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- 1. ASEA Metabolomics Results Nieman DC. Human Performance Laboratory, North Carolina Research Campus and Appalachian State University
- 2. Metabolomics Laboratory, North Carolina Research Campus, David H. Murdock Research Institute Slide 1 Appalachian state Human Performance Laboratory
- 3. • 75-km cycling 1-week ASEA • 75-km cycling 1 week Placebo Crossover BASELINE TESTING for VO2max, body composition (N=20 subjects) Blood/urine : Pre-Ex Post- Ex 1-h • 75-km cycling 1-week ASEA • 75-km cycling 1-week Placebo Blood/urine : Pre-Ex Post- Ex 1-h 3-week washout Metabolomics: Goal is to measure the influence of ASEA on small molecules (metabolites) that shift in response to supplementation. The shift in metabolites, depending on the nutritional product, may represent effects on inflammation, oxidative stress, and physiologic stress. Slide 2 Appalachian state Human Performance Laboratory
- 4. Working Summary • Seven days ingestion of ASEA (relative to placebo) caused an extensive mobilization of free fatty acids from adipose tissue in male cyclists. • Athletes on ASEA for 7-days started the 75-km cycling trial with high blood free fatty acids leading to increased fat oxidation and a sparing of amino acids (and potentially muscle glycogen). • ASEA intake was associated with a large increase in serum ascorbic acid levels (probably from the adrenal cortex). • Serum creatinine and urea also increased post- exercise. Slide 3 Appalachian state Human Performance Laboratory
- 5. 2) Acute Effect: Increased fatty acid oxidation and mobilization during exercise (placebo condition was linked to a late mobilization). Finding 1: Ingestion of ASEA beverage for one week strongly increased serum fatty acids levels (most likely from adipose tissue). 1) Chronic Effect: Higher fatty acid levels pre- exercise (several types of fatty acids --- see slides). Triglyceride Mobilization: corresponding with the increase in free fatty acids, glycerol was higher at baseline (indicative of extensive adipose triglyceride hydrolysis). FFAs Glycerol TG Hydrolysis + with ASEA - 500 1,000 1,500 2,000 ASEA Placebo Least Square Mean Area Myristic acid 14C Saturated Fatty Acid FDR=6.49E-32 - 500 1,000 1,500 2,000 Pre Post 1H Post Least Square Mean Area Myristic acid 14C Saturated Fatty Acid FDR=2.61E-20 ASEA Placebo Slide 4 Appalachian state Human Performance Laboratory
- 6. Post 7-day Ingestion: Fatty Acids Higher in ASEA vs. Placebo - 200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 ASEA Placebo Least Square Mean Area Myristic acid 14C Saturated Fatty Acid FDR=6.49E-32 - 5,000 10,000 15,000 20,000 25,000 ASEA Placebo Least Square Mean Area Palmitic Acid 16C Saturated Fatty Acid FDR=1.86E-25 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 ASEA Placebo Least Square Mean Area Oleic Acid 18C Monounsaturated n9 Fatty Acid FDR=5.21E-18 0 1000 2000 3000 4000 5000 6000 7000 8000 ASEA Placebo Stearic Acid 18C Saturated Fatty Acid FDR=3.22E-12 Slide 5 Appalachian state Human Performance Laboratory
- 7. 0 20 40 60 80 100 120 140 160 ASEA Placebo Capric Acid 10C Saturated Fatty Acid FDR=5.39E-07 0 5000 10000 15000 20000 25000 ASEA Placebo Glycerol Backbone of Triglycerides FDR=9.49E-07 - 200 400 600 800 1,000 1,200 ASEA Placebo Least Square Mean Area Palmitelaidic Acid 16C Trans Fatty Acid FDR=1.13E-08 7-days Ingestion of ASEA Fatty Acids and Glycerol Backbone: Higher in ASEA vs. Placebo Slide 6 Appalachian state Human Performance Laboratory
- 8. - 5,000 10,000 15,000 20,000 Pre Post 1H Post Least Square Mean Area Oleic Acid 18C Monunsaturated n9 Fatty Acid FDR = 7.96E-10 ASEA Placebo - 200 400 600 800 1,000 1,200 Pre Post 1H Post Least Square Mean Area Palmitelaidic Acid 16C Trans Fatty Acid FDR=1.66E-16 ASEA Placebo - 5,000 10,000 15,000 20,000 25,000 Pre Post 1H Post Least Square Mean Area Palmitic Acid 16C Saturated Fatty Acid FDR=1.56E-20 ASEA Placebo - 500 1,000 1,500 2,000 Pre Post 1H Post Least Square Mean Area Myristic acid 14C Saturated Fatty Acid FDR=2.61E-20 ASEA Placebo Serum Fatty Acids During Exercise Slide 7 Appalachian state Human Performance Laboratory
- 9. - 50 100 150 200 Pre Post 1H Post Least Square Mean Area Capric Acid 10C Saturated Fatty Acids FDR=0.0059 ASEA Placebo - 200 400 600 800 1,000 1,200 Pre Post 1H Post Least Square Mean Area Lauric Acid 12C Saturated Fatty Acids FDR=0.0281 ASEA Placebo - 2,000 4,000 6,000 8,000 10,000 12,000 Pre Post 1H Post Least Square Mean Area Stearic Acid 18C Saturated Fatty Acids FDR=1.33E-06 ASEA Placebo - 100 200 300 400 500 600 700 800 900 Pre Post 1H Post Least Square Mean Area Glyercol Monosterate FDR = 0.0060 ASEA Placebo Serum Fatty Acids During Exercise Slide 8 Appalachian state Human Performance Laboratory
- 10. Acetyl Co-A Citrate Isocitrate alpha- Ketoglutarate Succinyl CoA Succinate Fumarate Malate Oxaloacetate Pyruvate Pre Post 1H Post Serum Leucine FDR=0.0004 Pre Post 1H Post Serum Aspartate FDR=0.0075 Pre Post 1H Post Serum Fumarate FDR=1.06E-06 In urea cycle Via Beta Oxidation Via Glutamate Pre Post 1H Post Serum Valine FDR=0.0089 Via Odd Chain Beta Oxidation Pre Post 1H Post Serum Proline FDR=0.0066 Pre Post 1H Post Serum Threonine FDR=0.0108 Graph Key ASEA Placebo Pre Post 1H Post Serum Glycine FDR=0.0162 Pre Post 1H Post Serum Serine FDR=0.0273 Pre Post 1H Post Serum Malate FDR= 0.0004 Pre Post 1H Post Serum Citrate FDR = 0.0037 Finding 2: High levels of blood free fatty acids were linked to a sparing of amino acid catabolism, and increased Krebs Cycle intermediates, post-exercise Slide 9 Appalachian state Human Performance Laboratory
- 11. - 5,000 10,000 15,000 20,000 25,000 30,000 Pre Post 1H Post Least Square Mean Area Leucine Krebs Entry: alpha Ketoglutarate FDR =0.0004 ASEA Placebo - 10,000 20,000 30,000 40,000 50,000 Pre Post 1H Post Lesat Square Mean Area Proline Krebs Entry: alpha Ketoglutarate FDR = 0.0066 ASEA Placebo - 100 200 300 400 500 Pre Post 1H Post Lesat Square Mean Area Aspartate Krebs Entry: Oxaloacetate FDR = 0.0074 ASEA Placebo - 10,000 20,000 30,000 40,000 50,000 Pre Post 1H Post Lesat Square Mean Area Valine Krebs Entry: Succinyl CoA FDR = 0.0089 ASEA Placebo Serum Amino Acids at Pre, Post, and 1H Post-Exercise “Sparing” of Amino Acids with ASEA Slide 10 Appalachian state Human Performance Laboratory
- 12. Serum Amino Acids at Pre, Post, and 1H Post-Exericse “Sparing” of Amino Acids with ASEA - 1,000 2,000 3,000 4,000 5,000 6,000 7,000 Pre Post 1H Post Lesat Square Mean Area Threonine Krebs Entry: Pyruvate FDR= 0.0108 - 2,000 4,000 6,000 8,000 10,000 12,000 Pre Post 1H Post Lesat Square Mean Area Serine Krebs Entry: Pyruvate FDR= 0.0273 - 1 1 2 2 3 3 4 4 5 5 Pre Post 1H Post Lesat Square Mean Area Glycine Krebs Entry: Pyruvate FDR= 0.0162 Graph Key ASEA Placebo Slide 11 Appalachian state Human Performance Laboratory
- 13. Serum Krebs Intermediate at Pre, Post, and 1H Post-Exercise Higher Levels with ASEA - 100 200 300 400 500 600 Pre Post 1H Post Lesat Square Mean Area Fumarate FDR = 1.06E-06 - 20 40 60 80 100 120 140 160 180 200 Pre Post 1H Post Lesat Square Mean Area Malate FDR= 0.0004 - 10.00 20.00 30.00 40.00 50.00 60.00 Pre Post 1H Post Least Squre Mean Area Citrate FDR = 0.0037 Graph Key ASEA Placebo Slide 12 Appalachian state Human Performance Laboratory
- 14. 3) Ascorbic Acid Metabolism: ASEA supplementation appears to be affecting ascorbic acid both acutely and chronically. 0 500 1000 1500 ASEA Placebo Least Square Mean Area Fructose FDR=1.12E-05 0 2 4 6 8 ASEA Placebo Least Square Mean Area Threonic Acid FDR=1.46E-98 Chronic Differences: ASEA group has lower baseline levels of fructose and lower levels of threonic acid. Fructose is broken down into ascorbic acid which is further metabolized into threonic acid. This could be suggestive of higher ascorbic acid production but no differences in groups were detected at baseline. Acute Differences: ASEA group has higher levels of ascorbic acid, an antioxidant, and lower levels of both fructose and threonic acid. - 500 1,000 1,500 Pre Post 1H Post Least Square Mean Area Ascorbic Acid FDR = 5.6E-06 - 500 1,000 1,500 Pre Post 1H Post Least Square Mean Area Fructose FDR = 0.0002 - 2 4 6 8 10 Pre Post 1H Post Least Square Mean Area Threonic Acid FDR=0.0031 Graph Key ASEA Placebo Slide 13 Appalachian state Human Performance Laboratory
- 15. - 100 200 300 400 500 600 700 800 900 Pre Post 1H Post Least Square Mean Area Serum Creatinine FDR=2.55 E-06 Graph Key ASEA Placebo - 50 100 150 200 250 300 350 400 Pre Post 1H Post Least Squre Mean Area Aminomalonic Acid FDR = 1.13E-05 Breakdown product of creatine Plays role in binding calcium to protein 4) Other Changes. Some other changes were found both acutely and chronically that require further investigation into implications. - 20 40 60 80 100 120 Pre Post 1H Post Least Square Mean Area Urea FDR = 0.0108 Formed in liver; Removal of nitrogen and ammonia Slide 14 Appalachian state Human Performance Laboratory
- 16. 0 5 10 15 20 25 Pre Post 1H mg/dl Blood Urinary Nitrogen (BUN) Normal Range: 8-20 mg/dl Placebo ASEA BUN levels did not differ between treatment (treatment x time p-value=0.9743) Slide 15 Appalachian state Human Performance Laboratory
- 17. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Pre Post 1H mg/dl Creatinine Normal Range: 0.7-1.2 mg/dl Placebo ASEA Creatinine levels did not differ between treatment (treatment x time p-value=0.7717) Slide 16 Appalachian state Human Performance Laboratory
- 18. 0 0.2 0.4 0.6 0.8 1 Pre Post 1H mg/dl Bilirubin Normal Range: 0.3-1.2 mg/dl Placebo ASEA Bilirubin levels did not differ between treatment (treatment x time p-value=0.9971) Slide 17 Appalachian state Human Performance Laboratory
- 19. 56 58 60 62 64 66 68 Pre Post 1H IU/L Alkaline Phosphatase Normal Range: 39-117 IU/L Placebo ASEA Alkaline Phosphatase levels did not differ between treatment (treatment x time p-value=0.8819) Slide 18 Appalachian state Human Performance Laboratory
- 20. 0 5 10 15 20 25 30 35 40 Pre Post 1H IU/L AST Normal Range: 15-41 IU/L Placebo ASEA AST levels did not differ between treatment (treatment x time p-value=0.9546) Slide 19 Appalachian state Human Performance Laboratory
- 21. 0 5 10 15 20 25 30 35 40 Pre Post 1H IU/L ALT Normal Range: 17-63 IU/L Placebo ASEA ALT levels did not differ between treatment (treatment x time p-value=0.9739) Slide 20 Appalachian state Human Performance Laboratory