This document outlines the results of a metabolomics study on the effects of Asea supplementation in male cyclists, highlighting its impact on free fatty acids, fat oxidation, and amino acid sparing during exercise. The findings suggest significant increases in various serum metabolites, particularly in free fatty acids and ascorbic acid levels, after a week of Asea ingestion compared to a placebo. Additionally, changes in other metabolites related to energy metabolism and antioxidative stress were noted, indicating potential benefits of Asea supplementation for athletic performance.

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
Appalachian state
Slide 1 Human Performance Laboratory

3. Blood/urine : Pre-Ex Post- Ex 1-h Blood/urine : Pre-Ex Post- Ex 1-h
Crossover
1-week • 75-km 1-week • 75-km
3-week washout
BASELINE
TESTING for ASEA cycling cycling
ASEA
VO2max,
body
1 week • 75-km
composition
(N=20 1-week • 75-km
Placebo cycling Placebo cycling
subjects)
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.
Appalachian state
Slide 2 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. Appalachian state
Slide 3 Human Performance Laboratory

5. Myristic acid
Finding 1: Ingestion of ASEA beverage for one 14C Saturated Fatty Acid
week strongly increased serum fatty acids FDR=6.49E-32
Least Square Mean Area
levels (most likely from adipose tissue). 2,000
1,500
1) Chronic Effect: Higher fatty acid levels pre- 1,000
exercise (several types of fatty acids --- see slides). 500
-
ASEA Placebo
2) Acute Effect: Increased fatty acid oxidation Myristic acid
14C Saturated Fatty Acid
and mobilization during exercise (placebo
Least Square Mean Area
FDR=2.61E-20
condition was linked to a late mobilization). 2,000
1,500
1,000
500
-
Triglyceride Mobilization: corresponding Pre Post 1H Post
with the increase in free fatty acids, glycerol ASEA Placebo
was higher at baseline (indicative of extensive
adipose triglyceride hydrolysis).
TG Hydrolysis
FFAs + Glycerol with ASEA
Appalachian state
Slide 4 Human Performance Laboratory

6. Post 7-day Ingestion:
Fatty Acids Higher in ASEA vs. Placebo
Myristic acid Palmitic Acid
14C Saturated Fatty Acid 16C Saturated Fatty Acid
FDR=6.49E-32 FDR=1.86E-25
Least Square Mean Area
2,000 25,000
Least Square Mean Area
1,800
1,600 20,000
1,400
1,200 15,000
1,000
800 10,000
600
400 5,000
200
- -
ASEA Placebo ASEA Placebo
Oleic Acid Stearic Acid
18C Monounsaturated n9 Fatty Acid 18C Saturated Fatty Acid
FDR=5.21E-18 FDR=3.22E-12
8000
18000
7000
Least Square Mean Area
16000
6000
14000
12000 5000
10000 4000
8000 3000
6000
2000
4000
2000 1000
0 0
ASEA Placebo ASEA Placebo
Appalachian state
Slide 5 Human Performance Laboratory

7. 7-days Ingestion of ASEA
Fatty Acids and Glycerol Backbone: Higher in ASEA vs. Placebo
Palmitelaidic Acid Capric Acid
16C Trans Fatty Acid 10C Saturated Fatty Acid
FDR=1.13E-08 FDR=5.39E-07
1,200 160
Least Square Mean Area
1,000 140
120
800
100
600 80
400 60
40
200
20
- 0
ASEA Placebo ASEA Placebo
Glycerol
Backbone of Triglycerides
FDR=9.49E-07
25000
20000
15000
10000
5000
0
ASEA Placebo Appalachian state
Slide 6 Human Performance Laboratory

8. Serum Fatty Acids During Exercise
Myristic acid Palmitic Acid
14C Saturated Fatty Acid 16C Saturated Fatty Acid
Least Square Mean Area
FDR=2.61E-20 FDR=1.56E-20
Least Square Mean Area
2,000 25,000
1,500 20,000
15,000
1,000
10,000
500
5,000
- -
Pre Post 1H Post Pre Post 1H Post
ASEA Placebo ASEA Placebo
Palmitelaidic Acid Oleic Acid
16C Trans Fatty Acid 18C Monunsaturated n9 Fatty Acid
Least Square Mean Area
Least Square Mean Area
FDR=1.66E-16 FDR = 7.96E-10
1,200 20,000
1,000
15,000
800
600 10,000
400
5,000
200
- -
Pre Post 1H Post Pre Post 1H Post
ASEA Placebo ASEA Placebo
Appalachian state
Slide 7 Human Performance Laboratory

9. Serum Fatty Acids During Exercise
Stearic Acid Capric Acid
18C Saturated Fatty Acids 10C Saturated Fatty Acids
FDR=1.33E-06 FDR=0.0059
Least Square Mean Area
Least Square Mean Area
12,000 200
10,000
150
8,000
6,000 100
4,000
50
2,000
- -
Pre Post 1H Post Pre Post 1H Post
ASEA Placebo ASEA Placebo
Lauric Acid Glyercol Monosterate
12C Saturated Fatty Acids FDR = 0.0060
FDR=0.0281
Least Square Mean Area
900
Least Square Mean Area
1,200 800
1,000 700
600
800
500
600 400
400 300
200
200 100
- -
Pre Post 1H Post Pre Post 1H Post
ASEA Placebo ASEA Placebo
Appalachian state
Slide 8 Human Performance Laboratory

10. Serum Aspartate Serum Serine
Serum Glycine
FDR=0.0075 FDR=0.0273
FDR=0.0162
Pyruvate
Pre Post 1H Post Pre Post 1H Post
Pre Post 1H Post
In urea cycle
Acetyl Co-A Serum Citrate
Serum Fumarate FDR = 0.0037
FDR=1.06E-06
Oxaloacetate Citrate
Pre Post 1H Post
Pre Post 1H Post Serum Threonine
FDR=0.0108
Serum Malate Malate Isocitrate
FDR= 0.0004 Graph Key
Pre Post 1H Post
ASEA Placebo
Pre Post 1H Post Via Beta Oxidation
alpha-
Fumarate
Ketoglutarate
Serum Leucine
FDR=0.0004
Succinate Succinyl CoA
Pre Post 1H Post
Finding 2: High levels of
blood free fatty acids were Via Odd Chain Beta Oxidation Via Glutamate
linked to a sparing of amino Serum Valine Serum Proline
FDR=0.0089
acid catabolism, and FDR=0.0066
increased Krebs Cycle
intermediates, post-exercise
Pre Post 1H Post
Pre Post 1H Post
Appalachian state
Slide 9
Human Performance Laboratory

11. Serum Amino Acids at Pre, Post, and 1H Post-Exercise
“Sparing” of Amino Acids with ASEA
Leucine Proline
Krebs Entry: alpha Ketoglutarate Krebs Entry: alpha Ketoglutarate
FDR =0.0004 FDR = 0.0066
30,000 50,000
Least Square Mean Area
Lesat Square Mean Area
25,000 40,000
20,000
30,000
15,000
20,000
10,000
5,000 10,000
- -
Pre Post 1H Post Pre Post 1H Post
ASEA Placebo ASEA Placebo
Aspartate Valine
Krebs Entry: Oxaloacetate Krebs Entry: Succinyl CoA
FDR = 0.0074 FDR = 0.0089
500 50,000
Lesat Square Mean Area
Lesat Square Mean Area
400 40,000
300 30,000
200 20,000
100 10,000
- -
Pre Post 1H Post Pre Post 1H Post
ASEA Placebo ASEA Placebo
Appalachian state
Slide 10 Human Performance Laboratory

12. Serum Amino Acids at Pre, Post, and 1H Post-Exericse
“Sparing” of Amino Acids with ASEA
Threonine Serine
Krebs Entry: Pyruvate Krebs Entry: Pyruvate
FDR= 0.0108 FDR= 0.0273
7,000 12,000
Lesat Square Mean Area
Lesat Square Mean Area
6,000 10,000
5,000
8,000
4,000
6,000
3,000
4,000
2,000
1,000 2,000
- -
Pre Post 1H Post Pre Post 1H Post
Glycine
Krebs Entry: Pyruvate
FDR= 0.0162
5
Lesat Square Mean Area
5
4
4
3
3 Graph Key
2
2
1
ASEA Placebo
1
-
Pre Post 1H Post
Appalachian state
Slide 11 Human Performance Laboratory

13. Serum Krebs Intermediate at Pre, Post, and 1H Post-Exercise
Higher Levels with ASEA
Fumarate Malate
FDR = 1.06E-06 FDR= 0.0004
600 200
Lesat Square Mean Area
Lesat Square Mean Area
180
500
160
400 140
120
300 100
80
200
60
100 40
20
- -
Pre Post 1H Post Pre Post 1H Post
Citrate
FDR = 0.0037
60.00
Least Squre Mean Area
50.00
40.00
30.00
20.00 Graph Key
10.00
ASEA Placebo
-
Pre Post 1H Post
Appalachian state
Slide 12 Human Performance Laboratory

14. 3) Ascorbic Acid Metabolism:
ASEA supplementation appears to be affecting ascorbic acid both acutely and chronically.
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.
Fructose Threonic Acid
FDR=1.12E-05 FDR=1.46E-98
Least Square Mean
Least Square Mean
1500 8
6
1000
Area
Area
4
500
2
0 0
ASEA Placebo ASEA Placebo
Acute Differences: ASEA group has higher levels of ascorbic acid, an antioxidant, and lower levels of
both fructose and threonic acid.
Ascorbic Acid Fructose Threonic Acid
FDR = 5.6E-06 FDR = 0.0002 FDR=0.0031
Least Square Mean
Least Square Mean
1,500 1,500 10
Least Square Mean
8
1,000 1,000
Area
Area
6
Area
500 500 4
2
- -
Pre Post 1H Post Pre Post 1H Post -
Pre Post 1H Post
Graph Key
Appalachian state
Slide 13 ASEA Placebo Human Performance Laboratory

15. 4) Other Changes. Some other changes were found both acutely and
chronically that require further investigation into implications.
Aminomalonic Acid Serum Creatinine
FDR = 1.13E-05 FDR=2.55 E-06
Least Square Mean Area
400 900
Least Squre Mean Area
350 800
300 700
250 600
500
200
400
150
300
100 200
50 100
- -
Pre Post 1H Post Pre Post 1H Post
Plays role in binding calcium to protein Breakdown product of creatine
Urea
FDR = 0.0108
Least Square Mean Area 120
100
80
60
40
20
Graph Key
-
Pre Post 1H Post
ASEA Placebo Formed in liver; Removal of nitrogen and ammonia
Appalachian state
Slide 14 Human Performance Laboratory

16. Blood Urinary Nitrogen (BUN)
Normal Range: 8-20 mg/dl
25
20
15
mg/dl
Placebo
10 ASEA
5
0
Pre Post 1H
BUN levels did not differ between treatment
(treatment x time p-value=0.9743)
Appalachian state
Slide 15 Human Performance Laboratory

17. Creatinine
Normal Range: 0.7-1.2 mg/dl
1.4
1.2
1
0.8
mg/dl
Placebo
0.6
ASEA
0.4
0.2
0
Pre Post 1H
Creatinine levels did not differ between treatment (treatment
x time p-value=0.7717)
Appalachian state
Slide 16 Human Performance Laboratory

18. Bilirubin
Normal Range: 0.3-1.2 mg/dl
1
0.8
0.6
mg/dl
Placebo
0.4 ASEA
0.2
0
Pre Post 1H
Bilirubin levels did not differ between treatment
(treatment x time p-value=0.9971)
Appalachian state
Slide 17 Human Performance Laboratory

19. Alkaline Phosphatase
Normal Range: 39-117 IU/L
68
66
64
IU/L
62 Placebo
60 ASEA
58
56
Pre Post 1H
Alkaline Phosphatase levels did not differ between
treatment (treatment x time p-value=0.8819)
Appalachian state
Slide 18 Human Performance Laboratory

20. AST
Normal Range: 15-41 IU/L
40
35
30
25
IU/L
20 Placebo
15 ASEA
10
5
0
Pre Post 1H
AST levels did not differ between treatment
(treatment x time p-value=0.9546)
Appalachian state
Slide 19 Human Performance Laboratory

21. ALT
Normal Range: 17-63 IU/L
40
35
30
25
IU/L
20 Placebo
15 ASEA
10
5
0
Pre Post 1H
ALT levels did not differ between treatment
(treatment x time p-value=0.9739)
Appalachian state
Slide 20 Human Performance Laboratory