Quantification of blood metabolites from 4 volunteers during fasting

Blood samples were obtained from four young, healthy, non-obese volunteers. Obese people are not included in the present study, as obesity is known to affect the levels of some fasting markers, BCAAs and acylcarnitines17. Their ages, genders, and BMIs are shown in Fig. 1a. Phlebotomy was performed in the hospital at 10, 34, and 58 hr after fasting (Fig. 1b), to facilitate rapid preparation of metabolome samples. Immediately after blood collection, metabolome samples for whole blood, plasma, and RBCs were prepared separately, followed by metabolomic measurements by LC-MS6. Levels of ATP, an essential energy metabolite, did not change significantly in whole blood, plasma, or RBCs of the four volunteers throughout the fast (Fig. 1c). Plasma ATP levels were much lower than in RBCs or whole blood. All participants remained healthy and manifested no adverse symptoms during the study. Blood glucose levels of participants remained within the normal range (70–80 mg/dl) (Fig. 1d).

Figure 1 Quantification of blood metabolites from 4 volunteers during prolonged fasting. (a) Experimental procedures employed to study metabolomic changes during human fasting for 58 hr. Four, healthy young volunteers joined the study. The right panel shows age, gender, and BMI for each of four volunteers. (b) Blood samples from each person were taken at the indicated timepoints. Samples were immediately quenched in 50% methanol at −40 °C. Resulting extracts were used for metabolomic analysis. (c) Levels of ATP remained constant. (d) Blood glucose levels determined with a glucose tester during fasting. Whole blood glucose levels remained within the normal range (70–80 mg/dL) due to gluconeogenesis during fasting. (e) Levels of vital metabolites remained essentially constant during fasting. Profiles of ATP, glutathione disulfide (GSSG), and NADP+ in whole blood are shown. (f) Scatter plot of 120 metabolites between 10 and 58 hr of fasting. Average whole blood data are shown for the four volunteers. Compounds that displayed minor shifts (within 1.5x~0.66x) in abundance are placed between two purple lines. In each panel, p-values are presented to show the significance of serial change until 58 hr by Friedman test. Full size image

Comprehensive, quantitative analyses of blood metabolites were performed. Previously we identified 126 metabolites in human whole blood, approximately half of which were enriched in RBCs6,7. During 58 hr of fasting, the majority (62%) of these compounds were maintained at roughly constant levels. For example, levels of essential compounds, such as gluthathione, and NADP+ remained roughly constant, as in the case of ATP (Fig. 1e). The abundance of metabolites in three components (blood, plasma, and RBCs) are summarized in Table S1. Among 120 metabolites, five RBCs enriched metabolites (carnosine, NADP+, opthalmic acid, S-methyl-ergothioneine, trimethyl-tyrosine) were not detected in plasma, due to their low abundance in plasma.

Increase of 44 blood metabolites and decrease of 2 during fasting

We employed non-targeted, comprehensive analysis of whole blood, plasma, and RBC metabolites using the software, MZmine 218 for metabolite identification.

Overall metabolite changes between 10 and 58 hr are seen in a scatter plot (Fig. 1f), in which metabolite abundances that were significantly affected are shown as dots displaced from the diagonal line. Levels of ~37% of detected metabolites increased significantly (>1.5x). Table 1 lists 46 compounds that displayed statistically significant (<0.66x or >1.5x) shifts in abundance during 58 hr of fasting, whose calculated formal powers were shown in Table S2: among 46 metabolites, the statistical power for 38 compounds was found to be rather high (more than 0.8). In addition, the effect size f of Cohen’s logic19 obtained (Table S2) was large for the majority of metabolites. Among them, 32 metabolites have not previously been reported as fasting markers. Non-targeted analysis thus enabled us to find many new candidate fasting markers.

Table 1 Most metabolites changed peak areas significantly during fasting. Full size table

The abundance of blood metabolites at 10 hr was categorized as H (high), M (medium), or L (low)6. None of the metabolites listed was comparable in abundance to ATP (H) in RBCs. All of the 46 compounds increased, except for aspartate (0.4-fold decrease) and gluconate (0.6-fold decrease) (Fig. S1a). All four volunteers showed similar patterns of decrease.

Fasting-induced increase of four butyrates

We found four compound peaks that were nearly invisible 10 hr after fasting, but which later increased greatly, two of them becoming major peaks after 34 and 58 hr of fasting (Fig. 2a). Identifying these peaks using standards, the four were identified as aminobutyrate, 2- and 3-hydroxybutyrate (2-HB and 3-HB), and 2-ketobutyrate (KB). The first three have previously been reported as fasting markers16,20, but KB is novel. In plasma, KB increased 4.9- and 11.7-fold at 34 and 58 hr, respectively. In Rubio et al.20, 2-HB and 3-HB increased in concentration, respectively, from 0.03 mM (12 hr) to 0.16 mM (36 hr after fasting) and from 0.07 mM (12 hr) to 1.17 mM (36 hr after fasting). Our measurements (Table 1) closely corroborate those of Rubio et al. 2-aminobutyrate (2-AB) increased 2-fold in the study of Rubio et al., while our measurement was 3.7-fold. Note that after 58 hr of fasting, the average levels of 2-HB and 3-HB in four volunteers showed further increases (13.7- and 55.4-fold for 2-HB and 3-HB, respectively), indicating that blood concentrations of 2- and 3-HB after prolonged fasting reach exceedingly high concentrations (~5 mM 3-HB).

Figure 2 Four butyrates increased significantly during 58 hr of fasting. (a) Titers of four butyrates (indicated by arrows) increased strikingly during fasting. Importantly, these compounds were negligible before fasting and were not listed in our previous non-fasting study7. Peak areas increased sharply during fasting, becoming major peaks after 58 hr. Identification of compounds was verified using standards with MS/MS6. (b) Peak area changes of the four butyrates in whole blood samples of four volunteers. (c) Increases of 2- and 3-hydroxybutyrate in plasma and RBCs during fasting. In each panel, p-values are presented to show the significance of serial change until 58 hr by Friedman test. Full size image

The striking increase of these four butyrates was observed in all 4 individuals (Fig. 2b,c), although absolute abundances varied among them. Curiously, the further increase of 2-HB and 3-HB from 34 to 58 hr did not occur in volunteer 2 (see Discussion section).

Fasting-induced increases of BCAAs and carnitines

Branched-chain amino acids (BCAAs) are known as fasting markers21,22,23. Because BCAAs are converted to CoA compounds and used for energy generation via the Krebs cycle, they are implicated in mitochondrial activation. In our analysis, we found a novel BCAA fasting marker, ketovaline, in addition to isoleucine, keto(iso)leucine, leucine, and valine, which were previously known (Figs 3a and S2). These compounds are detected in blood before fasting and the degree of increase after 58 hr of fasting was moderate (2.0–3.4-fold increase; Table 1). After 58 hr fasting, ketovaline and ketoisoleucine increased the most (average 3.4-fold, Table 1) in all four volunteers (Fig. S3).

Figure 3 Profiles of BCAAs and carnitines abundant during fasting. (a) Profiles of BCAAs in blood during fasting. Ketovaline is identified as a novel fasting marker, although increases of isoleucine, leucine, valine, and keto(iso)leucine were reported previously. (b) Profiles of four carnitines. Left-hand panels display profiles for hexanoyl- and tetradecanoyl-carnitine (C6 and C14, respectively) in whole blood, while righthand panels show isovaleryl-carnitine (C5) in plasma. (c) Profiles of four other carnitines. Acetyl-, decanoyl-, dodecanoyl-, and octanoyl-carnitine have previously been identified as fasting markers. In each panel, p-values are presented to show the significance of serial change until 58 hr by Friedman test. Full size image

We also found that hexanoyl-, isovaleryl- and tetradecanoyl-carnitines may be novel fasting markers (Fig. 3b). Acylcarnitines are also major fasting metabolites, as are butyrates (Figs 3b,c, S2b,c and Table 1)20,23,24. Acylcarnitines function as lipid carriers, supporting lipid metabolism and reflecting β-oxidation activity in mitochondria25,26. Even though all four volunteers were young and healthy, the degree of increase (1.6~14-fold) reflected individual variability in carnitine functions in blood and tissues. The 15 metabolites displaying the most significant changes are listed in Fig. S3, in order of magnitude of change. This list largely contains butyrate derivatives, acylcarnitines, and BCAAs, consistent with previous work. However, volunteers 1, 3, and 4 displayed more prominent changes in butyrates and acylcarnitines than in BCAAs, while volunteer 2 showed greater increases in BCAAs than in acylcarnitines. Because volunteer 2 had Body Mass Index (BMI) below the lower limit (18.5) of the normal range (only 16.86), his lipid stores may not have been sufficient, so that his supplies of 2- and 3-HB were also lower than normal (Fig. 2c). In addition, the changes of GSSG (Fig. 1e), tetradecanoyl-carnitine (Fig. 3b), dodecanoyl-carnitine (Fig. 3c) and malate (Fig. 4a) from 34–58 hr in volunteer 3 were different from those in volunteer 1 and 4, by unknown reasons.

Figure 4 Profiles of organic acids and vitamins during 58 hr of fasting. (a,b) Increased levels of organic acids during fasting. 2-oxoglutarate, malate, cis-aconitate, and succinate are newly identified as fasting markers (a) in addition to confirmation of a previously reported increase of citrate (b). (c) Increased levels of nicotinamide in RBCs and pantothenate (a precursor for CoA) in whole blood. In each panel, p-values are presented to show the significance of serial change until 58 hr by Friedman test. Full size image

Increase of organic acids and coenzymes

In the present study, we identified other classes of compounds that underwent significant changes, such as purines and pyrimidines, coenzymes, organic acids, anti-oxidants, and sugar metabolites (in plasma, not RBCs), revealing hitherto unrecognized aspects of fasting.

Roughly 2-fold increases in several organic acids (cis-aconitate, malate, 2-oxoglutarate, and succinate) were observed (Fig. 4a). These are involved in the TCA cycle. Taken together with the increase of citrate, which has been previously reported (Fig. 4b)20, mitochondrial activity in tissues may be activated during fasting. As human RBCs have neither mitochondria nor TCA cycle activity, these organic acids are likely to have been derived from tissues. Notably, coenzymes (nicotinamide and pantothenate, a precursor for acetyl-CoA) were also upregulated (Fig. 4c). Nicotinamide is essential for production of NADH and NADPH, while pantothenate serves as a precursor for production of Coenzyme A (CoA).

Increases of pyrimidines and purines

Urate and uridine are known to increase during fasting2,27, as we confirmed (Fig. 5a). Urate was the most abundant nucleoside or nucleotide detected during fasting (Table 1 and Fig. 5). All four volunteers clearly showed 1.5~1.7-fold increases in urate. In addition, GTP, CTP, ADP, IMP, cytidine, adenine, and xanthine (a precursor of urate) showed statistically significant increases (Fig. 5b). Increases of GTP, IMP, and CTP were found only in plasma (Fig. 5b). The 1.5-fold increase of ADP in blood was significant, judging from the peak area (~108). Increases of GTP (2.4-fold in plasma), uridine, and xanthine (2.8- and 4.0-fold in blood, respectively) were also prominent (Table 1).

Figure 5 Profiles of pyrimidines and purines during 58 hr of fasting. (a) Increased concentrations of urate and uridine in blood were observed. (b) Upper panels show changes of ADP, xanthine (precursor for urate) in whole blood, and adenine, cytidine in RBCs, while lower panels indicate those of CTP, IMP, and GTP (in plasma). In each panel, p-values are presented to show the significance of serial change until 58 hr by Friedman test. Full size image

Increased levels of pentose phosphate pathway (PPP) metabolites and antioxidants

An additional new finding is that six sugar phosphates (6-phosphogluconate, diphosphoglycerate, glucose-6-phosphate, glycerol-phosphate, pentose-phosphate, phosphoglycerate and sedoheptulose-7-phosphate) increased more in plasma than in whole blood (Fig. 6a,b). Even small increases could be detected in plasma as their plasma titers were rather low under non-fasting conditions, whereas their levels in RBCs and consequently in whole blood were much higher. Significantly, of these, 6-phosphogluconate, glucose-6-phosphate, pentose-phosphate, and sedoheptulose-7-phosphate are generated in the pentose phosphate pathway (PPP), and are reported to be essential for redox maintenance and nucleic acid synthesis28.

Figure 6 Profile of sugar phosphates and anti-oxidants during 58 hr of fasting. (a) Previously unreported changes in sugar phosphates were observed in plasma. 6-phosphogluconate, glucose-6-phosphate, pentose phosphate, and sedoheptulose-7-phosphate are intermediates in the pentose phosphate pathway. (b) Profile of sugar phosphates in whole blood. (c) Anti-oxidants increased during fasting: ergothioneine in plasma (left panel) and ophthalmic acid (OA) in whole blood (centre panel), and carnosine in RBCs (right panel). OA is produced by the same enzymes required to generate glutathione. Aminobutyrate, the precursor of OA, also increased (Fig. 2b) in parallel with upregulation of OA, while the level of glutathione disulfide (GSSG) remained constant (Fig. 1e). In each panel, p-values are presented to show the significance of serial change until 58 hr by Friedman test. Full size image

Consistent with increases in PPP metabolites, the anti-oxidants, ergothioneine, and carnosine also greatly increased (Fig. 6c). Another interesting example is that of a tripeptide analog of glutathione, L-γ-glutamyl-L-α-aminobutyrylglycine, also known as ophthalmic acid (OA) (Fig. 6c). Synthesis of OA employs the same enzymes utilized for glutathione production29,30. Interestingly, the level of OA significantly increased, while that of glutathione remained constant (Fig. 1e). Although not previously reported, we also observed increases of lysine, dimethyl-arginine, and N-acetyl-(iso)leucine (Fig. S4). The physiological significance of these changes remains to be determined.