ASEA Metabolomics Results Nieman DC. Human Performance Laboratory, North Carolina Research Campus and
Appalachian State University
Metabolomics Laboratory, North Carolina Research Campus,
David H. Murdock Research Institute
Slide 1 Appalachian state
Human Performance Laboratory
• 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-w
eek
was
hout
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
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
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 PlaceboLeas
t S
qu
are
Mea
n A
rea
Myristic acid 14C Saturated Fatty Acid
FDR=6.49E-32
-
500
1,000
1,500
2,000
Pre Post 1H Post
Leas
t S
qu
are
Mea
n A
rea
Myristic acid 14C Saturated Fatty Acid
FDR=2.61E-20
ASEA Placebo
Slide 4 Appalachian state
Human Performance Laboratory
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
Leas
t S
qu
are
Mea
n A
rea
Myristic acid 14C Saturated Fatty Acid
FDR=6.49E-32
-
5,000
10,000
15,000
20,000
25,000
ASEA Placebo
Leas
t S
qu
are
Mea
n A
rea
Palmitic Acid 16C Saturated Fatty Acid
FDR=1.86E-25
02000400060008000
1000012000140001600018000
ASEA PlaceboLeas
t S
qu
are
Mea
n A
rea
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
020406080
100120140160
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
Leas
t S
qu
are
Mea
n A
rea
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
-
5,000
10,000
15,000
20,000
Pre Post 1H Post
Leas
t S
qu
are
Mea
n A
rea
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
Leas
t S
qu
are
Mea
n A
rea
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
Leas
t S
qu
are
Mea
n A
rea
Palmitic Acid 16C Saturated Fatty Acid
FDR=1.56E-20
ASEA Placebo
-
500
1,000
1,500
2,000
Pre Post 1H Post
Leas
t S
qu
are
Mea
n A
rea
Myristic acid 14C Saturated Fatty Acid
FDR=2.61E-20
ASEA Placebo
Serum Fatty Acids During Exercise
Slide 7 Appalachian state
Human Performance Laboratory
-
50
100
150
200
Pre Post 1H PostLeas
t S
qu
are
Mea
n A
rea
Capric Acid 10C Saturated Fatty Acids
FDR=0.0059
ASEA Placebo
-
200
400
600
800
1,000
1,200
Pre Post 1H Post
Leas
t S
qu
are
Mea
n A
rea
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
Leas
t S
qu
are
Mea
n A
rea
Stearic Acid 18C Saturated Fatty Acids
FDR=1.33E-06
ASEA Placebo
- 100 200 300 400 500 600 700 800 900
Pre Post 1H PostLeas
t S
qu
are
Mea
n A
rea
Glyercol Monosterate FDR = 0.0060
ASEA Placebo
Serum Fatty Acids During Exercise
Slide 8 Appalachian state
Human Performance Laboratory
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
-
5,000
10,000
15,000
20,000
25,000
30,000
Pre Post 1H Post
Leas
t S
qu
are
Mea
n A
rea
Leucine Krebs Entry: alpha Ketoglutarate
FDR =0.0004
ASEA Placebo
-
10,000
20,000
30,000
40,000
50,000
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
Proline Krebs Entry: alpha Ketoglutarate
FDR = 0.0066
ASEA Placebo
-
100
200
300
400
500
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
Aspartate Krebs Entry: Oxaloacetate
FDR = 0.0074
ASEA Placebo
-
10,000
20,000
30,000
40,000
50,000
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
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
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
Lesa
t S
qu
are
Mea
n A
rea
Threonine Krebs Entry: Pyruvate
FDR= 0.0108
-
2,000
4,000
6,000
8,000
10,000
12,000
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
Serine Krebs Entry: Pyruvate
FDR= 0.0273
- 1 1 2 2 3 3 4 4 5 5
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
Glycine Krebs Entry: Pyruvate
FDR= 0.0162
Graph Key
ASEA Placebo
Slide 11 Appalachian state
Human Performance Laboratory
Serum Krebs Intermediate at Pre, Post, and 1H Post-Exercise Higher Levels with ASEA
-
100
200
300
400
500
600
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
Fumarate FDR = 1.06E-06
- 20 40 60 80
100 120 140 160 180 200
Pre Post 1H Post
Lesa
t S
qu
are
Mea
n A
rea
Malate FDR= 0.0004
-
10.00
20.00
30.00
40.00
50.00
60.00
Pre Post 1H Post
Leas
t S
qu
re M
ean
Are
a
Citrate FDR = 0.0037
Graph Key
ASEA Placebo
Slide 12 Appalachian state
Human Performance Laboratory
3) Ascorbic Acid Metabolism: ASEA supplementation appears to be affecting ascorbic acid both acutely and chronically.
0
500
1000
1500
ASEA Placebo
Leas
t S
qu
are
Mea
n
Are
a
Fructose FDR=1.12E-05
0
2
4
6
8
ASEA Placebo
Leas
t S
qu
are
Mea
n
Are
a
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
Leas
t S
qu
are
Mea
n
Are
a
Ascorbic Acid FDR = 5.6E-06
-
500
1,000
1,500
Pre Post 1H Post
Leas
t S
qu
are
Mea
n
Are
a
Fructose FDR = 0.0002
-
2
4
6
8
10
Pre Post 1H PostLeas
t S
qu
are
Mea
n
Are
a
Threonic Acid FDR=0.0031
Graph Key
ASEA Placebo Slide 13 Appalachian state
Human Performance Laboratory
- 100 200 300 400 500 600 700 800 900
Pre Post 1H Post
Leas
t S
qu
are
Mea
n A
rea
Serum Creatinine FDR=2.55 E-06
Graph Key
ASEA Placebo
-
50
100
150
200
250
300
350
400
Pre Post 1H Post
Leas
t S
qu
re M
ean
Are
a
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
Leas
t S
qu
are
Mea
n A
rea
Urea FDR = 0.0108
Formed in liver; Removal of nitrogen and ammonia
Slide 14 Appalachian state
Human Performance Laboratory
0
5
10
15
20
25
Pre Post 1H
mg/
dl
Blood Urinary Nitrogen (BUN) Normal Range: 8-20 mg/dl
PlaceboASEA
BUN levels did not differ between treatment (treatment x time p-value=0.9743)
Slide 15 Appalachian state
Human Performance Laboratory
00.20.40.60.8
11.21.4
Pre Post 1H
mg/
dl
Creatinine Normal Range: 0.7-1.2 mg/dl
PlaceboASEA
Creatinine levels did not differ between treatment (treatment x time p-value=0.7717)
Slide 16 Appalachian state
Human Performance Laboratory
0
0.2
0.4
0.6
0.8
1
Pre Post 1H
mg/
dl
Bilirubin Normal Range: 0.3-1.2 mg/dl
PlaceboASEA
Bilirubin levels did not differ between treatment (treatment x time p-value=0.9971)
Slide 17 Appalachian state
Human Performance Laboratory
56
58
60
62
64
66
68
Pre Post 1H
IU/L
Alkaline Phosphatase Normal Range: 39-117 IU/L
PlaceboASEA
Alkaline Phosphatase levels did not differ between treatment (treatment x time p-value=0.8819)
Slide 18 Appalachian state
Human Performance Laboratory
05
10152025303540
Pre Post 1H
IU/L
AST Normal Range: 15-41 IU/L
PlaceboASEA
AST levels did not differ between treatment (treatment x time p-value=0.9546)
Slide 19 Appalachian state
Human Performance Laboratory
05
10152025303540
Pre Post 1H
IU/L
ALT Normal Range: 17-63 IU/L
PlaceboASEA
ALT levels did not differ between treatment (treatment x time p-value=0.9739)
Slide 20 Appalachian state
Human Performance Laboratory