Preface

Metabolic Role of Glutamine
Review of the Current Literature on Glutamine in TPN

Glutamine-Supplemented TPN Supports
Protein Metabolism

Glutamine-Supplemented TPN Supports
Intestinal Function

Glutamine-Supplemented TPN has
Favourable Effects on the Immune System

Summary

Preface

Numerous in vivo and in vitro experimental studies have, especially over the past ten years, substantially increased our knowledge of glutamine metabolism. Human studies have demonstrated reduced glutamine concentrations in skeletal muscle after minor surgical procedures as well as in critically ill septic patients and in acute hemorrhagic pancreatitis. While some evidence suggests that the reduction in muscular glutamine concentration is primarily due to increased glutamine delivery, decreased glutamine synthesis and/or uptake and increased glutamine consumption may also be involved to variable extents in the development of glutamine deficiency. The physiochemical properties of glutamine had foiled attempts at parenteral glutamine supplementation prior to the recent advent of stable synthetic glutamine dipeptides.

L-Glutamine or glutamine dipeptide has since been used in many different patients to study its effects on nitrogen balance, muscular glutamine content, intestinal permeability, extracellular fluid, muscular protein synthesis, and length of hospitalization. Some authors observed a favorable impact of glutamine on specific study variables, while others found no treatment effect.

New medicinal products should nowadays be documented not only for safety but also for clinical efficacy in the format of studies designed and analyzed in line with current, stringent standards. These considerations along with aspects of health economics argue against the use of glutamine dipeptides in the current state of knowledge. More studies are needed to define specific patient populations, clinical conditions, and/or degrees of injuries that stand to benefit from glutamine supplementation as a nutrient or drug*).

The purpose of this booklet is not only to provide an overview of individual glutamine studies but also to foster an interest in current glutamine research, so doctors can draw their own conclusions regarding the utility of L-glutamine or glutamine dipeptides in clinical practice.

Prof. Dr. med. J. Eckart
(Augsburg, Germany)

*) see also: Hornsby-Lewis (p. 26), Powell-Tuck (p. 53 ff.),
Schloerb (p. 41 ff.), Ziegler (p. 37 ff.)

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Metabolic Role of Glutamine

- Glutamine is the most abundant intracellular and extracellular free amino acid

- Glutamine is a nonessential proteinogenic amino acid

- Glutamine is a nitrogen and carbon carrier

- Glutamine modulates the cellular hydration state

- Glutamine is a substrate for gluconeogenesis

- Glutamine is an intestinal fuel

- Glutamine is a substrate for RNA and DNA precursor synthesis

Changes in Glutamine Metabolism Following Trauma and During Sepsis

Open Questions:
a) Is glutamine indeed a conditionally essential amino acid?
b) If so, what would be the required daily dose?
c) What manifestations of glutamine deficiency may occur?

Schematic representation of glutamine flow between organs in trauma and sepsis (in the rat model).

Gln release from muscle (and lung) tissue and to some extent also from liver and gut; Gln consumption in the cells of the immune system, gut, kidneys, and wound area.

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Review of the Current Literature on:

Glutamine in TPN (Major Clinical Studies)

Below follows a discussion of major clinical trials of glutamine-supplemented TPN.

Glutamine supplementation:

a) Reduces glutamine losses in muscle

b) Prevents muscle loss

c) Reduces postoperative catabolism

Effects of Glutamine (Ala-Gln)* Supplementation on Amino Acid Metabolism

- Medical ICU patients receiving TPN for at least 9 days

- There were no statistically significant differences in the above variables between
the two treatment groups (Gln-free standard TPN vs. TPN + 13 g of Gln)

* L-Alanyl-L-Glutamine dipeptide

Effects of Parenteral Glutamine (Ala-Gln) Supplementation on Protein Metabolism

- Patients with acute necrotizing pancreatitis first receiving standard TPN and then glutamine-supplemented TPN.

TPN for at least 9 days

Glutamine reference values:

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Effects of Parenteral Glutamine (Gly-Gln)* Supplementation on Nitrogen Balance, Acute Phase Proteins and Interleukin-6

- Patients who underwent stomach or bowel resection and received postoperative (total) parenteral nutrition for 5 days.

- Also no statistically significant differences could be demonstrated for the following variables:

* L-Glycyl-L-glutamine dipeptide

Reference: D. Ysebaert et al., Clinical Nutrition 12 (1993) 12, Sp. Suppl. 2

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Effects of Parenteral Glutamine (Gly-Gln) Supplementation on Nitrogen Balance

- 18-80-year-old patients undergoing colorectal resection for cancer
- TPN for at least 5 days

Nitrogen excretion was determined daily.

Reference: M. G. O'Riordain et al., Ann. Surg. 220 (1994) 212-221

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Effects on Nitrogen Balance

Total daily urinary nitrogen excretion

Control TPN: vs. Glutamine-supplemented TPN:

- No between-group differences were found.
- The authors expect no effect of glutamine supplementation on nitrogen balance in minor catabolism.

Reference: M. G. O'Riordain et al., Ann. Surg. 220 (1994) 212-221

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Effects of Glutamine (Ala-Gln) Supplementation on Protein Metabolism

- Patients undergoing elective colon/rectal resection
- Postoperative TPN for about 5 days

- This publication showed that Ala-Gln dipeptide supplementation had a favourable effect on nitrogen balance and muscle glutamine concentration.

Reference: P. Stehle et al., Lancet (1989) 231-233

Effects of Glutamine-Supplemented TPN on Protein Metabolism

- Patients who underwent elective cholecystectomy
- TPN for 3 days

- The mean plasma Gln concentration was unchanged from day 0 to day 3 in the control group but fell significantly in the Gln group.

- Both treatment groups showed a significant decrease in muscle glutamine concentration, which was more pronounced in the control group than in the Gln group.

- The cumulative (day 1 - 3) nitrogen loss was significantly different in the two treatment groups, but the nitrogen loss on the individual study days was not.

Reference: F. Hammarqvist et al., Ann. Surg. 4 (1989) 455-461

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Effects of Glutamine Supplementation (TPN, Ala-Gln) on Protein Metabolism

- Patients who underwent elective cholecystectomy
- TPN for 3 days

- The mean plasma Gln concentration was unchanged from day 0 to day 3 in the control group but fell in the Gln group (NS).

- The muscle glutamine concentration decreased significantly in the control group but was essentially unchanged in the Gln group.

- The cumulative (days 1 - 3) nitrogen loss was significantly different in the two treatment groups, but the nitrogen loss on the individual study days was not.

Reference: F. Hammarqvist et al., Ann. Surg. 5 (1990) 637-644

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Effects of Glutamine (TPN, Gly-Gln) Supplementation on Protein Metabolism

- Patients undergoing elective stomach resection or gastrectomy
- Postoperative TPN for at least 6 days

- There were no statistically significant differences in the above variables between the two treatment groups (Gln-free standard TPN vs. TPN + 13 g of Gln/day for a 70-kg individual)

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Effect of parenteral L-Glutamine on Muscle in the Very Severely III

- ICU patients (APACHE II score > 10, range 11-34)
- TPN for 6 days (median)
- age: 22-89 years

- No influence on the muscle gln level; in both groups 4 patients with increasing muscle-gln-levels, no statistical significant difference.

Control TPN: Gln-suppl TPN:

- "Between biopsies no consistent pattern of change was seen with or without exogenous Gln. It also proved difficult in these very sick patients to correct a low plasma
Gln with L-Gln-TPN¨"
- TPN supplementation with 25 g/24 h, L-glutamine appears inadequate in the acute
periode to counteract the muscle and plasma biochemical changes seen in
these patients.

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- Consumption of glutamine as a fuel is increased following trauma and during sepsis.
- Plasma and muscle glutamine concentrations are reduced following trauma and
during sepsis.
- Insufficient glutamine supply to the gut produces muscosal atrophy, facilitating bacterial and/or endotoxin translocation.

Glutamine supplementation preserves the barrier function of the intestine, thus reducing the occurrence of bacterial or endotoxin translocation.

Intestinal Glutamine Metabolism without Stress (Metabolism)

Gln provides the carbon skeleton (C) for subsequent metabolic processes and is also a supplier of nitrogen (N).

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Effects of Glutamine on the Rat Intestinal Mucosa

Effects of Glutamine Supplementation (TPN, Ala-Gln) on the Intestinal Mucosa

- ICU patients receiving TPN for 9 days.

- Although the day 9 D-xylose values indicating intestinal absorptive capacity were lower in the control group than in the Gln group, baseline (day 0) values would have been required for a meaningful evaluation of therapeutic effect of glutamine supplementation.

Reference: H. Tremel et al., Gastroenterology 107 (1994) 1595-1601

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Effects of Glutamine Supplementation (Gln*, HPN) on Gut Mucosa and Intestinal Absorption

- 7 patients receiving supplementary home parenteral nutrition (HPN) for at least one year because of insufficient intestinal absorptive capacity were used to study the safety and effects of glutamine-supplemented HPN on intestinal absorption.

Results:

- Glutamine supplementation adversely affected hepatic metabolism ("3 of 7 patients showed liver enzyme elevations").

- Glutamine supplementation had no measurable effect on plasma glutamine concentration.

- Under the conditions selected for the study, glutamine supplementation failed to improve residual gut absorptive capacity (determined by the D-xylose test).

* Gln as L-glutamine solution

Reference: L. Hornsby-Lewis et al., JPEN 18 (1994) 268-273

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Effects of Glutamine Supplementation (Gly-Gln) on the Intestinal Mucosa

- Patients receiving TPN for 10 - 14 days.
- No between-group comparison but merely within-group pre-post comparison.

- Under the conditions selected for the study, glutamine supplementation had no favourable effect on gut-associated lymphoid tissue.

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Effects of Glutamine Supplementation on the Intestinal Mucosa

Intestinal permeability in healthy volunteers (control, n = 12) and patients (Gln-TPN, n = 10; glutamine-free standard TPN (STPN), n = 8). Lactulose/ mannitol ratio before and after 2 weeks' TPN.

- The lactulose/mannitol ratio was not normalized, neither in the Gln-supplemented TPN group nor in the standard TPN group.

Reference: R.R.W.J. van der Hulst et al., Lancet 341 (1993) 1363-1365

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The prevalence of Bacterial Translocation (BT) from the Gut
Findings of Sedman et al.¹⁾ (evalutation of 267 patients)

Reference: P. C. Sedman¹⁾ et al., Gastroenterology 107 (1994) 643-649

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- Lymphocytes and macrophages use glutamine as substrate for RNA and DNA precursor synthesis.

- Immune system activation increases glutamine consumption.

- Plasma glutamine levels fall after trauma and during stress.

- Decreased plasma glutamine levels produce lymphocyte and macrophage glutamine deficiency and therefore a reduction in lymphocyte and macrophage activity.

Glutamine supplementation normalizes lymphocyte and macrophage activity.

Lymphocyte and Macrophage Glutamine Metabolism

Reference: E.A. Newsholme et al., Q. J. Exp. Physiol. 70 (1985) 473-489

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Plasma Glutamine Concentration in Various Conditions

- The plasma Gln concentration is virtually unchanged after surgical procedures but significantly reduced in burn patients and increased in sepsis.

1) M. Parry - Billings et al., Arch. Surg. 127 (1992) 1237-1240
2) M. Parry - Billings et al., Lancet 336 (1990) 523-525
3) M. Planas et al., JPEN 17 (1993) 299-300
4) P. Stehle et al., The Lancet (1989) 231-233
5) F. Hammarquist et al., Ann. Surg. (1989) 455-461
6) F. Hammarquist et al., Ann. Surg (1990) 637-649

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Effects on Immune Cells

Lymphocytes and Macrophages

Conclusions

- Glutamine and glucose metabolism in lymphocytes and macrophages is almost exclusively anaerobic.
- the preferred fuel for lymphocytes is glutamine.
- the preferred fuel for macrophages is glucose.

¹⁾ M. S. M. Ardawi et al., Biochem. J. 212 (1983) 835-842
²⁾ P. Newsholme et al., Biochem. J. 239 (1986) 121-125
³⁾ P. Newsholme et al., Biochem. J. 242 (1987) 631-636

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"Glutamine-Supplemented Total Parenteral Nutrition Enhances
T-Lymphocyte Response in Surgical Patients Undergoing Colorectal Resection."

A. Study Design:

- 18- to 80-year-old patients undergoing colorectal resection for cancer
- TPN for at least 5 days

Determination of Study Variables:

- pre-op (before surgery)
- on day 1 (after surgery before TPN)
- on day 6 (after surgery immediately after stopping TPN)

Reference: M. G. O'Riordain et al., Ann. Surg. 220 (1994) 212-221 (cf. pp. 13 and 14)

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B. Effect on Immune Function

Day 1 (before TPN) and day 6 (immediately after stopping TPN) T-cell DNA synthesis measured in terms of thymidine uptake.
o control, patients receiving glutamine-free standard TPN; • glutamine-supplemented TPN. Values are reported in % of individual pre-op baseline readings.
On day 1 there was no significant between-group difference, but on day 6 DNA synthesis was significantly greater in the glutamine-supplemented group than in the glutamine-free group (* p < 0.05, Tukey-Duckworth two-sample test).

Reference: M. G. O'Riordain et al., Ann. Surg. 220 (1994) 212 - 221

B. Effect on Immune Function

- The favourable effects of glutamine supplementation on cytokines
(IL-2 and IL-6) and tumor necrosis factor described in animal studies could not be confirmed in this human study.

Reference: M. G. O'Riordain et al., Ann. Surg. 220 (1994) 212-221

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Hypothesis 4:

Glutamine-Supplemented TPN
Improves Clinical Outcome

A) Effect of Glutamine (Gln*) Supplementation on Outcome in Cancer Patients/BMT Recipients

- Allogeneic bone marrow transplant recipients.
- Start of TPN on the first post-BMT day (i. e., day 1)

TPN Regimen:

- Glutamine supplementation in a 70-kg patient: 40 g/day.

Reference: T. R. Ziegler et al., Ann. Int. Med. 116 (1992) 821-828

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- The 7-day (days 18 - 25) cumulative nitrogen balance was balanced in both groups.

Reference: T. R. Ziegler et al., Ann. Int. Med. 116 (1992) 821-828

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Results with significant between-group differences
(mean ± SEM, p < 0.05)

Reference: T. R. Ziegler¹⁾ et al., Ann. Int. Med. 116 (1992) 821-828

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Comments on Study A

- The authors failed to justify why they calculated the nitrogen balance for only half the patients (50 %) and only on days 4 - 11 and 18 - 25.

- The publication failed to report the data of the second nitrogen balance
measurement period.

- 'Clinical infection was defined as follows':
"Clinical infection was defined by the presence of positive blood cultures or by signs and
symptoms compatible with localized infection (with or without positive microbial cultures of
the affected site) that prompted the initiation or alteration of antibiotic administration and
adjunct care.

- Although there were differences in the number of clinical infections
(for definition see above), the typical infection markers (total no. of days with antibiotics,
no. of antibiotic doses, no. of patients receiving amphotericin B, time [days] to mean neutrophil count = 0.5 3 109/l, time [days] to recontamination) showed no
between-group differences.

- The Gln dose was comparatively high: 0,57 g Gln/kg/day.

Reference: T. R. Ziegler et al., Ann. Int. Med. 116 (1992) 821-828

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Effect of Glutamine-Supplemented TPN on Outcome in Cancer Patients/BMT Recipients

- 29 bone marrow transplant recipients (15 autologous, 14 allogeneic)
- Protocol similar to Study A, but without measurement of nitrogen balance

Comparison of Results of Studies A and B

A) T. R. Ziegler et al., Ann. Int. Med. 116 (1992) 821-828
B) P. R. Schloerb et al., JPEN 17 (1993) 407-413

Comments on Study B

- Although the protocols of Studies A and B were similar, the infection rate data reported by Ziegler et al. (Study A) could not be confirmed by Schloerb et al. (Study B).

- As in Study A, all documented patients should have been included in the analysis to allow evaluation of the length of hospitalization.

- There were three deaths in the Gln group versus one in the standard TPN group (NS).

Reference: P. R. Schloerb et al., JPEN 17 (1993) 407-413

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Effects of Glutamine-Supplementation (TPN, Ala-Gln) on Chemotherapy Toxicities in Patients with Haematologic Malignancies

- Patients on intensive chemotherapy
- (Total) parenteral nutrition plus oral food intake (documented separately)

- No between-group differences were found for any of the haematologic and clinical variables under study, including days with fever (> 38.5 °C), microbial infection, and antibiotic therapy.

- Although the mean duration of hospitalization was 13 days shorter in the control group than in the Gln group, this difference was not statistically significant (NS).

- The authors concluded: "Supplementation of glutamine dipeptide was safe but had no significant positive clinical effect".

Reference: H.C.T. van Zaanen et al., Cancer 74 (1994) 2879-2884

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Survival of patients who received glutamine-supplemented parenteral nutrition at 6 months following ICU admission

- ICU patients (APACHE II score > 10, range 11-34)
- TPN for 6 days (median)
- age: 22-89 years

R.D.Griffiths, et al., Abstract presented at BAPEN 1995
R.D.Griffiths, et al., Yearbook of intensive care and emergency medicine (1997) 715-723, Springer
R.D. Griffiths, et al., Nutrition 13 (1997) 295 - 302
see also page 19 ff. Palmer et al.

Outcome at 6 months

- The stay in ICU and post ICU was double to five fold the total TPN time. Total TPN time
was 6 days (median); the longest duration was 91 days.

- There is no information concerning the nutrition (enteral/oral) received by patients
following the conclusion of TPN, which lasted an average of 7 days. Study measurements were first made 170 days later at day 180.

- Survival was similar for the first 20 days. Nutritional intervention seems to do little
to affect early mortality in ICU

R.D.Griffiths, et al., Abstract presented at BAPEN 1995
R.D.Griffiths, et al., Yearbook of intensive care and emergency medicine (1997) 715-723, Springer
R.D. Griffiths, et al., Nutrition 13 (1997) 295 - 302
see also page 19 ff. Palmer et al.

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Mortality

1. In the Gln-group 15 patients died from MOF; three deaths not due to MOF:
   2x bronchopneumonia and 1x sudden death with intracerebral haemorrhage.
2. In the control-group 22 patients died from MOF in ICU.
3. In the control-group 3 patients died post ICU discharge in hospital: 1x bronchopneumonia, 1x congestive cardiac failure, 1x pneumonia.
4. The deaths in the control-group post-hospital discharge were:
   1x massive GI-haemorrhage, 1x chronic obstructive pulmonary disease and pneumonia and 1x widespread metastases.
   There is no proof for a correlation between the early nutritional intervention and the causes of the late deaths.
   

R.D.Griffiths, et al., Abstract presented at BAPEN 1995
R.D.Griffiths, et al., Yearbook of intensive care and emergency medicine (1997) 715-723, Springer
R.D. Griffiths, et al., Nutrition 13 (1997) 295 - 302
see also page 19 ff. Palmer et al.

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TPN with Glutamine Dipeptide (Ala-Gln) after Major Abdominal Surgery

- Patients undergoing elective abdominal surgery.
- continous TPN for 5 days.

- no information available regarding post TPN nutrition/treatment

References: B. J. Morlion et al., Annals of Surgery 229 (1998) 302 - 308

- the lymphocyte count curve runs nearly in parallel in both groups, on a different level.

- the post operative recovery was normal in all patients.

References: B. J. Morlion et al., Annals of Surgery 229 (1998) 302 - 308

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Results

- plasma free concentrations of glutamine, glutamate and alanine did not differ between the groups.

- "It might be asked whether a mean stay of 22 days in the control group could be regarded as usual practice in these patients, particular as no major complication occured. Furthermore, the surgeon decided the time of discharge, criteria for which were not clearly defined, and factors that determined discharge included nursing considerations" (S. D. Heys and F. Ashkanani, British Journal of Surgery 86 [1999] 289 - 290).

References: B. J. Morlion et al., Annals of Surgery 229 (1998) 302 - 308

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Oral and parenteral Glutamine* in Bone Marrow Transplantation (BMT)

- Patients with BMT (43 hematologic, 23 solid malignancies)
- Enteral - if necessary - parenteral nutrition

Outcome variables after marrow transplantation:
hematologic malignancies-survivors

Outcome variables after autologous marrow transplantation:
solid tumor malignancies-survivors

- the length of time patients required enteral Nutrition and TPN, time after BMT, and total hospital days were similar in both groups.

- both groups were also similar for incidence of positive blood cultures, sepsis, mucositis, diarrhoea and graft-vs.-host disease.

- patients with hematopoetic malignancies who received oral glutamine were less
apt to need TPN (p = 0.03).

- in the subgroup of patients with hematologic malignancies a trend (n. s.) indicates possible improvement of long-term survival in the gln-group after 3 months.

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Effect of Glutamine (Gln*) Supplementation on outcome
(length of hospital stay or mortality).

- patients with indication for TPN
- minimum Nitrogen (N-)intake 11 g/day
- median TPN duration 8 days

- In the subgroup of surgical patients gln-supplementation was associated with a borderline sig. reduction in length of hospital stay.

- Over all patients no difference between the groups was detected. In discrepancy
to Griffiths (1997) the six months survival was identical for both groups.

- Until much larger clinical trials in special subgroups of patients (e. g. surgical, haematological malignancy) will be achieved "Glutamine supplementation cannot be recommended for routine use in parenteral feeding"¨

- "The benefit from glutamine supplementation of parenteral feeds as used in this
trial has not been proved".

Reference: J. Powell-Tuck, et al., Gut, 45 (1999) 82 - 88

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Summary

Hypothesis 1: Glutamine-Supplemented TPN Supports Protein Metabolism

- Studies carried out to date have produced conflicting evidence so that Hypothesis 1 could not be confirmed.

Hypothesis 2: Glutamine-Supplemented TPN Supports Intestinal Function

- Human evidence generated to date regarding:

1. trophic effects,
2. effects on absorptive capacity,
3. effects on intestinal permeability,
4. effects on translocation, and,
5. effects on the lymphatic system,

still needs to be validated.

- This has been studied inadequately so far.

Hypothesis 4: Glutamine-Supplemented TPN Improves Clinical Outcome

- Two studies using almost identical designs in an attempt to document clinical benefit
from glutamine-supplemented TPN show database inconsistencies (cf. pp. 33 - 38). Moreover, studies along these lines have so far been conducted exclusively in BMT patients receiving very high dose glutamine supplementation.

The study of van Zaanen (cf. p. 39) concluded with: "Supplementation of glutamine dipeptide was safe but had no significant positive clinical effect".

From the current literature reviewed in this booklet we conclude that the role of glutamine supplementation in TPN is still a fancy rather than a fact.

There are still important open questions to be answered:

- who/which patient will indeed require exogenous glutamine?
- which glutamine-dosage might be effective and how does glutamine work?
- does it work as a nutrient/substrate and/or a drug?
- when to start with a defined glutamine application and how long?

The current state of medical knowledge suggests that the most promising approach to clinical nutrition, as a function of indication, is to use both parenteral and enteral nutrition whenever possible, even in situations where the amount of enterally administered substrate may be quite small. Being the more physiological source of nutrition, enteral feeding should be the preferred route wherever possible so as to make use of its favourable effects on the integrity of overall intestinal function.

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