Fat Loss Principles That Work: 10g+ of EAAs W/ Every Meal. Do Energetic Costs of Protein Synthesis Trigger This Effect?

EAAs beyond whey: It may not necessarily look like this, but this salad (repicecorner) is an EAA power horse with cheddar cheese (25% protein, 0.49 EAA / P ratio), tuna (in oil, 29%,  0.45) and kidney beans (9%, 0.45). You see, it does not always have to be chicken breasts or whey to get beyond the 10g+ EAA threshold, I have repeatedly suggested as one of the fundamental rules of dieting for weight loss, maintenance and muscle gain.

Many people take it for granted that you become fat, when you get old. If you look at the statistics, you could even make a point that obesity has some protective effects with esp. with respect to CVD mortality. Scientists call this the "obesity paradox" (Kastorini. 2013). What's particularly paradox, at least in my humble opinion, is yet not the phenomenon itself, but rather the fact that it gets smart scientists derailed from working on useful dietary and exercise interventions to prevent the development of heart disease, cancer, metabolic syndrome etc. in early years. Instead, they argue ex-post, i.e. when the baby has already been thrown out with the bathtub by comparing sick lean (in parts even cachectic) and sick "obese" people, why their statistical shenanigan that's based on the useless BMI produces paradoxical results. And that, when studies such as the one Jacobs et al. did in 2010 clearly show that 50+ year old men and women with waist circumference >120cm and >110cm, respectively, have 2x higher all-cause mortality risk than their lean peers - irrespective of BMI (Jacobs. 2010)!

To get lean and stay lean, yet not thin and skinny fat is therefore a challenge everyone...

... from the child in the Kindergarten to the obese granny in the nursing home is facing. Against that background a previous study by Loenneke et al. comes to mind. The results of their analysis, which were published in Nutrition and Metabolism in January 2013 clearly show that the amount of times people eat meals with a 10g+ EAA content per day was inversely related to percent central abdominal fat (Loenneke. 2013). In previous studies EAAs have also been shown to improve glucose clearance without increases in insulin and in the absence of effects on the fat burnin and health promoting expresion of AMPK-alpha2 in skeletal muscle tissue (see "EAAs Stimulate Muscle Glucose Uptake by Exponentiating Insulin's Effect on GLUT4 Expression"). With the advanced publication of a study by Coker, Miller, Schutzler, Deutz and Wolfe in the online verison of the Nutrition Journal a couple of days ago, the notion that EAAs have a particularly beneficial effect on fat loss - in this case in obese elderly individuals - gets further support from a well-controlled randomized trial (Cooker. 2013).

EAA-rich protein increases fat loss to a greater extent than low EAA protein

The researchers from the Center for Translational Research in Aging and Longevity and the University of Arkansas for Medical Sciences in Little Rock, AR, USA randomized 12 elderly individuals (mean age 69 years) to an 8 week, caloric restriction diet utilizing equivalent caloric meal replacements (~850 kcal/day; the exact nutrient composition can be found in figure 1) + ~400kcal from solid foods (total intake: ~1,250kcal/day; the subjects were free to chose their solid meals but were provided with a list of examples the should pick from, if possible).

Figure 1: Macronutrient composition of the meal replacements used in the study (Cooker. 2013)

The diet was designed to induce a 7% weight loss in two months. And while both,  the rate of weight loss (~1.6lbs per week), as well as the relatively high caloric deficit are certainly appropriate for someone with a 30+ BMI and ~40% body fat, leaner people will fare better with a less pronounced kcal deficit or (alternatively) have to add some strategically planned refeeds to the equation in order to minimize the loss of lean mass and, more importantly, avoid the ensuing reduction in energy expenditure (for the obese, the latter is actually less of the problem, because the downsides of being calorically deprived are at least partly counglucose tolerance and leptin sensitivity with every gram of body can actually help the body recognize that there is still plenty of energy that has just not been available (glucose) or "visible" (fat) before).

Figure 2: Changes in lean and fat mass (kg, left) and fractional protein synthesis rates (FSR) in participants receiving iso-caloric meal replacements with identical macronutrient compositions (see figure 1), but different amounts of essential amino acids (EAAs) content (Coker. 2013)

As the data in figure 2 goes to show you even the obese individuals in the study at hand lost a non-negligible amount of lean mass - unfortunately the body composition was measure with a sophisticated, but still body impedance based device, the trends are still accurate, but it is questionable in how much we are actually talking about ~2 and 2.5kg of muscle mass (figure 2, left), because somebody's "lean body mass" does obviously include more than just skeletal muscle.

When it comes to supplements, we are often like children on Christmas eve. About all the new stuff we get we tend to forget our former favorite and often way more fun to play with toys. Don't make this mistake and ditch your PWO whey (personally, I like a ~1.5:1 whey + micellar casein mixture) for EAAs, they don't come close... read more

Be that as it may - since the before and after values were taken with the same device the changes should be correct, so that both the slightly yet not statistically significantly ameliorated loss of lean body mass and, more importantly, the significantly higher degree of body fat loss in the EAA meal replacement (EAAMR) group speak in favor of the 5 servings of a the 170 kcal, 6g EAA per day. Moreover, "the sparing influence of muscle loss might have been demonstrated with a larger sample size", so that you can take it for granted that the preservation of precious muscle mass is an advantage of being choosy with your protein sources and preferring those with higher over those with lower essential amino acid contents.

On a related note: I don't know if you noticed, but with a total energy content of 850kcal and 30g EAA these 5 meal replacements did in fact have exactly those 10g+ of essential amino acids, I have repeatedly recommended to have with each of the 3 meals most people consume in the course of the day.

In all fairness, it should also be mentioned that despite not being significantly different at baseline, the body fat percentage of the subjects in the EAA meal replacement group was ~3% higher to begin with.This may seem irrelevant, since figure 2 compares lean mass and fat mass as absolute changes and not their percentages, but in the end, the amount of fat you you can drop within a given time-frame decreases with lower body fat percentages.

Do the energetic costs of protein synthesis drive fat loss?

Another interesting observation Coker et al. made is the close association between fat loss, on the one hand, and increased protein synthesis (55%), on the other hand. The researchers take this as an incentive to do one of the of the much loved calories in vs. calories out calculation and come up with the following hypothesis:

"Acute administration of EAAMR did promote a significant increase in skeletal muscle protein FSR compared to CMR. Assuming that the energy cost of protein synthesis is 3.6 kJ/g and the baseline GAIA-derived lean tissue mass was 56.4 kg for EAAMR and 54.4 kg for the CMR, we can extrapolate that the overall energy discrepancy between the two groups was roughly equivalent to 27,170 kcal or 3.5 kg of weight loss across the entire caloric restriction-based weight loss paradigm. Based on the amount of total lean mass in each group, this value takes into account a consistent intervention structure of five servings/day across an eight week period. In short, these calculations suggest that differences in the source of intact protein/formulation of EAA may have a significant influence on diet-induced energy expenditure that coincides closely with the greater reduction of adipose tissue in EAAMR compared to CMR." (my emphasis in Coker. 2013)

I usually discard fallacious calculations like this one if they are not highlight the stupidity of trying to eat exactly as much as some funky formula + the figure on your treadmill, pedometer, heart rate monitor or whatever fancy tool you may use to "measure" your energy expenditure suggest you would have burned in the last 24h. In this case, however, I made an exception, because I feel that the notion that protein quality is one of the myriad of parameters that are missing from this foolish calculation is important, for lean and obese people from all age groups who are trying to shed body fat.

Bottom line: The take away message of the study is in the end identical to the previously mentioned study by Loenneke et al.: Make sure you hit the 10g EAA threshold with each and every of your meals, if being lean and muscular not skinny yet fat is your goal.

References:

• Coker RH, Miller S, Schutlzer S, Deutz N, Wolfe RR. Whey protein and essential amino acids promote the reduction of adipose tissue and increased muscle protein synthesis during caloric restriction-induced weight loss in elderly, obese individuals. Nutr J. 2013 Dec 11;11(1):105. [Epub ahead of print]
• Jacobs EJ, Newton CC, Wang Y, Patel AV, McCullough ML, Campbell PT, Thun MJ, Gapstur SM. Waist circumference and all-cause mortality in a large US cohort. Arch Intern Med. 2010 Aug 9;170(15):1293-301.
• Kastorini CM, Panagiotakos DB. The obesity paradox: methodological considerations based on epidemiological and clinical evidence--new insights. Maturitas. 2013 Jul;72(3):220-4.
• Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2013 Jan 27;9(1):5.

The Dipeptide Advantage!? 43% More Muscle Glycogen With Whey Hydrolysate Compared to Matched Amino Acid Mixture

The bigger the choice, the harder it is to choose. If you had to pick just one, go for the concentrate, if you want to afford two, get an isolate or hydrolysate and a casein protein.

"Whey is still the way to go." I believe I wrote, or at least thought just that only a couple of days ago, when I wrote about the wheat gluten hydrolysate in the last installment of the SuppVersity Science Round Up Seconds (click here to read all previous installments). Unfortunately, there is not just one way... ah, pardon me, I do of course mean "whey", there are many! And in order to completely confuse their customers supplement companies will spike their concentrates, isolates and even hydrolysates with free form amino acids, or - which is even more confusing - advertise their BCAA and EAA products as being made from whey protein. I mean, who cares if the damn molecules are unbound and indistinguishable, anyway? But let's get to the point, a soon to be published study from Japan could yield at least some insights into demonstrable and purported benefits of one over the other.

Whey hydrolysate or simply its aminos, what gets pro-glycogen job done?

Based on results from previous experiments in the course of which the researchers had found that the acute provision carbohydrate + whey protein hydrolysate (WPH) had superior effects on muscle glycogen repletion compared to iso-nitrous amounts of BCAAs (Morifuji. 2010a) Kanda et al. speculated that chronic supplementation with whey protein hydrolysate (WPH) should elicit similarly beneficial effects and could help them clarify the underlying mechanism of this glycogen boosting effects of WPH. To this ends, the researchers put a group of mice on diets that differed only terms of the protein composition of the diet.

• 

  Table 1: Composition of the test diets

  The control group's exclusive protein source was the casein from the standard chow.
• The whey amino acid (WAA) group received a chow, where 50g of the casein protein was replaced by an iso-nitrogenous amount free amino acids that was matched to the amino acid composition of the whey hydrosolate.
• The whey hydrolysate group received 20% of the original casein from the standard chow in form of a whey hydrolysate from Meiji Co., Ltd.

In the course of the 4-week study period, body weight and food intake of the animals were measured on a weekly basis. The mRNA expression, protein levels, and phosphorylation of glycoregulatory enzymes were measured in the gastrocnemius muscle. All rodents performed a regular 30-min swimming exercise protocol  in a fancy adjustable-current water pool five times per week and had to 'survive' a weekly (endurance-)performance test in the course of which they had to swim to absolute failure (defined as being drowning for 7s, already).

Figure 1: Food intake, liver weight, and visceral fat (left), as well as time to total fatigue during swimming exercise (right) of the rodents on control, WAA and WPH diets  (data adapted from Kanda. 2013)

Contrary to the data from this test, the body composition markers in figure 1 do not show any significant inter-group differences (positive or negative) the same goes for the food intake and the liver weight, as well as for the total body weight of the mice, which was totally identical (not shown in figure 1). The said swimming performance of the WAA and WPH group, on the other hand, were 32% and 48% higher than in the control group.

More glycogen synthetase = more glycogen content = more endurance

Now, despite the fact that this increase did - for whatever reason - not reach statistical significance, Kanda et al. are convinced that this increase in endurance must be a direct consequence of the increase in glycogen storage, which has been observed by Evans and Hughes in 1985 (Evans. 1985), already, and has been confirmed numerous times thereafter.

Figure 2: Glycogen content and glycogen synthetase levels (GS), as well as mRNA expression of glycogen synthase I and the ratio of phosphorylated to unphosphorylated GS (data based on Kanda. 2013)

And, as you can see, the actual data in figure 2 clearly confirms this hypothesis. The mice who had received whey protein hydrolysate (WPH) in their diets for the whole 4-week study period had significantly (p < 0.05) higher muscle glycogen levels than their peers in the control group (73%) and still more than 40% more total glycogen than the mice who had been fed the amino acid enriched chow (WAA). Quite impressive, right? And all that is just a consequence of a peptide induced elevation in glycogen synthetase and it's activity, which is indicated by the lower ratio of phosphorylated (=incative) to un-phosphorylated (=active) levels of this tightly regulated enzyme.

There is more to whey than BCAAs

The total amount or activity of the glucose transporter (GLUT-4) as well as the hexokinase activity (which figures in the phosphorylation of sugars) were not different between treatments and though the dreaded gluconeogenesis in the liver was not measured it is, given the high amount of carbohydrates in the diets of the rodents, very unlikely that the higher susceptibility of "fast" protein sources to be oxidized, when no other nutrients are available, played a significant role in the 'pro-glycogenic' of whey hydrolysate (after all the rodents consumed the protein as part of their chow), so that the most likely explanation for the superiority of the whey protein hydrolysate over the iso-nitrous amino acid mixtures remains their peptide content. In this regards, the authors of the study remark:

"The amino acid compositions of the two diets used in this study contained equal amounts of BCAA and leucine; however, muscle glycogen accumulation varied between diets. This result strongly suggests that not only the BCAA content but also the molecular form of BCAA found in the protein source might be important for muscle glycogen storage." (Kanda. 2013)

Kanda et al. do then refer to a previous study, in which his group had been able to demonstrate that BCAA-containing peptides in WPH, which have been shown to be markedly elevated (meaning they are not digested) after the consumption of whey protein hydrolysates (Morifuji. 2010) in a follow up study on human beings, do actually have the ability to stimulate the rate of glucose uptake in vitro (Morifuji. 2009).

Figure 3: Insulin response after the ingestion of 12.5g of either soy or whey protein or their respective hydrolysates (Morifuji. 2010)

"And what about insulin?"

I know that this question is now on your minds and in way you are right the only slight caveat you have to keep in mind before you blindly follow the scientists' outspoken advice to consume "carbohydrates mixed with WPH[to] enhance sport performance by increasing glycogen storage" (Kanda. 2013) would in fact be be the increased insulin response. Of the latter, the scientists found in the aforementioned 2010 human trial (Morifuji. 2010) that it is ~70% more pronounced in the first hour after the the ingestion of 12.5mg of whey protein hydrolysate and, once more compared to regular whey protein, still ~17% higher over the whole 2h period (the different proteins you see in figure 3 were all ingested on an empty stomach after an overnight fast by the 10 normal-weight subjets; the data I mentioned refers to the AUC values on the bottom of figure 3).

The insulin 'spike' is not necessarily a problem. At the right time in the right person it can even be highly beneficial.

Nevertheless, the results of the study at hand to actually confirm that before you invest in all sorts of useless pills, it may make more sense to make sure that you a) have more than just a single protein powder in your supplement arsenal and that you b) have been reading enough SuppVersity articles to be able to use them properly ;-) ... What? You still don't know how? Well assuming you have no problems with high insulin levels and are mainly interested in building muscle, you could start out with 20-30g whey + 15-25g casein (depending on your body size and needs) after a workout (see "Whey & Casein Work Hand in Hand for Muscle Protein Anabolism") and a 40g casein shake pre-bed (see "3.2kg of Lean Mass Overnight").

By the whey *lol*, I suppose you will see similar benefits from a whey isolate, although this would have to be tested. What does not need any tests, on the other hand is that you better make sure you don't forget the carbs! I mean, what is your body supposed to use as a substrate for the increased glycogen synthetase activity, if you are depriving yourself of carbohydrates? The protein your liver converts to blood glucose? Yeah, what a glorious idea... whatare your brain and your other organs going to use then? Ketones? No way, if you are pounding tons of fast acting glucogenic amino acids in form of protein shakes.


References

• Evans, W. J.; Hughes, V. A. Dietary carbohydrates and endurance exercise. Am. J. Clin. Nutr.1985, 41 (5, Supplement), 1146−1154.
• Kanda A, Morifuji M, Fukasawa T, Koga J, Kanegae M, Kawanaka K, Higuchi M. Dietary Whey Protein Hydrolysates Increase Skeletal Muscle Glycogen Levels via Activation of Glycogen Synthase in Mice. J Agric Food Chem. 2013 Oct 31.
• Morifuji, M.; Koga, J.; Kawanaka, K.; Higuchi, M. Branched-chain amino acid-containing dipeptides, identified from whey protein hydrolysates, stimulate glucose uptake rate in L6 myotubes and isolated skeletal muscles. J. Nutr. Sci. Vitaminol. 2009, 55(1), 81−86.
• Morifuji, M.; Kanda, A.; Koga, J.; Kawanaka, K.; Higuchi, M. Post-exercise carbohydrate plus whey protein hydrolysates supple-mentation increases skeletal muscle glycogen level in rats.Amino Acids 2010a, 38(4), 1109−1115.
• Morifuji, M.; Ishizaka, M.; Baba, S.; Fukuda, K.; Matsumoto, H.; Koga, J.; Kanegae, M.; Higuchi, M. Comparison of different sources and degrees of hydrolysis of dietary protein: Effect on plasma amino acids, dipeptides, and insulin responses in human subjects. J. Agric. Food Chem. 2010b, 58(15), 8788−8797.

2x40g, 4x20g or 8x10g of Whey? Which Feeding Strategy Yields the Greatest Net Protein Retention? Plus: What the Results Can Tell Us About Intermittent Fasting on a "Bulk"

In know, after reading the headline you are probably already urgently waiting for the results of the latest study on protein timing, but before we get to the facts, let me briefly announce that this "bolus vs. intermittent vs. pulse" protein study, which is incidentally the result of an international cooperation between researchers from the Nestlé Research Centre in Lausanne, Switzerland, Canadian researchers from the University of Guelph, the Canadian Sport Centre and (you guessed it) Stuart M Phillips' group at the McMaster University, and their colleagues from the Australian Institute of Sport and the RMIT University in Melbourne, will be one of the topics of today's SuppVersity Science Round Up on Super Human Radio.

Other things I hope Carl Lanore and I will be able to squeeze into today's show, which airs, just as every Thursday live at 1PM EST and will also be available as a podcast later today, either right from the nav-bar on the right ("Physical Culture for Your Ears") or at www.superhumanradio.com, are ...

• the latest news on natural nitrate supplementation with beet root juice, 
• how stress and laziness increase breast cancer risk more than hormonal imbalances, and
• how you can prepare your own powerful stevia-based wound ointment  

There is obviously more to the list, but I have learned from past mistakes and won't announce all I have piled up, when I know that's simply not possible to squeeze all of them into a single 1h show ;-)

A note for those of you who are looking for Adelfo Cerame's weekly contest prep blog: Don't worry it's still alive! You must have over-read that he has switched to a bi-monthly format!

Ok, ok... but NOW tell me hod do I have to spread my protein across the day"

While we know already that more is not necessarily better, when it comes to protein intake and that timing plays a significant role with respect to the returns in protein synthesis, and more importantly net protein retention you get for each gram of additional protein you consume, the question how you best spread your roughly 1.5-2.0g of protein per kg body weight across the day is still a matter of contemporary research and bro-scientific debate.

Suggested read: Protein Synthesis "Beyond the 20g Limit: Study Shows Exercise Facilitates 32% Greater Increases in Fractional Protein Synthesis With 40g vs. of 20g of Whey PWO" (click here to read)

What appears to be widely accepted, though, is the notion that both, the ingestion of a slow digesting protein before, and the intake of a fast digesting protein after a workout can effectively increase protein synthesis and net protein retention. If we assume that the combination of both strategies will yield further benefits (this has to my knowledge not been shown yet and is certainly not necessarly the case!), and regard the peri-workout supplementation as a "stand alone" that's not part of the 1.5g-2.0g /kg body weight baseline protein intake, we still end up with at least 80g of high quality protein (for the real light-weights or ladies ;-) we would have to spread in one way or another across the rest of the day.

8 x 10g, 4 x 20g or 2 x 40g? What's "optimal"?

Now, Moore et al. obviously won't have had my allegedly botchy "real-world" scenario on their minds, when they came up with the exact experimental design of their latest study. Still, if we forget about the 20g+ of protein post-workout, I believe none of you will be willing to abandon, their experimental setup fits the framework pretty nicely. After all, the scientists deliberately picked the 12h period after a workout "to standardise and take advantage of the accentuated protein synthesis in the exercised muscle over this period" and investigate three archetypal means of spreading a total amount of 80g of protein across the day: In 2 x 40g servings, 4x 20g servings or 8x 10g serving (Moore. 2013).

Suggested read: "3.2kg of Lean Mass Over Night W/ 40g of Slow Digesting Protein 30min Before Bed!?" (click here to read more)

The 24 male subjects who were advanced trainees working out 4–6 times per week in what the researchers call a "high intensity resistance training regimen" (note: I don't think this denotes a classic low volume HIT regimen) had to

"[...] follow standardized diet for the 72h prior to the trial that provided an energy availability of 45 kcal/kg fat-free mass with a macronutrient contribution 1.5 g protein/kg/d and 4 g carbohydrate/kg/d, respectively. [Moreover, s]ubjects were instructed to refrain from training and other vigorous physical activity during the 72h period."

When the men reported to the laboratory on the testing day, they had refrained from training or performing any other vigorous activity during the 72h period leading to the intervention and had been fasting 10h (over night). In absence of any other information I assume they remained in the fasted state for the subsequent standardized acute bilateral leg extension exercise session (4x10 sets at 80% 1-RM with 3 min recovery between sets), after which they were randomly allocated to receive their 80g of protein from whey as

• pulsed feeding (PULSE), 8x10g every 1.5h; 
• intermediate feeding (INT), 4x20g every 3h ; or 
• bolus feeding (BOLUS), 2x40g every 6h. 

The supplementation regimen was started right after the workout and the protein synthesis, breakdown and net balance were determined based on previously tested and verified procedures (Hartmann. 2006).

Figure 1: Comparison of effect sizes, p-values (remember only p < 0.05 would be a statistical significant difference) and the scientists qualitative inference's based on the effect of feeding pattern on whole body net protein balance (left) and a detailed breakdown of the feeding specific effects on 12h protein synthesis expressed relative to the bolus group (based on data from Moore. 2013)

As the data in figure 1 goes to show you, the results clearly confirm that the pattern according to which you consume your daily allotment of protein does matter, what it does yet not really tell us is how this will translate into a real-world scenario, in which, as I have pointed out before, not having at least 20g of whey / other protein sources after a workout appears almost negligent. The provision of a 20g whey + 10g casein mix right after a workout could, for example, have undone the minimal (and statistically non-significant) advantage in net protein retention of the intermediate feeding group. And that may still have been the case if the latter had been "upgraded"  to a 4x25g whey pattern.

On the other hand, if we wanted to pick on the study design, the "workout" (leg extension) and the absence of other nutrients (or the lack of information about those in the paper?), which could easily have reduced the amino acid breakdown that nullified the advantage the pulse feeding had with respect to its ability to trigger and sustain (over 12h) protein synthesis, would be more relevant points of critique, anyway. That said the "study" at hand is actually only a "short communication", and I am pretty sure there is more to come in the future (it stands to reason that the SuppVersity is the place to go to read about that, right?)

Note: I still maintain that overnight fasting is healthy, and IF probably one of the best, r at least a very effective way to shed body fat, but that does not mean that it should be the only diet strategy in your "nutritional toolbox", in which other tools are probably better suited to pack on slabs of muscle!

(Preliminary) bottom line: The results Moore et al. present certainly don't provide a definitive answer on "the very best" way to time your protein intake (and even if there was an "optimal" way, no single study will ever be able to elucidate it). They do however make one thing pretty clear: My gut feeling that intermittent fasting and here especially those varieties with very long fasting and very short feeding windows, is probably not the best way of dieting to gain muscle. After all, there is no debating that the bolus regimen (2x40g 6h apart!) is trailing behind.

You can certainly tweak and thus optimize it by (a) adding a third meal in between and (b) cleverly using / combining fast and slow acting proteins (cf. "Whey and Casein Work Hand in Hand for Protein Anabolism"), but if you want level playing fields you would have to apply similar tweaks to the more frequent 4 x 20g and 8x 10g regimen as well... and I that would probably restore, if not magnify the difference.

Update on the real world significance of the advantage: I know that SuppVersity readers are smart and therefore was not suprised that only minutes after I posted this article, Steven Arcera objected that long-term studies don't show this advantage. Now, while Steven is right the implicit assumption that this implies that there is no advantage of spreading your protein across meals is false. If we simply take the exact figures from the study, which would be an added ~0.02g/kg body weight in protein retention over 12h, assume (which is obviously not valid) that the protein retention would be identical over the other 12h of the day in all groups and do the math for the study participants who weighed 80kg, this would be an additional 1.6g of protein retention for the whole body (remember this is whole body protein retention) and therfore even in a long-term study of 12 weeks only 134.4g! This would still be 134.4g more than with bolus feeding but would NEVER make a statistical significant difference in any study. And even the 584g "advantage" you would accumulate over a whole year would make it past the p < 0.05 line! So much about "optimal feeding strategies" and the real world outcomes of the latter :-)

References:

• Hartman JW, Moore DR, Phillips SM. Resistance training reduces whole-body protein turnover and improves net protein retention in untrained young males. Appl Physiol Nutr Metab. 2006 Oct;31(5):557-64.
• Moore DR, Areta J, Coffey VG, Stellingwerff T, Phillips SM, Burke LM, Cléroux M, Godin JP, Hawley JA. Daytime pattern of post-exercise protein intake affects whole-body protein turnover in resistance-trained males. Nutr Metab (Lond). 2013 Oct 16;9(1):91.

Pre Workout Protein Supplementation 101: Slow or Fast, Bolus or Pulse? Protein Synthetic Response is Identical!

Should she drink her protein shake all at once or in 33ml gulps every 15min, if she has it before her workout? And wait, wouldn't it be better to have the shake afterwards, anyway? A recent study provides some answers.

I don't know if you notices, but it has been a while since the last study from the Exercise Metabolism Research Group at the Department of Kinesiology of the McMaster University in Hamilton, Ontario, Canada, hit the SuppVersity news. Allegedly, I mentioned Stuart Phillips only a couple of days ago, when I referenced the West study on "Associations of exercise-induced hormone profiles and gains in strength" (West. 2013), in the context of the questionable significance of post-exercise increases in testosterone levels, as far as muscle and strength gains are concerned - for those who missed that, it was  in the "Anabolic Workouts Revisited" post from last Monday. That was however about it as far as the news of the last weeks are concerned. So I was already wondering, when the first of you would be showing initial signs of "acute protein synthesis in response to protein ingestion study withdrawal symptoms", when I hit onto the latest study Phillips' group at McMasters participated in. The lead author of the study is however Louise M. Burke who's currently working at the Australian Institute of Sport in Canberra, Australia.

What happens if you ingest your post-workout protein before the workout ;-) ?

The aim of the study, of which the authors explicitly state that it is a quasi-followup to previous results which have conclusively identified fast acting protein sources (mostly whey, in some instances EAAs) as superior triggers, or I should say, promoters of post-exercise protein synthesis, was

"[...] to investigate the effects of manipulating patterns of aminoacidemia from protein sources consumed before a bout of resistance exercise bout." (Burke. 2013)

To this end, Burke et al. simulated the ingestion of slow or fast protein sources by bolus vs. pulse feeding of a leucine-enriched (+5g) whey protein drink (Nestec by Nestlé, sponsor of the study; the reason for the enrichment was to make sure that there would be a decent amount of leucine in each pulse serving).

• Bolus (B) - 1 x 500ml w/ 25g whey + 5g leucine drink, 14 x 33ml placebo every 15 min
• Pulse (P) - 1 x 500ml placebo drink,  14 x 33ml w/ 1.79g whey + 0.36g leucine each
• Placebo - 1 x 500ml placebo drink, 14 x 33ml placebo drink

The subjects, 12 resistance-trained men (age: 27y; body mass: 94.3kg; 1 RM single leg ext.: 42.8kg) with greater than 2 yr of experience of regular (at least twice per week) strength training, who had followed a standardized diet before each of the testing sessions (energy content of 80kcal/kg BM; 45%/34%/21% of the energy from carbs/fats/protein), started consuming their large 500ml beverage and the subsequent 14 small 33ml servings (every 15 min) 45min before they performed a standardized leg training session:

"This bout consisted of a  standardized warm-up, followed by 10 sets of 8–10 repetitions of leg extension at a workload equivalent to 80% of the specific leg 1 RM with 2-min recovery between sets. The leg that performed exercise was alternated for each trial. The duration of the resistance bout was approximately 45 min." (Burke. 2013)

Before during and after the exercise bout blood samples were collected. Muscle biopsies from the vastus lateralis of the exercised leg were taken 45min before, as well as after 1 h and 5 h of recovery.

Figure 1: Serum leucine and insulin levels after bolus and pulse ingestion, expressed relative to placebo group (left) and fractional protein synthesis rate (in %/h) during the 5h following the rest period after the workout (right; based on Burke. 2013)

As you can see in figure 1 the results basically confirmed the scientists expectation that the provision of protein before a workout would work just as well, because - more than anything else - it is the availability (or should I say abundance?) of amino acids in the blood stream that is the main determined of post the actual fractional protein synthesis rate (FSR) after moderate  volume workout like the one the participants conducted in the study at hand. In addition it did, at least as far as the protein synthetic response goes, neither make a significant difference, whether the subjects had all their protion at once before the workout or consumed it in 15min intervals before and during the workout.

Bolus or pulse = fast or slow? Pulsed whey does not equal casein (imho)

What I am personally not happy with, though, is the way Burke et al. equate the "pulse" protocol to the ingestion of a "slow" protein source, such as a micellar casein protein, for example. While they put that into perspective in the discussion of the results (see below) and despite the fact that do see the rationale of Burke et al. not to use a real slow digesting protein like casein in order to have absolutely identical amino acid compositions and to exclude other confounding factors, it is at least in my humble opinion somewhat confusing for the "average" reader. And while it is likely that the results for a "real" slow digesting protein would be similar, this would warrant direct experimental evidence. A fact the authors only hint at indirectly towards the end of the discussion of their results, when they state:

"[...] A specific issue in interpreting the finding of these previous studies and in increasing the utility of the concept of ‘‘fast’’ and ‘‘slow’’ dietary proteins is the difficulty of determining the individual and interactive contributions of the different AA composition of protein-rich foods and the digestibility of proteins or protein-rich meals to the pattern of delivery of these AAs. Our protocol, in which the same (fast) protein was consumed to achieve its traditional AA profile or as a series of small divided feed-ings to replicate the plasma leucine response associated with a slow protein, provides an opportunity to differ-entiate these effects." (Burke. 2013)

But hey, who knows, maybe that's going to be the research question of the next paper... and in the mean time it will spare you to buy two or even more different protein powders ;-)

"So what's better Dr. Andro? Pre- or post-workout protein supplementation?"

I guess most of you won't care anyways, as there is another question that's now preying on your minds... but to be honest, I can't provide you with a definite answer to it (see headline). What I can do, however is compare the study by West et al. (West. 2011) which used 25g of whey protein post workout to the one at hand (Burke. 2013).

Figure 2: Fractional protein synthesis (FSR in %/h) after the workout with post workout protein ingestion (West. 2011) and pre workout protein ingestion (Burke. 2013) on the left; relative increase in FSR in the two trials (compared to fasted for West. 2011 vs. Placebo for Burke. 2013) on the right (please mind that this is by no means a scientifically valid comparison, it's more of a "food for thought illustration"!)

I've done just that for you in figure 2, and what this comparison tells you is that it does not make a difference, whether you ingest all your protein as a bolus after a workout or start "pulse ingesting" (mind my words in the previous paragraph wrt to "slow != pulse") your protein 45min before the workout - at least, if you take the relative increase in fractional protein synthesis as a measure (figure 2, right). 

Unfortunately, both the composition of the protein supplement (25g whey in West. 2011 vs. 25g whey + 5g leucine in Burke. 2013), as well as the exact outcome variables that were measured (1-3h and 3-5h FSR in West. 2011 vs. 5h post workout FSR in Burke. 2013) were different, so that the comparison of the relative increases in protein synthesis I plotted on the right hand side of figure 2 is actually not 100% valid.

Moreover, and this is something I know a couple of you will now be thinking of, this comparison does by no means allow for any quantitative predictions with respect to the question of ...

What would happen, when you do both: Pulse ingest before and bolus ingest afterwards?

If  you don't remember or - even worse ! - have not read the previous SuppVersity post "Protein Synthesis Beyond the '20g Limit': Study Shows Exercise Facilitates 32% Greater Increases in Fractional Protein Synthesis With 40g Instead of 20g of Whey PWO" click on the image and (re-)read it ;-)

In view of the fact that the existence of a "threshold level" of protein intake, where the addition of even more protein won't yield any further benefits appears to be self-evident, the exact amount of this limit has yet still to be determined. At least after a workout, it seems that this threshold would be higher than the 25g and 20g of protein the subjects ingested in the Burke and West studies, respectively (please read my previous post "Protein Synthesis Beyond the 20g Limit" for a more detailed discussion of this topic).

That being said, it is reasonable to assume, but would likewise warrant experimental verification, that the combination of both protocols could increase the fractional protein synthesis even further. Yet while I am 100% sure that they won't simply add up, I would hesitate to bet money that the difference would actually reach statistical significant... at least with a low volume leg extension workout as it was used in the studies at hand.

References:

• Burke LM, Hawley JA, Ross ML, Moore DR, Phillips SM, Slater GR, Stellingwerff T, Tipton KD, Garnham AP, Coffey VG. Preexercise aminoacidemia and muscle protein synthesis after resistance exercise. Med Sci Sports Exerc. 2013 Oct;44(10):1968-77.
• West DW, Burd NA, Coffey VG, et al. Rapid aminoacidemia enhances protein synthesis and anabolic intramuscular signal-ling responses after resistance exercise. Am J Clin Nutr. 2011; 94:795–803. 
• West DW, Phillips SM. Associations of exercise-induced hormone profiles and gains in strength and hypertrophy in a large cohort after weight training. Eur J Appl Physiol. 2013 Jul;112(7):2693-702.  

Whey or Casein, Pulse or Spread Evenly Across the Day? Does it Even Make a Difference in Terms of Fat Loss and Lean Mass Retention on a Diet? New + Old Empirical Data!

Image 1: Instinctively right? Milk contains soluble (=whey) proteins and casein. Are we overthinking things, when we rip them apart and does it even make a difference? Or is timing all that counts?

It's funny "overthinking", right next to overtraining and overdieting, has become one of the most common problems among the health and fitness enthusiasts who spend equal (or even more) time online as in the gym. "Would it be better if I take my BCAAs at a 3:1:1 or 2:1:1 ratio?", "Does it matter if my protein powder is 10% hydrosolate, 50% isolate and 40% concentrate or has a 30/50/20 ratio?" All that may well make a difference, but let's be honest: Look at the things 80% of these people are eating day in and day out and the way they throw the weights around in the gym and contrast that to a question like "Will my post-workout protein synthesis be 5% greater, when I switch from concentrates to hydrosolates?" ... enough of the ranting, though. After all this post is actually about one of the more sensible among these world-shattering questions:

Q

Will it make a difference, whether I use casein or whey protein on a diet and... what's the significance of having my daily allotment of protein spread evenly across the day vs. mostly (80%) in one sitting, when I am dieting?

In order to find the answer to this question a group of French scientists recruited 41 healthy, but chubby subjects (BMI ~32kg/m²; age ~33y) and put them on a relatively moderate caloric deficit that was calculated based on their basal energy requirement (what you would need lying around all day). In all four arms of the study, the macro-nutrient composition (25% as proteins, 25% as lipids, and 50% as carbohydrates) and energy content per pound of lean body weight (average energy intake 5.87 MJ per day) of the meals, which were prepared according to personalized menus the subjects received from trained dietitians, were identical.

Figure 1: It did not make a difference if the protein was ingested either spread equally across the day or as a pulse mostly (80%) in one sitting (top), fat and weight loss after the 6 week study period were virtually identical (data based on Adechian. 2013)

The little information on the exact menu choices the scientists offers includes a list of stable foods, such as various proportions of spinaches, broccoli, lentils, or green beans, butter, bread, fruits, soy yogurt, rice cakes and gingerbread and suggest that we are dealing with the typical "your dietitian recommends diet", here. With one exception, of course, the main protein source of all four experimental diets were dairy proteins (~80g; >80% of total protein). Casein and whey aka "milk soluble protein"* (see red box above), which were to be ingested either spread equally across or in a "pulsed" fashion (see figure 1, left):

*Note: the scientists refer to whey as "milk soluble protein, I stuck to the terminology in the graphs, but in essence these are mainly β-lactoglobulin, α-lactalbumin, as well as serum albumin, immunoglobulins, lactoferrin, and other minor fractions and thus the same you would find in your average whey concentrate which is, as you may have notices "more soluble" than casein (cf. Lacroix. 2006)

• casein spread- subjects consumed ~20g of a casein protein supplement 4x a day
• milk spread - subjects consumed ~20g of milk protein supplement 4x a day
• casein pulse - subjects consumed the lions share, i.e. 80% of their ~80g of casein, as part of their 2nd meal, so that the protein intake over the day was 6.4g / 64g / 3.2g / 6.4g (see figure 1)
• milk pulse - same as above, but with milk instead of casein protein

In view of the overemphasisze nutrient timing has gotten as of late withing the physical culture and the assumption that you would expect to see profound differences based on when you consume how much of fast or slow, high (milk) or average (casein) leucine protein etc., it may be disappointing that the weight loss was absolutely identical in all four arms of the study (-7.5 ± 0.4 kg).

Differences are few and far between: Weight loss, fat loss, muscle loss - NOT different! 

What may yet surprise even you, a seasons SuppVersity veteran, who will probably already have expected the non-significant (in fact non-existent) differences in terms of weight loss, could be surprised that the changes in body composition (see figure 1, bottom), i.e. -5.1 ± 0.2 kg reduction in body fat mass and -2.2 ± 0.2 kg reduction in lean body mass, were identical.

Since the same goes for the changes in the fat "liberating" proteins lipoprotein lipase (LPL) and adipose triacylglycerol lipase (AGTL), the fat "forming" protein fatty acid synthase (FAS), and three of the usual subjects, i.e. leptin, the adipoQ gene which is responsible for encoding adiponectin, of which recent research suggests it may be even more important than leptin for your metabolic health (Li. 2013; Hickman. 2013), and the reduction in the pro-inflammatory monocyte chemotactic protein-1  (MPC-1), the slightly more pronounced meal-induced postprandial protein synthetic response in the casein group at the end of the study period is actually the only difference based on which you could argue for one over the other protein source:

Figure 2: While the changes in LPL, AGTL, FAS, leptin, AdipoQ and MCP expression were identical (left); the post 6-week protein synthetic response to identical meals was slightly more pronounced in the casein group (right), the overall significance of this finding is yet questionable in view of identical lean mass losses - it could yet become important on a diet + exercise regimen as in the Demling study discussed in the bottom line box  (data based on Adechian. 2013).

Whether the measurable advantage of casein during this test (the evaluation was carried out by leucine tracer infusion, by the way) is just an experimental artifact or

Adherence is the key to success: While there was no difference in terms of the hunger the subjects felt when they were on the diet, the fact that only 23 of the initially 41 subjects did make it through the 6- week on ~ 1,500kcal/day is quite telling, also in view of the perceived inability to lose weight - if you can't stick to a by no means crazy caloric restriction for 6 weeks, how can you expect to get lean and stay lean, when the inevitable prerequisite for the latter is that you totally revamp your dietary habits for the rest of your life not just six, eight, or twelve weeks.

• maybe something like "leucine resistance" in response to the higher leucine concentrations after the ingestion of the milk protein supplement in the course of the study period, or
• alternatively, the greater IGF-1 response to casein (cf. Hoppe. 2009, a study which compares whey vs. casein, but would obviously suggest an advantage of casein over milk = whey + casein, as well); unfortunately IGF-1 wasn't measured, but the insulin levels which were minimally higher in the casein group could support that hypothesis,

... is questionable. Since the same is true for the practical relevance of the ~10-13% larger leucine balance during the postprandial phase of the post-diet whole body protein metabolism test in week 6, I would not fret about this difference too much, though.

Maybe, just maybe, the adipocyte morphology could make a difference

What I would consider significant, though it did not reach that status (probably due to the low number of participant that actually made it to the end of the study, see red box on the right), is the slight but in my eyes potentially important superiority of the equally spread protein ingestion in terms with respect to the before vs. after adipocyte diameter in the casein group:

Figure 3: The difference did not reach statistical significance, but if we take for granted that greater reductions in adopcyte sizes are associated with healthier metabolic profiles, you would be better advised to take your casein protein equally spaced across (15% reduction in adipocyte size vs. 7%, only, for pulsed casein intake) the day... for whey, aka "milk soluble protein", on the other hand it does not seem to matter (data calculated base on Adechian. 2013)

Now, even if we assume that this made a difference and a greater reduction in adipocyte size was a significant advantage, which it probably is from a health perspective, as Skurk et al. state that there is

"[...] a differential expression of pro- and antiinflammatory factors with increasing adipocyte size resulting in a shift toward dominance of proinflammatory adipokines largely as a result of a dysregulation of hypertrophic, very large cells." (Skurk. 2006)

and a recently conducted human trial, by Rizkalla et al. the main message this study should be sending out is not that it does not make a difference whether you use casein or milk protein as your main protein source on a diet, but that a high protein diet with a mediocre caloric reduction of ~20-25% and supplemented with high quality dairy protein (whey or casein) works: After all, more than -1kg of weight loss per week, 68% of the weight loss from fat in the absence of exercise is more than your average celebrity XYZ diet will do for you ;-)

Whey or casein? It's high cysteine content that can help to replenish your glutathione (=the master antioxidant) pools would be another factor that speaks in favor of whey. Whether normal-weight individuals on an already optimized dietary regimen would benefit to the same extend as the obese young men in the 6-week whey supplementation trial, Vatani et al. describe in the August issue of Appetite, is however questionable. After all, the increases in HDL the total antioxidant capacity and glutathione is as questionable as any possible negative influence of the starchy placebo the researchers used in that study (some of you may have seen the link on the SuppVersity Facebook Wall, already).

Figure 4: Fat loss and lean mass gains in formerly overweight police officers after 12 weeks of training and dieting with or without casein / whey hydrosolate (Demling. 2000)

Moreover, one of the few long-term (=non acute protein synthesis) studies investigating the differential effects of concomitant whey vs. casein hydrosolate protein supplementation, found statistically significant higher body fat reductions and lean mass gains in those 33-34 year-old police officers who supplemented their 12-week diet + strength training regimen with 2x37g of casein hydrosolate (8h apart; for the exact data see figure 4; Demling. 2000).

Note: since both the whey (Pro-Score Champion Nutrition) and the casein protein (MET-Rx USA) in this study were hydrosolates the differences in lean mass gains and fat loss are depend primarily on the amino acid composition of the proteins, and not, as it would be with micelle casein vs. whey, the absorption kinetics!

Bottom Line: Against that background the study at hand supports previous findings of the importance of a threshold intake of protein. Interestingly, it did not confirm the notion that this threshold intake should be spread equally across the day, which is something most commenters (me included) read into the seminal paper by Loenneke et al., which found a statistically significant negative correlation not between total protein intake, but between the number of meals with 10g or more essential amino acids in them and abdominal obesity (Loenneke. 2013). So, does timing matter, or does it not? 

1. It does matter, when you work out, there is ample evidence to support that the ingestion of protein in the vicinity of the workout cannot just amplify the protein synthetic response but will also results in an increase in real world muscle gains.
2. It appears that it does not matter, when you are dieting (only), though; not just the study at hand, but also the success many people report on intermittent fasting regimen, would support the notion that the more sustained anabolism you may be able to achieve by ingesting say 4x25g of protein instead of 1x80 + 2x10g has, compared to the total amount of protein you eat, relatively little influence on the conservation of lean body mass, when you are dieting.

And as far as the choice between casein and milk soluble protein, aka whey (see first red box), is concerned (see box on the right, as well), it would appear prudent to assume that a combination of both - just like nature intended it - would be the best choice as a "standalone" protein source (cf. "Whey and Casein Work Hand in Hand for Protein Anabolism, but Scientists Overlook Fat, When They Reassemble Milk"), while the higher leucine content and faster digestibility render whey the better candidate for classic "supplementation", as in having an additional shake before you start preparing your whole-foods post-workout meal, which should - and I hope it's not really necessary that I say that - obviously include a significant amount of protein (fish, eggs, meats, and if you will even more dairy ;-), as well. The usefulness (again, not necessarily the superiority!)  of slow digesting protein is something you should be aware of, anyway, right? If not re-read the "3.2kg of Lean Mass Over Night W/ 40g of Slow Digesting Protein 30min Before Bed!?" post from February 22, 2013.

References:

• Adechian S, Balage M, Remond D, Migné C, Quignard-Boulange A, Marset-Baglieri A, Rousset S, Boirie Y, Gaudichon C, Dardevet D, Mosoni L. Protein feeding pattern, casein feeding or milk soluble protein feeding did not change the evolution of body composition during a short-term weight loss program. Am J Physiol Endocrinol Metab. 2013 Aug 14.
• Demling RH, DeSanti L. Effect of a hypocaloric diet, increased protein intake and resistance training on lean mass gains and fat mass loss in overweight police officers. Ann Nutr Metab. 2000;44(1):21-9.
• Hickman IJ, Whitehead JP. Structure, signalling and physiologic role of adiponectin - dietary and exercise-related variations. Curr Med Chem. 2013 Aug 9.
• Hoppe C, Mølgaard C, Dalum C, Vaag A, Michaelsen KF. Differential effects of casein versus whey on fasting plasma levels of insulin, IGF-1 and IGF-1/IGFBP-3: results from a randomized 7-day supplementation study in prepubertal boys. Eur J Clin Nutr. 2009 Sep;63(9):1076-83. 
• Lacroix M, Bos C, Léonil J, Airinei G, Luengo C, Daré S, Benamouzig R, Fouillet H, Fauquant J, Tomé D, Gaudichon C. Compared with casein or total milk protein, digestion of milk soluble proteins is too rapid to sustain the anabolic postprandial amino acid requirement. Am J Clin Nutr. 2006 Nov;84(5):1070-9.
• Li FY, Lam KS, Xu A. Therapeutic perspectives for adiponectin: an update. Curr Med Chem. 2013 Aug 9.
• Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2013 Jan 27;9(1):5. 
• Rizkalla SW, Prifti E, Cotillard A, Pelloux V, Rouault C, Allouche R, Laromiguière M, Kong L, Darakhshan F, Massiera F, Clement K. Differential effects of macronutrient content in 2 energy-restricted diets on cardiovascular risk factors and adipose tissue cell size in moderately obese individuals: a randomized controlled trial. Am J Clin Nutr. 2013 Jan;95(1):49-63.
• Skurk T, Alberti-Huber C, Herder C, Hauner H. Relationship between adipocyte size and adipokine expression and secretion. J Clin Endocrinol Metab. 2007 Mar;92(3):1023-33.
• Vatani DS, Golzar FA. Changes in Antioxidant Status and Cardiovascular Risk Factors of Overweight Young Men after Six Weeks Supplementation of Whey Protein Isolate and Resistance Training. Appetite. 2013 Aug 10.

Are Camels the Better Cows? Cancer, Heart Disease, High LDL and Triglycerides, Diabetes, High Blood Pressure, Allergies, Viral and Bacterial Infections and Trace Mineral Deficiencies, Camel Milk Prevents or Fixes Them All!

Image 1: To the average inhabitant of the Western hemisphere camels are probably not the most beautiful animals under the sun; but hey, cows aren't either, hah?

When muscle-heads think of dairy, they think of whey, they think of casein, they think of cottage cheese... but I bet few of them will think of camels! Even if you just went by the mere amino acid composition (see. figure 2 at the end of the article), of which you, as an educated SuppVersity student should by now be aware that it does not give you the 'whole picture', as far as the biological effects of a given protein and peptide containing foodstuff is concerned, it appears that camel milk would at least make an excellent alternative for cows milk, in case global warming is progressing and Europe and the US turn into desert wastelands... but all jokes aside, muscle is not everything and I bet that after reading this article you will be interested to register for the US' first official camel milking seminar *rofl*

7+1 reasons you may want to slaughter your grass fed cows and replace them with camels

While the idea of drinking the milk of an ugly desert ship may appear hilarious at first, I guess my compilation of purported and scientifically established benefits of camels milk will have you reconsider if the Sheikh Hamdan bin Mohammed bin Rashed Al-Maktoum, Crown Prince of Dubai, may not have made a very good investment, when he spent $16.5 million dirham ($4.5 million USD) on the winner of a 2008 beauty pageant in the United Arab Emirates’ capital city of Abu Dhabi (FYI, the guy in image 1 is not the Sheikh, just tom make sure I don't get sued, here ;-):

Image 2 (DrCate.com): Homogenization makes milk more convenient, but it disrupts the natural structure of the fat globules and releases the otherwise bound xanthine oxidase of which scientists hypothesized that it could trigger heart disease, a hypothesis, btw., that was not disproven, but simply kept quiet for the past 25+ years (Deeth. 1983)

• Little to no xanthine oxidase (=reductase) - While the idea that XOR (xanthine oxidoreductase), which is supposedly released during homogenization of bovine milk, could be a potential contributor to overall inflammation and cardiovascular disease has disappeared from the 'scientific radar' within the past couple of years (cf. Deeth. 1983; Berry. 2004) , it may still be of interest (and for certain populations such as people with increased gut permeability even of great importance) that camel milk apparently contains little to no xanthine oxidase - irrespective of whether you drink it raw, pasteurized or homogenized (Baghiani. 2003).
• Anticancer effects - Camels milk has been shown to trigger apoptosis (controlled cell death) in human breast cancer and liver cancer cells via epigenetic mechanisms (Korashi. Feb 2013; Korashi. May 2013).
• Antibacterial & antiviral effects -Camel milk prevents gram positive bacteria from growing and reduces the amounts and activity of gram-negative cultures (el Agamy. 1992). With the latter being among the primary drivers of lipopolysaccharide (LPS) and endotoxin induced inflammation (Ulevitch. 1999), camel milk could thus help to reduce local and systemic inflammation. Aside from its activity against rotavirus, the lactoferrin faction from camel milk appears to have protective effects against hepatitis C, as well (Redwan. 2007).
• Camel milk whole- & beta-caseins act as natural anti-oxidants and ACE-inhibitors - As Salami et al have shown the whole casein and beta-casein (β-CN) faction(s) of camel milk exert Angiotensin Converting Enzyme (ACE)-inhibitory (=blood pressure reducing) and antioxidant activity after they were hydrolyzed in the stomach (Salami. 2011). In a previous study, the same researchers had already determined that the whey fraction of camel milk exhibits significant anti-oxidant and antimicrobial activities, as well, and that those were up to 100% greater (depending on the essay and fraction the scientists used) than those of bovine whey protein (Salami. 2010).



Figure 1: A certain part of the population in Rajasthan (India) who consumes camel milk on a daily basis has been found to have a significantly reduced incidence of diabetes (not a single one!), impaired fasting glucose (-6%/-11%) and impaired glucose tolerance (-10%/-10%) than both non-camel milk drinking parts of the Raica community or other non-camel milk drinkers from the same region (based on Agrawal. 2007)

• Profound and long-lasting anti-diabetic effects - Camel milk has a long history of being used to tread type 1 diabetes in the Middle East (see figure 1; cf. Mohamad. 2009). Studies from animal models (dogs, Sbou. 2010) and humans (Agrawal. 2009; Mohamad. 2009) improved blood glucose, microalbumenia and secondary symptoms such as diabetic neuropathy. Probably also as a consequence of the small, but biologically active natural insulin content of camels milk (Malik .2013), the type 1 diabetics in a 2009 study by Agrawal could even reduce their insulin medication by 32% from 41µ/day to 28µ/day. In the 2-year follow up, the researchers report that "out of 12 subjects receiving camel milk, insulin requirement in 3 subjects reduced to zero" (Agrawal. 2011) - try that with metformin, let alone some of the other 'diabetes prolongation drugs'.
• Improved lipid metabolism - The 24 type one diabetics who consumed 500ml of plain camel milk per day for 6 months in the aforementioned 2009 study by Agrawal et al. for example exhibited -30% decreases in LDL and -66% decrease in triglycerides.
• Camel milk is an extraordinary good source of trace minerals - According to Al-Awidi et al. Camel milk contains 7-20x and 1-10x higher levels of manganese and iron than human milk, more zinc and comparable amounts of selenium, copper and other protein bound and thus highly bioavailable trace minerals (Al-Awadi. 2001).

And best of all, based on studies on people with cow's milk allergy, we know that the incidence of allergic reactions to camel milk is not only much lower, but also that 80% of cow's milk allergy sufferers can actually ingest camel milk without any unwanted side-effects (Cardoso. 2010; Ehlayel. 2011).

Figure 2: Even if you go solely by the amino acid composition (here expressed relative to the total amino acids), camel milk protein could be a valid replacement for bovine proteins (data based on Davis. 1994 & Beg. 1987)

This is also worth mentioning, because the host of great effects I listed above do - at least in parts - also occur with the unwarrantedly vilified bovine (=cow's) milk, which also contains ACE precursors (Saito. 2008), exert anti-cancer effects (Gill. 2000), and so much more (see "suggested readings" at the end of this article).

So, in the unfortunate case that you "ain't got no camel handy" at the moment and, due to "the current economy" (I hate when people say that) lack the $1300 to $1700 (Debacle. 2006) to buy your own, obviously not  pageant winning $16.5 million dirham camel, and tolerate bovine milk, just stick to the milk of the farmer you trust. After all, even if Camels were the better cows, you better have a gallon of cows milk in the fridge than a camel in the Arabian desert, right? Ah, wait that was a bird in the hand, right? ... ah, whatever ;-)

Suggested readings:

• Ask Dr. Andro: Are Colostrum and Milk Products in General Healthy Muscle Builders, a Waste of Money or Toxic Waste?
• Childhood Asthma: Children Born to Mothers Who Consume Semi-Skimmed Milk 8% More Likely, Kids of Whole Milk Drinking Mothers 15% Less Likely to Develop Asthma
• The Fat Truth Behind the Dairy Weight Loss Miracle: MUFA and PUFA Impair, Saturated Fat and Plenty of Micronutrients Drive Full-Fat Dairy-Powered Fat Loss.
• Higher Calcium Intake Greater Fatty Acid Oxidation!? True: Chronic & Acute Effect Size Comparable to Caffeine
• Read all about milk here at the SuppVersity

References:

1. Agrawal RP, Budania S, Sharma P, Gupta R, Kochar DK, Panwar RB, Sahani MS. Zero prevalence of diabetes in camel milk consuming Raica community of north-west Rajasthan, India. Diabetes Res Clin Pract. 2007 May;76(2):290-6.
2. Agrawal RP, Dogra R, Mohta N, Tiwari R, Singhal S, Sultania S. Beneficial effect of camel milk in diabetic nephropathy. Acta Biomed. 2009 Aug;80(2):131-4. 
3. Agrawal RP, Jain S, Shah S, Chopra A, Agarwal V. Effect of camel milk on glycemic control and insulin requirement in patients with type 1 diabetes: 2-years randomized controlled trial. Eur J Clin Nutr. 2011 Sep;65(9):1048-52. doi: 10.1038/ejcn.2011.98. Epub 2011 Jun 1.
4. Al-Awadi FM, Srikumar TS. Trace elements and their distribution in protein fractions of camel milk in comparison to other commonly consumed milks. J Dairy Res. 2001 Aug;68(3):463-9.
5. Baghiani A, Harrison R, Benboubetra M. Purification and partial characterisation of camel milk xanthine oxidoreductase. Arch Physiol Biochem. 2003 Dec;111(5):407-14.
6. Beg OU, von Bahr-Lindström H, Zaidi ZH, Jörnvall H. Characterization of a heterogeneous camel milk whey non-casein protein. FEBS Lett. 1987 Jun 1;216(2):270-4.
7. Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol. 2004 Mar 16;555(Pt 3):589-606.
8. Cardoso RR, Santos RM, Cardoso CR, Carvalho MO. Consumption of camel's milk by patients intolerant to lactose. A preliminary study. Rev Alerg Mex. 2010 Jan-Feb;57(1):26-32.
9. Davis TA, Nguyen HV, Garcia-Bravo R, Fiorotto ML, Jackson EM, Lewis DS, Lee DR, Reeds PJ. Amino acid composition of human milk is not unique. J Nutr. 1994 Jul;124(7):1126-32.
10. Deeth HC. Homogenized milk and atherosclerotic disease: a review. J Dairy Sci. 1983 Jul;66(7):1419-35.
11. Gill HS, Cross ML. Anticancer properties of bovine milk. Br J Nutr. 2000 Nov;84 Suppl 1:S161-6. Review.
12. Ehlayel MS, Hazeima KA, Al-Mesaifri F, Bener A. Camel milk: an alternative for cow's milk allergy in children. Allergy Asthma Proc. 2011 May-Jun;32(3):255-8.
13. el Agamy EI, Ruppanner R, Ismail A, Champagne CP, Assaf R. Antibacterial and antiviral activity of camel milk protective proteins. J Dairy Res. 1992 May;59(2):169-75. 
14. Gill HS, Cross ML. Anticancer properties of bovine milk. Br J Nutr. 2000 Nov;84 Suppl 1:S161-6. Review.
15. Korashy HM, El Gendy MA, Alhaider AA, El-Kadi AO. Camel milk modulates the expression of aryl hydrocarbon receptor-regulated genes, Cyp1a1, Nqo1, and Gsta1, in murine hepatoma Hepa 1c1c7 cells. J Biomed Biotechnol. 2013;2013:782642. Epub 2013 Feb 27.
16. Korashy HM, Maayah ZH, Abd-Allah AR, El-Kadi AO, Alhaider AA. Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism. J Biomed Biotechnol. 2013;2013:593195. Epub 2013 May 13.
17. Debacle J. Long or Short Capital. Camels, The Next Big Thing. August 1, 2006. < http://longorshortcapital.com/camels-the-next-big-thing.htm > received on July 13, 2013
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19. Mohamad RH, Zekry ZK, Al-Mehdar HA, Salama O, El-Shaieb SE, El-Basmy AA, Al-said MG, Sharawy SM. Camel milk as an adjuvant therapy for the treatment of type 1 diabetes: verification of a traditional ethnomedical practice. J Med Food. 2009 Apr;12(2):461-5.
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80% Greater Protein Synthesis 3-5h After Workout: 20g+ PWO Protein Threshold Holds. Spiking Lower Amounts With Leucine or EAAs Will Still Yield Sub-Optimal Results

Image 1: Milk (proteins) are not just leucine or EAA - try doing that with half the amount of free-form aminos in water - the results will certainly be "suboptimal", I can vouch for that  ;-)

It has been a while since the last study from Stuart Phillips group at the McMaster University has made it to the SuppVersity news. Their latest publication does yet have the potential to pour oil on troubled waters, because the results appear to confirm that even when every other supplement appears to be failing you, you can always rely on your postworkout whey protein (Churchward-Venne. 2013). And while the study confirms that with some free form amino acid witchcraft, you can actually illicit identical post-exercise increases in protein synthesis, the previously determined threshold dosage of 20g of high quality protein is still the gold standard, for everyone whose interest is to actually build muscle which is, as you as a seasoned SuppVersity veteran know, not happening only in the first hour after a workout but within a 24h+  "window of opportunity" that has the size of barn door (cf. "Opening the 'Anabolic Barn Door' With the Key of Exercise and Nutrition Science!")!

You won't get a-whey without 20g+ of whey!

To elucidate whether the increasingly popular practice of pimping whole proteins with amino acids does make any sense in terms of being able to get away with less total protein, yet identical increases in post-workout protein synthesis Churchward-Venne et al. recruited 24 recreationally active, young adult male volunteers (22±0.6 years; 1.80±0.02m; 76.4±2.0 kg; BMI 24.3kg/m²), who had to perform a standardized 4x4 unilateral leg-workout with 3 min rest between sets consisting of

• 4x 10-12 reps of seated knee-extension and 
• 4x 10-12 reps of leg-press

at ~95% of their individual 10-rep max, which had been determined in a testing session 14 days prior.

Note: The reason Churchward-Venne et al. decided to use a unilateral exercise protocol was that this allowed them to take biopsies from both the exercised and non-exercised leg and thus determine the individual influence of exercise and supplementation.

You better make sure you get your protein, not just EAAs or leucine

Figure 1: Amino acid compositions of the test drinks (Churchward-Venne. 2013)

The study participants, who had consumed a standardized, prepackaged relatively low-protein diet (15% protein,. 55% carbohydrate, 30% fat) the day before the exercise intervention, were randomly assigned to consume one of the following drinks

• whey protein - 25 g whey protein isolate (total leucine: 3g)
• whey + leucine: 6.25 g whey protein isolate supplemented with free-form leucine (total leucine: 3g)
• whey + EAA: 6.25 g whey protein isolate supplemented with free-form EAAs (total leucine: 0.75g)

The 300ml of fluid which contained identical tracers, were consumed immediately post workout, blood and muscle biopsies were taken at regular intervals pre- and post workout and MPS, signaling through mTOR, and amino acid transporter (AAT) mRNA abundance were determined.

Figure 2: Relative expression of p-mTOR (left) and p70S6K (right) compared to baseline (Churchward-Venne. 2013)

Now what is interesting is that the "classic" markers of protein anabolism, p-akt (not shown, but exhibited significant differences between treatment), mTOR and P70S6K (figure 2 & 3) do not show a clear-cut advantage of either of the treatments. Immediately post exercise, the increase in mTOR in the exercised leg, for example, is significantly more pronounced in those subjects who consumed a whey protein shake. The "downstream" activation of p70S6K, which supposedly controls protein synthesis at the ribosome, however, is identical in all groups.

Figure 3: Pseudo (=simply weighed by the timespan) area under the curve (a.u) for mTOR and p70S6K, AUC for leucine (a.u.) and fractional protein synthesis in the exercised leg 3-5h after the workout (based on Churchward-Venne. 2013)

The same is true for the protein synthetic response measured as fractional protein synthesis in the whole post exercise period in the untrained, and up to 3h post exercise in the trained leg. Then, however, we see a markedly higher influx of protein into the trained muscle in the whey protein group, which is - and this is somewhat remarkable - not in accordance with the p70S6K levels, which would suggest that the protein influx should be maximal in the leucine and not in the whey group.

A protein pump without protein is useless

Based on the data we have, it is difficult to say whether it is the lack of an individual, a certain combination or the total amount of (non-)essential amino acids that is responsible for this affect. If you take a look at the amino acid composition of the test solutions in figure 1, it does yet appear likely to assume that it is the absence of non-essential amino acids...what? Glutamin? No, I thought so as well, but when you come to think about it, glutamine, of which we have recently seen that it does play a hitherto under-appreciated role in protein synthesis, is unlikely to exert this effect on its own. After all, Chiu et al. based their conclusions with respect to the necessity of glutamine to maximize protein synthesis on increases in mTOR expression (cf. "A New Role for Glutamine in Protein Synthesis?"). 3-5h after the workout the initially increased mTOR levels in the whey protein group had yet returned to baseline and the the leucine, BCAA and EAA levels in the blood of the subjects were identical in all groups (data not shown); and still, the influx of protein into the exercised leg musculature of the whey group was ~80% higher than that in the EAA group.

Image 2 (dormtainment.com): Subjects from the EAA, the leucine and the whey group (from left to right) after ingestion of the respective fluids - just kiddin' *rofl*

But let's be honest, in the end, these results only what common sense should have told us all along: You can push the gas pedal as much as you want (leucine group) and still won't get very far if your protein tank is half empty. Similarly, you can ingest as much leucine as you want and it will still have little effect on total protein synthesis, regardless of whether you train or not. For the practitioner, any further speculations about the minimal amount of leucine, a given persons in a given age-group would need to maximally stimulate protein synthesis, as the authors make them in their discussion of the results are non-significant compared to the following straight forward take home messages:

1. 20-25g of whey protein are still the go to post-workout protein source
2. building a better post workout protein from free form EAAs is not feasible
3. the importance of the non-essential amino acids in "real" protein is probably under-appreciated
4. the importance or I should say potency of leucine is probably much over-estimated
5. muscle protein synthesis and thus skeletal muscle hypertrophy is not a 2h post workout game

In essence, it would suffice to remember just (1) and (5) and to follow the simple yet effective maxime to get 20g+ of quality protein (not 20g leucine ;-) with every meal to get big and muscular and, as all of you who read yesterday's news or one of the many previous posts in which I envoked the findings of Loenneke et al. which show just that: People with a frequent intake of quality protein have the lowest body fat levels (Loenneke. 2013).

References:

1. Chiu M, Tardito S, Barilli A, Bianchi MG, Dall'asta V, Bussolati O. Glutamine stimulates mTORC1 independent of the cell content of essential amino acids. Amino Acids. 2013 May 8. [Epub ahead of print]
2. Churchward-Venne TA, Burd NA, Mitchell CJ, West DW, Philp A, Marcotte GR, Baker SK, Baar K, Phillips SM. Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men. J Physiol. 2013 Mar 25.
3. Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2013 Jan 27;9(1):5.
4. Moore DR, Robinson MJ, Fry JL, Tang JE, Glover EI, Wilkinson SB, Prior T, Tarnopolsky MA, Phillips SM. Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. Am J Clin Nutr. 2009 Jan;89(1):161-8.