallowed. A basic recipe guide was also provided. It is important to note that subjects purchased and prepared their own food during the entire fast.

The investigators simply provided initial instruction and guidance when needed. In contrast to studies in which subjects are provided with all meals in an attempt to enhance compliance, our method placed the responsibility of food purchase, preparation, and intake directly on the subject.

We believe that this approach has greater practical application with regards to a dietary intervention. Following this initial screening visit, subjects returned to the lab weeks later to have baseline assessments performed and to begin the 21 day fast.

Outcome variables were measured before baseline: day 1 of the fast and after the fast day All data collection was done in the morning hours am and with subjects in a 12 hour post-absorptive state.

Following collection, blood collected in tubes containing ethylenediaminetetraacetic acid EDTA was immediately separated to plasma by centrifugation at g for 15 minutes at 4°C.

Blood collected in tubes containing no additives was allowed to clot at room temperature for 30 minutes and then separated to serum by centrifugation at g for 15 minutes at 4°C.

Plasma and serum samples were immediately stored in multiple aliquots at °C until analyzed. Malondialdehyde was analyzed in plasma following the procedures of Jentzsch et al. Specifically, 75 μL of plasma was added to microcentrifuge reaction tubes with the addition of 3 μL of butylated hydroxytoluene in methanol to minimize ex vivo lipid peroxidation.

Samples and reagents were incubated for 60 minutes at 60°C. Following incubation, the reaction mixture was transferred to a microplate, and the absorbance was read using a spectrophotometer at both and nm to correct for baseline absorption.

Malondialdehyde equivalents were calculated using the difference in absorption at the two wavelengths. Quantification was performed with a calibration curve using tetramethoxypropane in a stabilizing buffer. Hydrogen peroxide was analyzed in plasma using the Amplex Red reagent method as described by the manufacturer Molecular Probes, Invitrogen Detection Technologies, Eugene, OR.

In the reaction mixture, H 2 O 2 , in the presence of horseradish peroxidase, reacts with Amplex Red reagent to generate the red-fluorescence oxidation product, resorufin. Quantification was performed with a calibration curve. Thawed plasma samples were centrifuged at 10,g for 5 minutes in a refrigerated centrifuge 4°C.

Following the addition of nitrate reductase co-factor to each diluted sample, nitrate reductase was added and the mixture was incubated for three hours to allow for the full conversion of nitrate to nitrite.

Greiss reagent was then added, which converts nitrite into a deep purple azo compound. The absorbance was then detected photometrically at nm. Antioxidant capacity was analyzed in serum via the TEAC assay using procedures outlined by the reagent provider Sigma Chemical, St.

Louis, MO. Antioxidant capacity was also analyzed in serum following a fold dilution via the Oxygen Radical Absorbance Capacity ORAC assay using procedures outlined by the reagent provider Zen-Bio, Inc. It should be noted that several methods are available to assess the " total" antioxidant capacity of blood.

These include the TEAC assay which appears to be primarily influenced by urate , the ORAC assay, the ferric-reducing ability of plasma FRAP assay, and the total radical-trapping antioxidant parameter TRAP assay.

Of these, ORAC and FRAP have been noted to be well-correlated, while TEAC has little correlation with either ORAC or FRAP [ 45 ]. Therefore, we chose to include more than one antioxidant capacity marker, which has been suggested previously [ 46 ].

All subjects were instructed to maintain their normal diet until they began the fast. During the seven days immediately prior to the fast, subjects recorded all food and beverage intake.

Subjects were also asked to record food and beverage intake during the final seven days of the fast. Records were reviewed with each subject for accuracy and then analyzed using Food Processor SQL, version 9.

Regarding physical activity, subjects were instructed to maintain their normal habits during the entire study period with one notable exception: subjects were instructed not to perform strenuous exercise during the 48 hours immediately preceding the two assessment days.

Subjects were also instructed to refrain from alcohol consumption during the fast, as well as during the 48 hours that preceded day 1.

Pre and post assessment data aside from those indicated within the present manuscript - including anthropometric, blood lipid, and other hematological measures - are presented elsewhere [ 41 ].

Biochemical data were analyzed using a paired t-test. In addition, sex, exercise training status trained or untrained , and weight status normal weight, overweight, or obese were used in an analysis of variance ANOVA to determine the influence of these variables on the changes in our biomarkers from pre to post fast.

Analyses were performed using JMP statistical software version 4. The data are presented as mean ± SEM. The overall response of subjects to the fast was positive.

Subjects noted that they enjoyed the ad libitum nature of the fast as well as the wide variety of food choices. Most subjects reported that they would continue implementing many components of the eating plan into their usual diets.

Self-rated compliance to the fast was reported to be Although previously presented elsewhere [ 41 ], it should be noted that body weight was reduced by an average of 2.

Waist and hip circumference measures were decreased by approximately 1. As expected, several differences existed in dietary intake from pre to post fast.

Data are presented in Table 3. Related to the bloodborne variables, we were able to obtain blood samples from 42 of the 43 subjects. Therefore, bloodborne data presented within are specific to a sample of 42 subjects 13 men and 29 women.

Plasma malondialdehyde A and plasma hydrogen peroxide B of men and women before and after a 21 day Daniel Fast. Values are mean ± SEM. Results from the present study indicate that a 21 day Daniel Fast increases NOx and improves selected measures of antioxidant status and oxidative stress.

The fast was well-tolerated, as evidenced by a self-rated compliance of To our knowledge, this is the first scientific investigation to assess the impact of the Daniel Fast on biomarkers of oxidative stress. Moreover, this study extends the findings of other plant-based diets by documenting the impact of a strict vegan diet on multiple measures of oxidative stress and antioxidant capacity.

It is important to note that our findings are in reference to relatively healthy, young to middle age men and women age range: years , with a wide BMI range Interestingly, we noted similar findings across subject groups when comparing data from pre to post fast.

However, while the overall response to the fast was not different between subject groups i. women, exercise- trained vs. untrained, obese vs. overweight vs. normal weight , it should be noted that some overall differences across subject groups did occur with regards to MDA and TEAC, and to a lesser extent H 2 O 2.

For example, MDA was lower for women 0. With regards to the antioxidant capacity in exercise-trained compared to untrained subjects, mixed results are available; some supporting our finding of lower antioxidant capacity in trained individuals [ 50 ] and some refuting this [ 51 , 52 ].

Those findings in support of our data may be linked to the acute increase in ROS experienced by exercise-trained individuals, which may partially deplete endogenous antioxidants over time [ 50 ].

Of course, in accordance with the principle of hormesis [ 53 ], the opposite effect may occur as a result of acute elevations in ROS. This is an area of investigation that requires further attention and is likely dependent on the volume and intensity of the exercise sessions, coupled with post exercise recovery strategies.

This reduction is greater than those noted in studies incorporating other plant-based diets [ 29 , 54 ], which may be due to the omission of many other food components e. Collectively, these findings indicate that a 21 day Daniel Fast can reduce blood oxidative stress biomarkers, which may potentially be associated with improved health [ 55 ] and increased longevity [ 56 ].

Regarding the discrepancy in the TEAC and ORAC findings, it has been noted that these two measures are not well correlated [ 45 ] despite the fact that both provide an indication of antioxidant capacity.

When investigating changes in other biomarkers for these subjects from pre to post fast, they followed the overall findings of the group e. Further study pertaining to the effects of a Daniel Fast on serum TEAC, ORAC, and other antioxidant and oxidative stress biomarkers is needed.

Nitric oxide is well-recognized as an important signaling molecule involved in numerous biological processes including, but not limited to, smooth muscle relaxation, attenuation of smooth muscle cell proliferation, inhibition of platelet and leukocyte aggregation, and immune defense [ 33 , 34 ].

As such, it is possible that an increase in NOx may be associated with improved cardiovascular health [ 58 ]. It is interesting to note that the changes highlighted above were observed in a sample of subjects who, for the most part, were considered to be relatively healthy at the onset of the fast.

Regardless of weight classification, all subject groups demonstrated improvements in the measured variables as indicated in the Results section.

Considering that our subjects may have had less "room for improvement" as compared to many individuals beginning a new dietary program, it is possible that we may have observed more robust effects if we had recruited a homogenous sample of individuals who were classified as unhealthy to begin with e.

Also of interest, we noted similar mean changes between vegetarians and omnivores in our sampled biomarkers.

These results suggest that the exclusion of meat from the diet is not the only dietary factor responsible for altering oxidative stress, antioxidant capacity, or nitric oxide metabolites.

It is likely that multiple dietary factors - particularly the exclusion of additives, preservatives, and high-glycemic, processed carbohydrate foods, in addition to the focused inclusion of fresh and frozen fruits and vegetables - positively affected the biochemical variables measured in the present design.

Related to the above, while it is obvious that the change in subjects' dietary intake is responsible for our findings, we are uncertain as to which specific variables had the greatest impact.

Because we have included multiple variables in this study, in addition to the measurement of several dietary variables, a complete analysis of predictor and response variables deserves attention. As we have recently completed an additional study of the Daniel Fast, greatly increasing the needed sample size for multiple regression analysis of this scope, this is now the focus of a separate manuscript.

Because variables such as body mass, sex, exercise training status, and dietary variables, as well as changes in certain anthropometric and biochemical variables e. With only a cursory view, we believe that the combination of decreased calorie and saturated fat intake, together with an increase in nutrient- and fiber-rich fruit, vegetable, and whole grain intake, contributed to our findings.

It is also possible that the elimination of food additives, preservatives, and processing agents, in addition to the decrease in protein intake methionine included [ 59 ] , could be responsible for our findings.

Considering our collective results, it is important to point out some limitations of this work. First, while it is possible that our data may have clinical implications, particularly for individuals prone to oxidative stress-related ill-health and disease, we cannot state this with confidence.

The majority of our subjects did not have known disease and actually had relatively low levels of oxidative stress biomarkers at the start of the fast. Although elevated oxidative stress appears related to disease progression [ 8 ], it is unknown what impact a slight reduction in oxidative stress would have on relatively healthy individuals who do not have elevated oxidative stress to begin with.

Further work with the inclusion of subjects with known cardiovascular and metabolic disease, in particular those with elevated oxidative stress biomarkers, would be needed to determine the clinical relevance of these potential changes.

This could be done with the inclusion of healthy subjects within the design, to allow for the comparison of this fast between those with known disease and those who are without disease.

It is possible that the findings may differ between the two groups. Second, because our protocol merely followed subjects over a 21 day period, long-term compliance to this eating plan needs to be determined.

While it is possible that more favorable results may be observed with a longer intervention period, subjects would first need to have success in compliance for such results to be realized. Third and related to the above, disease progression involves multiple components and typically follows an extended time course.

Therefore, future studies should be longer in length- possibly several months in duration- in order to more completely determine the effects of this eating plan on biomarkers of human health. Melhorn SJ, Krause EG, Scott KA, Mooney MR, Johnson JD, Woods SC, et al.

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CTRL: 0. T 0. CTRL T0: 0. Abundance of the genus Bacteroides family Bacteroidaceae, phylum Bacteroidetes, Figure 3F increased in OB in response to the dietary treatment T0: 2.

T However, all phosphate values were within the normal range. Table 4. There was instead no correlation between CRP and antioxidant capacity biomarkers in OB. Table 5. Full details of weight loss outcomes of OB dogs are given in Table 6. All dogs lost weight, and 3 out of 16 reached their target weight.

The median overall percentage of weight loss was The median overall rate of weight loss, expressed as a percentage of SBW lost per week, was 0. The median overall energy intake, expressed as kcal of ME per kg 0. Table 6. Table 7. Similarly, alpha-and beta-diversity indices, as well as selected bacterial populations detected by qPCR were not influenced by caloric restriction Table 8 ; Figure 4A ; Supplementary Table 4 , respectively.

Also, DI was not affected by caloric restriction in OB dogs Figure 4B. Table 8. Figure 4. A PCoA plot based on the unweighted UniFrac distance metric of the fecal microbiota of obese dogs. Negative values the grey area are indicative of a health microbiota, while values between 0 and 2 are considered equivocal.

Among the five most abundant bacterial phyla recovered in OB dogs in Phase 1, only Actinobacteria showed a shift in their community composition during caloric restriction Figure 5 : in particular, the median abundance of Actinobacteria was lower at T than as at T30 0.

Figure 5. Moreover, a tendency to a decrease was observed between T30 vs. T and T vs. T for Proteobacteria T 2. T 1. Differences in terms of bacterial class, order, family, genera and species in OB dogs during caloric restriction, are available in Supplementary File 1 , while significative results are presented in Figure 6.

At the family level Figure 6A , fecal microbiota composition of OB dogs was characterized by an increase of the family Coriobacteriaceae after the second half of the caloric restriction period T 0.

The same trend was observed at genus and species levels for the genus Collinsella T 0. stercoris T 0. T 8. Figure 6. All serum biochemical parameters are presented in Supplementary Table 5 , and significant changes found in OB dogs during caloric reduction are showed in Figure 7.

Figure 7. Significant changes in the concentration of selected serum metabolites. Caloric restriction did not result in any changes in inflammatory and oxidative status biomarkers Table 4 ; moreover, there was no correlation between biomarkers of antioxidant capacity TEACH, TEACA and Thiol and inflammatory markers CRP and Hp; Table 5.

Obesity is the most commonly occurring metabolic disease in dogs, causing severe concurrent clinical conditions, such as functional Mosing et al.

When it comes to an excess of body fat in dogs, the protracted imbalance between food intake and energy expenditure must be considered Courcier et al. Such factors might have affected energy expenditure in client-owned OB dogs in this study, which had different age and neuter status.

However, the daily activity level of OB dogs was not assessed in the present study. Excess body fat is also related to endocrine and inflammatory profile changes in dogs with insulin resistance, modification of adipokine patterns, and lipid metabolism changes being reported Antonio Brunetto et al.

In this study, we observed significant differences in TT4 and acute phase proteins CRP and Hp concentrations between a population of 16 client-owned obese and 15 lean dogs. Thyroid hormones are among the main factors involved in the regulation of energy expenditure Reinehr, ; Mullur et al.

Although thyroid function is usually normal in obese dogs, they are frequently tested for hypothyroidism before starting a weight-loss protocol. Differences in thyroid function have been previously evaluated in obese and lean dogs showing that obese dogs have higher total triiodothyronine TT3 and TT4 serum concentrations than lean animals, even if without clinical importance Daminet et al.

In accordance with these findings, in the present study, TT4 concentrations of OB dogs before caloric restriction tended to be higher than in CTRL animals, but remained within the normality range.

During two previous studies Daminet et al. Similarly, fasting and weight loss have been associated with a decrease of thyroid hormone levels in humans, probably as a result of the decline of circulating leptin Kok et al.

In contrast with these findings, in the present study, TT4 serum concentrations unexpectedly significantly increased during caloric restriction, albeit remaining within the range of normality.

In humans, the interaction between total calories and carbohydrate intake on thyroid hormone response has been previously investigated.

An increase of TT4 was also reported in humans consuming a low-carbohydrates diet, resulting in a decrease of fat mass and an increase in lean mass Volek et al. A previous study reported a decrease TT3 and no change in TT4 in response to reduced carbohydrate intake Mathieson et al. The significant increase in TT4 observed by Volek et al.

However, this speculation should be made with caution since, as in the previously cited study, TT3, free T3, or free T4 were not investigated in the present study. In the present study, serum concentrations of creatinine decreased during caloric restriction, in accordance with what was previously observed in Beagle dogs during a weeks weight loss study, where only a reduction of body fat with no loss of lean mass was confirmed Salas-Mani et al.

Serum creatinine concentration is used as a marker of muscle mass in dogs, beyond kidney function, with higher concentration being measured in dogs with great muscle mass, such as sighthound dogs, while lower concentrations are seen in small dogs Braun et al.

In the present study, we did not assess changes in body composition by dual-energy X-ray absorptiometry DEXA , so that some degree of lean mass loss cannot be completely ruled out. However, considering the slow rate of weight loss that we recorded, and according to what has been observed by other authors German et al.

In line with our results, obese dogs can show higher serum total protein concentrations than lean dogs Piantedosi et al. However, in the present study, we did not observe any differences in the serum concentrations of urea, a biochemical marker of muscle and protein catabolism Gunst et al.

Progress in human obesity indicates that adipose tissue plays a major role concerning metabolism and inflammation, and it is involved in the release of inflammatory cytokines influencing systemic inflammatory processes Wozniak et al.

Advances in canine obesity is still in its early stage; however, a decrease of inflammatory markers, such as CRP and Hp, has been observed after weight loss in previous studies, suggesting that also dogs may suffer from a compelling association between low-grade inflammatory state and obesity German et al.

Interestingly, experimentally induced overfeeding in laboratory dogs failed to stimulate an increase of inflammatory markers Tvarijonaviciute et al.

To date, only two studies have explored differences in systemic concentrations of pro-inflammatory markers and cytokines in obese and lean dogs, with a lack of evidence of clear differences Veiga et al.

In the present study, we observed higher serum concentrations of acute-phase proteins CRP and Hp in obese dogs compared to lean subjects and these findings seem to be in accordance with what has been previously observed in some human studies Das, ; Ste¸pień et al.

However, serum concentrations of pro-inflammatory markers were not affected by caloric restriction in this study, and this finding was in agreement with previous reports in dogs, regardless of the type of obesity, short-term experimentally induced or long-term spontaneous disease Tvarijonaviciute et al.

This finding may indicate that canine obese-related inflammatory condition is not responsive to changes in energy balance, despite the fact that the degree of weight loss that we observed was in line with the weight loss degrees that are known to improve inflammatory condition in obese humans Forsythe et al.

Butyrylcholinesterase BChE is an enzyme secreted by the liver under the stimulation of free fatty acids flux originating from adipose tissue Cucuianu et al. In our study, BChE did not differ between obese and lean dogs and was not affected by caloric restriction, in contrast with previous research in canine obesity; in fact, higher BChE activity was assessed in dogs with obesity-related metabolic dysfunction and in those where obesity was induced Tvarijonaviciute et al.

Contrary to previous findings, however, our study was not conducted in experimental conditions, and the use of antiparasitic collars were not investigated. In fact, some antiparasitic treatments act as acetylcholinesterase inhibitors, and could therefore have affected the levels of BChE in our dogs Birdane et al.

Obesity has also been associated with oxidative stress, as a result of an imbalance between oxidant and antioxidant molecules Sánchez-Rodríguez and Mendoza-Núñez, ; moreover, the oxidant components may increase the risk for systemic low-grade chronic inflammation, which may be related to obesity-associated metabolic disorders Trayhurn, ; Donath and Shoelson, ; Lumeng and Saltiel, Interestingly, in our study, OB dogs showed higher markers of total antioxidant capacity TAC , such as TEACH and Thiol, than lean dogs.

A possible interpretation for this finding might be a compensatory system carried out by obese dogs to restore homeostatic balance by enhancing endogenous antioxidants levels, as previously described in obese humans, where a positive correlation between CRP and TAC was observed Petelin et al.

Interestingly, we observed that TEACH and Thiol were inversely correlated with Hp in obese dogs before the caloric restriction, suggesting that a high degree of low-grade inflammation may result in the partial loss of antioxidant capacity. Nonetheless, the association between antioxidant status and canine obesity remains intriguing and warrants further research.

It is currently well defined that the use of a low-energy, high-protein, high-fiber diet is the most effective strategy to favor weight loss in dogs, reducing voluntary food intake, minimizing muscle loss and attenuating signals of hunger Blanchard et al.

The dietary intervention not only helps dogs to lose weight appropriately, but may also impact the gut microbiota and the connection existing between this complex ecosystem and the host; in fact, modulation of the macronutrient content of the diet may affect the intestinal bacteria which in turn may influence the host in many ways, mainly through the release of metabolites that have been acknowledged as beneficial for the host gastrointestinal tract and beyond it.

Among gut microbial metabolites, VFA mainly acetate, propionate, and butyrate are produced by anaerobic bacteria in the colon and distal small intestine through the fermentation of resistant starch, dietary fiber, and other low-digestible polysaccharides Alexander et al.

In our study, fecal VFA concentrations did not differ between obese and lean dogs and were not affected by dietary treatment. It has to be remembered that different fecal sample collection, preservation, and processing methods may all be significant sources of variation in the quantification VFA in feces Sowah et al.

Nevertheless, the potentially higher production and turnover of VFA in obese mice could be related to differences in microbiota composition and function, with an increased potential for energy harvest among obese Turnbaugh et al. The fecal VFA concentrations have been found to be reduced in obese humans after weight loss Duncan et al.

In line with the reported findings in humans, in a study with obese dogs Kieler et al. Biogenic amines include gut microbial metabolites such as cadaverine, putrescine, spermidine, and spermine, and are putrefactive compounds produced by intestinal bacteria from the fermentation of undigested amino acids; biogenic amines are required for cells growth and differentiation, and for the synthesis of DNA, RNA, and proteins Delzenne et al.

Also, biogenic amines have been correlated in humans with increased fecal odor and increased incidence of colon cancer Johnson, In this study, fecal biogenic amines of OB and CTRL dogs were determined, and fecal spermine concentration resulted decreased in CTRL after dietary treatment; it has been previously reported that fecal biogenic amines concentrations may be linked to fecal microbiota Matsumoto and Benno, In line with our results, other studies have shown that the diet can impact the fecal biogenic amines concentrations in dogs, as fiber addition decreased fecal proteolysis and biogenic amines production Jackson and Jewell, , while biogenic amines increased in feces of working dogs fed a grain-free high-protein petfood Chiofalo et al.

The increase in dietary fiber intake is, therefore, the most likely explanation for the reduction of fecal spermine concentration observed in this study in CTRL dogs after dietary treatment, and, even though not significant, a decrease in fecal spermine concentration was also observed in OB dogs fed the same diet.

The effects of spermine on host health remain controversial so far; in fact, spermine seems to possess significative physiological activity and toxicity in mice, being strictly controlled by both colonic microbiota and colonocytes Matsumoto et al.

Results from studies conducted with mouse models suggest that biogenic amines metabolism could be dysregulated in the presence of obesity and other metabolic disorders, with the result of impaired glucose regulation, and lipid and energy homeostasis Ramos-Molina et al.

To date, this is the first study evaluating fecal biogenic amines in obese dogs, and even though their concentrations were affected by neither nutrition status nor caloric restriction, further research is warranted in order to clarify the effects and influence of microbial metabolites in canine obesity.

Studies on the composition of the gut microbiota have provided evidence of existing differences in the bacterial taxa found in lean and obese dogs, with sometimes controversial results Handl et al. In this study, Firmicutes were the dominant phylum in obese as well as in lean dogs, as previously reported in a study involving pet dogs Handl et al.

However, when obesity was induced in dogs, the proportion of Proteobacteria was increased and Firmicutes were decreased, with the result of Proteobacteria being the dominant phylum in the obese animals Park et al. In the present study, we did not observe any difference in the proportions of the major phyla between obese and lean dogs; conversely, inconsistently with our results, two previous researches showed that Actinobacteria were more abundant in obese than in lean pet dogs Handl et al.

In this study, the dietary treatment had a significant impact on the fecal microbiota composition of OB dogs, mainly within the predominant phyla Firmicutes and Bacteroidetes. For instance, the fecal abundance of Bacteroides spp.

increased, and Firmicutes decreased, in obese in response to the diet fed at maintenance requirement, while no difference was seen between obese and lean dogs; this finding appears to be in agreement with what was observed in a previous study that aimed to evaluate the effects of diets dissimilar in macronutrient composition on dogs of diverse body conditions, finding no differences in bacterial abundances among groups Li et al.

The genus Bacteroides consists of bile-tolerant microorganisms associated with the consumption of diets rich in protein and fat in humans and dogs De Filippo et al. Bacteroides has been negatively correlated with energy intake and adiposity in humans and dogs Yatsunenko et al.

uniformis CECT was able to reduce weight gain and serum triglycerides and cholesterol concentrations in mice fed high-fat diets Gauffin Cano et al.

In this study, Firmicutes, which include species known to metabolize dietary plant polysaccharides and produce VFA Pilla and Suchodolski, , significantly decreased in the feces of OB dogs after dietary treatment.

Consistently with our results, in a previous study Mori et al. These findings may suggest that feeding a high-protein, high-fiber diet may play a role in the modulation of fecal microbiota in canine obesity. Conversely, in the present study, Firmicutes and Bacteroidetes were not affected by diet in lean dogs.

It has been seen that the composition of intestinal microbiota in healthy dogs can be unaffected Bresciani et al. The effectiveness of persistent changes in the intestinal microbiota induced by dietary interventions has been associated, in recent studies, to prolonged experimental set-ups, as well as to the shift to diets that were extremely different in terms of macronutrients composition Mori et al.

However, obese dogs do not seem to show a clear state of intestinal dysbiosis: in fact, in the present study, the DI of obese dogs remained within the established reference interval for healthy dogs both before and after caloric restriction, in accordance with results previously reported by other authors Bermudez Sanchez et al.

Similarly, a relative abundance of microbial populations, as well as diversity of the microbial community, did not differ between OB and CTRL, in accordance with results from a previous study involving a number of lean and obese colony dogs similar to ours Handl et al.

The 16S rRNA gene profiling revealed that OB dogs had a lower abundance of Erysipelotrichi, Erysipelotrichales, Erysipelotrichaceae, Eubacterium and E. biforme data not shown for class and order , compared with CTRL dogs.

The same findings were observed in a previous study which enrolled pet dogs and compared microbial abundances in obese and normal weight dogs Forster et al. Hence, Erysipelotrichaceae and Eubacterium spp. abundance seem to be negatively correlated with obesity in dogs, while a positive correlation has been described in humans, where high levels of E.

dolichum have been associated to increased visceral fat mass Pallister et al. Eubacterium spp. are associated with the production of organic acids, from carbohydrates or peptone, including butyric, acetic and formic acids Louis and Flint, ; Mukherjee et al.

In a study by Forster et al. and E. biforme and reduced concentrations of VFA. In fact, the abundance of Eubacterium spp. and other butyrate-producing bacteria in the gut is strongly correlated with VFA concentrations, and the ingestion of dietary fibers has been seen to increase VFA concentrations and abundance of Eubacterium spp.

Duncan et al. However, in the current study, fecal VFA concentrations were not affected, suggesting that the relationship between the fecal abundance of Eubacterium spp. and VFA concentrations in obese dogs needs further investigation. In a study with elderly humans, the abundance of Eubacterium spp.

was negatively correlated with CRP Ghosh et al. and lower concentrations of inflammatory markers CRP and Hp , compared with OB. Erysipelotrichaceae consists of a bacterial family that has been identified in the fecal microbiota of healthy dogs Garcia-Mazcorro et al.

Interestingly, members of this family have been shown to change in abundance in response to changes in dietary macronutrient composition; in a study performed with dogs fed either kibbles or a raw-meat based diet, Erysipelotrichaceae were positively correlated with dietary fat content and markers linked to carbohydrate fermentation such as VFA on one hand, and negatively correlated with crude protein content of the diet on the other Bermingham et al.

One possible explanation for the decrease in Erysipelotrichaceae abundance that we observed in lean dogs after the dietary treatment may therefore be associated with the high protein content of the weight loss diet.

Similarly, low levels of Eubacterium spp. have been associated with increased protein, fat, and fructose intake in humans and the consumption of sugar-rich diets in mice Duncan et al.

An expected result of weight reduction in humans is an increase in the diversity of bacterial communities. However, conflicting results have been obtained depending on the method by which the weight loss was achieved Damms-Machado et al.

In a previous study, a reduction of Bacteroidetes abundance was observed in human patients that underwent obesity surgery, while, on the contrary, an increase of this phylum was seen secondary to treatment with a low-calorie diet Damms-Machado et al.

Other studies found no alteration of Bacteroidetes after weight reduction, challenging the reversibility of reduced Bacteroidetes abundance in obese humans Remely et al. In the present study, the caloric restriction did not have a significant impact on bacterial diversity and this finding is consistent with the results from a previous research, in which the fecal microbiota of 6 obese Beagle dogs was not affected by a weeks weight loss program Schauf et al.

Conversely, in a recent study, 20 obese pet dogs showed an increase in bacterial diversity when they reached their ideal body weight; in the same study, bacterial diversity was not improved in a small group of dogs with less effective weight loss Bermudez Sanchez et al. In the study by Bermudez Sanchez et al.

However, in the study by Bermudez Sanchez et al. Collinsella spp. belong to the Actinobacteria phylum and have been described as fiber degraders and H 2 consumers, resulting in the production of mainly lactate and acetate. and C. aerofaciens have been proposed as biomarkers of obesity in humans, as they are positively associated with body mass index and insulin resistance Companys et al.

in humans was significantly reduced during a weight loss program Frost et al. In the present study, abundance of Collinsella spp.

According to our findings, several human studies have previously shown that changes in the diversity and composition of the gut microbiome rapidly occur during dietary intervention e.

On the contrary, in this study, the family Clostridiaceae was consistently associated with weight loss, increasing its abundance at the end of the study compared with trial start.

To date, results on Clostridiaceae abundance in obese dogs are not univocal, and the degree of caloric reduction may represent an important factor; in fact, in previous studies with obese dogs, the genus Clostridium showed a decrease after a weight loss program Salas-Mani et al.

On the contrary, in other studies with dogs, the use of a high-protein weight-loss diet, fed without any caloric restriction, increased Clostridiaceae Zentek et al. Our results seem to suggest that the increase in Clostriadaceae was caused by caloric restriction, rather than by the diet, because no changes in their abundance were observed during the first phase of the study.

The study has some limitations. The use of private-owned animals, rather than research dogs introduced variables, both dogs and owners related. Factors affecting populations variability included signalment and different environmental conditions.

In addition, the small study populations might not be able to show some significant differences between compared groups. However, results from the present study are plausibly more representative of the overall canine population. A second limitation was related to ethical limitations: in fact, given the requirement for sedation, DEXA scanning cannot be performed in obese dogs, therefore the changes of lean mass or fat mass, before and after the weight loss, were not assessed.

Finally, it should be also noted that fT4 was not measured neither the use of antiparasitic collars were investigated in obese dogs, therefore only careful conclusions have been formed regarding thyroid homeostasis and BChE activity in obese dogs.

The present study has provided evidence that obese dogs suffer from a subclinical inflammatory state, characterized by higher levels of some inflammatory markers and a concomitant higher total antioxidant capacity.

However, caloric restriction did not influence the inflammatory status of obese dogs. Caloric restriction resulted in a few changes in the abundance of some bacterial populations but failed to affect bacterial diversity, DI, and metabolites in obese dogs.

However, the decrease of Bacteroides spp. and the increase of Clostriadiaceae family, were the only changes consistently associated with caloric restriction and weight loss throughout the study. This study has provided insights into the involvement of the intestinal microbiota, inflammatory and antioxidant status as well as thyroid homeostasis in canine obesity.

Further research is warranted to better clarify the influence of these factors on canine obesity, since the mechanism of these connections is somewhat far from being conclusive. The datasets presented in this study can be found in online repositories. Written informed consent was obtained from the owners for the participation of their animals in this study.

FF and GB conceived and designed the study. CV, SG, and ED carried out the clinical study and collected all data.

CV, CP, RP, AT, CR, ED, CD, and CS carried out laboratory work. CV and RP performed statistical analyses. CV wrote the original draft with assistance and feedback from SG, CP, RP, JS, AT, CR, CS, EP, FF, and GB.

All authors contributed to the article and approved the submitted version. Results were presented, in part, at the Congress of the European Society of Veterinary and Comparative Nutrition, Basel, Switzerland. A, Monasterolo di Savigliano, Italy.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funder had the following involvement in the study: assistance during manuscript preparation.

The reviewer CWC declared a shared affiliation with the authors RP, JS to the handling editor at the time of review. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Rejuvenation Res. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Richard J Bloomer. MMK was responsible for coordination of the study. MMK, JFT, REC, and TMF were responsible for subject recruitment, screening and retention, data collection and entry, and blood collection and processing.

This article is published under license to BioMed Central Ltd. Reprints and permissions. Bloomer, R. et al. A 21 day Daniel Fast improves selected biomarkers of antioxidant status and oxidative stress in men and women.

Nutr Metab Lond 8 , 17 Download citation. Received : 06 December Accepted : 18 March Published : 18 March Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.

Skip to main content. Search all BMC articles Search. Download PDF. Download ePub. Abstract Background Dietary modification via both caloric and nutrient restriction is associated with multiple health benefits, some of which are related to an improvement in antioxidant status and a decrease in the production of reactive oxygen species.

Methods 43 subjects 13 men; 30 women; 35 ± 1 yrs; range: yrs completed a 21 day Daniel Fast following the guidelines provided by investigators. Results A decrease was noted in MDA 0. Conclusion Modification of dietary intake in accordance with the Daniel Fast is associated with an improvement in selected biomarkers of antioxidant status and oxidative stress, including metabolites of nitric oxide i.

Background Reducing daily caloric intake has been reported to improve overall health in both humans [ 1 ] and animals [ 2 ], and to increase lifespan in a variety of species [ 3 ]. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: MSB was supported in part by grant DK, from the National Institutes of Health NIH. Additional support came from a NIH MERIT Award GM awarded to LJR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Obesity is associated with increased oxidative stress and chronic low-grade chronic inflammation [1] — [4].

Both events contribute to metabolic abnormalities occurring in the obesity-associated metabolic syndrome [1] , [5] — [7] and play a critical role in the pathogenesis of various diseases such as atherosclerosis [8] , [9] , cancer [10] , [11] , cardiovascular disease [12] , and diabetes type 2 [13].

Recent research has shown that weight loss attenuates inflammation and leads to improvement in adipokine profiles [14] — [16].

Although associations of overweight and obesity with increased oxidative stress have been reported, the effects of weight loss on oxidative stress markers are rarely described in literature [17]. Moreover, many clinical studies and intervention studies with diets or supplements have employed single measurements of F 2 -isoprostane before and after the intervention to estimate the oxidative stress without exploring intermittent or interval changes.

To our knowledge, no prior study has investigated the association between caloric restriction CR and systemic short-term changes in markers of oxidative stress. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.

The protocol flow diagram is in Figure 1. Forty premenopausal overweight and obese women, to years-old; body mass index - BMI: Participants classified their own ethnicity according to investigator-defined options. At a screening visit, all volunteers were measured for height, weight, and waist circumference, and completed a lifestyle questionnaire.

Approximately women were screened for eligibility based on the above criteria and 40 were enrolled. All applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research, in accordance with the ethical principles of the Helsinki-II Declaration.

The trial Clinicaltrials. gov Identifier: NCT was approved by the Institutional Review Board at Vanderbilt University and all participants provided written informed consent. The study consisted of an experimental group that received a CR diet and a control group that remained on a habitual diet throughout the study.

The ratio of women in CR group to the control group was 4 to 1 allocated via blocked randomization by a statistician not involved in patient recruitment. The study was divided into a 7-day lead in period Day -6 to Day 0 , immediately followed by a day intervention Day 1 to Day 28 , and a 3-month follow-up Day 29 to Day The National Health and Nutrition Examination Survey NHANES protocols were followed for all anthropometrical measurements [18].

Body weight was measured at baseline and daily for the experimental group within 0. Daily weight measurements were not revealed to participants and served as the criterion measure for dietary adherence.

Height was measured at baseline within 0. The average of two readings was used for analysis. All measurements were performed by the same investigator.

Body composition including fat mass and fat free mass was measured at baseline Day 0 , during intervention Days 14 and 28 , and during the follow up visits Days 42 and using dual energy x-ray absorptiometry Lunar instrument Lunar Prodigy, GE Medical Systems, Madison WI, adult software v. For quality assurance and equilibration, a calibration block was scanned each morning and a spine phantom was scanned on a weekly basis the coefficient of variation was 0.

The accuracy and precision of our metabolic chamber for measuring EE as determined by routine alcohol combustion test was Participants in the CR group received individualized energy and nutrient controlled diets provided by the CRC metabolic kitchen for consumption at home.

Energy needs were calculated as the sum of resting energy expenditure REE and energy expenditure of physical activity. All participants received a multivitamin supplement daily Nature Made, Mission Hills, CA.

No restrictions were imposed on the amounts of energy-free foods ingested. Each participant received a written list of foods at daily pick-up. Any uneaten foods and any additional foods eaten by the participants were reported on sheets collected daily.

The study dietitian met with each participant weekly to discuss the diet, resolve any barriers or concerns related to food or specimen collection, and encourage compliance. Energy and nutrient intake calculations were performed using the Nutrient Data System for Research NDSR software version developed by the Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN, Food and Nutrient Database [20] , [21].

Adherence to the protocol was monitored by urinary biomarkers sodium, potassium, nitrogen [22] , [23]. Participants in the control group were asked to follow their habitual diet for the study duration and they received a multivitamin supplement daily Nature Made, Mission Hills, CA.

Their dietary intake was assessed during the intervention Days 1—28 from three dietary recalls 2 weekdays and 1 weekend day performed using automated multi-pass method and NDS-R software [24] , [25].

Daily physical activity was assessed using an RT3 accelerometer StayHealthy, Monrovia, CA, US. Participants were instructed to maintain their habitual physical activity level and wore an activity monitor on their right hip while awake for the duration of the intervention and twice for 7 days during the follow-up.

Total and physical activity energy expenditure was calculated using energy calculated from the monitor-measured movement and measured REE. Physical activity levels PAL for each monitored day were calculated by dividing total energy expenditure by REE.

Blood pressure was measured 3 times a week in the reclining position after 10 min rest with automatically inflating cuff Dynamap, General Electric, Milwaukee, WI, USA using a standard protocol.

Venous blood samples were drawn after an overnight fast at baseline and on days 1, 3, 5, 7, 14, 21, 29, 42, and , centrifuged immediately at 1 g for 10 minutes at 4°C, and the serum was stored in cryovials at —80°C until the assays were performed in batches.

Samples obtained at baseline and during the study for each participant were included in the same assay run to avoid inter-assay variability among participants. F 2 -isoprostane assessing oxidation of lipids [29] has been shown to provide one of the most accurate assessments of oxidative stress status [27] , [30].

Other methods not used in this study are targeting protein oxidation by measuring carbonyl groups in serum [31] , DNA damage by measuring hydroxyl radical-induced products of DNA bases [32] , and activity of several antioxidative enzymes including catalase, superoxide dismutase SOD , glutathione S-transferase, and glutathione peroxidase GPx [33] — [35].

A basic metabolic panel, hematological indices hemoglobin concentration, hematocrit, red and white blood cell counts , and C-reactive protein were analyzed in the Vanderbilt University Hospital Laboratory using standard methodologies. Plasma triglyceride TG , total cholesterol TC , low-density lipoprotein LDL , and high-density lipoprotein HDL levels were measured using enzymatic kits from Cliniqa Corporation San Marcos, CA.

Free fatty acids FFA were measured using the NEFA-C kit by Wako Nneuss, Germany and by gas chromatography. Glucose was measured using the Vitros Chemistry analyzer.

Insulin and leptin measurements were performed using RIAs. Adiponectin was measured using a kit from Millipore Billerica, MA and Luminex multiplexing technology.

Complete h urine samples were collected once weekly. Urinary calcium, sodium, and potassium were measured using Vitros Analyzer Ortho-Clinical Diagnostics, Rochester, NY, USA.

Urinary nitrogen content was measured using nitrogen analyzer Antek Instrument Nitrogen System NS, Antek Instruments, Inc. The nitrogen excretion in the urine was used as a biological marker for protein intake by multiplying the content of nitrogen in the urine by the factor 7.

The urine sodium and potassium contents were used as biological markers of sodium and potassium intake, respectively. Urinary creatinine was measured on a Sirrus Clinical Chemistry analyzer Stanbio Laboratory, Boerne, TX. Descriptive statistics were presented as mean and standard deviation SD or median and IQR or percentage, as appropriate.

Daily caloric intake and REE were expressed as the absolute and deficit number of kilocalories per day. Continuous endpoints were compared between the control and intervention group using Wilcoxon rank sum test.

The change within group was assessed using Wilcoxon signed rank test. Spearman correlation coefficient was used to assess the correlation between two continuous variables. Multivariable linear model was used to assess the treatment effect at single time point while adjusting for the baseline measures.

We performed a linear model using generalized least squares with autocorrelation structure of order 1 AR1 for the within-subject correlation to assess the F 2 -isoprostane change within 28 days of study period.

The main effects included baseline age, BMI, and F 2 -isoprostane, time, group, and time by group interaction. Time was modeled as nonlinear relationship to the F 2 -isoprostane using restricted cubic splines.

Residual plot and quantile-quantile QQ plots were used to check the model assumptions. All analyses were done with STATA 11 StataCorp, College Station, TX and the statistical programming language R, version 2. Thirty of 32 participants in the CR group and all controls completed the intervention part of the study i.

Twenty-six CR and 7 control group participants completed the entire study. The participants who did not complete the CR protocol did not differ from the completers in regards to age, body weight, body fat, CRP, or insulin, but did have lower baseline F 2 -isoprostane plasma concentrations Baseline demographic and anthropometric characteristics are shown in Table 1.

Adherence to the protocol in completers was good as measured by urinary biomarkers calculated as ratios of reported intake and excretion for protein 1. We fit a linear model of F 2 -isoprostane within the 28 days study period using generalized least squares.

F 2 -isoprostane was log transformed since the distribution was skewed. Figure 2 , Table 3. The average caloric intake was significantly lower in the CR than in the control group with an average intake of By the end of the dietary intervention Day 29 , total body weight and body fat weight decreased in the CR more than in the control group difference 2.

However, differences in body weight and body fat between CR and control groups were non-significant at baseline, end of intervention, and day follow-up Table 5. There was no significant between group difference in REE adjusted for fat free mass. The CR diet did not have a significant effect on either systolic or diastolic blood pressure or serum concentrations of insulin, leptin, adiponectin, total cholesterol, LDL cholesterol, HDL cholesterol, CRP and triglycerides Tables 8 and 9.

The novelty of the present study is that we investigated serial changes in a marker of oxidative stress induced by days of a caloric restriction diet.

The magnitude of this change was significant in comparison to baseline F 2 -isoprostane plasma concentrations. This suggests that the potential benefits from reducing the oxidative stress level can be achieved rapidly without restricting caloric intake to a level that overweight and obese people might find difficult to sustain.

CR is hypothesized to lessen oxidative damage by reducing energy flux and metabolism with a consequential lowering of reactive oxygen species and rate of oxidative damage to vital tissues [36].

Several studies documented the association of CR with lowering of resting metabolic rate and thermic effect of food, and a decrease in cost of physical activity [37]. In the present study, we did not observe significant reductions in adjusted energy expenditure most likely because of a much shorter intervention period 1 month vs.

Other reported benefits of long-term CR, but not detected in our study, include reduction in fasting glucose and insulin, which are linked to decreased insulin resistance and risk for type 2 diabetes [39].

Our results showing decreased F 2 -isoprostane with weight loss are consistent with a previous Davi et al [40] study involving 11 obese women who participated in a diet-induced weight loss program for a week period and lost at least 5 kg of initial body weight.

In eight of these women, there was a significant reduction in urinary excretion of F 2 -isoprostane. In contrast, a randomized controlled trial with 42 participants with the metabolic syndrome assigned to 16 weeks of weight maintenance or a week weight-loss program followed by 4 weeks of weight stabilization, showed different results.

Relative to the weight-maintenance group, a 4-kg loss in weight resulted in a significant decrease in blood pressure but did not alter urinary or plasma F 2 -isoprostane [41].

The discrepancies between these studies could be explained, at least in part, by different methodology plasma or urinary isoprostanes , various experimental designs, and diverse study populations. Baseline F 2 -isoprostane correlated with body fat content.

However, the F 2 -isoprostane decrease during the CR intervention was not correlated with the concurrent changes in body fat. Although we used a clinical trial design, strict diet control, and validated biomarkers [42] , [43] , it was not possible to determine whether body fat itself was a source of oxidative stress.

As far as we are aware, F 2 -isoprostanes, although present in foods, are not absorbed through the gut [44] , [45]. A plausible explanation is that in addition to a decrease in energy content, changes in content and amount of macronutrients consumed affected systemic oxidative balance.

Lower concentrations of protein, carbohydrates, and lipids in the CR diet, when compared to habitual diets reported by Control diet participants, could have potentially shifted postprandial oxidative status towards decreased susceptibility to oxidative damage [46].

Indeed, previous research has shown that postprandial increases of lipid and carbohydrate concentrations lead to increased oxidative stress [47] and consequent hyperlipidemia and hyperglycemia [48]. Although our study did not test whether decreases in oxidative stress are linked to an improvement in risk factors for associated chronic diseases, previous data do provide such evidence.

Zacardi et al [49] reported that declines in adhesion molecules and improvement in endothelial function with sustained weight loss were related to decreases in IL-6 and TNF-α, independent of change in adiposity and body fat distribution. Despite the many studies that have examined the role of oxidative stress on cardiovascular health, mechanistic studies designed to abruptly reduce plasma oxidative stress and to subsequently determine the acute effects of this intervention on antioxidative capacity, oxidative stress, and vascular function have not been extensively reported.

For example, studies in young and older individuals demonstrated a dichotomous effect of antioxidant consumption on endothelial function with age [50].

Nevertheless, if the link between obesity and increased oxidative stress is confirmed, the potential for antioxidant therapy to decrease the risk of obesity-related co-morbid conditions such as cardiovascular disease warrants additional studies in diverse populations [51].

Our findings extend those of previous studies in several ways.