Transcript Haky 2012 Creatine Effects on Microbial Florax

Creatine Effects on Microbial
Flora
Andrew Haky
11th Grade
Pittsburgh Central Catholic HS
Microbial Flora
• The skin has a normal flora of eukaryotic fungi, protists, and
bacteria
• Not much is known about the association between humans
and their flora
• Effects can be mutualistic, parasitic, pathogenic, and
commensal
• Normal Flora provide nutritional and digestive benefits,
secrete vitamins, stimulate antibody production, and protect
against pathogenic microbes
• Supplements taken by humans might have unintended effects
on these important functions of the flora
• Bacteria capable of expressing specific enzymes to degrade
creatine have been found in the bacterial flora of the human
colon
Creatine
• An organic acid naturally synthesized
from amino acids (methionine, glycine,
arginine) primarily in the kidney and
liver, then transported in the blood for
use by muscles.
• Approximately 95% located in the
skeletal muscle.
• Direct relation to ATP production and
storage.
• For an adult 70 kg male, daily turnover
of creatine has been estimated at about
2g.
Supplemental Creatine
• Most popular bodybuilding supplement on the market.
• Has such an impact because it super-hydrates muscle cells with
water.
• Enhances muscle growth, and strengthens fibers.
• Increases energy levels, strength, and recovery rates.
Accelerates weight loss and builds lean body mass.
• Used to increase athletic performance, in the treatment of
heart disease, muscular dystrophy, and Parkinson’s Disease.
Two Types of Creatine:
• Free Creatine (Crf)
– One-third of skeletal muscle is present in Crf
form.
• Phosphorylated (Crphos)
– The rest is present in Crphos form.
“Studies have shown that both Crf and Crphos levels
in skeletal muscle can be increased, and
performance of high intensity intermittent exercise
enhanced, following a period of creatine
supplementation.”
Creatine
Increases
Phosphocreatine
storage
Increases ability
of muscles to
resynthesize ATP
from ADP
• Thus,
increased
energy
demands can
be met.
Previous Studies:
• Effects on Isokinetic Muscle Torque:
– “muscle peak torque production was greater in all
subjects, when compared with the data prior to
creatine ingestion.”
– Placebo effect showed no difference
• Increase in Creatine pool:
– “supplementation with 5g of creatine monohydrate
resulted in a significant increase in the total creatine
content of the quadriceps femoris muscle measured
in 17 subjects.”
Escherichia coli (E. coli)
• Cells are rod shaped and usually about 2 micrometers
in length
• Gram-negative
• Part of the human flora; found in the human colon
and digestive tract
• Reproduces rapidly, often within thirty minutes
• Commonly used as a prokaryote cell model
• Many strains, most non-pathogenic
• Free living, symbiotic or pathogenic
Staphylococcus epidermidis
• Part of our normal skin flora, gram-positive
• Anaerobic, but grows best in aerobic
conditions
• Opportunistic pathogen, requires major
breach in hosts defenses
• Non-pathogenic strain utilized
Purpose
• To determine the effects of
creatine on E.coli and Staph e.
survivorship/proliferation
Hypothesis
NULL Hypothesis
• Creatine will
significantly
increase Staph.
and E. coli
proliferation/
survivorship.
• Creatine will not
significantly affect
Staph or E. coli
proliferation/
survivorship.
Microbes may degrade
creatine into by-products
that can benefit cell
metabolism.
Materials
 Ethanol (sterilization of instruments)
 Latex gloves
 LB media (0.5% yeast extract, 1%
tryptone, 1% sodium chloride)
 E. coli DH5 alpha
 Bunsen burner
 Staphylococcus epidermidis
 Spreader bars
 Micropipette
 Matches
 Tube rack
 Sterile pipette tips
 Test Tubes
 Incubator
 SDF (per 1 liter) (100mM KH2PO4,
 Vortex
100mM K2HPO4, 10mM MgSO4, 1mM
NaCl)
 LB agar plates
 Klett spectrophotometer and sidearm
flasks
 Creatine
Procedure
1) E. coli and Staphylococcus epidermidis were grown
overnight in sterile LB media.
2) A sample of the overnight culture was added to
fresh media in a sterile sidearm flask.
3) The cultures were placed in an incubator (37°C)
until a density of 50 Klett spectrophotometer units
was reached. This represents a cell density of
approximately 108 cells/mL.
4) The culture was diluted in sterile dilution fluid to a
concentration of approximately 103 cells/mL.
5) The tubes were prepared as follows:
Table of Concentrations
LIQUID
EXPOSURE
5 replicates
per group
0%
Concentration
0.1%
Concentration
1%
Concentration
10 %
Concentration
Microbe
E.coli/Staph
0.1mL
0.1mL
0.1mL
0.1mL
SDF
9.9mL
9.89mL
9.8mL
8.9mL
Creatine
Solution
0mL
0.01mL
0.1mL
1mL
Final Volume
10mL
10mL
10mL
10mL
Procedure, con’t
6) The E. coli and Staphylococcus epidermidis were
exposed to the creatine concentrations for 20
minutes.
7) 100 µL aliquots were removed from the tubes
and spread on LB plates.(5 replicates)
8) The plates were incubated at 37 degrees
Celsius for 24 hours.
9) The resulting colonies were quantified. Each
colony is assumed to have arisen from one
cell.
Creatine Effects on E.coli
200
P-value= 0.000251
180
160
Colony Count
140
120
100
80
60
40
20
0
0%
0.10%
1%
Creatine Concentration
10%
Creatine Effects on Staph.
250
P-value=9.33E-06
Colony count
200
150
100
50
0
0%
0.10%
1%
Creatine Concentration
10%
Dunnet’s Test
Test Group
T-crit value=3.29
T-value
Interpretation
0.1% creatine
3.51
Significant
1% creatine
3.00
Insignificant
10% creatine
5.91
Significant
0.1% creatine
0.56
Insignificant
1% creatine
3.20
Insignificant
10% creatine
7.05
Significant
e.coli groups:
staph. groups
Analysis of Results
• Did liquid exposure to creatine significantly affect E.coli and
Staph. proliferation/survivorship?
– P-value= 0.000251 (E.coli), SIGNIFICANT
– P-value=9.33E-06 (Staph.), SIGNIFICANT
• Which concentrations of creatine significantly effected e.
coli proliferation/survivorship?
– 0.1% creatine, T-value: 3.51, SIGNIFICANT
– 1% creatine, T-value: 3.00, INSIGNIFICANT
– 10% creatine, T-value: 5.91, SIGNIFICANT
• Which concentrations of creatine significantly effected
staph. proliferation/survivorship?
– 0.1% creatine, T-value: 0.56, INSIGNIFICANT
– 1% creatine, T-value:3.20, INSIGNIFICANT
– 10% creatine, T-value:7.05, SIGNIFICANT
Conclusion
• The null hypothesis can be rejected because
the variable did have a significant effect.
• It appears that creatine did have a positive
survivorship effect on the microbial flora tested.
• There are many reasons why creatine could
have had this positive survivorship effect.
• However, microbial creatine degradation at
present is not completely understood.
• An evolutionary screening and characterization
of enzymes involved in digestion is necessary
for this understanding.
Limits:
•
•
•
•
Only one exposure time was used
Ways to better dissolve creatine
More replicates
Better synchronization of plating
Extensions:
•
•
•
•
•
A growth curve analysis
Other microbial models
Synergistic effects of multiple variables
Creatine effects on stressed microbes
Could test creatine on a model representing muscle cells
• Creatine effects on stem cell differentiation
• Other variables: whey, glutamine
Sources
•
•
•
•
•
•
•
•
Balsom PD, Söderlund K, and Ekblom B. "Creatine in Humans with Special Reference to Creatine
Supplementation." National Center for Biotechnology Information. US National Library of Medicine,
18 Oct. 1994. Web. 12 Nov. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/7817065>.
"Creatine Background : What Is Creatine?" Creatine Information Center. Web. 12 Nov. 2011.
<http://www.creatinemonohydrate.net/creatine_background.html>.
"Creatine." Wikipedia, the Free Encyclopedia. Web. 12 Nov. 2011.
<http://en.wikipedia.org/wiki/Creatine>.
Greenhaff PL, Hultman E, Soderlund K, Harris R, Short AH, and Casey A. "Influence of Oral Creatine
Supplementation of Muscle Torque during Repeated Bouts of Maximal Voluntary Exercise in Man."
Department of Physiology and Pharmacology, Queens Medical Centre, University of Nottingham,
U.K., May 1993. Web. 12 Nov. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/8504634>.
Harris RC, Söderlund K, and Hultman E. "Elevation of Creatine in Resting and Exercised Muscle of
Normal Subjects by Creatine Supplementation." PubMed.gov. Department of Clinical Chemistry II,
Karolinska Institute, Huddinge University Hospital, Sweden, Sept. 1992. Web. 12 Nov. 2011.
<http://www.ncbi.nlm.nih.gov/sites/entrez/1327657?dopt=Abstract>.
Http://www.euronet.nl/~jonkr/, Ron Jonk-. "Creatine." University of Maryland Medical Center |
Home. Web. 12 Nov. 2011. <http://www.umm.edu/altmed/articles/creatine-000297.htm>.
Hu, Amanda. "Looks Can Be Deceiving: the Case of Escherichia Coli." Journal of Young Investigators
6.5 (2002). Web. 12 Nov. 2011. <http://www.jyi.org/volumes/volume6/issue5/features/hu.html>.
"Staphylococcus Epidermidis." Medpedia. Web. 12 Nov. 2011.
<http://wiki.medpedia.com/Staphylococcus_epidermidis>.
E.coli Analysis of Variance
Anova: Single Factor
SUMMARY
Groups
Column 1
Column 2
Column 3
Column 4
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
Count
Sum
5
5
5
5
SS
625
793
769
908
df
Average
125
158.6
153.8
181.6
MS
8108.55
3
2702.85
3667.2
16
229.2
11775.75
19
Variance
109
161.8
192.2
453.8
F
11.79254
P-value
0.000251
F crit
3.238872
Staph. Analysis of Variance
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
5
652
130.4
46.8
Column 2
5
677
135.4
368.3
Column 3
5
796
159.2
318.7
Column 4
5
969
193.8
74.7
ANOVA
Source of
Variation
Between
Groups
Within
Groups
Total
SS
df
MS
12560.2
3
4186.733
3234
16
202.125
15794.2
19
F
20.71358
P-value
9.33E-06
F crit
3.238872