Douglas W. Wilmore1
In humans, glutamine has traditionally been thought of as a nonessential amino acid probably because of its abundance within the body's various amino acid pools. Almost all human cells contain the enzyme glutamine synthetase, which can, under appropriate conditions, produce glutamine. However, it recently has been postulated that during catabolism, the tissue demands for glutamine exceed the endogenous production of this amino acid, resulting in a state of glutamine deficiency (Lacey and Wilmore, 1990). It is thought that major illness such as injury, burns (Gore and Jahoor, 1994; Parry-Billings et al., 1990), infection (Shabert and Wilmore, 1996), and/or other disease states associated with a significant inflammatory response initiate this increased glutamine requirement. Exogenous glutamine may be helpful during these conditions to restore an adequate supply of this important nutrient.
Glutamine provides a ready source of energy through its conversion to citric acid cycle intermediates and the generation of ATP. It serves as a major substrate involved in the intraorgan transport of nitrogen and is highly efficient
because it contains two nitrogen moieties. It is important in the generation of purines and pyrimidines necessary for DNA biosynthesis (Martin, 1985) and serves as a precursor in some tissues for metabolically generated bases (Welbourne, 1995) (that is, endogenously synthesized purines and pyrimidines; those not from dietary sources) and glycoproteins. Glutamine is also a regulator (or co-regulator) of cell proliferation (Kandil et al., 1995), the generation of heat-shock proteins2 (Ehrenfried et al., 1995), and the expression of certain cell surface receptors (Spittler et al., 1995). It is not known if some of these specific activities involve direct or indirect genetic regulatory mechanisms.
Glutamine may also be rate limiting for the synthesis of glutathione, one of the most important intracellular antioxidants. Studies show that in the presence of cysteine, the provision of glutamine will enhance glutathione stores and reduce oxidant damage (Hong et al., 1992).
This chapter reviews the pertinent clinical studies that suggest an association between glutamine and the immune defenses of the body.
Although almost all tissues contain the enzymes for glutamine synthesis, most glutamine is synthesized in skeletal muscle and brain, and these are the major organs that export glutamine. Liver, however, has the capacity to both consume and produce glutamine, depending on a variety of controlling factors. Because of the large mass of skeletal muscle, most glutamine comes from this tissue and is exported via the bloodstream to visceral organs (Souba et al., 1985). Under normal conditions, glutamine is maintained in high concentrations within the skeletal muscle free amino acid pool. Excluding taurine, glutamine represents about 60 percent of the free amino acids in skeletal muscle and maintains an intracellular concentration of about 20 mmol/Liter intracellular water. With normal plasma concentrations ranging from 600 to 650 µmol/Liter, this large concentration gradient (about 30:1) favors rapid transfer of a large quantity of glutamine from this intracellular store into the bloodstream (Muhlbacher et al., 1984). Because skeletal muscle intracellular glutamine concentrations fall with starvation and the stress of illness, muscle biopsy followed by analysis of intracellular glutamine concentration has been used as a marker of nutritional status in depleted patients and may even be predictive of a fatal outcome (Roth et al., 1982). Other studies have demonstrated that the skeletal muscle intracellular concentration of glutamine is related to the rate of protein synthesis in skeletal muscle (Jepson et al., 1988; MacLennan et al., 1987). Finally, the exogenous administration (supplementation) of glutamine (by addition to total parenteral nutrition [TPN]) attenuates the usual fall in
skeletal muscle intracellular concentrations following stress (Hammarqvist et al., 1989) and improves net synthesis of skeletal muscle protein.
During catabolic states, elaboration (synthesis and secretion) of a variety of stress hormones, including glucocorticoids, occurs; this latter steroid has been shown to induce the expression of glutamine synthetase in skeletal muscle (Hickson et al., 1996) and thus initiate de novo glutamine synthesis and enhanced skeletal muscle glutamine production and release into the bloodstream. In normal humans in the postabsorptive state, approximately 40 percent of plasma glutamine is thought to be derived from other amino acids, and an additional 45 percent originates from its direct release from tissue protein (Perriello et al., 1995). The remainder of glutamine comes from the conversion of glucose and glutamate to glutamine. Studies have not yet been performed in stressed individuals to determine the relative contribution of various disease states to the accelerated rate of glutamine production during stress, but data from animal models (Muhlbacher et al., 1984) suggest that all pathways are accelerated to enhance glutamine production during catabolic states.
The glutamine produced by skeletal muscle is transported via the bloodstream and taken up by various visceral organs (Souba et al., 1985). The distribution of blood glutamine is concentration dependent but also relies on membrane transporters that are distributed throughout the various visceral tissues. These transporters are regulated by a variety of metabolic factors that modify the rate of glutamine transported into the cell (Fischer et al., 1995). During stress states, organs compete for glutamine, and a hierarchy of priorities is established among tissues to determine glutamine uptake and subsequent utilization. Organs or tissues such as liver, gastrointestinal mucosa, kidney, and immunological tissue are the major consumers of glutamine. As blood concentrations fall, cell transport along with blood flow to specific organs become the rate-limiting factors that determine cell uptake and subsequent utilization. These regulating events and the intraorgan competition for glutamine have major impact on cell protection, proliferation, and function.
Glutamine is consumed by the kidney to aid acid-base homeostasis; as the amide nitrogen is cleaved, it joins with a H+ (hydrogen ion/proton) to excrete NH4+ (ammonium) in the urine; simultaneously, a bicarbonate group (HCO3-) is released into the bloodstream (Pitts et al., 1972). Tissues like the enterocytes (Windmueller, 1982), colonocytes (Ardawi and Newsholme, 1985), lymphocytes, and macrophages (Parry-Billings et al., 1990) utilize glutamine as a primary fuel, but glutamine also serves as a molecule that supports the proliferative response (Kandil et al., 1995). Finally, the liver utilizes this amino acid in a host of metabolic functions, depending on the requirements of the body. Glutamine plays an active role in gluconeogenesis, and recently it has been demonstrated (Perriello et al., 1995) that in postabsorptive individuals, glutamine, not alanine or lactate, is the predominant precursor for the transfer of new carbon to the glucose pool. Because glutamine is such an efficient molecule for the shuttling of nitrogen throughout the body, it serves as a major nitrogen
donor for the hepatic synthesis of amino acids and/or hepatic proteins. Efficient pathways also exist for excess glutamine nitrogen to be converted to urea, which is then excreted from the body. Finally, glutamine is extracted by the liver from the bloodstream and used for the synthesis of glutathione (Welbourne et al., 1993).
Glutamine and Immune Function
In the 1950s, it was realized that glutamine was an essential nutrient in vitro necessary for the growth of some bacteria and almost all cultured cells. Eagle and coworkers (1956) reported that both mouse fibroblasts and HeLa cells died in culture unless the media was supplemented with glutamine. When this amino acid was added to the culture media, cell proliferation occurred in a dose-responsive manner with increasing concentrations of glutamine. Ardawi and Newsholme (1983) studied lymphocytes harvested from rat mesenteric lymph nodes to determine the influence of glutamine on cell function. Glutamine addition caused a fourfold increase in [3H] thymidine incorporation, a marker of cell proliferation. This effect was not observed when glutamine was substituted by other amino acids or by ammonia.
Glutamine uptake in these and other experiments far exceeded the requirements for oxidative metabolism of the cells studied. In proliferative cells, glutamine yields such compounds as ammonia, glutamate, asparate, and lactate, a process termed glutaminolysis (McKeehan, 1982; Newsholme et al., 1988a, b). This pathway makes available essential precursors—ammonia, glutamine, and aspartate—for purine and pyrimidine biosynthesis. Glutamine also provides the nitrogen for the formation of glucosamine, guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD), all important substances necessary for normal cell function.
A variety of in vitro experiments have demonstrated the importance of glutamine in maintaining or improving immunological function. Parry-Billings et al. (1990) demonstrated that glutamine was necessary for the proliferative response of lymphocytes. In addition, a dose-response relationship was found between in vitro glutamine concentration and the rate of phagocytosis achieved by mouse macrophages.
Others have isolated neutrophils from burn patients and studied the ability of these cells to kill Staphylococcus aureus in the presence or absence of glutamine. Glutamine enhanced bactericidal function in normal neutrophils and generally restored this function to normal levels in neutrophils taken from burn patients (Ogle et al., 1994). Others have demonstrated that glutamine plays a supportive role in the generation of lymphokine-activated killer cells (LAK cells), which are also important for effective host defense (Juretic et al., 1994). Finally, in vitro studies demonstrated an important role for glutamine in the upregulation and/or maintenance of specific cell surface antigens of human
monocytes, which may be important in the host response to infection (Roth et al., 1982).
Glutamine has also been administered to patient populations to evaluate the effect of this amino acid on clinical outcome, specifically the impact of supplementing this amino acid on infection. Ziegler et al. (1992) studied 45 adult patients receiving allogeneic bone marrow transplant for hematologic malignancies. After a week of intensive chemotherapy and total body radiation, parenteral nutrition was initiated the day after bone marrow transplantation. Patients were randomized to receive glutamine-supplemented (0.57 g/kg/d) or standard (glutamine-free) isonitrogenous, isocaloric, intravenous, nutritional formulas for the next 3 to 4 weeks, when oral intake was resumed.
MacBurney and coworkers (1994) found that hospital stays were shorter in the patients receiving glutamine supplementation (29 vs. 36 days, p = 0.017), and this was primarily due to the reduction in clinical infection (three compared with nine in the control group, p = 0.041). The incidence of bacterial contamination was also significantly reduced. This resulted in a cost savings to the hospital of about $10,700 per patient, plus the revenues gained from the increased bed availability.
Ziegler and coworkers (1994) also evaluated circulating white blood cells in the glutamine-treated and control bone marrow transplant patients. Lymphocytes were isolated and subjected to flow cytometry using monoclonal antibodies. The glutamine-treated subjects demonstrated a significant increase in total lymphocytes, CD3, CD4, and CD8 cells when compared with the patients receiving standard therapy. These data are consistent with a more rapid recovery in lymphocytes of the patients receiving glutamine.
Two other trials have been performed in similar populations. One demonstrated a decreased length of stay in the treatment group, but retrospective analysis did not identify a relationship between glutamine administration and reduced infection rate (Schloerb and Amare, 1993). The other trial was performed in Europe using a glutamine dipeptide (Van Zaanen et al., 1994). Patient selection and treatment protocols varied from the initial reported studies. These findings showed no difference between groups, although the glutamine administered was only about two-thirds the amount given in the other two studies.
A final study has evaluated the effect of glutamine-supplemented parenteral nutrition solutions on the immunological effects following an elective operation (O'Riordain et al., 1994). Patients were randomized to receive postoperative standard or glutamine-supplemented TPN. After 5 days of infusion, T-cell DNA synthesis was increased in the glutamine-supplemented group but did not change in the control group. Other outcome variable were not evaluated in this study.
These data, when taken together, suggest that the in vitro proliferative response mediated by glutamine can be translated to whole body experiments. Studies in bone marrow transplant patients and postoperative patients support
the concept that glutamine is a specific growth factor for lymphocytes. Whether these effects can be universally translated to all critically ill individuals is not known; to date the populations studied are highly specific and results are dependent on the dose and duration of glutamine administered.
Glutamine and the Gastrointestinal Tract
The gut is another important target organ for glutamine metabolism, and the maintenance of normal function of this organ may be invaluable to host defense against intestinal flora and/or their by-products. Animal studies demonstrate that a variety of stresses—starvation, infection, injury—result in the increased movement of bacteria from the bowel lumen to local and regional lymph nodes (Deitch et al., 1989). This process, termed bacterial translocation, is well characterized in animals, particularly rodents. It is not known if this process occurs in normal bowel in humans sustaining a similar stress. However, a second process also occurs in the intestinal tract of stressed animals, and this change has clearly been demonstrated in humans. This process involves changes in the permeability of the small bowel to small intraluminal molecules that enter the body during various diseases. Channels exist between the enterocytes, and the entrances to these pericellular pathways are highly regulated and energy dependent. During hypoperfusion, hypoxia, malnutrition, or injury, these pathways become more permeable to luminal molecules that would otherwise be excluded from the body. This enhanced intestinal permeability is well documented in patients with burns (Deitch, 1990), infection (Ziegler et al., 1988), and inflammatory bowel disease (Hollander et al., 1986) and raises the possibility that endotoxin, or other bacterial factors that are found in the intestinal lumen, may gain entrance to the body during these disease states. Thus, strategies that will maintain bowel mucosal vitality and barrier function also may contribute to the enhanced immune defenses of patients.
Glutamine is able to enhance mucosal growth and improve gut barrier function during certain situations. Windmueller (1982) demonstrated that glutamine provided a major portion of the energy required by the enterocytes, and Ardawi and Newsholme (1985) showed similar effects in colonocytes. Rhoads and colleagues demonstrated that glutamine activates a variety of early response genes, essential to the proliferative response of the enterocyte (Kandil et al., 1995). In addition, glutamine enhances the effect of growth factors on enterocyte DNA synthesis (Jacobs et al., 1988) and stimulates ornithine decarboxylase activity in a dose- and time-dependent manner. This latter enzyme regulates the rate-limiting step in polyamine biosynthesis, which is critical for intestinal cell generation and repair.
When glutamine was added to parenteral nutrition solutions and administered to animals as their sole nutrient source, the villus atrophy that is ordinarily associated with intravenous nutrition was greatly attenuated (O'Dwyer et al., 1989). Similar support of villus growth has been observed by
van der Hulst and coworkers (1993) in humans. Patients requiring preoperative intravenous feedings were randomized into two groups, one receiving glutamine-supplemented and the other receiving standard (glutamine-free) parenteral nutrition. Intestinal biopsies were taken before and at the end of the parenteral infusions, and tests of intestinal permeability also were performed. After 2 weeks of parenteral glutamine, villus height was unaltered in the glutamine-supplemented group, and it decreased significantly in the group receiving standard parenteral feedings. In addition, the patients receiving glutamine had no changes in intestinal permeability, whereas permeability increased in the group receiving glutamine-free nutrition.
Other studies in humans have demonstrated improvement in bowel function with glutamine administration. For example, when oral glutamine was administered to a small group of patients with Crohn's disease, body weight increased and bowel permeability significantly improved (Zoli et al., 1995). Glutamine administration to premature infants enhanced their ability to take full enteral feeds, compared with a nonsupplemented control group (Lacey et al., 1996). Finally, glutamine administered to patients in an intensive care unit enhanced absorption from the gastrointestinal tract when compared to that of patients receiving glutamine-free intravenous solutions (Tremel et al., 1994).
Taken together, these studies demonstrate that glutamine enhances normal structure and function of the gastrointestinal tract in humans. Additional trials are under way to evaluate the effect of administering this amino acid to populations at risk for infectious diarrhea and those with known bowel disorders.
Author's Discussion and Conclusions
Glutamine serves many important functions in the body that may be beneficial and support the host's immunologic defenses. For example, glutamine supports skeletal muscle protein synthesis and also enhances bicarbonate production, which may neutralize the acid load that is generated by moderate to severe exercise or catabolism (Welbourne, 1995). Glutamine also supports glutathione biosynthesis, and this antioxidant attenuates tissue damage associated with free-radical production. Over the past several years, glutamine has been studied in several groups of critically ill patients, for whom a specific effect may not be able to be identified, but the multiple effects of this amino acid may benefit the individual patient. For example, Griffiths et al. (1997) randomly divided 84 intensive care patients who were admitted to their unit into two groups: one group received glutamine supplementation (25 g) and the second received standard glutamine-free feedings. The groups were well matched in terms of their general characteristics and received similar quantities of calories and protein. Mortality was significantly greater at 6 months in the patients receiving standard therapy when compared with the glutamine group (67% vs. 43%). Although the pattern of early deaths was similar, length of stay
and increased late mortality were observed in the group receiving standard therapy. It is not known how glutamine supplementation prevented these later deaths. Effects could occur via enhanced immunologic function, improved repair of bowel mucosa associated with augmented absorptive and barrier function, enhanced skeletal muscle function, augmented acid-base homeostasis, or improved antioxidant activity. Whatever the mechanism, glutamine appears to serve an essential function in selected patient groups.
Because of the favorable cost-benefit ratio of this amino acid, other populations are currently being evaluated in an effort to improve outcome and quality of life with glutamine supplementation. Over the next several years, data should be forthcoming to direct the use of this therapy to specific groups of highly responsive individuals.
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GAIL BUTTERFIELD: Should weight lifters take glutamine supplements to increase their muscle mass?
DOUGLAS WILMORE: If they really want to increase their muscle mass, anabolic steroids and growth hormone are much better. They are also much more expensive, and you can walk down to the corner store and get [glutamine]. So, glutamine is a very inexpensive and really safe agent.
GAIL BUTTERFIELD: But are there data to suggest that in a normal healthy person, increase in glutamine intake is going to have an anabolic effect?
DOUGLAS WILMORE: There is only anecdotal data in normals. There are some randomized trials going on in AIDS patients following weight loss, for example, where there is an early increase in body weight, which in fact may be due to water because glutamine helps transport water across the gastrointestinal tract.
Realize that with all the anabolic agents, protein synthesis is associated with water retention and cell swelling. So, you always have to see water going into a person or water uptake increasing, and this at least fits that criterion so far. However, I don't think the data are available to show that a normal individual will increase their muscle mass.
SIMIN NIKBIN MEYDANI: How much of the effect on the immune system is due to glutamine itself and how much is due to [glutathione]?
DOUGLAS WILMORE: I don't know. It is quite clear that if you deplete those immune cells of glutathione that you will really cause cellular dysfunction.
The other thing I don't know is the role that glutamine plays as an acid-base buffer system within cells. Clearly this may be a major role that glutamine plays in the gastrointestinal tract because tight junctions are quite sensitive to acidosis of the enterocyte. So, I think we just don't know the answers to these issues, and really in vitro studies would have to provide this information.
LEONARD KAPCALA: Is it known if there are any central effects of supplemental glutamine that would be influencing the glutamic acid and MDA [methyldopanine] receptor activation in brain?
DOUGLAS WILMORE: There are central effects from glutamine, and these are known effects. There is an old body of data in the European psychiatric literature where glutamine was used both in animals and in people to reduce addiction, and it has been used in alcoholics to reduce alcohol ingestion. It has been used in rats to reduce drug ingestion and alcohol ingestion, so that there may be central effects that can be perceived somehow in that role.
Nobody has moved further with that. We were quite concerned with the administration of glutamine in the premature infants because of their immature brains and actually did very careful dose-response studies to be sure that we didn't see toxicities in those infants.
So, yes, there are known central effects. The brain makes glutamine and glutamic acid and exports them. Brain and lung and muscle are the big exporters.
JEFFERY ZACHWIEJA: This is a follow-up on Gail [Butterfield]'s question. When I was at Washington University, we infused normal healthy males with either an amino acid solution without glutamine or an amino acid solution supplemented with glutamine and looked at the effects on muscle protein synthesis by carbon-13 level and leucine incorporation in skeletal muscle and found no difference in those young healthy males.
So, at least in terms of what we did, it appears that unless the muscle glutamine pool has become rapidly depleted by some condition, the extra glutamine won't have an effect on the muscle.
DOUGLAS WILMORE: I think that that is the point that always has to be reemphasized, particularly if you propose a hypothesis that this is a conditional essential amino acid. The point is that you have to have the condition, and the condition clearly isn't [present in] well-nourished people, because it is not an essential amino acid in well-nourished people, and in fact, even in the military troops that take a large amount of protein and amino acids in their diet, it may in fact not be conditional under those circumstances. Of course, we really need the skeletal muscle biopsy data and concentration data and things of that sort to prove that fact.
However, there may be some functional outcome data that enhance performance, and there are a number of Olympic teams that are now using glutamine during training. So, we can see how that works out.
NED BERN: Is the mechanism by which glutamine stimulates growth hormone release known?
DOUGLAS WILMORE: Not specifically, no. I suspect it is quite similar to the same mechanism by which leucine stimulates hormone release.
NED BERN: Have you any idea how long that stimulation lasts?
DOUGLAS WILMORE: It is very short. A glutamine concentration curve with oral ingestion, if you ingest enough, looks very much like a glucose tolerance curve. So, you will get your peak stimulation at a couple of hours, something like that.
RANJIT CHANDRA: The question is, do you have any idea about the threshold for both deficiency and where the effects of glutamine occur?
DOUGLAS WILMORE: The question is thresholds, and I guess that implies or asks about dose-response curves and at what levels can we achieve those kinds of responses? In the hospital, we are moderately aggressive with glutamine administration, and it is not unusual to give one-third of the amino acid load as glutamine. That comes from the fact that that is what your skeletal muscle is producing. If in fact, we mimic what skeletal muscle does under stressed states, it puts out one-third of glutamine, one-third of alanine, and the rest a variety of amino acids.
If we were to feed an adult patient under a stressed state 1 1/2 g of protein per kilogram body weight, 1/2 g of that would be glutamate. So, a 70 kg person would receive about 30 g of glutamate.
Now with babies, we give 20 percent of their amino acid load as glutamine, and their total amino acid load is about 2 1/2 to 3 g/kg.
BRUCE BISTRIAN: I was interested when you talked about what was essential. In most of the conditions you mentioned, potential essentiality is also characterized by increased acid production. Has anyone done anything to see what the effect of just providing an equivalent amount of base would be?
DOUGLAS WILMORE: When we first started the studies with uptake across the bowel, the control was actually to give bicarbonate. That is really the proper control under those conditions.
You don't achieve those same effects. There are much greater effects of glutamine than [would be achieved by] just giving an equivalent amount of bicarbonate, but you know, those were stable physiologic preparations that we used, and the question has come up in the audience from time to time about people exercising and diminishing splanchnic blood flow, and that clearly is a
phenomenon that I think Loren B. Rowell looked at in the 1960s or early 1970s. He ran students from the University of Washington so hard, he could make their liver enzymes go up and had simultaneous splanchnic blood flow measurements, which demonstrated splanativic ischemia.
So, that is a phenomenon that occurs, and acidosis of the gastrointestinal epithelium is probably a real thing. It appears to be important in clinical practice because the tonometry data has suggested that the mucosa becomes acidotic. One of the effects of glutamine is to neutralize this intracellular acidosis.
BRUCE BISTRIAN: I was thinking, also, about the effect of the base on Philmidge. When used in acidosis conditions like renal failure, it has profound effects on muscle metabolism where the effect on the muscles may be its base effect.
DOUGLAS WILMORE: I don't think we know the answer to that. There are some Swedish data looking at that in postoperative patients that could possibly address that with intracellular pH probes, but I don't think anyone has done that study.