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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




                                          Appalachian state
Slide 1                               Human Performance Laboratory
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
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
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
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-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
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
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
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
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
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
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
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
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
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
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
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
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
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
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

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ASEA Research Summary Presentation 2012

  • 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