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.
3. BASELINE
TESTING for
VO2max,
body
composition
(N=20
subjects)
1-week • 75-km
cycling
ASEA
1 week • 75-km
Placebo cycling
1-h
Blood/urine :
3-week washout
Blood/urine : Pre-Ex Post- Ex
Crossover
Pre-Ex Post- Ex
1-h
1-week • 75-km
cycling
ASEA
1-week • 75-km
Placebo cycling
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 postexercise.
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).
Triglyceride Mobilization: corresponding
with the increase in free fatty acids, glycerol
was higher at baseline (indicative of extensive
adipose triglyceride hydrolysis).
Least Square Mean Area
1) Chronic Effect: Higher fatty acid levels preexercise (several types of fatty acids --- see slides).
Least Square Mean Area
Finding 1: Ingestion of ASEA beverage for one
week strongly increased serum fatty acids
levels (most likely from adipose tissue).
Myristic acid
14C Saturated Fatty Acid
FDR=6.49E-32
2,000
1,500
1,000
500
-
ASEA
Placebo
Myristic acid
14C Saturated Fatty Acid
FDR=2.61E-20
2,000
1,500
1,000
500
-
Pre
ASEA
Post
1H Post
Placebo
TG Hydrolysis
FFAs
Slide 4
+
Glycerol
with ASEA
Appalachian state
Human Performance Laboratory
6. Post 7-day Ingestion:
Fatty Acids Higher in ASEA vs. Placebo
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
-
Palmitic Acid
16C Saturated Fatty Acid
FDR=1.86E-25
Least Square Mean Area
Least Square Mean Area
Myristic acid
14C Saturated Fatty Acid
FDR=6.49E-32
ASEA
Placebo
Least Square Mean Area
Oleic Acid
18C Monounsaturated n9 Fatty Acid
FDR=5.21E-18
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Slide 5
25,000
20,000
15,000
10,000
5,000
-
ASEA
Placebo
Stearic Acid
18C Saturated Fatty Acid
FDR=3.22E-12
8000
7000
6000
5000
4000
3000
2000
1000
ASEA
Placebo
0
ASEA
Placebo
Appalachian state
Human Performance Laboratory
7. 7-days Ingestion of ASEA
Fatty Acids and Glycerol Backbone: Higher in ASEA vs. Placebo
Least Square Mean Area
Palmitelaidic Acid
16C Trans Fatty Acid
FDR=1.13E-08
Capric Acid
10C Saturated Fatty Acid
FDR=5.39E-07
1,200
160
1,000
140
120
800
100
600
80
400
60
40
200
-
20
ASEA
Placebo
0
ASEA
Placebo
Glycerol
Backbone of Triglycerides
FDR=9.49E-07
25000
20000
15000
10000
5000
0
Slide 6
ASEA
Placebo
Appalachian state
Human Performance Laboratory
8. Least Square Mean Area
Myristic acid
14C Saturated Fatty Acid
FDR=2.61E-20
2,000
1,500
1,000
500
-
Pre
Post
Least Square Mean Area
ASEA
20,000
15,000
10,000
5,000
-
Pre
Placebo
Post
ASEA
1,200
1H Post
Placebo
Oleic Acid
18C Monunsaturated n9 Fatty Acid
FDR = 7.96E-10
20,000
1,000
15,000
800
600
10,000
400
200
Pre
Post
ASEA
Slide 7
25,000
1H Post
Palmitelaidic Acid
16C Trans Fatty Acid
FDR=1.66E-16
-
Palmitic Acid
16C Saturated Fatty Acid
FDR=1.56E-20
Least Square Mean Area
Least Square Mean Area
Serum Fatty Acids During Exercise
Placebo
1H Post
5,000
-
Pre
Post
ASEA
1H Post
Placebo
Appalachian state
Human Performance Laboratory
9. Capric Acid
10C Saturated Fatty Acids
FDR=0.0059
12,000
10,000
8,000
6,000
4,000
2,000
-
Pre
Post
Least Square Mean Area
ASEA
1H Post
Least Square Mean Area
Stearic Acid
18C Saturated Fatty Acids
FDR=1.33E-06
1,000
800
600
400
200
Post
ASEA
Slide 8
100
50
-
Pre
Placebo
Post
1H Post
Placebo
Glyercol Monosterate
FDR = 0.0060
1,200
Pre
150
ASEA
Lauric Acid
12C Saturated Fatty Acids
FDR=0.0281
-
200
Placebo
1H Post
Least Square Mean Area
Least Square Mean Area
Serum Fatty Acids During Exercise
900
800
700
600
500
400
300
200
100
-
Pre
Post
ASEA
1H Post
Placebo
Appalachian state
Human Performance Laboratory
10. Serum Serine
FDR=0.0273
Serum Aspartate
FDR=0.0075
Serum Glycine
FDR=0.0162
Pyruvate
Pre
Post
Pre
1H Post
In urea cycle
Post
Oxaloacetate
Pre
Malate
Post
Succinate
1H Post
1H Post
Serum Threonine
FDR=0.0108
Pre
Post
1H Post
Via Beta Oxidation
alphaKetoglutarate
Fumarate
Serum Leucine
FDR=0.0004
Succinyl CoA
Pre
Via Odd Chain Beta Oxidation
Serum Valine
FDR=0.0089
Pre
Slide 9
Post
Placebo
1H Post
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
Post
Isocitrate
Graph Key
ASEA
Pre
Pre
Citrate
1H Post
Serum Malate
FDR= 0.0004
1H Post
Serum Citrate
FDR = 0.0037
Acetyl Co-A
Serum Fumarate
FDR=1.06E-06
Pre
Post
Post 1H Post
Post 1H Post
Via Glutamate
Serum Proline
FDR=0.0066
Pre
Post
1H Post
Appalachian state
Human Performance Laboratory
11. Serum Amino Acids at Pre, Post, and 1H Post-Exercise
“Sparing” of Amino Acids with ASEA
Proline
Krebs Entry: alpha Ketoglutarate
FDR = 0.0066
30,000
25,000
20,000
15,000
10,000
5,000
-
Pre
Post
ASEA
1H Post
Lesat Square Mean Area
Least Square Mean Area
Leucine
Krebs Entry: alpha Ketoglutarate
FDR =0.0004
50,000
40,000
30,000
20,000
10,000
-
Placebo
300
200
100
ASEA
Slide 10
Placebo
1H Post
Lesat Square Mean Area
Lesat Square Mean Area
400
Post
1H Post
Placebo
Valine
Krebs Entry: Succinyl CoA
FDR = 0.0089
500
Pre
Post
ASEA
Aspartate
Krebs Entry: Oxaloacetate
FDR = 0.0074
-
Pre
50,000
40,000
30,000
20,000
10,000
-
Pre
Post
ASEA
1H Post
Placebo
Appalachian state
Human Performance Laboratory
12. Serum Amino Acids at Pre, Post, and 1H Post-Exericse
“Sparing” of Amino Acids with ASEA
Serine
Krebs Entry: Pyruvate
FDR= 0.0273
7,000
Lesat Square Mean Area
Lesat Square Mean Area
Threonine
Krebs Entry: Pyruvate
FDR= 0.0108
6,000
5,000
4,000
3,000
2,000
1,000
-
Pre
Post
1H Post
12,000
10,000
8,000
6,000
4,000
2,000
-
Pre
Post
1H Post
Lesat Square Mean Area
Glycine
Krebs Entry: Pyruvate
FDR= 0.0162
5
5
4
4
3
3
2
2
1
1
-
Slide 11
Graph Key
ASEA
Pre
Post
1H Post
Placebo
Appalachian state
Human Performance Laboratory
13. Serum Krebs Intermediate at Pre, Post, and 1H Post-Exercise
Higher Levels with ASEA
Fumarate
FDR = 1.06E-06
Malate
FDR= 0.0004
200
Lesat Square Mean Area
Lesat Square Mean Area
600
500
400
300
200
100
-
Pre
Post
1H Post
180
160
140
120
100
80
60
40
20
-
Pre
Post
1H Post
Citrate
FDR = 0.0037
Least Squre Mean Area
60.00
50.00
40.00
30.00
Graph Key
20.00
10.00
-
Slide 12
ASEA
Pre
Post
Placebo
1H Post
Appalachian state
Human Performance Laboratory
14. 3) Ascorbic Acid Metabolism:
ASEA supplementation appears to be affecting ascorbic acid both acutely and chronically.
Fructose
FDR=1.12E-05
Least Square Mean
Area
Least Square Mean
Area
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.
1500
1000
500
0
ASEA
Placebo
Threonic Acid
FDR=1.46E-98
8
6
4
2
0
ASEA
Placebo
Acute Differences: ASEA group has higher levels of ascorbic acid, an antioxidant, and lower levels of
both fructose and threonic acid.
1,000
500
Pre
Post
1H Post
1,500
1,000
500
-
Pre
Graph Key
Slide 13
ASEA
Placebo
Post
1H Post
Least Square Mean
Area
1,500
-
Threonic Acid
FDR=0.0031
Fructose
FDR = 0.0002
Least Square Mean
Area
Least Square Mean
Area
Ascorbic Acid
FDR = 5.6E-06
10
8
6
4
2
-
Pre
Post
1H Post
Appalachian state
Human Performance Laboratory
15. 4) Other Changes.
Aminomalonic Acid
FDR = 1.13E-05
400
Serum Creatinine
FDR=2.55 E-06
Least Square Mean Area
Least Squre Mean Area
Some other changes were found both acutely and
chronically that require further investigation into implications.
350
300
250
200
150
100
50
900
800
700
600
500
400
300
200
100
-
-
Pre
Post
Pre
1H Post
Plays role in binding calcium to protein
Post
1H Post
Breakdown product of creatine
Graph Key
ASEA
Slide 14
Placebo
Least Square Mean Area
Urea
FDR = 0.0108
120
100
80
60
40
20
-
Pre
Post
1H Post
Formed in liver; Removal of nitrogen and ammonia
Appalachian state
Human Performance Laboratory
16. Blood Urinary Nitrogen (BUN)
Normal Range: 8-20 mg/dl
25
mg/dl
20
15
Placebo
10
ASEA
5
0
Pre
Post
1H
BUN levels did not differ between treatment
(treatment x time p-value=0.9743)
Slide 15
Appalachian state
Human Performance Laboratory
17. Creatinine
Normal Range: 0.7-1.2 mg/dl
1.4
1.2
mg/dl
1
0.8
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)
Slide 16
Appalachian state
Human Performance Laboratory
18. Bilirubin
Normal Range: 0.3-1.2 mg/dl
1
mg/dl
0.8
0.6
Placebo
0.4
ASEA
0.2
0
Pre
Post
1H
Bilirubin levels did not differ between treatment
(treatment x time p-value=0.9971)
Slide 17
Appalachian state
Human Performance Laboratory
19. Alkaline Phosphatase
Normal Range: 39-117 IU/L
68
66
IU/L
64
62
Placebo
60
ASEA
58
56
Pre
Post
1H
Alkaline Phosphatase levels did not differ between
treatment (treatment x time p-value=0.8819)
Slide 18
Appalachian state
Human Performance Laboratory
20. IU/L
AST
Normal Range: 15-41 IU/L
40
35
30
25
20
15
10
5
0
Placebo
ASEA
Pre
Post
1H
AST levels did not differ between treatment
(treatment x time p-value=0.9546)
Slide 19
Appalachian state
Human Performance Laboratory
21. IU/L
ALT
Normal Range: 17-63 IU/L
40
35
30
25
20
15
10
5
0
Placebo
ASEA
Pre
Post
1H
ALT levels did not differ between treatment
(treatment x time p-value=0.9739)
Slide 20
Appalachian state
Human Performance Laboratory