DS Craze - An Obituary Notice From the Lab: Highly Variable Amounts of "Amphetamine-Like" Compounds in DS Craze & Gaspari's Detonate. Plus: Phenetylalamine in MP's Assault

2013's fitness craze was about Craze

Let me first get this straight: I have no intention whatsoever to comment on the reasons of the "amphetamine contamination" of Driven Sport's flagship pre-workout product Craze - I'll leave that to better informed gossipers.

I guess, I would even have let the whole issue rest forever, if Mahmoud A. ElSohly and Waseem Gul hadn't published a peer-reviewed paper earlier this week that contains much more information than the previous short lab report ElSohly sent back to his clients over at Thermolife who had commissioned his lab to test a sample of DS Craze for amphetamines and decided that it was in everyone's best interest to publicize the results immediately.

Craze is not alone, Detonate & Assault join the party - but you already knew that, right?

The paper was published under advanced access in the Journal of Analytical Toxicology on December, 15 and the main result, i.e. the fact that the researchers "found an amphetamine-like compound that's not disclosed on their labels and puts athletes at risk of being banned from competition" (USAToday.com) did even make it into the online edition of USAToday.

What is phenethylamine? Phenethylamine (PEA) is a natural monoamine alkaloid that belongs to a class of chemicals with many compounds of known psychoactive and stimulant effects (Glen. 2005). It functions as a neuromodulator or neurotransmitter (Sabelli. 1976) with similar actions as amphetamine: Norepinephrine + dopamine release (Parker. 1988; Paterson. 1993). PEA and its substituted forms NDP and ETH is rapidly metabolized when taken orally (Shulgin. 1995-2009).

I guess by now few of you will be surprised that Craze was not the only supplement, of which  ElSohly and his colleague, Waseem Gul, from the University of Mississippi found that it contains "undisclosed amounts" of "amphetamine like substances".

What's the cause of the random fluctuations in "amphetamine-like" compounds

If you take a look at the data in Table 1, it does however become obvious that the total amount of "amphetamine-like" compounds in one cap of Gaspari Nutrition's fat burner / energy supplement Detonate is significantly lower than the varying amounts of N-ethyl-a-ethylphenethylamine aka ETH and phenethylamine aka PEA (also b-phenethylamine, or phenylethylamine) in a 5.5g serving of the tested LOTs of DS Craze - in the worst (or best?) case the latter contained ~80mg of either one or a combination of both agents.

Table 1: List of the analyzed products + ETH, phenethylamine & N, N-diethylphenethylamine contents (ElSohly. 2013)

If we compare the different lots of Craze, it does also become obvious that both, the ratio of ETH : phenethylamine, as well as their total value varies. There does yet not appear to be a distinct pattern, like ever-increasing or decreasing PEA levels. It's thus unlikely that someone changed the formula purposefully. Rather than that, the following explanations come to mind:

• 

  On a side note: A big belly is a greater threat to our brains than Craze | more

  The source of "contamination" is actually a source of contamination, i.e. PEA and ETH occur randomly in some batch of raw ingredients DS got from God knows where in China.
• The assay the researchers developed is not accurate, or at least not as accurate as their (albeit sophisticated) tests would suggest, i.e. the day-to-day variability was significantly higher than the 9.8%, and 3.1% the researchers measured using a low and high dose control sample.
• Someone at DS was super smart and thought: "If the effects of these ingredients are similar, I will just randomize the exact dosage, to make sure that it looks as if it was accidentally contaminated".

I guess you could come up with more speculations, but actually those three are already more than I had intended to present. I will thus close this somewhat inglorious chapter of supplement history with a brief hint on the red circle in the utmost right column of Table 1.  The circle indicates that the two known suspects, i.e. Detonate and Craze were not the only PEA offenders; and I wonder, if MusclePharm knew that, when they suddenly reformulated their best-selling preworkout product Assault a couple of weeks...

What? Oh, yes, I start gossiping, again. Sorry! But that's just human; or, as R.I.M. Dunbar from the University of Liverpool has it, it's evolutionary preserved and an "important a component of human interaction" and displays "a mechanism for bonding social groups, tracing these origins
back to social grooming among primates." (Dunbar. 2004)

PEA Alternatives: "Theanine or Caffeine? Soda, Black or Green Tea? What's Going to Get Your Brain Going?" | more

Bottom line: As the last lines of today's SuppVersity article tell you, it's just human that there was such a craze about Craze. If we are honest with ourselves, it's "much ado about nothing", anyway.

As far as I know nobody got physically harmed and the amounts of PEA and ETH in Craze are not exactly so high that you'd have to be afraid that you could have killed a couple of brain cells with it. And unless he or she found a way to inject the PEA equivalent of ~100g of Craze right into his /her veins, it's unlikely that he or she'd die (the figure is calculated based on the LD50 in rodents, so I would not rely on its accuracy; cf. Lands. 1952 ;-) ...

Don't get me wrong, I don't intend to play the whole case down, but let's not get carried away, here.

References:

• Dunbar, R. I. (2004). Gossip in evolutionary perspective. Review of general psychology, 8(2), 100. 
• ElSohly, M. A., & Gul, W. (2013). LC–MS-MS Analysis of Dietary Supplements for N-ethyl-α-ethyl-phenethylamine (ETH), N, N-diethylphenethylamine and Phenethylamine. Journal of Analytical Toxicology, bkt097.
• Glen, R., Hanson, P.J., Venturelli, A., Fleckenstein, E. (2005) Drugs and Society.9th edition. Jones and Bartlett Publishers: Sudbury, MA. ISBN 978-0-7637-3732-0 Retrieved 2011-04-19. 
• Lands, A. M., & Grant, J. I. (1952). The vasopressor action and toxicity of cyclohexylethylamine derivatives. Journal of Pharmacology and Experimental Therapeutics, 106(3), 341-345.
• Parker, E. M., & Cubeddu, L. X. (1988). Comparative effects of amphetamine, phenylethylamine and related drugs on dopamine efflux, dopamine uptake and mazindol binding. Journal of Pharmacology and Experimental Therapeutics, 245(1), 199-210.
• Paterson, I. A. (1993). The potentiation of cortical neuron responses to noradrenaline by 2-phenylethylamine is independent of endogenous noradrenaline. Neurochemical research, 18(12), 1329-1336.
• Sabelli, H. C., Mosnaim, A. D., Vazquez, A. J., Giardina, W. J., Borison, R. L., & Pedemonte, W. A. (1976). Biochemical plasticity of synaptic transmission: a critical review of Dale's Principle. Biological psychiatry, 11(4), 481-524.
• Shulgin, A., Shulgin, A.Erowid Online Books: ‘PIHKAL’ - #142 PEA. Transform Press: Berkeley, CA. < http://www.erowid.org/library/books_online/pihkal/pihkal142.shtml > (20 Dec 2013, date last accessed)

Branched Chain Amino Magic: Study Takes Another Step Towards a Better Understanding of the Anabolic & Anticatabolic Effects of BCAAs and Their Essential Cousins

Image 1: Without the other essential amino acids (EAAs), the branched chain amino acids, leucine, isoleucine and valine (BCAAs) have nothing to "build" your muscle from ;-)

Usually, I do not get very excited, when I hit upon another study into the "protein-synthetic response" that is triggered by the ingestion of branched chain amino acids (BCAAs). I mean, let's be honest... we all know that their ingestion will trigger the phosphorylation of the mammalian target of rapamycin and thusly increase protein synthesis, so why would we need another study where instead of a 17.5% increase in protein synthesis, we would see a 18.3% increase? Actually, we don't... the data Marcus Borgenvik, William Apró and Eva Blomstrand from the  Åstrand Laboratory, Swedish School of Sport and Health Sciences and the Karolinska Institutet, in Stockholm, Sweden (Borgenvik. 2011), collected goes yet well beyond what we have seen in most of the previous studies and is thus well worth an individual blogpost here at the SuppVersity.

BCCAs work! How? Little do we know...

If we are honest, we must concede that our (=the scientific) understanding of the complex processes that are triggered when "large" amounts of BCAAs hit our bloodstream, is very limited. What we know is that we can measure increases in mTOR-expression that correlate with likewise measurable increases in protein synthesis. What we do not really know is how exactly one leads to the other and where the influences of amino acid supplementation and exercise training overlap. This is even more true for the complementary side of the protein synthetic equation of which Borgenvik et al. state that

[w]hereas extensive evidence for the stimulatory effect of amino acids, either alone or in combination with exercise, on protein synthesis has been reported, their effect on protein breakdown is elusive.

In that, it is particularly confusing that "previous investigations involving ingestion of essential amino acids (EAA) in connection with resistance exercise have revealed no attenuating effect on protein breakdown", while studies which investigated the effect of BCAA or leucine in isolation, report reduction in protein degradation in subjects at rest or performing eccentric endurance exercise (MacLean. 1994). Reason enough for the Swedish scientists to recruit a group of seven healthy, recreationally active participants (5 men, 2 women; 27 (± 2) years; height 175 (± 5) cm; weight 67 (± 7) kg), put them on a standardized diet (17% protein; 25% fat; 57% carbs; ~2100kcal for women, ~2700kcal for men) for two days and, on the subsequent morning (subjects reported to the lab fasted) and after a thorough warm-up, have them perform

• 4 sets of 10 repetitions at 80% of their predetermined 1 RM, followed by another 
• 4 sets of 15 repetitions at 65% of their 1 RM of single-legged leg presses.

The subjects used the same leg on all exercises and rested ~5min after each set. Before the warm-up, during and immediately after and 15 and 45min after the exercise regimen the subjects consumed either

• 150 mL of BCAAs (2:1:1 ratio) in flavored water, or
• 150 mL flavored water alone.

The total amount of BCAAs was 85mg/kg or 5.695g for the "average" study participant. After four weeks the experiment was repeated with each participant receiving the opposite treatment.

Figure 1: Complete analysis of serum amino acid levels in the trained and untrained leg of subjects receiving BCAA or Placebo supplement before, during and after the completion of a standardized single-legged leg press exercise (data adapted from Borgenvik. 2011)

As far as the study protocol goes this is thus certainly not an extraordinary study. If you take a look at figure 1, where I deliberately plotted all the data the scientists gathered as far as serum amino acid concentrations are concerned, you will yet realize that what makes this study stand out is the sheer amount of parameters Borgenik et al. have analyzed. Similar data is also available for the amino acid concentrations in the exercised muscle and though, the scientists, who set out to investigate the effects of BCAA supplementation on protein breakdown, would probably disagree with me, here, I feel that this data actually has the most real world significance for physical culturists, like you and me.

Figure 2: Relative increase / decrease in intra-muscular BCAAs and other EAAs in BCAA supplemented subjects vs. placebo control at different time-points before, during and after single-legged leg presses (data adapted from Borgenvik. 2011)

After all, a brief glance at the effects the ingestion of ~6g of BCAA had on the respective tissue levels of leucine, isoleucine and valine (figure 2, BCAA) and the other, "missing" essental amino acids (figure 2, EAA - BCAA) should suffice to understand that though BCAAs may be the necessary to trigger protein synthesis, they are yet obviously not sufficient to "build muscle" - or how else would you explain the

pronounced reduction in the concentration of the aromatic amino acids, tyrosine and phenylalanine, in both plasma and muscle as well as muscle EAA (BCAA excluded) during the recovery period

Borgenik et al. observed in their study? The scientists at least conclude that

[s]ince tyrosine and phenylalanine are neither synthesized nor degraded in skeletal muscle, reduction in the levels of these amino acid could be indicative of an improved net muscle protein balance, i.e. an enhanced rate of synthesis and/or decreased rate of breakdown [and] could  be explained by incorporation into protein.

The accrual of muscle mass (whatever that may eventually mean, cf. yesterday's installment of the Intermittent Thoughts) thusly obviously relies on the presence of all essential amino acids and not just the "branched chained holy grail" of protein synthesis, of which the current study revealed that they (BCAA ingestion) reduced the expression of MAFbx, which regulates the protein transcription factor MyoD and the eukaryotic initiation factor-3f (eIF-3f), which, in turn is of importance in the mTOR-p70S6k signaling pathway, by 30% and 50% in the resting and exercising legs, respectively.

Figure 3: Relative (compared to placebo) mTOR and p70S6K phosphorylation in response to BCAA supplementation in exercised (EX) and non-exercised (Rest) leg at different time-points before, and after single-legged leg presses (data adapted from Borgenvik. 2011)

As figure 3 finally goes to show, we see the "usual" increases in mTOR and p70S6K phosphorylation that are commonly held responsible for the downstream increases in protein synthesis, and which were obviously more pronounced in the exercised compared to the non-exercised leg. The latter may be ascribed to what the scientists cautiously label a ...

[...] tendency for BCAA supplementation to attenuate the elevation in the level of Rheb mRNA in both resting (1.7-fold under the placebo versus 1.2-fold in the BCAA condition) and exercising muscle (2.4-fold versus 1.5-fold).

This ameliorative effect on Rheb, the low-molecular weight GTPase located immediately up-stream of mTOR, in combination with the exercise induced reductions in REDD2 expression (another negative regulator of mTOR) the scientists observed in the exercised leg are actually where we are currently at, as far as our understanding of the complex protein synthetic machinery goes. It is here at the gene-level where amino acid supplementation and its effect on Rheb and exercise and its effect on REDD synergize and facilitate those muscle gains trainees have been making for years often without any understanding of the biological underpinnings.

And though we may eventually be able to squeeze out another 5-10% more muscle mass, when we eventually get the "whole picture", I seriously doubt that even the most thorough understanding of the underlying biomolecular processes will change such basic recommendations as "take your 25g of fast digesting whey as a bolus immediately post workout" (cf. "Never Sip Your Whey!") - or what would you say?

Study Comparing Whey & Pea Protein Finds Superior Gains in Sleeve Size W/ Pea, But the Results May be Misleading

One study wouldn't be enough to prove that pea is superior to whey as muscle builder, anyways.

In a recent study, French scientist investigated the effects of two types of protein supplementation on muscle thickness and strength in 161 moderate physically active (2–6 hours per week) male participants (mean age 22 years). More specifically, the scientists compared the effects of vegetable Pea protein (NUTRALYS®) vs. Whey protein and Placebo on biceps brachii muscle thickness and strength after a 12-week resistance training program. And as the headline already reveals: The pea protein had the upper hand, when it comes to biceps brachii size, but the way the gains were measured and superior strength gains in the whey group raise questions about whether the results of this study are practically relevant - meaning you have to switch from whey to pea.

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These values are based on measurements of arm circumference of the right arm which were taken using a constant tension tape during maximal elbow extension at rest and during a maximal voluntary contraction (with maximal elbow flexion).

Figure 1: Overview of the study design.

"All subjects followed the same training routine, three times per week with a rest day between each session. Training was based on three exercises involving the elbow flexor and extensor muscles. The exercises soliciting the flexor muscles were arm curl and lateral pulldown. In the arm curl exercise, subjects sat with weights in their hands with a ~40° trunk/arm angle. They had to flex/extend the forearm over the arm. For the lateral pull-down, subjects sat with a bar in their hands above the head. They had to flex/extend the forearm over the arm with a vertical movement. The exercise soliciting the extensors was the bench press. Subjects were lying on their backs with a bar in their hands with a 90° trunk/arm angle, arms extended, and had to flex and extend their upper limbs vertically. Throughout the training program, the number of sets was progressively increased from 2 to 5 while the number of repetitions was reduced in parallel from 15 to 5 repetitions maximum (RM). In the final week, the subjects did three sets of 5 RM in order to preclude any fatigue for the D84 tests" (Babault. 2015).

Recovery between sets was identical for all workouts: 2–3 minutes. The load used for each exercise was regularly adapted during training depending on individuals’ maximum load (1-RM, one maximum repetition, evaluated every two weeks).

Pea protein (PE) is similarly satiating as whey (WH) (Lang. 1998).

Don't get me wrong: The fact that I put a huge "?" behind the assumption that pea protein was a better muscle builder than whey does not mean that I would go back on previous recommendation to use pea proteins if you don't want to use dairy protein supplements like whey or casein. They have a high amount of EAA and BCAAs and various of proven health benefits ranging from anti-inflammatory effects (albeit of yellow pea hydrolysate | Ndiaye. 2012) over similar gluco-regulatory effects as whey protein (Smith. 2012) to lipid reducing effects that have been confirmed in both rodent and human studies (Sirtori. 2012).

Figure 2: Muscle thickness in the 3 groups at baseline after 42 and 84 days. There was only a trend (£) for increased gains in the pea protein group (Babault. 2015).

Three measurements were made (at rest and contracted) along the length of the biceps, namely ¼, ½ and ¾ of the length of the upper arm (distance between the acromion process of the scapula and the lateral epicondyle of the humerus). Averaging was performed to obtain mean values for the circumference at rest and contracted.

Faulty measurements due to messed up timing?

That sounds great, but since the time of measurement is not mentioned, it may well be that all the values are fundamentally flawed. Why?

Well, as a SuppVersity reader you know that even in leg muscles, which are muss less prone to "the pump", "Cell Swelling Keeps Muscles "Pumped" For More Than 52h. Size Increases of Up to 16% After a Single Leg Workout!" (learn more).

Practically speaking, this means that the "size measurements" in the study at hand may be worthless if the scientists didn't wait for at least 72h-96h before they measured the sleeve sizes of their subjects.

Don't trust sleeve size measurements, unless you know that they were taken at least 72h after the last workout. If they are taken before, the "gains" may well be a mere result of cell swelling. You don't believe that? Well reread my article "Cell Swelling Keeps Muscles 'Pumped' For More Than 52h. Size Increases of Up to 16% After a Single Leg Workout!" (read it).

The absence of information on the time-point at which the sleeve sizes were measured (suggestive of immediately post) and the fact that the strength gains do not reflect the alleged superiority of pea protein as muscle builders (see Figure 3) put a huge question mark behind the assumption that pea protein tends to produce greater muscle gains than whey.

Figure 3: It is strange that the "superior muscle builder" triggers the lowest gains in maximal isometric contractile force. Even the placebo group had greater strength gains - despite a lack of sign. differences at baseline (Babault. 2015).

Rather than "gains", the increases could in fact be a result of increased inflammation and the reduced "gains" in the whey group a result of its anti-inflammatory effects (Buckley. 2010; Sugawara. 2012; Kerasioti. 2013). The existing evidence of anti-inflammatory properties of pea protein (Ndiaye. 2012) does yet render this assumption questionable.

On paper, the amino acid profile of whey looks more anabolic (higher BCAA content).

Overall, the study clearly indicates that pea protein is a good vegan replacement for whey protein. The assumption that it may be superior to whey protein, however, is questionable. First of all, the strength gains don't reflect these changes. In fact, the whey protein group saw the largest strength increases - as it was the case for the size gains, these differences were not statistical significant, though.

If we also take into consideration that the "size" gains are actually gains in sleeve size (not reliable DXA measurements) which may have been taken way too early after the workouts to reflect actual muscle gains, we don't even have to raise the question whether the fact that the French company Roquette, the producer of NUTRALYS® pea protein, sponsored the study may have constituted an obviously subliminal bias ;-) Anyways, I would not yet replace my whey protein with pea protein isolates | Comment Facebook!

References:

• Babault, et al. "Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein." Journal of the International Society of Sports Nutrition 12:3 (2015).
• Buckley, Jonathan D., et al. "Supplementation with a whey protein hydrolysate enhances recovery of muscle force-generating capacity following eccentric exercise." Journal of Science and Medicine in Sport 13.1 (2010): 178-181. 
• Lang, Vincent, et al. "Satiating effect of proteins in healthy subjects: a comparison of egg albumin, casein, gelatin, soy protein, pea protein, and wheat gluten." The American journal of clinical nutrition 67.6 (1998): 1197-1204.
• Ndiaye, Fatou, et al. "Anti-oxidant, anti-inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds." European journal of nutrition 51.1 (2012): 29-37.
• Kerasioti, Efthalia, et al. "Anti-inflammatory effects of a special carbohydrate–whey protein cake after exhaustive cycling in humans." Food and Chemical Toxicology 61 (2013): 42-46.
• Sirtori, Cesare R., et al. "Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals." British journal of nutrition 107.08 (2012): 1176-1183.
• Smith, Christopher E., et al. "The effect of yellow pea protein and fibre on short-term food intake, subjective appetite and glycaemic response in healthy young men." British Journal of Nutrition 108.S1 (2012): S74-S80.
• Sugawara, Keiyu, et al. "Effect of anti-inflammatory supplementation with whey peptide and exercise therapy in patients with COPD." Respiratory medicine 106.11 (2012): 1526-1534.