![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Pediatrics, Division of Gastroenterology, University of North Carolina, Chapel Hill, NC
 |
INTRODUCTION |
|---|
 TOP INTRODUCTION LITERATURE CITED |
|---|
Before opening the general discussion, I have been asked to review our laboratory’s studies on the potential role of glutamine in the therapy of diarrheal disease.
Diarrheal disease kills more infants and children worldwide than any
other condition except respiratory infections. That is why I became
interested in glutamine. Dr. Lobley raised the question whether
glutamine helps in diarrheal disease, and the answer is "yes" and
"no." We have studied two disease models, piglet
rotavirus infection, and piglet and calf
cryptosporidiosis. My first investigations of glutamine had
to do with super oral rehydration. Oral rehydration saves lives, but
only 90% of infants can be rehydrated orally. If an infant resides in
a developing country, there may be insufficient resources to provide
intravenous fluids, which is one of the reasons why 
2.5 million
children die annually of diarrheal disease.
Thus the concept was developed that adding amino acids to glucose in an
oral rehydration solution
(ORS)2
should maximize sodium absorption. "Super ORS" may be more
efficacious than standard ORS in human cholera, as was shown for
L-alanine + glucose ORS in a double-blind WHO-funded
trial in patients with cholera and enterotoxigenic Escherichia
coli diarrhea (Patra et al. 1989
). A problem is
that cooked rice plus ORS, or rice-based ORS, may be equally
effective and cheaper (Santosham et al. 1990
). We have
also found that not all amino acids are "created equal." Most of
the studies investigated glycine, an amino acid that is not
cotransported with Na+ in the piglet intestine.
We previously compared a number of amino acids in terms of their
effects on net sodium flux in the piglet jejunum. When we studied
glutamine, phenylalanine, alanine, proline, leucine and asparagine, the
only ones that stimulated a substantial amount of sodium absorption
were glutamine, phenylalanine and alanine. Phenylalanine was dismissed
as an ORS additive because it could be toxic to the neonatal brain and
children with phenylketonuria, and we focused on glutamine and alanine.
Glutamine and glucose were equally effective, and they had
additive effects on sodium absorption across the jejunum and
ileum. This observation of additivity applied to the damaged intestine
during piglet rotavirus infection. Even in severely infected tissues,
there was an additive response to glucose plus glutamine, or glucose
plus alanine. In another model, cryptosporidiosis of piglets and
calves, glutamine was additive to glucose in enhancing
Na+ absorption in the intestine (Argenzio et al. 1994
).
Additivity in stimulating absorption was attributable to the presence
of separate brush border amino acid- and glucose-coupled sodium
cotransporters, and also because glutamine stimulated electroneutral
sodium chloride absorption. In human infants, glutamine plus glucose
oral rehydration was investigated in studies funded by the WHO in India
and Brazil. These studies investigated infants with mild diarrhea.
Children often have diarrhea caused by a variety of pathogens, viruses
in particular. Ribeiro et al. (1994)
studied infants
with mild dehydration. Their body weights, on admission compared with
discharge, differed by only 2–3%. Additionally, the "Super ORS"
given was hyperosmolar (>380 mOsm). Recently a number of
investigations have shown that hypoosmolar oral rehydration is better
than isoosmolar ORS. Thus the studies of hypertonic "Super ORS" in
my opinion were inconclusive and should be repeated.
The other focus of our studies was whether glutamine would promote
intestinal repair in piglet rotavirus infection. To summarize our
studies, glutamine-supplemented ORS did not enhance repair
(Rhoads et al. 1996
). We have just completed another
study in calf cryptosporidiosis in which supplementation with glutamine
did not enhance the rate of recovery, although bovine serum concentrate
was efficacious (Rhoads et al., in press). All of our studies looked at
the duration and severity of diarrhea in well-nourished animals.
One may ask whether glutamine would be more beneficial in poorly
nourished subjects.
An important effect of glutamine in intestinal cells is that it
stimulates mitogen-activated protein kinases (MAPK) by a
metabolism-dependent but Raf-independent mechanism
(Rhoads et al. 2000
). This activation of MAPK is
inhibited by cyclic AMP. Glutamine additionally affects a
stress-activated protein kinase pathway, the c-Jun kinase
pathway, in two different ways. If the cells are glutamine-starved,
there is a progressive activation of Jun nuclear kinase (JNK) that
depends on the duration of starvation, resulting in apoptosis, as has
been shown by Papaconstantinou et al. (1998)
. But if the
cells are starved for a short period of time and then glutamine is
applied, JNK and the extracellular signal-regulated kinases (ERK)-1
and -2 are activated, and the cells undergo mitogenesis. A related
finding was that epidermal growth factor, when given to the cultured
intestinal cell line IEC-6, stimulates thymidine incorporation twofold
more when glutamine is present compared with when glutamine is absent.
Recently, we looked for evidence that glutamine levels might be
relevant to pediatric intestinal disease. We investigated glutamine and
arginine levels in newborn premature infants weighing <1800 g
(Becker et al. 2000
). It was a follow-up to two
other studies (Dallas et al. 1998
, Lacey et al. 1996
) that showed better outcomes in premature children
receiving glutamine supplementation. In a third related study by
Zamora et al. (1997)
, infants with necrotizing
enterocolitis (NEC) had lower serum levels of arginine, but not
glutamine at the time of onset of NEC. In our study, we immediately
stored serum from every infant that was premature and had each sample
analyzed by HPLC by Dr. Guoyao Wu at Texas A&M University. We
found that NEC occurred on mean day of life 14. This is the time at
which the infants were just beginning to reach full enteral feeding.
Before this, they were receiving primarily total parenteral nutrition
(TPN). There were 16 infants who developed NEC and 35 age-matched
normal premature infants. At 10 d before the infants developed NEC,
serum levels of specific amino acids, especially glutamine and
arginine, began to fall below the levels of those who did not develop
NEC (Becker et al. 2000
).
NEC was demonstrated on the abdominal flat plate X-ray, which
showed intramural air (air within the wall of bowel) or pneumatosis
coli. Also, physical findings indicated that the children were sick.
The control (no NEC) infants had a progressive increase in arginine and
glutamine concentrations as they grew older. Median values of serum
glutamine were 37–57% lower in NEC infants on d 7, 14 and 21,
compared with controls (P < 0.05). On d 7 and 14,
median values of arginine, alanine, lysine, ornithine and threonine
were also decreased 36–67% (P < 0.05) in NEC infants
compared with controls (Becker et al. 2000
).
The results were not fully explainable on the basis of intake. Arginine
(Arg) was especially interesting because Arg intake was actually
twofold higher in the children that developed NEC because of the higher
concentration of Arg in TPN than in formula. Because the children that
developed NEC were receiving less enteral feeding, they took in much
less glutamine compared with control infants. All of the children had a
low intake of glutamine because there was no glutamine in the TPN
(Becker et al. 2000
). Our study raises the question
whether low levels of serum glutamine or arginine could predispose to
NEC.
I will open the session at this point for questions. Dr. Lobley, when you discussed movement of the glutamine nitrogen—what is the significance of more glutamine nitrogen going to methionine than to phenylalanine?
Dr. Lobley.
It’s a preferred substrate as the oxo-acid for methionine, rather than that for phenylalanine. In the liver, we showed a very clear differential among glutamine and phenylalanine and methionine enrichment, with the glutamate being much higher. For the plasma proteins, where we had a lower enrichment level simply because they have a much lower fractional synthesis rate, we found that the glutamate was about the same as the methionine. So it’s a bit marginal as to whether it is coming from glutamate, and it was almost 100% transferred. I would often obtain equilibrium within the 6 h, which I find a bit suspicious. I hypothesize that it was transferred directly from glutamine, and did not go through glutamate.
Dr. Wernerman.
We have learned today that there is still much to learn about the physiology of glutamine. We have seen studies in which glutamine has and has not produced favorable effects. It strikes me that there are very few studies in the literature in which glutamine has a negative effect. Now this means that if these positive results come by chance, there should be as many negative results by chance. On the other hand, if we do not understand physiology, perhaps experimental design (e.g., insufficient numbers of subjects) in a lot of studies showing no results is the major problem.
Dr. John Alverdy.
Just as a follow-up to that comment, when we look at infection in humans, we think of it as occurring when the number or the virulence of a bacteria is balanced against the susceptibility or the resistance of the host. For example, opportunistic infections, infections that we create, are the worst infections in the hospital, certainly not the diarrheal infections resulting from contaminated food. It really is a unique set of circumstances in which the patients have lost lean body mass, or perhaps intracellular glutamine, and "we" have recolonized these patients with the most virulent hospital organisms. Those are the circumstances in which one might see the greatest benefit of glutamine, which is where you see some depletion in either intracellular or extracellular stores, and you see unique organisms colonizing the host. A "spritz," an infusion that evokes inflammation of lipopolysaccharides (LPS) and has a very short half-life in the blood, is not the same as a patient whose lungs are filled with gram-negative bacteria and who is on inadequate intravenous support. We went down this road 10 years ago where antiendotoxin antibodies were proposed as a magic bullet to treat these "end-stage" patients.
I believe the negative studies may belie the true effect that we are looking for, which is who are the most appropriate patients to target. They may be patients in whom there is unique susceptibility, and a pathogen that is not the meningitis pathogen, or the pathogens of pigs that produce diarrheal disease. The most feared pathogens may be the hospital pathogens.
Dr. Lobley.
Dr. Wernerman said in the laws of chance you would expect some results to go negative, as well as positive. I do not totally agree with that. If your experiments are good enough, then you really should get a null effect, or you should get an effect, although that effect may be negative.
I agree with the comment of Dr. Alverdy, but to some extent I think it depends on where ones research is directed. I’m trying to understand whether there is a role for amino acids in challenge situations. To do that, I need a challenge situation that I can control. That’s why we have gone for continuous infusion of LPS, rather than a single injection, because it gives us longer to investigate. It means that we are looking at a stable system. I’m not in a clinical situation. The other thing is I was forced by a home office inspector to start with well-fed sheep. We can actually study these animals under good conditions. It may well be that one of the experiments we will do in the future is to investigate animals under stressed conditions, when they have a low lean body mass. We may then find differential responses.
Dr. Alverdy.
There has been a lot of interest in the United States about why some college dormitory students develop fatal meningitis, and what is unique about these students when everybody in the dormitory is exposed. Work is showing that not only are they exposed, but they actually may be immunologically weakened, because they have had long days of sleep deprivation and a lot of alcohol use.
And so again, under the paradigm of infection, where there must be unique host susceptibility and then there must be an unusual exposure to a pathogen that is both virulent and high in number, it seems to me that giving glutamine may help. When there is glutamine sufficiency in the animal, introducing an organism that may or may not kill the animal and determining whether glutamine supplementation will change the course may not yield interpretable results. In a WHO trial, where you do not know what the organism is that is causing the diarrhea, looking at the effects of glutamine may not be the best study design, but it is certainly worth trying.
Participant.
I want to make a comment about study design with regard to enteral glutamine administration. Adibi and Matthews, and others showed many years ago that a significant proportion of enteral protein digestion products are taken up in the form of di- and tripeptides. These substrates are transported by PEPT1, which is a molecule that transports the small peptides along a hydrogen-dependent gradient. In fasting humans and certain catabolic animal models, L-amino acid transport is impaired, whereas small peptide transport mediated by PEPT1 is maintained. In our studies of enteral glutamine supplementation, perhaps we should do comparative studies with a variety of glutamine peptides such as those Dr. Fürst and others have synthesized, that are now available for use. Additionally, we need to realize that enteral glutamine does not necessarily always mean L-glutamine administration. Perhaps peptide forms of enteral glutamine may be more efficacious.
I want to ask Dr. Häussinger to comment with regard to the cell swelling concept in cell lines. Apparently, there are no in vivo data in humans. Glutamate, I think, also can be transported by a sodium-dependent pathway, is this correct?
Dr. Häussinger.
It’s a very restricted sodium-dependent pathway in the liver, localized at the cannalicular membrane. So it’s not clinically relevant.
Participant.
Other amino acids can be transferred with sodium. Is this a swelling process, a sodium-dependent process or is there something unique about the effect of glutamine?
Dr. Häussinger.
The hydration state of a cell in general is determined by the osmotic state that exists. And this osmotic state can be modified by all transport systems in the plasma membrane. This also means when a potassium channel is opened, potassium goes out of the cell and it shrinks. This is a mechanism of how oxygen radicals act by simply opening a potassium channel.
There are varied mechanisms by which cellular hydration is altered. There are many amino acids that are transported in a sodium-dependent way, and most of them induce cell swelling. As I hypothesized years ago, among the amino acids, glutamine is one of the "best swellers!"
Moreover, in contrast to transport systems like system A which has a lot of substrates, the N system only has one major substrate and this is glutamine. So it can be additive, and this was the reason why I emphasized sodium in my talk. I think this is one of the major reasons that glutamine is of interest.
Dr. Bode.
If the activity of all of the known transport systems is measured in isolated hepatocytes, system N is by far the most active. There is a lot of flux through this carrier. I think one of the reasons that glutamine is such a good sweller is that it is transported very rapidly into the hepatocyte.
I’m not certain about the other tissues. In human cells, the ATB0 carrier is incredibly active. So this too may play a role. It may not just be system N.
Participant.
With regard to the protein or the mRNA expression of system N in the membrane, is it regulated by the degree of swelling or shrinkage?
Dr. Häussinger.
It is regulated by this mechanism as shown by Drs. Bode and Kilberg.
Dr. Bode.
Actually, when swelling is induced artificially and plasma membrane vesicles are isolated, we found no evidence of additional carriers in the membrane. We believe that the swelling-induced activation in the carrier has to do more with post-translational modifications, or other driving forces within the cell that modulate its activity. In the kidney, there is evidence of withdrawal and reinsertion of different transporters. I believe they are ionic and not amino acid transporters. Thus there is precedent for osmotic regulation of protein trafficking. Dr. Häussinger pointed out that with the bile cannalicular carrier, this occurs as well. There is no evidence for additional system N insertion in response to cell swelling in the plasma membrane.
Dr. Häussinger.
If you induce swelling in a cell, it swells to a certain point, and then you activate a regulatory potassium efflux, which tends to hyperpolarize the cell membrane; this energizes other transporters.
Participant.
We investigated system N activation by glutamine. It’s an autoregulatory loop that actually stimulates itself. If the extracellular potassium concentration is increased, the activation of system N is inhibited. This suggests a potentially major role for many of the ionic transporters and their interplay with amino acid transporters in regulating cell swelling induced glutamine flux. The picture is quite complicated.
Dr. Rhoads.
The activation of MAP kinases does not always do the same thing to the cell. There are MAP kinases in the hepatocytes that are associated with changes in protein synthesis, transporters and proliferation. However, other amino acids, such as alanine, have major intracellular osmotic but not mitogenic effects in enterocytes. It may be that osmotic swelling of the intestinal cells activates MAP kinases without proliferation.
Dr. Häussinger.
In the liver, alanine is a poor sweller.
|
FOOTNOTES |
|---|
2 Abbreviations used: ERK, extracellular
signal-regulated kinase; JNK, Jun nuclear kinase; LPS,
lipopolysaccharides; MAPK, mitogen-activated protein kinase; NEC,
necrotizing enterocolitis; ORS, oral rehydration solution; TPN, total
parenteral nutrition.
|
LITERATURE CITED |
|---|
|
TOPINTRODUCTIONLITERATURE CITED |
|---|
1. Argenzio R. A., Rhoads J. M., Armstrong M. & Gomez G. (1994) Glutamine stimulates prostaglandin-sensitive Na+-H+ exchange in experimental porcine cryptosporidiosis. Gastroenterology 106:1418-1428.[Medline]
2. Becker R. M., Wu G., Galanko J. A., Chen W., Maynor A. R., Bose C. L. & Rhoads J. M. (2000) Reduced serum amino acid concentrations in infants with necrotizing enterocolitis. J. Pediatr. 137:785-793.[Medline]
3. Dallas M. J., Bowling D., Roig J. C., Auestad N. & Neu J. (1998) Enteral glutamine supplementation for very-low-birth-weight infants decreases hospital costs. J. Parenter. Enteral Nutr. 22:352-356.[Abstract]
4. Lacey J. M., Crouch J. B., Benfell K., Ringer S. A., Wilmore C. K., Maguire D. & Wilmore D. W. (1996) The effects of glutamine-supplemented parenteral nutrition in premature infants. J. Parenter. Enteral Nutr. 20:74-80.[Abstract]
5. Papaconstantinou H. T., Hwang K. O., Rajaraman S., Hellmich M. R., Townsend C. M. & Ko T. C. (1998) Glutamine deprivation induces apoptosis in intestinal cells. Surgery 124:152-160.[Medline]
6. Patra F. C., Sack D. A., Islam A. & Mazumder R. N. (1989) Oral rehydration formula containing alanine and glucose for treatment of diarrhoea: a controlled trial. Br. Med. J. 298:1353-1356.
7. Rhoads J. M., Argenzio R., Chen W., Graves L. M., Licato L. L., Blikslager A. T., Smith J. & Gatzy J. T. (2000) Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable, Raf-independent mechanisms. Gastroenterology 118:90-100.[Medline]
8. Rhoads J. M., Gomez G. G., Chen W., Goforth R., Argenzio R. A. & Neylan M. J. (1996) Can a "super" oral rehydration solution ("super ORS") stimulate intestinal repair in acute viral enteritis?. J. Diarrhoeal Dis. Res. 14:175-181.[Medline]
9. Ribeiro H., Jr, Ribeiro T., Mattos A., Palmeira C., Fernandez D., Sant’Ana I., Rodrigues I., Bendicho T. & Fontaine O. (1994) Treatment of acute diarrhea with oral rehydration solutions containing glutamine. J. Am. Coll. Nutr. 13:251-255.[Abstract]
10. Santosham M., Fayad I. M., Hashem M., Goepp J. G., Refat M. & Sack R. B. (1990) A comparison of rice-based oral rehydration solution and "early refeeding" for the treatment of acute diarrhea in infants. J. Pediatr. 116:868-875.[Medline]
11. Zamora S. A., Amin H. J., McMillan D. D., Kubes P., Fick G. H., Butzner J. D., Parsons H. G. & Scott R. B. (1997) Plasma L-arginine concentrations in premature infants with necrotizing enterocolitis. J. Pediatr. 131:226-232.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
