Haptoglobin polymorphism modulates cardiometabolic impacts of four consecutive weeks, dawn to sunset Ramadan intermittent fasting among subjects with overweight/obesity

Aims Haptoglobin (Hp) is a multifaceted marker of in�ammation, and mediates the interplay between obesity, in�ammation, and cardiometabolic dysfunction. However, the role of the Hp phenotype in modulating intermittent fasting (IF)-induced changes in cardiometabolic markers remains to be elucidated. Methods Hp phenotype was determined for the study subjects. Cardiometabolic markers were assessed before and at the end of four consecutive weeks, dawn to sunset IF. Results A total of 114 subjects (75 males and 39 females, 38.7 ± 11.7 years, body mass index (BMI) of 30.41 ± 5.09 kg/m 2 ) were recruited. Hp2-2 (n = 55, 48.2%) and Hp2-1 (n = 53, 46.5%) were the predominant phenotypes. Signi�cant reductions were observed in serum Hp, IL-6, TNF-α, triglycerides (TG), total cholesterol (TC), LDL, BMI, fat mass (FM), while the signi�cant elevation was observed in serum CD163, HDL, and IL-10 at the end of the IF month for the whole population. Based on the Hp polymorphism, signi�cant decreases in Hp, BMI, FM, TG, LDL, and TNF-α, with signi�cant increases in HDL and CD163 levels were observed among subjects with Hp2-2 and Hp2-1 phenotypes. A more pronounced reduction in FM was reported in subjects with Hp2-2 in comparison with Hp2-1.


Introduction
A growing body of evidence supports the role of weight-reducing regimens, intermittent fasting (IF), and physical activity in reversing (or protecting against) the adverse metabolic derangements associated with obesity [1].IF is a widely adopted dietary practice, used for both religious and non-religious (e.g., health improvement) purposes [2][3][4].Recent evidence supports the health-improving, disease-preventing effects of IF, such as: maintaining healthy aging; reducing insulin resistance and improving insulin sensitivity; decreasing cancer risk by improving DNA repair and mitochondrial health, and triggering autophagy; reducing body weight; and improving cardiovascular and cerebrovascular health [2,3,[5][6][7].Among the different regimens of IF, religious form of IF such as Ramadan IF (RIF) [2] is one of the most extensively examined types of IF, with various impacts, have been reported in anthropometric [8,9], dietary [10], in ammatory and oxidative stress [11][12][13], metabolic [14][15][16], hormonal [17] and lifestyle [18] aspects.Ramadan fasting month is the ninth month of the lunar calendar, through which adult Muslims are mandated to refrain from all food and drinks from dawn to sunset for 29-30 consecutive days.Fasting duration ranges from 12-20 hours depending on the geographical location and solar season of the fasting month [19].
Various single nucleotide polymorphisms (SNPs) are associated with the variable components of metabolic syndrome, and many of these SNPs have been shown to modify an individual's response to dietary and lifestyle interventions [20,21].Haptoglobin (Hp) is a plasma polymorphic glycoprotein that has an in ammatory potential and increases two-to ve-fold in response to in ammatory stimuli [22].The primary functions of Hp are to bind to the free hemoglobin (Hb) produced from red blood cell destruction, reduce the release of heme iron and the heme generation of reactive oxygen species (ROS), and consequently, inhibit oxidative tissue damage.Hp-Hb complex's uptake and release from circulation are regulated by the monocyte/macrophage scavenger receptor CD163 and hepatocytes [23,24].
The three main Hp phenotypes in humans (Hp2-2, Hp2-1, and Hp1-1) are derived from two alleles: Hp1 and Hp2 [25].Several studies have reported that the Hp phenotype has a crucial role in determining the antioxidant and anti-in ammatory responses, and hence the cardiometabolic risk [25,26].People with the Hp1-1 phenotype were found to be more resistant to oxidative stress (OS) than those with Hp2-2 and Hp2-1 phenotypes, by the better abilities to enhance the stability of the Hp-Hb complex, and prevent Hb oxidation and the formation of ROS.In contrast, Hp2-1 and Hp2-2 phenotypes have a lower Hp-Hb complex stability, which increases its association with higher OS and pro-in ammatory response, making Hp2-2 phenotype a risk factor for in ammatory disease [24,[26][27][28].This is reinforced by the fact that individuals with the Hp2-2 phenotype have increased levels of oxidized LDL-cholesterol and decreased clearance rate of free Hb compared to Hp1-1 [26,[28][29][30].These variations in the antioxidant activity and anti-in ammatory macrophage signaling between the three Hp phenotypes may explain the association between speci c Hp phenotypes and increased obesity and metabolic disorder risk factors [31].
With the growing attention directed toward IF in reducing body weight and improving cardiometabolic health, along with the existence of variable Hp phenotypes; it becomes rationalized to examine how people with different Hp phenotypes respond to IF regimen.Further, the extent of how anthropometric, metabolic, in ammatory markers change in response to IF among people with obesity is worth to be examined.Thus, the primary objective of the current work is to elucidate how people with different Hp phenotypes respond to four-week, dawn to sunset Ramadan IF (RIF), and how Hp modulates the anthropometric, in ammatory, and metabolic changes among subjects with overweight/obesity.The study stems from the hypothesis that subjects with overweight/obesity with different Hp phenotypes will respond differently to the observed RIF, and that the anthropometric, metabolic, and in ammatory responses will vary according to the Hp phenotype.

Study design
An observational, prospective cohort design was used to nd out how RIF-induced changes of anthropometric, metabolic, and in ammatory markers will differ among subjects with overweight/obesity with three distinct Hp phenotypes.This prospective study was conducted over two Ramadan fasting months over two consecutive years (during May-June 2017 and 2018).Data were collected at two-time intervals: baseline (T1, 2-7 days before commencement of RIF) and at the end of the fourth week of Ramadan month (T2, after completing 28-30 consecutive days of dawn-to-sunset RIF).During the fasting period (about 15 hours a day), individuals refrained from oral intake (including food and water) from dawn to sunset.Subjects received no particular dietary or physical activity regimens or recommendations during any stage of this study.According to Islamic laws of fasting, menstruating women are exempted from observing Ramadan fasting during their menstrual period; hence, the fasting period for participating women was less than that for men (23-25 days vs. 28-30 days).

Participant selection
A convenience sampling technique was followed.After announcing the research via social media, institutional emails, and personal communications, interested people who expressed their intention to observe Ramadan fasting, and visited the University Hospital Sharjah (UHS)/UAE for screening were recruited for this study.The study protocol was designed and conducted following the Declaration of Helsinki and approved by the UHS Research Ethics Committee (Reference no: REC-16-05-11-01).All enrolled subjects were provided with an information sheet describing the research plan, objectives, and requirements of participation.All subjects provided signed informed consent to participate in this study.Male and female subjects who were overweight/obese (Body Mass Index, BMI > 25 kg/m 2 ), willing to fast during Ramadan, and to participate in this study were considered eligible.Data were collected using a self-report questionnaire that covered the medical history and demographic information.The questionnaire was administered in individual face-to-face interviews.All interviews were conducted by trained research assistants.The exclusion criteria were: a history of metabolic syndrome, diabetes, cardiovascular disease; neuro-psychiatric patients taking regular medications; following a weight-reducing diet, a history of bariatric surgery within the last 9 months before commencing RIF; and being a pregnant or premenopausal woman.

Anthropometric assessment
Anthropometric measurements were taken at two-time intervals, T1 and T2.Body weight (BW), body mass index (BMI), fat mass (FM), body fat percent (BFP), fat mass (FM), fat-free mass (FFM), and visceral fat surface area (VFSA) were measured before and at the end of the fasting month using direct segmental multi-frequency bioelectrical impedance analysis machine (DSM-BIA; TANITA, MC-980, Tokyo/Japan) following the manufacturer instructions.Before taking the BIA measurements, all accessories, metals, and/or jewelry were removed according to the manufacturer's instructions, and each subject was requested to purge surplus bodily uids through urine.Because all people fasted for eight to ten hours before each of the two-time intervals, the impact of hydration and physical effort on BIA measurements was minimized, as was intra-individual variability.Furthermore, for T1 and T2, BIA and all other data were taken at the same time of day.The DSM-BIA machine measured the visceral fat rating (from 0 to 100); this value was converted into a VFSA by multiplying the obtained value by 10, consistent with the manufacturer's instructions.Height was measured using a xed stadiometer to the nearest 0.1 cm.BMI was calculated as weight (kg) divided by height in m 2 .Waist (WC) and hip circumference (WC) were measured to the nearest 0.01 m using a non-stretchable measuring tape (Seca, Hamburg/Germany), and their ratio was calculated accordingly.

Blood sampling
Alongside all anthropometric assessments, venous blood samples were collected from subjects after 8-10 hours of fasting at both time intervals (T1 and T2).
A volume of 10 ml of blood was collected at the two-time intervals.At both time intervals, the samples were collected between 11 am and 1 pm to eliminate the effect of timing and dietary intake on the measured biochemical parameters and ensure consistency in the duration of fasting.Collected blood samples were divided into two aliquots.One aliquot was centrifuged at 2500 rpm for 15 minutes within 1 hour of the collection; the serum was aliquoted, coded, and stored at − 80°C until it was used for biochemical analyses.The second aliquot was used for RNA extraction, as explained below.

Biochemical assays and determination of Hp polymorphism
Hp phenotype was determined for the subjects using G-vertical polyacrylamide gel electrophoresis [32].In this study, a fully automated clinical chemistry analyzer (Adaltis, Pchem1, Italy) was used to quantify fasting glucose (FBG), total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglycerides (TG) at the time points.Blood pressure was measured before blood sampling using a digital blood pressure monitor (GE, USA), with subjects in an erect, seated position after a 5-min resting period.Serum CD163 was measured using an ELISA kit (Elabscience, USA).The pro-and anti-in ammatory cytokines (IL-6 and TNF-α; and IL-10, respectively) were quanti ed using a multiplex assay (Luminex, Bio-Plex Pro™ Human Cytokine plex Assay).

Statistical analysis
Statistical analyses were performed using SPSS 26 (IBM, Armonk, NY, USA).The mean and standard deviation (SD) were calculated for continuous variables and percentage (%) for categorical variables.The normality distribution of the data was tested using Kolmogorov Smirnov.The change was calculated as [the endpoint value -baseline value], and the % change was calculated as [(endpoint value (T2) -baseline value (T1)) / baseline value] * 100%.The P-value for the trend was analyzed using a linear trend test.The Manne Whitney U-test was used to compare baseline data between two groups.The Wilcoxon signed-rank test for paired samples was used to compare changes within groups over the time course of the study.Changes in variables within groups over 4 weeks are presented as adjusted P values derived from a general linear model after adjusting for the baseline age, sex, waist circumference, and total caloric intake.The signi cance was considered at P-values < 0.05.

Results
Changes of anthropometric, lipid pro le, and in ammatory markers of the study population after RIF As presented in Table 1, subjects had signi cantly lost an average of 1.6% of their initial body weight (BW) and exhibited signi cant decreases in the BMI, BFP, FM, FFM, MM, VFSA, WC, HC, and WHR at the end of RIF.Moreover, signi cant reductions were observed in serum triglycerides (TG), total cholesterol (TC), LDL, while a signi cant elevation was observed in serum HDL at the end of the IF month for the whole population.Finally, a signi cant reduction in serum Hp, IL-6, and TNF was recorded at the end of the RIF month in comparison to the pre-fasting levels among the study subjects.The levels of serum CD163 and IL-10 of the study subjects, however, were signi cantly increased at the end of Ramadan.
Sex differences in the basic demographic, anthropometric, metabolic, and in ammatory indicators for the study subjects (females = 39, males = 75, total = 114) are presented in Supplementary Tables 1a,b,c, with most of the differences falling in the anthropometric measurements, with minimal differences in the metabolic and in ammatory parameters.Supplementary Table 2 shows that no differences were found in the prevalence of obesity classes among the three different haptoglobin phenotypes.

Changes in body weight, composition, and blood pressure after RIF based on Hp phenotype
The changes in anthropometric measurements in response to RIF based on the Hp phenotypes are shown in Table 3.The differences of the means at the baseline and end of Ramadan of all Hp phenotypes were calculated and analyzed to reveal the responses of each Hp phenotype individually to Ramadan and compare them to each other.The signi cance P-values were adjusted for the baseline age, sex, WC, and total caloric intakes (all adjusted P < 0.05).
Finally, all Hp phenotypes experienced signi cant changes in their SBP and DBP values over Ramadan, with decreases in SBP values of Hp2-1 and Hp2-2 phenotypes, and signi cant decreases in DBP values of Hp1-1 and Hp2-2 phenotypes.However, signi cant increases were reported in SBP and DBP for Hp1-1 and Hp2-1.No signi cant differences among the Hp phenotypes' blood pressure values, however, were present.

Changes in blood lipid and glucose pro le after RIF based on Hp phenotype
Fasting blood glucose and serum lipid pro le parameters were measured in the subjects according to their Hp polymorphism phenotype in response to RIF (Table 4).Serum levels of TC have signi cantly reduced in all types of Hp polymorphism at the end of RIF month compared to pre-fasting levels, according to the post-adjustment P-values.However, no signi cant difference was found between the three Hp phenotypes.Hp2-1 experienced the largest TC decrease among the three phenotypes.All three phenotypes experienced signi cant changes in their TG and LDL-C levels.In contrary to the other phenotypes, Hp1-1 experienced signi cant increases in both TG and LDL-C, while experiencing the highest numerical increase in HDL-C at the end of the Ramadan fasting month in comparison to the pre-fasting levels.Conversely, FBG and HDL-C serum levels were all signi cantly increased at the end of Ramadan for all of the three phenotypes, with Hp 2-2's HDL-C increase being signi cantly different from the other two phenotypes.

Changes in serum Hp, CD163, and in ammatory cytokines after RIF based on Hp phenotype
In ammatory cytokines, CD163, and serum Hp were measured for all subjects based on their Hp phenotype in response to RIF (Table 5).At the end of RIF, all of the three Hp phenotypes experienced a signi cant decrease in IL-6 and TNF-α, with Hp1-1's decrease of IL-6 and TNF-being the smallest and signi cantly different than the other two phenotypes.In contrast, serum IL-10 and CD163 levels were signi cantly elevated for all Hp phenotypes after RIF, with the least increase in CD163 was reported for the Hp1-1 phenotype group.Serum Hp was signi cantly lowered for Hp2-1 and Hp2-2, while signi cantly increased for Hp1-1, with a clear trend for differences between the three phenotypes.The change of the Hp2-2 phenotype in serum Hp was signi cantly different from the other two phenotypes, with the highest reduction was reported Hp2-2 (Table 5).

Discussion
The religious form of IF (i.e.: Ramadan model) was shown to mitigate low-grade systemic in ammation, reduce pro-in ammatory cytokines and OS markers, and confer a short-term transient cardiometabolic protection [11,15,33].
While more than one study examined the effect of observing IF regimens on genetic expression (e.g., FTO, Nrf2, TFAM, SOD2, SIRT1, SIRT3, and CLOCK) [34][35][36][37][38][39], few studies have examined the impact of genetic variations on the metabolic response to IF regimen among overweight and obese subjects.In these, the IF was associated with higher weight reduction in comparison to low-calorie diet among overweight/obese people with GG genotype of the UCP2 gene, while no differences in weight loss were found between the two regimes among people with AA + GA genotype [40].To our knowledge, this is the rst study investigating the impact of Hp phenotypes (Hp1-1, Hp2-1, and Hp2-2) in obesogenic (including bodyweight reduction), metabolic and in ammatory markers in response to IF regimen.Our results demonstrated that the Hp phenotype might independently in uence the outcomes of IF on abdominal obesity, lipid pro le, in ammatory cytokines, serum Hp, and CD163 in overweight and obese individuals.Our results con rmed the momentous role of IF in reducing low-grade systemic in ammation and enhancing anti-in ammatory mechanisms, thereby mitigating health deteriorations accompanying people with obesity.

Impact of RIF on serum Hp, CD163, and other measured parameters
The effect of IF and caloric restriction on body weight change has been the subject of several studies; numerous reviews and original research have been published in attempts to elaborate on this perpetuate effect [3,6,41,42].Given that fasting during Ramadan represents a form of IF and TRF [43] that is globally observed by at least 1.5 billion Muslim people each year, the impact of RIF on body weight loss and the associated metabolic and in ammatory effects needs further elaboration.
The present study suggests that RIF is associated with reduced body weight, body fat percentage, and visceral fat area, with improvements in several cardiometabolic risk factors.These ndings concerning body weight and composition are consistent with those of a previous study that reported signi cant reductions in body weight, fat mass, BMI, visceral fat area, and fat-free mass in healthy adults following Ramadan fasting during the summer [44,45].
Additionally, IL-6 and TNF-α levels had signi cantly decreased, whereas IL-10 levels had signi cantly increased at the end of the Ramadan month.These signi cant reductions in TNF-α and IL-6 after observing RIF were consistent with signi cant reductions reported in a similar previous study on the Ramadan model of IF [13].Furthermore and as discussed above, the secretion of pro-in ammatory cytokines IL-6, TNF-α, and anti-in ammatory cytokine IL-10 may be regulated by the Hp phenotype [46].Besides, pro-and anti-in ammatory cytokines play crucial roles in the upregulation and downregulation of CD163 expression, respectively [47].Hp expression depends on the Hp phenotype and the levels of the pro-in ammatory cytokines IL-6 and TNF-α, both of which have a crucial role in Hp concentration [48].Therefore, Hp phenotype and Hp-Hb complex have leading roles in the regulation of serum Hp and CD163 levels.
Several studies have shown that the pro-in ammation characteristics of serum Hp [30,49], and the anti-in ammation activity of CD163 [50] altered in response to different Hp polymorphisms.These ndings were consistent with our previous study that showed RIF had an ameliorating effect on proin ammatory cytokines and in enhancing the anti-in ammatory response [12,45,51].The present results con rmed a signi cant increase in the antiin ammatory serum CD163 and a signi cant decrease in the pro-in ammatory serum Hp following RIF when compared with pre-fasting levels.There may be, however, other unexamined factors that have an impact on these variables during the Ramadan month, such as changes in circadian rhythm that may in uence IL-6 production, as reported in non-fasting research [52].
It is well established that the increased ratios of anti-in ammatory (IL-10) to pro-in ammatory (TNF-α and IL-6) cytokines represent a protective factor against atherogenesis [53].In one study [54], the ratios of IL-10 to TNF-α and IL-10 to IL-6 showed preferred increments at the end of the fasting month.Their reported noticeable reduction in the IL-6:IL-10 ratio at the end of Ramadan suggests that RIF had a favorable protective effect against systemic in ammation and subsequent metabolic derangements [54].
Therefore, the lower level of IL-6 at the end of RIF in the present study could be explained by the decreased level of physical activity reported by most subjects during the month of Ramadan and in the literature [55].A recent study showed that RIF is associated with reduced activity and sleeping time without changes to the resting metabolic rate or total energy expenditure [56].This is consistent with the 0.5% reduction in muscle mass reported at the end of Ramadan.
HDL level was signi cantly increased at the end of RIF in this study.This nding was consistent with a previous study involving 81 fasting subjects, in which HDL decreased signi cantly at the end of RIF [57].This result was inconsistent with the ndings of the majority of previous studies on RIF, where HDL increased or remained unchanged during the Ramadan month [58].A systematic review and meta-analysis of 57 studies investigating RIF among healthy subjects (N = 2771) showed a signi cant, but small, pooled reduction in HDL at the end of Ramadan fasting [33].
This discrepancy in the effect of RIF on lipid pro le and HDL may be attributable to differences in cultural foods and dietary practices among different populations, especially in the types and amounts of dietary fats consumed.Further, the genetic makeup of study subjects affects the way their bodies respond to various changes during the Ramadan month, which in turn affects their lipid pro le at the end of the fasting month.
It has been proposed that the fasting state (i.e.: Ramadan IF) induces an elevation in free fatty acids and ketone bodies, such as beta-hydroxybutyrate, which in turn may impose damage on the metabolically active mitochondria in the neurons of the fasting organism.This damage, however, can be corrected by several mechanisms, including upregulated antioxidant defense genes and enhanced mtDNA repair [59].

The interplay between Hp polymorphism and IF in relation to CD163 and other measured parameters
The present study found that obese individuals with the Hp1-1 phenotype experienced a higher decrease in BW and BMI after RIF than those with the Hp2-1 or Hp2-2 phenotypes, with the differences in the change of BW being signi cantly different among the three Hp phenotypes.Overall, each Hp polymorphism underwent a signi cant decrease in BW and BMI due to RIF.Although expected, this result was interesting as there were signi cant changes in body weight and BMI in response to RIF in general.In addition, these results support the hypothesis of a previous study that suggested that the phenotype of Hp has a crucial role in modulating the oxidative-antioxidative status in both obesity and diabetes [60].Furthermore, our results showed a signi cant reduction in body fat percentage, fat mass, fat-free mass, and muscle mass for all of the three Hp phenotypes.Phenotype Hp1-1 had a tendency also to lose the greatest muscle/fat-free mass in comparison to the other two phenotypes, with the difference being signi cantly different, particularly against the Hp2-1 phenotype.
Previous studies showed that BMI is an indicator of total body fat, whereas waist circumference re ected visceral fat.Visceral fat deposition is known to have more metabolically adverse effects than subcutaneous fat [61].Therefore, waist circumference measurement is a better indicator of metabolic and in ammatory disorders related to obesity.Both waist circumference and BMI showed the strongest positive correlations with Hp among the various obesity measures [62,63].In a recent study, waist circumference and visceral fat area were found to signi cantly decrease in Hp2-1 after RIF compared with before RIF, whereas Hp1-1 and Hp2-1 showed non-signi cant reductions.
Interestingly, the WC reduction experienced by obese individuals withHp1-1 phenotype following RIF compared was signi cantly different from the ones of Hp2-1 and Hp2-2.Similarly, a recently published paper that investigated hypo-caloric dietary programs among obese women showed these dietary programs had a stronger positive in uence on abdominal obesity (waist circumference, total body fat, and fat mass) of the Hp1-1 phenotype compared with the Hp2-1 and Hp2-2 phenotypes [64].That study supported our nding that individuals with an Hp1-1 phenotype had higher expression of anti-in ammatory cytokines and were more resistant to OS than Hp2-1 and Hp2-2 individuals.Incidentally, Hp2-1 and Hp2-2 individuals have been reported to have a higher incidence of risk factors for obesity, diabetes, and cardiovascular diseases [27].
Due to its association with pro-in ammatory activity, higher levels of Hp have been associated with an increased risk for obesity, type 2 diabetes, and cardiovascular diseases [65-67].Many studies have suggested that Hp polymorphism not only affects serum Hp but also in uences lipid pro le levels.In other words, there is a positive correlation between serum Hp and TC, non-HDL-C, HDL-C, and TG concentrations in obese individuals [68].Our results are consistent with this nding.Moreover, several studies [67, 69], including the present study, demonstrated a higher concentration and positive association of LDL-C in obese Hp2-2 individuals compared with obese Hp1-1 individuals.Furthermore, the present study showed a signi cant decrease in TG and LDL in both Hp2-1 and Hp2-2 subjects after RIF.Additionally, HDL-C levels were signi cantly higher following RIF in all Hp polymorphisms.Conversely, TC was signi cantly reduced in all of the Hp phenotypes.The current results suggest that Hp2-2 individuals with obesity have more anti-in ammatory characteristics and will have a greater response to RIF and obtain optimum bene ts from IF than those with Hp2-1 and Hp2-2 phenotypes.
The present study investigated the role of the environmental factors in modulating anti-in ammatory markers (IL-10) and pro-in ammatory cytokines (IL-6 and TNF-α) in response to IF in people with different Hp polymorphism phenotypes.It also explored the expression of an essential anti-in ammatory factor, CD163, in both RIF and Hp polymorphism.With the observed interesting effect of RIF on signi cantly reducing levels of IL-6 and TNF-a in all Hp phenotypes, this con rms previous ndings demonstrating the positive impact of the Ramadan model of IF on the health of obese individuals [70].In contrast, IL-10 showed a signi cant increase in all of the Hp phenotypes after observing RIF.
As previously noted, serum Hp and CD163 levels are associated with and regulated by the pro-and anti-in ammatory cytokines (IL-6, TNF-α, and IL-10) alongside Hp polymorphism [50].There was a signi cant increase in serum of the anti-in ammatory CD163 and a considerable decrease of pro-in ammatory serum Hp in response to RIF.Additionally, serum CD163 levels were signi cantly increased in all of the Hp phenotypes after RIF, particularly Hp2-2 whose increase was signi cantly different from the Hp2-1 and Hp2-1 groups.Moreover, the serum CD163 mean difference values between Hp phenotypes revealed a trend in increment through Hp1-1, Hp2-1, and Hp2-2 respectively, but the changes were not signi cant between Hp1-1 and Hp2-2.In contrast, serum Hp showed signi cantly lower expression in the Hp2-1 and Hp2-2 phenotypes, yet an increase was experienced by the Hp1-1 phenotype after RIF.
Understandably, the Hp2-2 phenotype is mainly associated with increased risks for obesity, diabetes, and cardiovascular diseases, in addition to enhancing pro-in ammatory metabolites.RIF also has a positive impact on modulating and strengthening in ammatory cytokines.Interestingly, our ndings revealed that Hp2-2 had a better response in moderating and enhancing anti-in ammatory markers than Hp1-1, which highlighted the in uence of RIF on health outcomes for people with obesity, suggesting metabolites may offer a pathway to a healthier positive response.
Our results con rmed the decisive role of IF in reducing low-grade systemic in ammation and OS and enhancing anti-in ammatory mechanisms, thereby improving the health conditions of individuals with obesity.Moreover, this provided an example of the epigenetic factors of the different Hp phenotypes in response to RIF and con rmed that Hp1-1 individuals had a higher anti-in ammatory response than Hp2-1 and Hp2-2 individuals.Because of the positive impact of IF on the health of individuals with obesity in developing and enhancing adipocytokine pathways, Hp2-2 expressed a higher anti-in ammatory response to RIF compared with Hp1-1.Therefore, we propose that IF contributes to health bene ts for individuals with obesity in moderating and re ning their oxidative and in ammatory mechanisms.
In conclusion, four consecutive weeks of dawn-to-sunset IF was found to signi cantly and variably affect the anthropometric, metabolic, and in ammatory markers in relation to Hp polymorphisms.Our results con rmed the decisive role of IF in reducing low-grade systemic in ammation and OS and enhancing anti-in ammatory mechanisms, thereby improving the health conditions of individuals with obesity.Moreover, this provides an example of the epigenetic factors of the different Hp phenotypes in response to IF and con rmed that Hp1-1 individuals had a higher anti-in ammatory response than Hp2-1 and Hp2-2 individuals.

Declarations
Clinical Trial Registration number: ISRCTN18205186; https://trialsearch.who.int/?TrialID=ISRCTN18205186  a The P-value was analyzed by a general linear model after adjusting for baseline age, sex, waist circumference, and total caloric intake.
b P-value: The difference between baseline and 4 weeks was analyzed by Wilcoxon signed-rank test.
c P-value for trend was analyzed by a linear trend test.
d Change (%) was calculated as [(end point-baseline value)/baseline value] * 100.Continuous data are presented as the mean ± SD; categorical data are presented as frequencies (percentages).Bold denotes to statistically signi cant difference at P < 0.05.
a The P-value was analyzed by a general linear model after adjusting for baseline age, sex, waist circumference, and total caloric intake.
b P-value: The difference between baseline and 4 weeks was analyzed by Wilcoxon signed-rank test.
c P-value for trend was analyzed by a linear trend test.

Table 1
Changes in anthropometric, metabolic, and in ammatory markers between pre-fasting and the end of Ramadan fasting month.

Table 2
Baseline sociodemographic, anthropometric, biochemical, and in ammatory characteristics of subjects with overweight/obesity strati ed by haptoglobin phenotypes (n = 114) before the commencement of Ramadan intermittent fasting.P-value: the difference haptoglobin phenotypes was analyzed by the Manne-Whitney U-test.
a P-value for trend was analyzed by a linear trend test.bP-value: the difference between haptoglobin phenotypes was analyzed by the Manne-Whitney U-test.b

Table 3
Changes in blood pressure and anthropometric measurements among subjects with overweight/obesity strati ed by haptoglobin phenotypes before and after Ramadan intermittent fasting (n = 114).The P-value was analyzed by a general linear model after adjusting for baseline age, sex, waist circumference, and total caloric intake.
a b P-value: The difference between baseline and 4 weeks was analyzed by Wilcoxon signed-rank test.c P-value for trend was analyzed by a linear trend test.d Change (%) was calculated as [(end point-baseline value)/baseline value] * 100.a The P-value was analyzed by a general linear model after adjusting for baseline age, sex, waist circumference, and total caloric intake.b P-value: The difference between baseline and 4 weeks was analyzed by Wilcoxon signed-rank test.c P-value for trend was analyzed by a linear trend test.d Change (%) was calculated as [(end point-baseline value)/baseline value] * 100.

Table 4
Changes in blood glucose and lipid pro le among subjects with overweight/obesity strati ed by haptoglobin phenotypes before and after Ramadan intermittent fasting (n = 114).Continuous data are presented as the mean ± SD; categorical data are presented as frequencies (percentages).Bold denotes to statistically signi cant difference at P < 0.05.
a The P-value was analyzed by a general linear model after adjusting for baseline age, sex, waist circumference, and total caloric intake.b P-value: The difference between baseline and 4 weeks was analyzed by Wilcoxon signed-rank test.c P-value for trend was analyzed by a linear trend test.d Change (%) was calculated as [(end point-baseline value)/baseline value] * 100.

Table 5
Changes in in ammatory markers among subjects with overweight/obesity strati ed by haptoglobin phenotypes before and after Ramadan intermittent fasting (n = 114).