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JOURNAL OF THE AMERICAN COLLEGE OF

TRITION

Abstract 47

EFFECT ON SERUM MYQSTATIN LEVELS OF mGH-GRADE HANDLED FERTILE EGG YOLK POWDER

Calker, Co' Peak Wellness, Inc; Greenwich, CT

Results of a prior study on Folstaxan™ revealed that proper handling of a fertile egg yolk powder when ingested results in a significant reduction of serum myostatin at 24 hours; These seven (7) subjects ingested 20 grams of product and saw an average 24hr., reduction in serum myostatin of 11.32% with two non-responders (Colker C, Effect of DebulkedFertileEgg Yolk Ingestion On Serum Myostatin Levels. 2008 Conference on Post- Translational Modification: Detection and Physiological Evaluation for the American -Society jar Biochemistry and Molecular Biology; Category: Unusual Modifications; Abstract 10; October 2008). One of the limits of this earlier method, among a number of others issues, was the finding that irradiation destabilized the yolk membrane. This created a slurry, making any further debu..Ik:ing beyond simple breaking, 'and separating very challenging arid thus highly Unpractical from a commercial standpoint In addition, there was the problem of centrifuging as a relatively inefficient

. way to dubulk, A new generation of product (MYO-T12TM) was developed and tested for its comparative ability to suppress serum myostatin in ten (10) healthy adult male human subjects. After baseline testing (time 0). subjects received a single serving bolus of only ten (10) grams. This study was undertaken to test the hypothesis of whether high-grade handling eliminates the need for irradiation and centrifuging thus creating a more highly effective product at a much lower serving size: After initial blooddraw

and providing oral MYO-T12™ a single ten (10) gram serving size, serum myostatin levels were qual itiiti vely and quantitarivelymeasared as an indicator of pharmacokinetic acrivity, Testing utilized a purchasedmyostarin standardfer verification. Confirmations wererun by ELISA and quantitarions by Liquid Chromatography Tandem Mass Spectrometer with third degree fragmentation (Expertox, D~ Park. TX), Results showed an average baseline (time 0) myostatin level of27,5pg/ml; 12-18 hours after dosing average serum myostatin declined to 12.6pg/ml; Average serum myostatin completely normalized with a. 24-30 hour level of28,lpg/ml. All subjects responded. These results indicate thata high-grade handled fertile egg yolk powder, when orally ingested at only a. single 10 gram serving size) produces a staggering 46% reduction in serum myostatin in only 12-18 hours, with complete normalization after 24-30 hours .

VOLUME 28 • NUMBER 3

. JUNE 20'09

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JOURNAL OF THE AMERICAN COLLEGE OF

TRITION

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Abstract 46

EFFECT OF HIGH-GRADE HANDLING OF FERTll..E EGG YOLKS ON

FOLLISTATIN CONCENTRATION .

Calker,C,' PeakWellness.lnc.,Greenwtch, CT .

Prior.study has confirmed that biologically active follistatin occurs primarily in the yolk membrane of'fertile chicken eggs where it is producedatthe site of the germination. disc (bIastodisc). Usingthe previous method of separating and centrifuging yolk membranes a follistatinconceatration of0.48mcglmg was achieved (Calker C. Follistatin Resides Primarily In The Yolk Membrane. 2008 Conference on Post-Translational Modification: Detection and Physiological Evaluation for the

. American Society for Bioahemtstry and Molecular i3i~logy; Categozy: Unusual Modifications; Abstract 8; October 2008). In this study a method of high-grade handling designed to eliminate the need forcentrifuging Was examined to see if in fact a higher concentration offollistatin~s achieved using this technology. Follistatin was immobilized by binding toAetivin in a Cl8 HPLC column. The assay was standardized with Recombinant Pollistarin 288 and assayed in an LCIMS with third degree fragmentation (D.S. Tanetta, et, Al. 1977). Results revealed a sample concentration of 1.29mcglmg and thus clearly support the efficiency of this method and vast superiority over centrifuging.

VOLUME 28 • NUMBER .3

JUNE 2009

Colker C, Follistatin Resides PrimarilyIn The Yolk Membrane. Reviewed and accepted for poster presentation at the 2008 Conference on Post-Translational Modification:

Detection and Physiological Evaluation for the American Society for Biochemistry and Molecular Biology; Category: Unusual Modifications; Poster 5; Abstract 8; October 24, 2008.

FOLLISTATIN RESIDES PRIMARILY IN THE YOLK MEMBRANE

Colker, C; Peak Wellness, Inc., Greenwich, CT

Follistatin is found at higher levels in association with the yolk of fertile chicken eggs. We hypothesize that follistatin resides primarily in the vitelline (yolk) membrane and attached germination disc, as opposed to being uniformly dispersed in the yolk itself. In this test, yolks ware manually separated to test this hypothesis. Two sample groups were tested including a whole yolk control which included the entire yolk and yolk membranes (Sample A) verses the manually separated and centrifuged yolk membranes (Sample B). Individual membranes were homogenized before testing to insure the fo11istatin was evenly mixed before analysis. Follistatin was innnobilized by binding to Activin in a C 18 HPLC column. The assay was standardized with Recombinant Follistatin 288 and assayed in an LCIMS with third degree fragmentation (D.S. Tanetta, et. AI. 1977). Results showed a concentration of biologically active follistatin in the whole yolk control (Sample A) of 0.26mcglmg verses the separated and centrifuged sample (Sample B) with a follistatin concentration of0.48mcglmg. The fact that there was any measurable follistatin in the control Sample A can be accounted for by the included yolk membrane. The fact that the whole yolk concentration of follistatin was so low relative to the yolk membrane Sample B, can be accounted for by the dilution effect ofinc1uding the whole yolk contents in the sample which are bereft of any significant active follistatin. The presence of any follistatin in the bulk yolk content can be said to be due to a leaching effect offollistatin production from the area of the blastodicresiding on the membrane surface. In conclusion, this study confirms that biologically active follistatin occurs primarily in the yolk membrane of fertile chicken eggs where it is produced at the site of the germination disc (blastodisc).

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Colker C, Debulking of Fertile Chicken Egg Yolks Amplifies Available Follistatin. Reviewed and accepted for poster presentation at the 2008 Conference on PostTranslational Modification: Detection and Physiological Evaluation for the American Society for Biochemistry and Molecular Biology; Category: Unusual Modifications; Poster 5; Abstract 9; October 24,2008.

DEBULKING OF FERTILE CHICKEN EGG YOLKS AMPLIFIES AVAILABLE FOLLISTATIN

Colker, C; Peak Wellness, Inc., Greenwich, CT

We know that biologically active follistatin is found at higher levels in association with the yolk membrane of fertile chicken eggs. We also know that follistatin resides primarily in the vitelline (yolk) membrane and attached germination disc, as opposed to being uniformly dispersed in the yolk itself. The bulk yolk contents containing little or no folHstatin are primarily water and lipid in nature. In this study we tested the degree to which centrifuge debulking of the yolk water and lipid contents would amplify the concentration of yolk membrane and germination disc, which is the production site of biologically active follistatin. Twenty-four (24) fertile eggs (Trader Joe's, Upland, California) were manually yolk-separated and placed on individual dishes for weighing, lipid draining and washing with distilled water. Four (4) egg yolks were selected as control, the yolk membranes were then ruptured and mixed. A sample test tube loaded for a control sample (Sample A) was taken and frozen. The other twenty (20) yolks were ruptured and drained of lipid. Individual membranes were gently washed to remove excess lipids and weighed. Separated and washed membranes were then loaded in a test tube along with sodium acetate for centrifuging to remove remaining water and lipid. The supernatant was saved and frozen for testing (Sample B). Weights of the membranes were taken before and after centrifuging for later use in calculating membrane follistatin concentration and future yields while final samples were tested to confirm that concentrated, active follistatin remained in the debulked portion of egg yolk (vitelline membrane and germination disc) as opposed to the yolk itself. Samples of egg yolk debulked membranes were sent to test (ExperTox, Deer Park, TX). Results: Whole egg yolks had a weight range between 14.90 grams to 20.75 grams. The average whole yolk weight was 17.85 grams. The membranes, still containing excess wash water and lipids, had an average weight before centrifuging of 1.19 grams. After centrifuge debulking the average weight was 0.64 grams. This procedure successfully debulked the egg yolk by 28 times (17.85/0.64) while fully preserving the region containing active follistatin.

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Colker C~ Effect of Debulked Fertile Egg Yolk Ingestion On Serum Myostatin Levels. Reviewed and accepted for poster presentation at the 2008 Conference on PostTranslational Modification: Detection and Physiological Evaluation for the American Society for Biochemistry and Molecular Biology; Category: Unusual Modifications; Poster 5; Abstract 10; October 24,2008.

EFFECT OF DEBULKED FERTILE EGG YOLK INGESTION ON SERUM MYOSTATIN LEVELS

Colker, C; Peak Wellness, Inc., Greenwich, CT

Standard but properly handled fertile egg yolk powder contains significant concentrations offollistatin and other naturally occurring co-factors. We know that this source when properly processed to preserve. active follistatin, increased serum follistatin and decreased serum myostatin when orally ingested in a relatively high amount. The limiting factor was the bulk of the product (mainly accounted for by water and lipids) as well as the. still relatively large serving size. In an effort to reduce the bulk of the product while preserving naturally occurring follistatin, a newer centrifuged debulked version of the product (Folstaxanw) was created. Using this product at a significantly lower serving size we theorized that as little as 20grams of orally ingested product could still suppress myostatin. This study was undertaken to test this hypothesis. Seven (7) male subjects were recruited. Follistatin-rich, debulked fertile egg yolk powder properly handled to preserve active follistatin (FolstaxanTM) was obtained (Celldyne Biophanna, San Antonio, TX). After initial blood draw and providing oral Folstaxan™ at a single 20 gram serving size, serum myostatin levels were qualitatively and quantitatively measured as an indicator of ph anna co kinetic activity. Testing utilized a purchased myostatin standard for verification. Confirmations were run by ELISA and quantitations by Liquid Chromatography Tandem Mass Spectrometer with third degree fragmentation (Expertox, Deer Park, TX). Results showed an average baseline myostatin level of 27pglml; 12 hours after Folstaxan™ ingestion, average serum myostatin declined to 24,43pglm1; Average serum myostatin continued. to decline with a 24 hour level of23.86pglml. These results indicate that a debulked fertile egg yolk powder properly handled to preserve active follistatin, when orally ingested at only a single 20 gram serving size, can in fact still produce a major reduction in serum myostatin.

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JOURNAL OF THE AMERICAN COLLEGE OF

Abstract 66

ABSORPTION PROFILE AND HORMONAL lNFLVENCESOF FERTILE EGG YOLK INGESTION IN THE IIVMAN

CoUcer, C. Peak Wellnt'.3s, Inc; Greenwich1 cr ,

Fertile egg yolks contain signifioant concenearions offolllstati1l In an r;ffOrt to identifY whether this orally ingested seuree of aaturall)' occurring follistatin is actually absorbed and pharmaco.kinetica.lly active in the human model, this study wasundtmaken. A male subject waG chosen because: the nermal baseline male physiology does not regularly contain any ~casu.table concentration offolHstatin. Folli$tatin~tich fertile esg yo1k powder properly processed to preserve active fol1ista.tin (FolstaxanTM) was obtain¢d (CeUdyne Biopharma, San Antonio, TX), After initial blood dTaw and svbsequent oral Folstaxan™ ciosmg, serum fol1istatin tevels were qualitatively and quatltitatively measured. as an indicator of absorption. In addition, since we know fo1liscatin is a nesative modulator of myostatin, serum myostatin levels were qualitatively and quantitatively measured as 'an indicator hormonal inf]\lCf\ce and thus true pbannacokinetic; activity. Testing utilized a purchastld. fo11istatin and myostatin standard for verification. Continuations wc:re NO by ELTSA and quantitations by Liquid Chromatography Tandem Mass Spectrometer with third degree fragmentation.(Ex.perto~ De~ Park, TX). Result8 showed a predicted zero lovel offol1istatin at baseline w1dl a myostatin level of 46pglmL 12 hours after :F'olstaxan™ dosing. serum fo1li~~tin measured 57. 1 pglmJ with a decline of myostatin to 34pg/ml. 24 hours· after theirl'itial d08Sa& follistatin levels began to predicUlbly drop from the dme ofirtitiaJ dosmg to 11.4pWml. ¥et myostadn continued to decline $·Hghtly with a 24 hour level of 31pg/ml These results clearly indicate that 8. fertile egg yolk powder properly processed to. preserve active follistatin, when orally ingested, results in detectable serum foUistatin, Furthermore. this resultant follistatin presence has signjAcant pharm8oo~kinetic activity 8S shown by the hormonal dO'Wl1 .. regulation of serum myostatin.

VOLUME 25 • NUMBER 5 OCTOBER 2006

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JOURNAL OF THE AMERICAN COLLEGE OF

NUTRITION

Abstract 65·

RANDOMIZED BLIND CO~ARISON or FOLLISTATlN IN STANDARD STORE .. BOUGHT UNFERTTLIZED CHICKEN EGGS VERSES STANDARD STORE·BOtJGKT FERTILE. EGGS Collce" C. Peak WeJllIess~ [nc., Greenwich, CT

Both standard. store bOUght chicken eggs and fertile chicken eggs have long ·been availab1e for mass consumption and traditionally·believed by experts

and. the USDA to be DQ different in nutritive value .

{mtw. (sis. usda. goy/OAlpubsLshcUew.ium) Nevertheless, a sipificlDt number of people maJntain the bClicfthat the fertile egg offers a holistic mellor possibJy a nutritional advantage, Thus f~le qgs have long .since

bMl available to the consumer in order to satisfY~is demand Th~sh minor ccmc:rasts have: been cited, unfjl this study, no signit1~t hormon~ differenCes had been identified. In this randomized, blinded comParison oftbe two egg 1.')'peS, 6 standard grade A store bought eggs were tested:.·.Of~esc 4 were . fertile (New England Produce Center Brand ·purcbased at Stop &. Sbop, POrt Chester~ New York) and 2 unfertilized (Stop & Shop Brand purchased at Stop & Shop, Port Chester, New York). FoUiseatin levels were qualitatively ~d quantitnrively detm:nined via lDUllobHizing follistatin by binding to ACtivin in a CIS HPLC coJumn(JhperTox, Inc., Deer Park, Tcxa9). The assay was sumdardizedw;tb Recombinant Follistatin 288 and W85 asSEiyed in an·LCIMS with a third desret fragmentation (D.S., Tanerra, et at, 1977), Results revealed that.all4 fertile eu sample$ contained significant CODccntnltions offolli&tatin (O.l ] I 1.05. O.~7. and 0.81 mcg/mg), while the 2 unfertilized eggs contained no detectable fellistatin . (0 and 0 mcs!mg). Those results clearly contTadict what was thCprC~1Sting conventional wisdom oTwhlit wejJoughr we knew about commercially available eggs. Further studies need to reveal the tote aDd the advUltaga, j(any, ,ofbaving greater amounts offOllistatin in the diet.

VOLUME 25 • NUMBER 5

OCTOBER 2006