ONLINE DRIVERS MEDIA

Post-Workout High Isoleucine AA+CHO Decreases Glucose Spikes, But Impairs Musclular Glyocogen Resynthesis - Reason Enough to Skip Amino Acids?

If you put any faith into the promises of the supplement industry, amino acid supplements are the solution to all your problems - including those you havent even known about, yet. Against that background its always interesting if scientists study the real world effects of amino acid supplements in a realistic scenario like after strenuous exercise.

In their latest study Wang and colleagues from the University of Texas at Austin and the Shanghai Research Institute of Sports Science did just that: They studied the effects isoleucine and four additional amino acids, on blood glucose homeostasis and glycogen synthesis after strenuous exercise.
As the scientists point out, the results of their study "could provide a practical and safe means of increasing the rate of muscle glycogen synthesis after exercise and enhancing the rate of recovery" (Wang. 2015).

Table 1:  Subjects’ characteristics (Wang. 2015).

Ten healthy active adults volunteered for the study. All subjects were accustomed to cycling for prolonged periods of 3–5 h during an exercise session. The ,aximum oxygen uptake (VO2max) was measured in all subjects on a cycle ergometer by using a TrueOne 2400 metabolic measurement system (ParvoMedics, Sandy, Utah) to verify adequate aerobic fitness levels (results see Table 1).

Figure 1: Basically the AA supplement contained almost exclusively isoleucine. It was administered in the dosage shown above and at twice that amount in the LAA and HAA trials (Wang. 2015)

"Two to three days after the VO2max test, the subjects reported to the laboratory to perform a practice ride to familiarize them with the laboratory environment and the experimental protocol. The practice ride was also used to adjust and verify appropriate workloads for the experimental trials. The practice rides simulated the protocol ride but without blood samples or muscle biopsies being taken. The ride consisted of cycling at 70 % VO2max for 2 h, which was followed by five 1-min sprints at 85 % VO2max. The sprints were separated by 1 min cycling at 45 % VO2max. During the first 15 min of each hour, oxygen uptake was measured for 5 min to verify workload.

Water (250 mL) was provided every 20 min of exercise. Heart rate (HR) was monitored and ratings of perceived exertion (RPE) on a Borg-scale (ranging from 6 to 20) were collected every 30 min of exercise. The practice ride and each of the following three experimental trials were separated by a minimum of 7 days and maximum of 12 days" (Wang. 2015).

The actual tests consisted of cycling on an ergometer to deplete muscle glycogen. Blood sampling and a muscle biopsy were performed immediately on cessation of exercise. After the muscle biopsy, subjects were given the first of two supplement doses. More specifically they received either...

• 1.2 g carbohydrate/kg body weight (CHO), 1.2 g carbohydrate/kg body weight plus 6.5 g AA (CHO/LAA) or 
• the same carbohydrate supplement plus 6.5g (CHO/LAA) or 13 g AA (CHO/HAA) 

immediately after the first muscle biopsy and at 120 min of recovery. The carbohydrate base consisted of simple dextrose dissolved at a ratio of 100g/296 mL in an orange flavored drink (SUN-DEX, Fisher Healthcare, Houston, Texas). The additional amino acids contained 0.046 g cystine 2HCl, 0.023 g methionine, 0.045 g valine, 6.342 g Isoleucine, and 0.044 g leucine per person, or twice that amount in the CHO/HAA trial. The amino acids were simply added to the dextrose drink.

Why would you even believe that there may be benefits from AA supplementation?

As Wang et al. point out, "this amino acid mixture was selected as it was previously reported to be more effective in lowering the blood glucose response to a glucose challenge than isoleucine alone" (Wang. 2015) by Bernard et al. (2011).

Figure 2: Blood glucose AUC during the oral glucose tolerance test (OGTT). Sprague-Dawley rats were gavaged with either glucose (CHO), glucose plus a 5-amino acid mixture (CHO-AA-1), glucose plus a 5-amino acid mixture with increased leucine concentration (CHO-AA-2), or placebo (PLA). Blood was taken from the tail immediately before the gavage and 15, 30, 60, and 120 min afterward (Bernard. 2011).

The three test beverages were similar in color, taste, and texture to allow a double-blinded and counter-balanced study design. All test drinks were randomly assigned and dispensed by a laboratory technician who was not involved in the data collection.

Figure 3: Blood glucose postexercise and during the 4-h recovery. Treatments were with CHO (circle), CHO/LAA (triangle), and CHO/HAA (filled circle) supplements provided immediately after and 2 h after exercise. Values are mean ± SE. CHO/HAA vs. CHO (*p < 0.05). CHO/LAA vs. CHO (# p < 0.05) - left; Blood glucose area under the curve (AUC) during the 4-h recovery. Treatments were CHO, CHO/LAA, and CHO/HAA supplements provided immediately after and 2 h after exercise. AUC was calculated with baseline (pre). Values are mean ± SE. CHO/HAA vs. CHO (*p < 0.05). CHO/LAA vs. CHO (# p < 0.05) - right (Wang. 2015).

As the data in Figure 3 indicates,There was a similar effect in humans as it has previously been observed in rodents. An effect of which you as a SuppVersity reader know that it is probably mostly ascribable to isoleucine (see "The Glucose-Repartitioning Effects of Isoleucine" | more).

Figure 4: Total muscle glycogen storage in the vastus lateralis during the 4-h recovery from intense cycling. Treatments were CHO, CHO/LAA, and CHO/HAA supplements provided immediately after and 2 h after exercise. Values are mean ± SE. CHO/HAA vs. CHO (*p < 0.05 | Wang. 2015)

What is a bit disappointing is the fact that the decrease in blood glucose did not come with an increase in glycogen storage.

As the data in Figure 4 shows, the exact opposite was the case. After 4h of recovery the muscle glycogen levels were not higher, but lower in the amino acid supplemented trials.

For diabetics this wouldnt be a problem. For athletes its yet clearly a disadvantage that the 4-g recovery glycogen levels were lower and significantly lower in the low and high dose amino acid supplement trials.

Eventually this result is surprising because specifically in the high amino acid group (a) the insulin levels, (b) the AS160, a protein that controls insulin mediated glucose uptake, (c) the mTOR & p-AKT levels, (d) the "exercise hormon" levels of serum irisin  and (e) the levels of glycogen synthase which stores carbs in forms of glycogen in the high dose AA trials were significantly elevated.
References:

• Armstrong, Jane L., et al. "Regulation of glycogen synthesis by amino acids in cultured human muscle cells." Journal of biological Chemistry 276.2 (2001): 952-956.
• Bernard, Jeffrey R., et al. "An amino acid mixture improves glucose tolerance and insulin signaling in Sprague-Dawley rats." American Journal of Physiology-Endocrinology and Metabolism 300.4 (2011): E752-E760.
• Doi, Masako, et al. "Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes." Biochemical and biophysical research communications 312.4 (2003): 1111-1117. 
• Ivy, John L., et al. "Early postexercise muscle glycogen recovery is enhanced with a carbohydrate-protein supplement." Journal of Applied Physiology 93.4 (2002): 1337-1344.
• Ivy, J. L., et al. "Post exercise carbohydrate–protein supplementation: phosphorylation of muscle proteins involved in glycogen synthesis and protein translation." Amino acids 35.1 (2008): 89-97.
• Morifuji, Masashi, et al. "Dietary whey protein increases liver and skeletal muscle glycogen levels in exercise-trained rats." British journal of nutrition 93.04 (2005): 439-445.
• Morifuji, Masashi, et al. "Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats." Amino acids 38.4 (2010): 1109-1115.
• Wang, Bei, et al. "Amino acid mixture acutely improves the glucose tolerance of healthy overweight adults." Nutrition Research 32.1 (2012): 30-38.
• Zawadzki, K. M., B. B. Yaspelkis, and J. L. Ivy. "Carbohydrate-protein complex increases the rate of muscle glycogen storage after exercise." J Appl Physiol 72.5 (1992): 1854-9.

Ever since the Vinson study showed that green coffee bean extracts can help overweight women lose weight, green coffee is sexier than roasted one.

Only weeks after Vinson et al. were able to show that "Green Coffee Bean Extract Helps Pre-Obese Men and Women Shed 16lbs in 22 Weeks" (read more), green coffee bean supplements began to swamp the market - a great financial success for the supplement producers, and that despite the fact that follow-up studies that would confirm the amazing effects Vinson et al. report are still lacking. Two recent studies, one from the Queen Margaret University in the UK (Revuelta-Iniesta. 2014), the other from the  Jiangxi Agricultural University in the Peoples Republic of China (Zheng. 2014) could now shed a little more light on the weight loss effects of green coffee beans and the combination of caffeine and cholorgenic acid (CGA), the allegedly most important active ingredient in unroasted, green coffee beans.
In their 24-week rodent study, Zhang et al. (2014) tried to elucidate the mechanism by which CGA and caffeine regulate lipid metabolism. To this ends, they randomized their forty hairy subjects to diets containing no CGA or caffeine, CGA, caffeine, or CGA + caffeine.

Figure 1: Rel. (vs. control) body weight changes and liver and intraperitoneal adipose tissue weight (Zhang. 2014)

Over the course of the 24-weeks study, the scientists recorded body weight, intraperitoneal adipose tissue (IPAT) weight, and serum biochemical parameters of the rodents, tracked the the activities and mRNA and protein expression of lipid metabolism-related enzymes and analyzed the effects of caffeine, CGA and the combination of the two. What they found was...

1. Figure 2: Effects of chlorogenic acid (CGA) and caffeine on the hepatic protein expression levels of AMP-activated protein kinase (AMPK), adipose TAG lipase (ATGL) and fatty acid synthase (FAS; Zhang. 2014)

   decreases in the body weight and IPAT weight of mice fed the CGA + caffeine diet,
2. significant decreases in the serum and hepatic concentrations of total cholesterol, TAG and leptin of mice fed the CGA + caffeine diet,
3. increases of the activity of carnitine acyltransferase (CAT) and acyl-CoA oxidase (ACO), 
4. decreased levels of fatty acid synthase (FAS) and the respective mRNA levels
5. significantly upregulated mRNA expression levels of AMP-activated protein kinase (AMPK), CAT and ACO
6. pronounced reductions of PPARg2

If we group these findings as follows 3 + 5 and 4+6, we could say that they triggered 1 + 2 by (a) increasing the oxidation of fatty acids (3+5) and (b) decreasing the synthesis and storage of fatty acids (4+6) - an observation that could certainly explain the benefits Vinson et al. observed in their human study which has been retracted in October 2014, though. On the other hand, the amount of CGA and caffeine in the rodent diets (0.2 % CGA + 0.03 % caffeine) was quite significant and previous rodent studies on other allegedly promising fat burners were not replicable in human beings either (example: CLA). Against that background its good to have study #2 by R. Revuelta-Iniesta and E. A. S. Al-Dujaili, who investigated the effects of green coffee (GC), rich in chlorogenic acid, and black coffee (BC) on cardiovascular markers.

New human data with surprising results

The researchers designed a randomised pilot crossover study with healthy subjects who consumed both coffees for 2 weeks.

• The green coffee (GC) used in this project was Ethiopian Harrar 4 (100% Arabica) and the black coffee (BC) was Sainsbury’s Original Blend Cafetitère Coffee.
• The BC was  a blend of Brazilian, Colombian, Mexican, Nicaraguan, Peruvian, and Rwandan beans.

The study participants were asked to have 40 g of GC and BC per day distributed throughout the day into four cups of coffee. Thus, the scientists tried to ensure that high plasma coffee antioxidant concentrations were maintained over a period of time, "allowing effects to take place and the body to develop tolerance to caffeine, which can take 2-3 days." (Revuelta-Iniesta. 2014).

Table 1: Concentration of total polyphenols and antioxidant capacity determined in GC and BC as compared by the three methods of  coffee preparation (Revuelta-Iniesta. 2014).

The GC beans were grounded to powder using an electric coffee grinder. Instructions on how to make the coffee were provided and the Italian cafetière, the French cafetière, or the filter coffee machine was used to prepare the coffee drink.

The researchers measured anthropometry, blood pressure, and arterial elasticity after each intervention and collected urine samples to monitor antioxidant capacity. The free cortisol and cortisone levels you see in Table 2 were obtained from urine and analysed by specific ELISA methods.

Table 2: Comparison of results obtained (mean±SEM) after 14 days of green coffee vs. black coffee intervention (2-week cross over study); F: cortisol; E: cortisone; orange = almost bordeline significant; green = statist. significant inter-group difference (Revuelta-Iniesta. 2014).

In view of the short duration of the study (remember 2 weeks on each coffee), its not really surprising that we didnt see changes in any of the anthropometric measures. On the other hand, the mere fact that the cortisol/cortisone ratio (indicating 11beta-HSD1 activity) was reduced after GC (from 3.5 +/- 1.9 to 1.7 +/- 1.04, P = 0.002) does not suggest that you will see beneficial effects on body composition in the absence of significant reductions in energy intake. A reduction in glucocorticoid activity can in fact hamper not accelerate weight loss (learn more) and the researchers hypothesis that "GC can play a role in reducing cardiovascular risk factors" (Revuelta-Iniesta. 2014) is a possible, but unconfirmed hypothesis.
References:

• Jaquet, Muriel, et al. "Impact of coffee consumption on the gut microbiota: a human volunteer study." International journal of food microbiology 130.2 (2009): 117-121.
• Revuelta-Iniesta, R., and E. A. S. Al-Dujaili. "Consumption of Green Coffee Reduces Blood Pressure and Body Composition by Influencing 11?-HSD1 Enzyme Activity in Healthy Individuals: A Pilot Crossover Study Using Green and Black Coffee." BioMed Research International 2014 (2014).
• Vinson, Joe A., Bryan R. Burnham, and Mysore V. Nagendran. "Randomized, double-blind, placebo-controlled, linear dose, crossover study to evaluate the efficacy and safety of a green coffee bean extract in overweight subjects." Diabetes, metabolic syndrome and obesity: targets and therapy 5 (2012): 21.
• Zheng, et al. "Chlorogenic acid and caffeine in combination inhibit fat accumulation by regulating hepatic lipid metabolism-related enzymes in mice." British Journal of Nutrition (2014). Ahead of Print.

Even Arnold benefited from ?-hydroxy-isocaproic acid aka HICA - the HICA his body produced and the HICA he got from his diet, whenever he ate cheese and other fermented foods.

You know that I am not the kind of person who likes to tell others what to do. After reading my summary of the contemporary research on ?-hydroxy-isocaproic acid aka HICA, you should yet be able to decide whether its worth a try or not.

If you take a look at the pertinent databases you will realize that there are more patents than papers on ?-hydroxy-isocaproic acid - usually, this is a good indicator we are dealing with another industry scam, but in contrast to the many funky forms of creatine, ?-hydroxy-isocaproic acid does actually have a handful of studies to back up that it does... or I should say "that it could" help you getting big and buffed.
That being said, it may be worth taking a look at what ?-hydroxy-isocaproic acid actually is. Just like HMB which is about to make a comeback in liquid form, these days, HICA is a metabolite of the mTOR and thus protein synthesis triggering branch-chained amino acid leucine. It is also known as "leucic acid" or "DL-2-hydroxy-4-methylvaleric acid" and is formed by ?-hydroxylaction from leucine. Its one of the end-products of leucine metabolism in muscle and connective tissue (Walser. 1978). Its usual concentration in our blood is about 0.2560.02 mmol/L - thats ~100x less than the amount of ?-keto-isocaproic acid (KIC), the corresponding keto acid of leucine of which youll find more than 21 mmol per liter in your blood.

Figure 1: Relative changes in lean mass (%) during 4 weeks of intense soccer training on 1.5g/day HICA (Mero. 2010)

Cheese, wine, soy sauce, etc. - the recently celebrated fermented foods, they all contain HICA, which appears to be the anti-catabolic counterpart to leucine. While the latter is a potent promoter of protein synthesis, the former appears to make sure that the work of its predecessor leucine is not lost.

It is thus no wonder that the promising results of a 2010 study by Mero et al. were recorded during an intensive and thus potentially catabolic training period in soccer athletes. In contrast to the placebo group, where only one individual gained a significant amount of lean mass, while 4 lost muscle, the subjects who had been consuming 1.5g/day of ?-hydroxy-isocaproic acid gained 300g of lean mass, on average, in the course of the 4-week study.

Thats not much and it was not fat free (ca. 150g of fat), but the data in Figure 1 shows that this is a difference between minimal muscle loss and gain... and I guess most athletes would prefer a marginal muscle gain over a marginal loss of lean mass.

HICA, a potent anti-catabolic? I dont think so!

The notion that HICA is, above all, a muscle loss inhibitor appears questionable, if we take a look at the results Charles H. Lang, Hugues Magne, Elizabeth Offord and Denis Breuille presented at a 2013 FASEB meeting. In the abstract to their presentation they cite the results of a rodent study in the course of which the rodents were immobilized for two full weeks. The consequence, an increase in the expression of catabolic hormones and a profound loss of muscle mass was identical in both the HICA and placebo supplemented groups, but in spite of the fact that "?HICA did not alter the immobilization-induced increase in proteasome activity and atrogene expression", the muscle mass had returned to control values only in ?HICA-fed rats after 14 days.
The fast recovery in the HICA group was associated with increased muscle protein synthesis and higher levels of the "protein synthesis pump initiator proteins" S6K1 and 4EBP1 in the previously immobilized muscle. The results Lang et. al. have not yet published in a full paper (at least I couldnt find it) put a huge questionmark behind the long-heralded hypothesis that HICA supplementation would slow muscle loss and puts it in line with its cousin HMB and its precursor leucine as a purported pro-anabolic muscle builder.

Figure 2: Gastrocnemius weight (rel. to control) immediately before and 14-days after the immobilization (Lang. 2013).

The data from the corresponding full paper the authors published a couple of month later in the American Journal of Physiology - Endocrinology and Metabolism you see in Figure 2 are even more impressive, though. According to this rodent data HICA is a more potent muscle (re-)builder than leucine; and, importantly, neither of the two does what the industry keeps promising: prevent muscle catabolism in response to disuse.
Reference:

• Chow K., and Walser M. "Effects of substitution of methionine, leucine, phenylalanine, or valine by their alpha-hydroxy analogs in the diet of rats." J Nutr 1975;105(3):372 8
• Lang, Charles H., et al. "Chronic ?-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy." American Journal of Physiology-Endocrinology and Metabolism 305.3 (2013): E416-E428.
• Mero, Antti A., et al. "Effects of alfa-hydroxy-isocaproic acid on body composition, DOMS and performance in athletes." Journal of the International Society of Sports Nutrition 7.1 (2010): 1.
• Walser, Mackenzie. "Therapeutic compositions comprising alpha-hydroxy analogs of essential amino acids and their administration to humans for promotion of protein synthesis and suppression of urea formation." U.S. Patent No. 4,100,160. 11 Jul. 1978.
• Woods M., and Goldman P. "Replacement of L-Phenylalanine and Leucine by a-Hydroxy analogues in the diets of germ-free rats." J Nutr 1979;709:738 43.

Shouldnt it be obvious that the "happy medium" must be the solution, when high protein leads to brittle bones, and low protein to frail muscle? Sure! But where is this "happy medium"?

Some of you may remember my recent Facebook post "High Protein Diet in the Firing Line. Rodent Study Says: Kidneys Are at Risk". It was based on a press release you could read on all the major science-news outlets on the Internet; a press release that will give the average reader the impression that the corresponding study by Aparicio et al. would "prove" that high protein diets will ruin your kidneys and eventually jeopardize your health (read more).

Another paper (Cao. 2014), Jose Antonio, the CEO of the ISSN and the editor of the ISSNs journal posted on Facebook yesterday, didnt get as much media attention, though.

No wonder, the message of this study is after all not in line with one of the fundamental arguments you will hear, whenever you question the allegedly necessary restriction of total protein intake to 0.8g/kg, maximally 1.2g/kg protein per kilogram body weight day in the current nutritional guidelines:

"[...S]hort-term consumption of high-protein diets does not disrupt calcium homeostasis and is not detrimental to skeletal integrity."

Thats not what you will learn at med-school and it is certainly not in line with the hysteria about protein intakes that are 2x or even 3x higher than the 0.8g protein per kilogram body weight we are supposed to consume. Apropos RDA, the subjects in the control group of the said study by Jay J Cao et al. consumed a diet that contained exactly those 0.8g/kg body weight thats supposed to be good for us. The 21 human guinea pigs in the treatment groups, on the other hand, consumed 2x and 3x more than the average dietitian would recommend and they did so for 31 days (Cao. 2014).

Figure 1: Protein intake (in g/day; left), mineral intake (in mg/day; middle)  and calculated renal acid load (in mEq; right) of 49 normal weight, healthy men (n=32) and women (n=7) who consumed normal (0.8g/day), high (1.6g/kg per day) and very high protein (2.4g/kg per day) energy restricted (40%) diets for 4 weeks (Cao 2014)

If you take a look at the PRAL values in Figure 1, you can see that math (not bio- or physiology!) tells us that this reckless practice could compromises the acid-base balance of the healthy, normal-weight subjects, whose energy restricted diets were modeled on the increasingly popular high protein weight loss diets.

Equations vs. experiments | PRAL vs. urinary calclium loss | theory vs. practive

The urinary analysis the scientists conducted does yet speak a very different language. There is, as the scientists emphasize in the discussion of the results no evidence that

Suppversity Suggested Read: "High protein diet = high protein loss" | more

"habitual consumption of dietary protein at levels above the RDA [would] significantly alter urinary calcium excretion, dietary calcium retention, or markers of bone turnover or BMD, despite increased urinary acidity. These results indicate that diets that are 2 or 3 times the RDA for protein are not detrimental to calcium homeostasis when calcium and vitamin D are consumed at recommended intake"

In that I would like to emphasis the importance of adequate calcium (min. 800mg/day) and vitamin D intakes (800-1000IU/day) and the fallacy of the word "habitual". The study at hand did not test the effects of "habitual" high protein consumption. It tested the effects of short-term (28 days) high protein consumption in a low calorie scenario, which is by definition less prone to produce adverse inflammatory and thus potentially pro-osteoporotic side effects (Mundy. 2007).

Not eating enough protein could increase bone loss, when youre dieting

In view of the fact that the evidence I am about to cite, stems from rodent model of postmenopausal bone metabolism, I deliberately used the word could in the headline of this paragraph. And still, the way in which the low protein diet  "negatively impacted bone mass and magnified the detrimental effects of vitD and/or estrogen deficiencies" (Marotte. 2013) in the pertinent study from the Buenos Aires University is particularly disturbing.
The (postmenopausal) women the scientists try to model with their ovariectomized rats (=rats whose ovaries have been removes) are after all one of the many patient groups who are advised to carefully control their protein intake to make sure that the additional acid load will not compromise their bone health even further and that in spite of the fact that there is ample evidence that the current RDA for protein is inadequate to maintain optimal health, particularly when the total energy intake is restricted and especially in populations who are susceptible to bone loss (Kerstetter. 2005; Chernoff. 2004).

Figure 2: We know for quite some time not that low protein diets decrease the absorp- tion of protein (Kerstteter. 2005). Its not certain if this is "just" a homeastatic me- chanism to stabilize the net/acid balance.

In their 2005 study, Kerstetter et al. were in fact able to show that protein intakes that are 2.6x higher than the RDA increase the effective absorption of calcium from the diet (see Figure 2).

This increase stands in contrast to the significant decrease in calcium absorption the researchers observed in the healthy young (age: 26y) women in the low protein arm (0.7g protein per kg body weight) of the study and should remind us that a reduction in protein intake is not going to stop the insidious loss of bone thats caused by the triage of low estrogen, no exercise and a diet that may be low in protein, but high in acid producing grains (Remer. 1995) and devoid of alkaline fruit and vegetables.

I could now go more into details, but I will just leave you with the notion that the "paleo diet" is, despite its high meat content, among the most kidney-, and above all bone-friendly diets we know. In fact, its fruit and vegetables content yield a net alkaline renal load, and will lead to significant improvements in urinary calcium excretion rates (Appelet. 1997; Frassetto. 2013).   

? Note: If you want more about the "Paleo connection" - let me know this (best on Facebook) and what you would be most interested in and I will address that in a future SuppVersity article.

References

• Aparicio, V. A., et al. "High-protein diets and renal status in rats." Nutrición hospitalaria: Organo oficial de la Sociedad española de nutrición parenteral y enteral 28.1 (2013): 232-237.
• Appel, Lawrence J., et al. "A clinical trial of the effects of dietary patterns on blood pressure." New England Journal of Medicine 336.16 (1997): 1117-1124. 
• Cao, Jay J., et al. "Calcium homeostasis and bone metabolic responses to high-protein diets during energy deficit in healthy young adults: a randomized controlled trial." The American journal of clinical nutrition 99.2 (2014): 400-407.
• Chernoff, Ronni. "Protein and older adults." Journal of the American College of Nutrition 23.sup6 (2004): 627S-630S. 
• Frassetto, L. A., et al. "Established dietary estimates of net acid production do not predict measured net acid excretion in patients with Type 2 diabetes on Paleolithic–Hunter–Gatherer-type diets." European journal of clinical nutrition 67.9 (2013): 899-903.
• Kerstetter, Jane E., et al. "The impact of dietary protein on calcium absorption and kinetic measures of bone turnover in women." Journal of Clinical Endocrinology & Metabolism 90.1 (2005): 26-31.
• Mundy, Gregory R. "Osteoporosis and inflammation." Nutrition reviews 65.s3 (2007): S147-S151.
• Remer, Thomas, and Friedrich Manz. "Potential renal acid load of foods and its influence on urine pH." Journal of the American Dietetic Association 95.7 (1995): 791-797.

Trp and its metabolite 5-HTP may be particularly useful for female sugar cravings and binges.

Can l-tryptophan help you lose body fat? If you look at the results of the latest study from the University for Health Sciences, Medical Informatics and Technology it would seem that the answer to this question may be "Possibly, yes, but..." Before we come to the implications I would yet like to take a closer look at said study which shows that a lack of tryptophan (Trp) during diets does not just affect the biosynthesis of serotonin, but may also be associated with increased susceptibility for mood disturbances and carbohydrate craving. Accordingly, "strategies to supplement Trp while dieting could be highly useful in treating uncontrolled weight gain or in preventing neuropsychiatric symptoms" (Strasser. 2014).
As Strasser et al. point out, both overweight and obesity go hand in hand with significant increases in low-grade inflammation. The latter is not just the reason that obesity increases the risk of cardiovascular disease, though. Recent evidence suggests that it is also associated with errors in the kynurenine (Kyn) pathway, in which tryptophan is broken down to kynurenine which in turn has been associated with increased risk of depressive symptoms, cognitive deficits in schizophrenia, Alzheimers and, as mentioned before, cardiovascular disease. Weight loss, on the other hand,

"[...] has been shown to improve or prevent many of the aforementioned conditions. Bariatric surgical intervention in patients with adiposity was found not to improve tryptophan breakdown rates and other signs of immune activation and inflammation [4], whereas caloric restriction is known to be a strong activator of protective metabolic pathways, thereby leading to lower blood pressure, improved blood lipids, and reduced inflammatory markers, including CRP [9]. Still, little is known about the effects of an extreme short-term hypocaloric diet on Trp metabolism and changes in inflammatory biomarkers" (Strasser. 2014).

The study Barbara Strasser, Ken Berger and Dietmar Fuchs conducted was thus designed to assess the effect of a 2-week caloric restriction weight loss diet on Trp breakdown, leptin, and inflammatory biomarkers in over weight adults.
The scientists randomized 27 overweight and 11 obese participants (22 men and 16 women, mean age 52.8 ± 9.1 years) from the health center Lanserhof, Innsbruck–Lans, into two diet groups:

• a very low kcal diet group (VLCD; Ø 600 kcal/ day) and 
• a low kcal diet group (LCD; Ø 1,200 kcal/day). 

Only healthy subjects with BMI [25 kg/m²] between the ages of 35 and 70 years were accepted for the study. A physician performed physical examinations on all subjects before the study. Subjects were excluded if they consume any anti-inflammatory drugs (e.g., ibuprofen or aspirin) or supplements (such as antioxidants or fish-oil capsules). None from either group was involved in regular training programs.

Figure 1: Changes in body composition pre- vs. post (Strasser. 2015).

As the measurements of body composition, which were just like the energy intake and biologic markers conducted in all subjects before and after the 2-week energy restriction intervention period, indicate, both diets lead to significant reductions in body mass - and that almost exclusively in form of body fat.

Table 1: Biologic markers before and after a 2-week very low kcal diet (VLCD) or low kcal die (LCD) in 38 overweight subjects (mean ± SD)

"Data for biologic markers are shown in Table [1]. Fasting blood glucose declined significantly (P < 0.05) in the LCD group with no significant changes in insulin sensitivity in both groups after 2 weeks of caloric restriction. Weight loss diet lowered leptin levels in both groups, although not reaching the level of significance. Inflammatory biomarkers were not significantly altered during the trial, although there was a tendency toward an increase in IL-6 and TNF-a in the LCD group" (Strasser. 2015).

In contrast to what the researchers expected, both the Trp and Kyn concentrations decreased significantly by 21 and 16 % for VLCD and by 15 and 17 % for the LCD group, respectively, with no significant difference between groups. Practically speaking, this means that the ratio of Kyn/Trp concentrations did not change significantly in both groups.
A significant reduction in Phe concentrations was only seen after VLCD. Neopterin and Tyr levels remained unchanged during the trial. Which leaves us with only one significant finding:

"Trp concentrations decreased significantly with a caloric restriction weight loss diet, and lowest Trp concentrations were observed in the group of individuals with the lowest calorie intake." (Strasser. 2015)

This reduction in Trp levels may well induce a disturbance in the biosynthesis of neurotransmitter 5-hydroxytryptamine (5-HT | Anderson. 1990), and appears to be associated with an increased susceptibility for depression (Widnet. 2002; Raison. 2009). Strasser et al. highlight:

Figure 2: The consumption of tryptophan-free amino acid supplements leads to highly significant increases in hunger ratings in healthy female subjects (Rieber. 2010).

"Because Trp is precursor in various biochemical pathways, e.g., it is hydroxylated by tryptophan-5-hydroxylase (T5H) into the intermediate product 5-hydroxy-tryptophan, which by decarboxylation is further converted to neurotransmitter 5-HT (serotonin), and because substrate saturation of T5H is only about 50 % (Dantzer. 2011), changes in plasma Trp levels may have an immediate impact on brain serotonin levels" (Strasser. 2014).

Experiments in which Trp was acutely depleted (in many studies by administering BCAAs | see red boy) support this assumption. Young et al. (2013), for example, confirmed that the acute depletion of tryptophan will lead to low serotonin and subsequently lower mood and increased aggression, although results vary somewhat between studies with similar participants.

Figure 3: Correlations between changes in tryp:LNAA ratio and appetite ratings (Gendall. 2000).

For the link to obesity, though, the correlation (r-values in Figure 3) between high Trp:LNAA (BCAAs, tyrosine, phenlylanine) and a reduction carbohydrate cravings, general hunger and binge eating is yet way more important - and that specifically for women, who appear more vulnerable than men both to the diet-induced reductions in Trp and to its consequences for brain serotonin function (Anderson. 1990).

Ah, and in case you are asking yourself why carbohydrate / sugar binges are a common consequence of low tryptophane:LNAA ratios, its important to know that increases in glucose and insulin in response to high carbohydrate meals will trigger an increase in brain tryptophan and serotonin synthesis (Benton. 2002). This is why the effects of low tryptophan or high LNAA (BCAA, tyrosine, phenylalanine) levels are more pronounced if you avoid dietary carbohydrates.
References:

• Anderson, I. M., et al. "Dieting reduces plasma tryptophan and alters brain 5-HT function in women." Psychological medicine 20.04 (1990): 785-791. 
• Benton, David. "Carbohydrate ingestion, blood glucose and mood." Neuroscience & Biobehavioral Reviews 26.3 (2002): 293-308.
• Cangiano, Carlo, et al. "Eating behavior and adherence to dietary prescriptions in obese adult subjects treated with 5-hydroxytryptophan." The American journal of clinical nutrition 56.5 (1992): 863-867.
• Cangiano, Carlos, et al. "Effects of oral 5-hydroxy-tryptophan on energy intake and macronutrient selection in non-insulin dependent diabetic patients." International journal of obesity and related metabolic disorders: journal of the International Association for the Study of Obesity 22.7 (1998): 648-654.
• Ceci, F., et al. "The effects of oral 5-hydroxytryptophan administration on feeding behavior in obese adult female subjects." Journal of neural transmission 76.2 (1989): 109-117.
• Cui, Y., T. F. Lee, and L. C. H. Wang. "Thermoregulatory responses following injection of 5-hydroxytryptamine into the septohippocampal complex in rats." Pharmacology Biochemistry and Behavior 45.4 (1993): 935-939.
• Dantzer, Robert, et al. "Inflammation-associated depression: from serotonin to kynurenine." Psychoneuroendocrinology 36.3 (2011): 426-436. 
• Fernstrom, John D. "Branched-chain amino acids and brain function." The Journal of nutrition 135.6 (2005): 1539S-1546S.
• Gendall, Kelly A., and Peter R. Joyce. "Meal-induced changes in tryptophan: LNAA ratio: effects on craving and binge eating." Eating behaviors 1.1 (2000): 53-62. 
• Le Feuvre, R. A., L. Aisenthal, and N. J. Rothwell. "Involvement of corticotrophin releasing factor (CRF) in the thermogenic and anorexic actions of serotonin (5-HT) and related compounds." Brain research 555.2 (1991): 245-250.
• Nieuwenhuizen, Arie G., et al. "Acute effects of breakfasts containing ?-lactalbumin, or gelatin with or without added tryptophan, on hunger,‘satiety’hormones and amino acid profiles." British journal of nutrition 101.12 (2009): 1859-1866.
• Raison, Charles L., et al. "CSF concentrations of brain tryptophan and kynurenines during immune stimulation with IFN-?: relationship to CNS immune responses and depression." Molecular psychiatry 15.4 (2009): 393-403.
• Rieber, N., et al. "Acute tryptophan depletion increases experimental nausea but also induces hunger in healthy female subjects." Neurogastroenterology & Motility 22.7 (2010): 752-e220.
• Strasser, Barbara, Ken Berger, and Dietmar Fuchs. "Effects of a caloric restriction weight loss diet on tryptophan metabolism and inflammatory biomarkers in overweight adults." European journal of nutrition (2014): 1-7.
• Widner, Bernhard, et al. "Neopterin production, tryptophan degradation, and mental depression—What is the link?." Brain, behavior, and immunity 16.5 (2002): 590-595.
• Young, Simon N. "The effect of raising and lowering tryptophan levels on human mood and social behaviour." Philosophical Transactions of the Royal Society B: Biological Sciences 368.1615 (2013): 20110375.