The significant reductions in body anthropometry and improvements in the lipid profile reported in the current work are consistent with the established improvements in body weight and cardiometabolic risk factors reported during Ramadan, as revealed by several previously published systematic reviews and meta-analyses [8; 9; 13; 15]. The main finding of the current work involves that a great majority of altered metabolites decreased during fasting and only 4 increased significantly.
According to the available literature and the best knowledge of the authors, none of the previously published works had used non-targeted liquid chromatography-mass spectrometry (LCMS) in exploring the metabolomic changes upon Ramadan fasting in subjects with overweight/obesity. Such advanced technology is characterized by its power to detect large numbers of metabolites and intermediary compounds in human blood. The PCA analysis in Fig. 1 clearly shows that fasting causes changes in metabolic plasma profiles, distinguishing samples from two populations before and after fasting, with few outliers on opposite sides e.g. Pre fasting sample numbers 02, 07, 23, and 50 and post-fasting samples 25, 26, 30, 42 and 44 (see Fig. 1). The main findings of the current work included significant increases in the blood levels of several metabolites that include lipids (PC(18:1(9Z)/18:1(9Z)), amides ( 2-pyrrolidinone), and Carboxylic acids (traumatic acid) (table 4). However, a larger number of metabolites were decreased by the end of Ramadan fasting, including cortisol, aspartame, valeric acid, isobutyric acid, L-aspartyl-L-phenylalanine, and other metabolites (Table 3).
The lack of detection for the branched-chain amino acids (valine, leucine, and isoleucine) in the current work is consistent with what was mentioned earlier about the lack of chronic energy shortage during the fasting month. Considering the intermittent nature of Ramadan fasting that extends from 12–17 hours, with the free eating pattern practiced during the night hours, along with the decreased physical activity and increased sedentary lifestyle during the day hours observed in many people, the release of BCAA becomes less demanded. This contradicts what was found upon prolonged fasting during 34–58 hour fasting of four healthy volunteers, where a significant increase was detected in the blood levels of BCAA as a response to the chronic, stressful shortage in energy supply [6]. It is well evident that BCAAs are mainly released from skeletal muscles, followed by uptake into the citric acid cycle for the energy metabolism pathway, or hepatic lipogenesis pathway after prolonged fasting or starvation [27].
Among the few studies that examined the metabolomic changes associated with Ramadan fasting is that of Mathew et al. (2014)[28]. In their study, a targeted metabolomics approach was applied to blood samples of eleven healthy male volunteers, taken directly before and two hours after consumption of a controlled meal in the evening at the beginning and the end of Ramadan, and after an over-night fast several weeks after Ramadan. The targeted metabolomics applied therein resulted in the detection and quantification of a total of 202 metabolites. These metabolites included amino acids, bile acids, acylcarnitines, and polyamines [28].
We observed that aspartame, an artificial sweetener, decreased after fasting by using non-targeted metabolomics. In contrast, previous studies revealed upon targeted and non-targeted metabolomics that sugar and its metabolites were increased after long- (58 hours) and short-term fasting (15 hours) confirming the expected physiological response to food intake [6; 28]. These physiologically plausible responses were further attributed to an increase in bile acid and amino acid levels and a decrease in long-chain acyl-carnitine and polyamine levels. Intriguingly, we have identified a decrease in the fatty acid isobutyrate, which is supported by a previous study that showed the concentrations of the number of phospholipids were decreased after 26 days of fasting [28]. In contrast, other studies demonstrated that short-chain fatty acids (SCFAs) like butyrates (2-hydroxybutyrate, 3-hydroxybutyrate, 2-keto-butyrate, and 2-amino-butyrate) were increased in the plasma after fasting [6; 29]. The subjects in these studies, however, were lean, and hence the decrease in isobutyrate we observed could be specific to overweight/obesity subjects. Since SCFA are known to regulate the function of several innate immune cells as well as modulate antigen-specific immunity, lower levels of these fatty acids could help regulate the chronic low-grade inflammation associated with obesity. Although purines such as urate and uridine are known to increase upon fasting for the regulation of energy homeostasis [30; 31], additionally supported by another study which revealed that purines (xanthan, cytidine) were significantly increased in the plasma of four study volunteers [6], still in our study we observed a decrease in the level of purines such as oxypurinol. These discrepancies may be due to differences in sample size and fasting duration. Upon prolonged fasting, various metabolites were associated with the metabolic switching after glycogen storage depletion, including branched-chain amino acids (BCAAs), butyrates, and carnitines. More recently, other metabolites have been identified after prolonged fasting including increased levels of anti-oxidative metabolites and metabolites of the pentose phosphate pathway as well as transcriptional modulators such as signaling metabolites (3-hydroxybutyrate and 2-oxoglutarate) and purines/pyrimidines [27]. However, such metabolomic exacerbations are not expected to be exhibited during Ramadan IF, during which ad libitum eating is allowed during the night hours, without restriction on any of the Halal foods, with an eating window of 7–12 hours (corresponding to 12–17 hours of daily fasting based on the solar season that crosses with the lunar month of Ramadan).
Cortisol is a glucocorticoid hormone and high levels thereof are associated strongly with stress-related disorders [32; 33]. In this study, we showed that the hormone-like cortisol was significantly reduced after practicing 30 days of RDIF. Similarly, a recent study also demonstrated a significant decrease in the serum cortisol level after 30 days of DIRF in 34 healthy individuals [34]. This will most likely be translated into better overall surveillance immunity. In addition, Vasaghi-Gharamaleki’s study also found that cortisol output and concentration significantly reduced after RDIF compared to baseline and this decrease lasted for three weeks after Ramadan [35]. However, such a finding contradicts the lack of changes in salivary cortisol among Ramadan fasting people [36].
Traumatic acid, a monounsaturated dicarboxylic acid, is originally a plant-derived “wound healing hormone”. However, this dicarboxylic acid metabolite could be detected in the human serum as part of the dicarboxylic acid derivatives that are increased upon ketogenesis. Furthermore, our study elucidated the positive effect of intermittent fasting on elevating levels of traumatic acid after RDIF. The previous study has revealed the antioxidant characteristics and collagen biosynthesis stimulating effect of traumatic acid, and it has been considered a potential agent in the treatment of many skin diseases [37; 38]. Moreover, our study shows a significant increase in the rare monosaccharide sorbose, which is a poorly digestible sugar. So far, no human studies have investigated sorbose. However, many studies illustrate the effect of dietary intake of sorbose on animals. Sorbose feeding caused body weight loss in rats and mice as described by Furuse et al.(1989). It was also shown to ameliorate hyperglycemia, hyperinsulinemia, and hyperphagia in gold thio glucose (GTG)-injected obese mice (K. Kita et al., 2002). Taking into consideration these findings will demonstrate the decisive impact of RDIF on the sugar metabolites.
Our study reveals a decreased level of L-phenylalanine which plays a main role in the biosynthesis of other amino acids. Despite the crucial role of phenylalanine in the synthesis of protein and many other molecules, the toxic effect and the neural cell damage caused by phenylalanine accumulation are well-identified [39]. Our study discloses the potential positive impact of RDIF on the early prevention of cognitive decline via showing the reduced levels of phenylalanine after RDIF. These findings are supported by the observation of increased levels of phenylalanine metabolites in the non-fasting group of a Mild Cognitive Impairment in the elderly study when compared to the fasting group [40]. Also, tyrosine and other aromatic chain amino acids were decreased after RDIF in the current study. Tyrosine metabolites are elevated in incident diabetes in south Asian individuals [41], therefore, the reduced level of this amino acid after RDIF attests to the diabetes-preventative characteristic of intermittent fasting.
Tryptophan and phenylalanine are essential for immune cells, especially T cell, activation. The observed decrease of these amino acids in the blood of overweight/obese subjects following fasting could help in manipulating the chronic low-grade inflammation associated with obesity. The current study reported an elevated level of glycerylphosphorylcholine (GPC) after the RDIF. While GPC was not itself significantly dysregulated (P < 0.1), glycerophospholipid metabolism, as a pathway, was significantly enriched for dysregulation (see Fig. 4). GPC is a natural brain choline compound that influences Alzheimer's disease and dementia treatment [42; 43]. Also, GPC accumulation has a protective effect from the high interstitial concentrations of NaCl and urea in renal medullary cells during the renal concentrating mechanism [44]. Moreover, GPC metabolites play a role in modulating the risk of cardiovascular disease via the atherosclerosis pathway [45].