Alanylglutamine

L-alanyl-L-glutamine (Alanylglutamine) is a dipeptide consisting of the amino acids L-Glutamine and L-alanine, which can be synthesized using bioengineered bacteria.^[1][2] It is often used intravenously in clinical nutrition for tissue repair, since L-glutamine requirements are increased in rapidly dividing cells.^[3] Because L-glutamine is readily hydrolyzed to glutamic acid in aqueous solution, dipeptides such as alanylglutamine are often used instead due to their increased stability.^[3][4]

In animal models, alanylglutamine has been shown to help increase the absorption of electrolytes more than glutamine.^[5]This suggests potential for use as an endurance-enhancing agent during extended cardiovascular exercise where hydration stress is a factor, a hypothesis that has been explored in humans.^[6] Alanylglutamine is also better absorbed in the intestines than L-glutamine itself,^[4] which underlies claims of increased bioavailability.

Alanylglutamine is a more stable and better orally absorbed form of L-glutamine that is often used intravenously in clinical nutrition.

Alanylglutamine is more stable than the free amino acid L-glutamine under acidic conditions and high temperatures. This is an important property for enteral and paternal solutions, where the high temperatures that are often employed for sterilization readily break down free-form L-glutamine.^[7] Alanylglutamine also allows for better absorption of electrolytes and water,^[5] which could underlie benefits over glutamine in instances of secretory diarrhea.^[5] Alanylglutamine is highly water-soluble, (568g/L) greatly exceeding that of free-form glutamine (35g/L at 20°C).^[7]

Dipeptides including alanylglutamine are absorbed in the intestines via the proton-dependent dipeptide transporter Pept-1^[8][9] which is also expressed to a small degree in colonocytes.^[10] While exhibiting increased stability in solution, this dipeptide retains the benefits of free-form glutamine in nutritionally supporting the intestines in the rat.^[11][12]

Alanylglutamine is absorbed by the Pept-1 dipeptide transporter in the intestines and colon.

In subjects who ingested either 60 mg/kg of L-glutamine or 89 mg/kg of alanylgutamine (equivalent to 60 mg/kg glutamine) in a fasted state, the average AUC of plasma glutamine was 124% higher than that of L-glutamine over the course of 4 hours post-ingestion, suggesting that alanylgutamine is more readily absorbed than free-form L-glutamine.^[4]

In humans under fasted conditions, alanylglutamine is better absorbed than equivalent amounts of L-glutamine.

When exercising rats given oral alanylglutamine for three weeks (1.5g/kg) were compared to those given a solution containing the free amino acids Glutamine (1g/kg) and L-alanine (0.67g/kg), both groups showed equivalent increases in serum glutamine levels (59-62% relative to water).^[13]Similar observations were noted elsewhere in rats both at rest and with exercise when comparing alanylglutamine dipeptide versus the free amino acids^[14][15] and when compared to equivalent doses of glutamine alone.^[16]

Chronic ingestion studies in rats have noted that equivalent amounts of alanine and glutamine in free-form increase serum glutamine concentrations to a similar extent to alanylglutamine dipeptide.

One study in humans using oral solutions of alanylglutamine in water noted that 0.05g/kg failed to increase plasma glutamine any more than water alone, while a higher dose of 0.2g/kg increased plasma glutamine approximately 45 minutes after consumption (from approximately 700µM to 950µM; data estimated from graph) which was maintained during the estimated 15 minutes of exercise.^[6] Elsewhere a peak in serum glutamine levels has been noted under fasting conditions with 0.09g/kg alanylglutamine increasing from 475µM to 735+/-147µM after 15-45 minutes, then returning to baseline after four hours in a time course paralleling that of L-glutamine (0.6g/kg) but reaching a higher C_max value.^[4]

One study has noted that low doses of alanylglutamine (50mg/kg) failed to increase serum glutamine while higher doses (90-200mg/kg) seem to increase glutamine in serum; the one acute comparative study noted better absorption with alanylglutamine but no chronic studies exist (to compare against the rat studies).

In exercising rats, orally administered alanlylglutamine (1.5g/kg) for three weeks increased glutamine concentration in the fast-twitch gastrocnemius muscle to a larger degree than a solution of the free amino acids Glutamine (1g/kg) and L-alanine (0.67g/kg).^[13] This differential effect was not noted in the soleus or in plasma,^[13] where both alanlylglutamine and equivalent amounts of the individual amino acids increased glutamine levels to a similar extent. Other studies have also failed to note any differences in glutamine levels in soleus or gastrocnemius with alanlylglutamine vs. glutamine supplementation,^[14] suggesting that both may be effective at increasing tissue glutamine levels.

When compared to glutamine (1g/kg) alone in trained rats who swam to exhaustion, a similar dose of glutamine via the dipeptide (1.5g/kg) has been noted to increase muscular glutamine to a higher level in both soleus and gastrocnemius again with no differences in serum nor exercise performance.^[16]

Both free form L-glutamine and alanylglutamine appear to midly increase concentrations of glutamine in the muscle of active rats, with alanylglutamine showing a trend to increase these concentrations slightly more than free glutamine.

Liver glutamine concentrations did not differ between chronic treatment groups.^[16] In another study, liver glutamine levels only differed after exercise (where a drop was attenuated with the dipeptide relative to free amino acids^[14]) whereas glutamate has been noted to be higher with the dipeptide compared to a combination of L-glutamine (1g/kg) and L-alanine (0.67g/kg) at rest only, the difference fading when exercise is utilized.^[14]

The peak concentration of liver glutamine does not appear to differ between alanylglutamine and the free form amino acids when delivered orally in similar amounts. Alanylglutamine may increase glutamine levels to a greater extent than L-glutamine when prolonged exercise is introduced, however.

In rats exercised to fatigue who previously had three weeks continuous supplementation of either L-glutamine (1g/kg) or alanylglutamine (1.5g/kg), a higher plasma glutamate concentration with the dipeptide was noted,^[16] although elsewhere no difference between the dipeptide and free glutamine was observed when L-alanine (0.67g/kg) was also used in the free peptide group.^[15] Since glutamate is taken up from the blood and transformed in muscle to glutamine in the glutamine-glutamate cycle^[17] via enzymatic conversion by glutamine synthetase,^[18] increased glutamate levels with alanylglutamine have been proposed to reflect a higher intramuscular glutamine concentration; with increased tissue levels of glutamine there may be and less of a need for glutamate uptake to support intramusuclar glutamine stores.^[16]

There may be an increase in serum glutamate levels secondary to an increased accumulation of glutamine in muscle tissue upon supplementation with alanylglutamine

Infusion of alanylglutamine under fasting conditions in humans appears to reverse a general efflux of glutamine and alanine from the muscle, instead causing a minor influx. While both the kidneys and splanchnic (liver and intestinal) tissues have a role in metabolizing alanylglutamine, it seems that renal elimination is preferred with the dipeptide.^[19] This is in contrast to administration of the free amino acids L-glutamine and L-alanine, which are heavily metabolized by splachnic tissue.^[19] The preference for renal elimination of the dipeptide is thought to be due to the fact that dipeptidase hydrolysis activity (which breaks alanylglutamine into L-glutamine and L-alanine) is primarily localized to the kidneys.^[20][21]

Alanylglutamine appears to be more metabolized by the kidneys than the liver when compared to the free amino acids, which are heavily metabolized by the liver. This is thought to be due to the enzyme that hydrolyzes the dipeptide bond primarily existing in kidneys.

Three weeks supplementation of alanylglutamine (1.5g/kg), relative to glutamine itself (1g/kg) or water in rats subject to exhaustive exercise did not influence glycogen content of the gastrocnemius muscle while both amino acid groups noted an increase in liver glycogen relative to water.^[16]

Limited animal evidence on glycogen levels in skeletal muscle fail to note any differences between alanylglutamine and the free form amino acids, although liver glycogen stores are increased with supplementation of both the dipeptide and free amino acid.

Rats given either 0.1 or 0.5 g/kg alanylglutamine immediately post-exercise altered muscle cell signalling in a way that theoretically could lead to reduced muscle breakdown, while muscle synthesis pathways were relatively unaltered.^[22] Specifically, phosphorylation was reduced for both AMPK and NF-kB p65 at both doses. The latter pathway has been seen to lead to muscle wasting in mice,^[23], and the former is a major energy sensor in cells, which can signal muscle breakdown when activated^[24].

Heat shock proteins are special stress-response proteins that function as molecular chaperones, protecting proteins from toxic aggregation to preserve function during the type of metabolic and oxidative stress^{[25][26][27][28]} that is often experienced during intense exercise^[29] In rats subjected to physical training given 1.5g/kg of either alanylglutamine or the free amino acids (1g/kg Glutamine and 0.67g/kg L-alanine) orally for three weeks, supplementation of the dipeptide increased expression of the heat shock proteins (HSP72 and HSP73) more than the free amino acids.^[13] Differential effects on HSP subcellular localization were also noted with the dipeptide vs. free amino acids. Whereas the dipeptide increased cytosolic HSP levels, the individual amino acids appeared to be associated with lower cytosolic HSP levels and increased nuclear localization.^[13] In the fast twitch gastrocnemius the opposing effects occurred, with the dipeptide causing more nuclear localization than the free amino acids, while both groups improved glutathione status to a similar degree.^[13]

Heat shock proteins are special stress-response proteins that function as molecular chaperones, protecting proteins from toxic aggregation during metabolic and oxidative stress. Both alanylglutamine dipeptide and its constituent amino acids in free-form are robust activators of heat-shock proteins. Although potential differential effects on heat shock protein cellular localization have been noted with the dipeptide vs. free amino acids, this requires further investigation to be substantiated.

Rat studies where the dipeptide was administered at 1.5g/kg for three weeks along with an endurance test failed to note any performance differences when the dipeptide was compared to either an equivalent amount of L-glutamine (1g/kg)^[16] or that dose of L-glutamine paired with L-alanine (0.67g/kg).^[13][14] A decrease in ammonia has been noted in rats subject to long duration exercise compared to water control, but the dipeptide again performed equally to a mixture of L-glutamine and L-alanine^[15] with the only difference being a reduction in plasma glutamate (a serum reserve for glutamine production in muscle cells^[17]).

In rodent research there is currently no noticeable improvement in performance seen with alanylglutamine that differs from taking the free forms of the amino acids.

In athletic women playing a competitive basketball game where the provided water included either a low or high concentration of alanylglutamine (1g or 2g per 500mL) it was noted that the inclusion of the low dose of the supplement relative to water improved shooting performance by 11.1% when tested after the game (alongside improved visual reaction time). This effect was not observed with the higher dose, which may be due to the dipeptide's abiliy to enhance rehydration.^[30] Notably, self-reported fatigue appeared to be reduced with only the higher dose.^[30] In another human study, a 75% VO2 max test to fatigue in trained men did failed to note an increase in time until failure with solutions of alanylglutamine (0.05g/kg and 0.2g/kg) relative to water.^[6]

Human studies involving alanylglutamine have had mixed results; a high (but not low) dose of the dipeptide as been noted to reduce self-reported fatigue, with no effect on overall performance. Another study in trained men failed to note any effects on fatigue or otherwise.

In a study where trained athletes reported to the lab in a dehydrated state and then rehydrated (over the course of 45 minutes) with either water or one of two solutions containing alanylglutamine (0.05g/kg or 0.2g/kg) it was found that, relative to water alone, alanylglutamine failed to increase performance on a 75% VO2 max test to fatigue.^[6] Hormones involved in water regulation (vasopressin and aldosterone), serum electroytes, and overall plasma volume did not differ between groups.^[6]

Another study involving trained female basketball players where dehydration was not present at the start of the study, alanylglutamine solutions (1g and 2g per 500mL) failed to increase weight acutely (attributed to water retention) any more than water alone. The low dose of the dipeptide did result in a modest increase in shooting performance, however.^[30] Since dehydration has been shown to effect shooting performance in basketball players in several studies,^[31][32][33] and alanylglutamine can increase water absorption,^[6] it is possible that enhanced rehydration may account for the increased shooting performance in the alanylglutamine group.^[30]

Although some studies have noted indirect evidence that is suggestive of a possible hydration-enhancing effect with alanylglutamine, current studies have failed to find any hydration increases over water alone. More evidence is required on this topic.

A study with rats fed either alanylglutamine (1.5g/kg) or the combination of L-glutamine (1g/kg) with L-alanine (0.67g/kg) for three weeks alongside exercise noted that both groups performed equally in reducing creatine kinase and TNF-α levels in serum, with alanylglutamine reducing PGE₂ to a slightly greater extent.^[14]

In response to a 75% VO2 max test to fatigue, a rehydration protocol of alanylglutamine (0.05g/kg or 0.2g/kg) failed to differ from water in altering C-reactive protein or interleukin-6 concentrations either immediately after the trial or when tested a day after.^[6]

The subtle immunological/antiinflammatory effects of alanylglutamine do not appear to be significant in vivo.

The alterations in testosterone concentrations seen during a test (75% VO2 max cycle to fatigue) did not differ in groups given 0.05g/kg or 0.2g/kg alanylglutamine relative to water.^[6]

Acute and 24 hour increases in cortisol and its stimulating hormone (ACTH) resulting from a 75% VO2 max cycle to fatigue do not appear to be influenced by 0.05g/kg or 0.2g/kg alanylglutamine relative to water.^[6]

The acute spike in growth hormone concentrations resulting from a 75% VO2 max cycle to fatigue does not appear to be influenced by 0.05g/kg or 0.2g/kg alanylglutamine relative to water.^[6]

Oral ingestion of high doses of alanylglutamine (24g) over ten days in HIV-infected subjects appeared to improve intestinal absorption relative to glycine placebo in subjects who reported diarrhea in the last two weeks.^[34]