Alanyl-Glutamine or Alanine + Glutamine? Dipeptide or Free Form Aminos? What Offers Maximal Muscle Protection?

"Wouldn't have happened if she'd used alanyl-glutamine instead of regular that cheap alanine + glutamine combo!" - True or False? Recent study says: False!
If you combine your liver's favorite gluconeogenic amino acids, i.e. alanine and glutamine, into a single peptide the result is called alanyl-glutamine and marketed as the ueber-potent alternative to regular l-glutamine supplements. It goes without saying that a comparison like this is about as stupid as comparing french fries with mayo to regular french fries and saying that the former are worse because they contain more fat, or whatever. Even if we didn't care about the physiological significance of the effects of alanyl-glutamine, we would obviously have to compare the purported cryogenic effects of this "innovative" dipeptide to those of a simple combination of free form amino acids to deserve the bragging rights for having created an advanced form of glutamine.

Alanine + glutamine vs. alanyl-glutamine - fight!

By now you are probably asking yourselves why I am bothering you with things like this. Right? Well, the reason is that Éder Ricardo Petry and his colleagues from the University of Sao Paulo must recently have been pondering the same question. To answer it, they conducted an experiment that would allow them to verify if the oral supplementation with l-glutamine and l-alanine as dipeptide has more pronounced muscle protective effects than a simple mixture of l-glutamine and l-alanine (GLN+ALA, both in their free forms) in a group of Wistar rats that are subjected to intense aerobic training (treadmill).

I know what you are thinking now: "Not another rodent study...", but think about it: How many people are willing to pay $50 and more on supplements without any in vivo evidence of their efficacy let alone long-term safety? Against that background Petry's rodent is a major advancement - isn't it?
True or False: You can (ab-)use glutamine to replenish your glycogen stores!? True! It sounds strange, but according to a study from the late 20th century glutamine is a pretty effective glycogen replenisher, even in the absence of your bodies favorite nitrous glucose precursor alanine | learn more
Don't get me wrong, there are a few alanyl-glutamine studies in humans, but there is not a single one that would compare the dipeptide to a reasonable placebo in an exercise scenario. I mean, who tells me that the basketball players in the 2012 study by Hoffman et al. wouldn't have experience the same beneficial effects on basketball skill performance and visual reaction time if their rehydration solution had contained alanine and glutamine or even glutamine alone? Yes, I know... the increased absorption: Well, let's just look at a fair comparison, i.e. the study at hand, and see what happens when the dreams of supplement formulators and reality meet ;-)

Ok, back to the facts - the exercise & supplementation protocol

The male Wistar rats, the researchers used in their experiment were exercised 5x per week - at increasing intensities: Starting with 30 and 45 min of treadmill running (incline 3°) at 20 and 22.5 m/min in the first three weeks, the speed and duration of their treadmill runs increased to 60 min at a speed of 25 m/min in week four and remained like that for the rest of the 8-week study period.

The supplements were administered via oral gavage in the course of the last 3 weeks, only. The daily doses for the animals in the dipeptide (DIP) and free form amino acid groups (GLN+ALA) were...
  • 1.5g/kg alanyl-glutamine in the DIP group,
  • 0.67g/kg l-alanine + 1.0g/kg l-glutamine in the GLN+ALA group, and
  • plain water in the control group
The amount of of alanyl-glutamine the scientists used was calculated in such a way that the total amount of l-glutamine was the same as that of l-glutamine administered in its free form.

Changes? YES! Dipeptide benefits? Not really...

The gavage was provided 1 h after the end of each session of exercise, after which the animals had with free access to water and chow. To make sure that the results of the examinations on the last day of exercise would not reflect the acute effects of a single dose of the supplements, the animals were killed 10 h after the last exercise session.
Figure 1: Plasma glutamine, glutamate, ammonium, malondialdehyde, myoglobin, and creatine kinase activity in Wistar rats supplemented with alanyl-glutamine (DIP) or regular glutamine + alanine; data expressed rel. to control (Petry. 2013)
The virtually identical increases in l-glutamine and l-glutamate, you see in Figure 1 should thus represent the baseline and not the 'immediately post supplementation level' of these amino acids. For the exercise-induced accumulation of ammonium, malondialdehyde (MDA; indicates lower lipid oxidation), myoglobin and creatine kinase (both indicate lower muscle damage) the timing is not that important, anyway. What is important, however, is the fact that there were no physiologically relevant advantages for the "super glutamine".
DHEA & estrogen are alternative muscle protectors. Despite the fact that estrogen has repeatedly been shown to have muscle protective-effects, I would not suggest you steel your granny's HRT medication. DHEA on the other hand, may be something to consider - specifically if you are about to overreach, like the male subjects in a 2012 study by Liao et al. (learn more)
If we take a closer look at the p-values and the statistical significance of these changes, it turns out that, the minor increase in glutamate aside, all of the difference to the placebo group were statistically significant. The DIP vs. GLN+ALA differences, on the other hand, were marginal and reached statistical significance only in the case of the marker of myoglobin. Where the dipeptide has a physiologically probably irrelevant edge of 9% over the GLN + ALA combination.
Figure 2: Glutathione (GSH) and glutathione disulfide (GSSG = used glutathione) levels in soleus and gastrocnemius skeletal muscles of the rodents; data expressed relaitve to control (Petry. 2013)
For the muscular GSH levels, it does not look much different. In this case, there is however not even a statistical difference between alanyl-glutamine and the simple l-alanine + l-glutamine mix - neither for the universal anti-oxidant glutathione (GSH), nor for its "used form" glutathione disulfide (GSSG).
Does that mean that alanyl-glutamine is another supplemental rip-off? I would say that it's too early to use such harsh words. There was after all one statistically, and maybe even physiologically relevant difference between the two groups I didn't mention, yet: The dipeptide group presented with a different heat-shock protein response: They had higher HSP-70 and lower HSF-1 levels in the soleus and lower HSP-70 and lower HSF-1 levels in the gastrocnemius.

"Will training your biceps, heal your heart & protect your brain!?" - a study on the effects of exercise induced HSP increases suggests so | more
In view of the fact that the subsequent "deficit in HSP70 expression" is supposed to "impair recovery from these injuries" Petry et al. are probably right to point out that
"one cannot discard the possibility that part of the beneficial effects of high-intensity exercise training may be due to the enhancement of HSP70 expression which is exacerbated by glutamine supplementation."
In view of the fact that the total amount of proteins from the HSP70 and HSF1 family was increased in both groups, and the differences appear random, it is impossible to tell, whether the slight differences in HSP expression actually matter and whether this is an advantage for alanyl-glutamine or rather for the cheap free form amino acids.

Before future studies provide additional data based on which we can decide whether these differences are relevant and why they differ between slow- (soleus) and fast-twitch (gastrocnemius) skeletal muscle fibers, I'd say that the study at hand would suggest that alanine and glutamine have muscle protective effects irrespective of whether they are bound or not, when you ingest them.

  • Cruzat VF, Rogero MM, Tirapegui J. Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise. Cell Biochem Funct. 2010 Jan;28(1):24-30. 
  • Cruzat VF, Tirapegui J. Effects of oral supplementation with glutamine and alanyl-glutamine on glutamine, glutamate, and glutathione status in trained rats and subjected to long-duration exercise. Nutrition. 2009 Apr;25(4):428-35.
  • Hoffman JR, Williams DR, Emerson NS, Hoffman MW, Wells AJ, McVeigh DM, McCormack WP, Mangine GT, Gonzalez AM, Fragala MS. L-alanyl-L-glutamine ingestion maintains performance during a competitive basketball game. J Int Soc Sports Nutr. 2012 Mar 7;9(1):4.
  • Petry ER, Cruzat VF, Heck TG, et al. Alanyl-glutamine and glutamine plus alanine supplements improve skeletal redox status in trained rats: Involvement of heat shock protein pathways. Life Sciences. 20 November 2013 [ahead of print]
  • Rogero MM, Tirapegui J, Pedrosa RG, Castro IA, Pires IS. Effect of alanyl-glutamine supplementation on plasma and tissue glutamine concentrations in rats submitted to exhaustive exercise. Nutrition. 2006 May;22(5):564-71.
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