Glutamine
Metabolism
After Small Intestinal
Resection
in Humans
Dominique Darmaun, Bernard Messing, Bernard Just, Monique Rongier, and Jehan-Fransois
Desjeux
Glutamine and leucine kinetics were measured using stable isotopes in five enterectomized patients (residual small bowel, 80 * 25 cm [mean f SE]) who were in a near normal nutritional status at distance from surgery. While parameters of leucine metabolism were normal, rates of whole body glutamine utilization were reduced by 20% in the patients. The data suggest that the small intestine plavs a prominent role in glutamine utilization in vivo in humans.
A
LTHOUGH THE prominent role of the small intestine in glutamine utilization has been demonstrated in animals,‘-4 it remains to be established in humans. If glutamine is indeed a major fuel and a nitrogen donor for the human small intestine, extensive resection of this organ may alter whole body glutamine metabolism in vivo; therefore, the present study assessed glutamine kinetics using stable isotope methodology. METHODS
Informed consent was obtained from five patients and 10 control subjects. Extensive bowel resection had been performed because of abdominal trauma (patient A), intestinal infarction (patients B and C), benign mesenteric fibroma (D), and radiation-induced enteritis (E). Residual small bowel length was estimated from x-rays obtained at least 6 months after resection. Three patients (A, B, and C) received parenteral nutrition (PN) in the postoperative period for 3, 4, and 60 months, respectively. All patients were weaned from PN for more than 3 months before inclusion in the study. Patients were free of inflammation, sepsis (C-reactive protein, .4 f .l mg/dL), or cancer. Although intestinal adaptation had probably occurred (patients were weaned from PN) the patients had short bowel syndrome, ie, moderate diarrhea and malabsorption; only 78% of ingested energy and nitrogen was absorbed (Table 1). Patients were instructed to maintain their usual levels of activity and dietary intake at home and to collect their stools for the 3-day period preceding the test. Absorbed dietary intake (Table 1) was calculated by subtracting measured nitrogen and caloric fecal losses from the intake, as estimated using a 3-day dietary history. Fat mass was estimated from four skinfold thickness measurements with a Harpenden calliper. Controls were 10 healthy adults studied after an overnight fast under a self-selected diet. The night before the study patients were admitted to the Nutrition Unit where they ate dinner at 7:00 PM and then remained fasting until completion of the test at 11:OOAM on the next day. The next morning, primed continuous infusions of sterile, pyrogen-free solutions of L-[l-*3C]leucine (90% ‘jC) and L-[2-“N]glutamine (97% 15N;CEA, Gif-sur-Yvette, France) providing 6 and 2 kmol kg-’ he’, respectively, were administered intravenously in the postabsorptive state as described.” Amino acid levels and stable isotope enrichments were determined in plasma by gas chromatography mass spectromet$ on a
From INSERMu-290, H&pita1St. Lazare, Paris, France. Supported in part by a Grant No. 8860121 from the Minist&e de la Recherche et de la Technologie France. Address reprint requests to Dominique Darmaun, MD, INSERM u-290 H6pital St. Lazare, 107Rue Fbg-St-Denis, 75010 Paris, France. Copyright 0 1991 by W.B. Saunders Company 0026-0495l9ll4001-0009$03.00/0
42
Nermag RlOlOT instrument (Nermag, Argenteuil, France) with a precision of approximately l%, as defined by the coefficient of variation (CV) = 100 x SD/mean of three measurements of same sample. Amino acid appearance rates (Ra) were calculated using equation 1: Ra = i [Ei/Ep - 11,
(I)
where i and Ra are the tracer infusion rate and the amino acid appearance rate, respectively (pmol kg-’ . h-l), and Ei and Ep are the tracer enrichments in the infusate and plasma at steady-state (mole % excess), respectively. Isotopic steady-state was defined by a CV < 10% in tracer enrichment over the last 2 hours of infusion (n = 5 to 7 arterialized plasma samples). Because leucine is an essential amino acid, leucine Ra (Ra,,) is entirely derived from protein breakdown in the postabsorptive state. In contrast, the nonessential amino acid glutamine has two inflow components to its flux? release from protein breakdown (BGln)and glutamine de novo synthesis (DGln);the former is estimated as follows: I&,, = k x RaLcu,
(2)
where k is the assumed ratio of glutamine to leucine content of body protein (8.0 and 13.9 g amino acid per 100 g protein, respectively); therefore, D,,, = Ra,,, - B,,,.
(3)
RESULTS
As expected from inclusion criteria, the patients were in a fair nutritional status (Table 1); besides, when corrected for fecal losses, their nutritional intake was close to the controls’ intake. Parameters of leucine metabolism, ie, plasma leucine level ([Leu]), leucine appearance rate, and leucine metabolic clearance rate (MCR,, = Ra,,/[Leu]) were similar in patients and controls (Table 2). While plasma glutamine level was normal, both glutamine turnover and metabolic clearance rate (MCR,,, = Ra,,,/[Gln]) were significantly depressed in the patients; the reduction was entirely accounted for by a smaller rate of glutamine de novo synthesis (Table 2). DISCUSSION
Based on infusion of stable isotope-labeled glutamine, this study demonstrates that whole body glutamine utilization is reduced in enterectomized patients. This reduction in glutamine turnover is unlikely to result from malnutrition since (1) the patients’ nutritional status was as close to normal as possible (Table 1); and (2) leucine appearance rate, an index of whole body protein turnover, was unaltered (Table 2). Similarly, neither surgical stress
Metabolism, Vol 40, No 1 (January), 1991: pp 42-44
GLUTAMINE
METABOLISM
43
AFTER ENTERECTOMY
Table 1. Clinical and Nutritional Characteristics
of the Patients
Nutrient Intake* Sexi
Residual Small
Delay Post
Age
Bowel
Surgery
WI
lcm)
(mol
A
Ml26
120
15
61
54
B
F/58
15
42
45
33
kcal in
Weight
g protein in
abs
% IBW
Fat Mass
Alb
Prealb
Hb
(% BWI
(g/dL)
lmg/dL)
(g/dL)
abs
kg
1.5
1.1
60
92
14
3.4
23
12.7
1.8
1.3
51
94
30
3.3
33
13.3
-
Patients
C
Ml49
30
108
54
38
2.1
1.7
54
81
10
4.0
25
12.2
D
Ml30
80
13
55
42
2.5
2.1
61
95
16
4.3
29
13.9
E
Ml34
150
148
56
43
1.8
1.4
60
95
nd
4.1
45
14.0
39
80
65
54
42
1.9
1.5
57
91
18
3.8
31
13.2
25 + SE 6 Controls (n = 10; 8M, 2F)
27
3
0.3
0.2
2
2
4
0.2
4
0.4
Mean
-
6
Mean
28
38
1.5
66
97
&SE
7
2
0.4
2
2
Abbreviations:
IBW. ideal body weight;/
BW. body weight;
“Daily nutrient intake per kg of body weight; intake minus fecal loss.
kcal,total
Alb, albumin;
Prealb, prealbumin;
energy (fat + carbohydrate
nor artificial nutrition can account for this finding, since patients were investigated several months after surgery and/or weaning from PN. Besides the small intestine, several organs such as liver, kidney, and lymphoid tissue contribute to glutamine consumption in animals.’ Because glutamine metabolism was assessed by means of whole body kinetics rather than through measurement of glutamine balance across the gut, the organ site of the reduction in glutamine utilization in the patients cannot be ascertained from our results. Since the short bowel patients studied were free of liver, kidney, or immune disease, the present findings nevertheless suggest that the reduction in glutamine turnover is related to enterectomy per se, ie, to a reduced intestinal cell mass. The fact that the depression in glutamine utilization reached only 10% to 25% despite a more than 5O%8 reduction in small bowel length suggests that additional factors can influence glutamine utilization: (1) the patients retained an intact duodenum and/or colon, and both tissues are known to utilize glutamine in animals’; (2) intestinal adaptation’“,”
Hb, blood hemoglobin.
+ protein);
in, dietary intake; abs, absorbed dietary intake =
had obviously occurred in our patients and may be associated with a compensatory increase in intestinal cell number or activity,‘” and, consequently, in glutamine requirement. The present findings point to the human small intestine as a “target organ” for glutamine metabolism in vivo and are in keeping with data obtained in preparations of isolated perfused intestine or whole animals.‘~’ As for the human species, although the capacity of human enterocytes to oxidize glutamine has been documented in vitro,” the only evidence previously obtained in vivo was the positive arterioportal venous gradient for glutamine concentrations (533 v 460 umol/L) reported in four patients studied peroperatively during cholecystectomy.” The present study is the first to document a reduced rate of glutamine turnover in short bowel patients and thus to provide quantitative evidence for the prominent role of the small intestine in glutamine utilization in vivo. Finally. as glutamine kinetics were assessed at steady state, the depressed turnover measured in our patients also reflects a depression in glutamine production, as a result of
Table 2. Leucine and Glutamine Metabolism in Enterectomized
Patients
Leucine
ILeul (~mol/U
-
Glutamlne
MCR (irmaI’
1-l
h-‘j
(mL. kg-‘.
IW (pmoNLl
h-‘1
(pmol
Ra kg-‘. h-‘1
MCR (pmol
i ‘. h
‘)
(mL.kg.‘-h
Patients
-
A
85
72
847
535
288
174
538
B
86
70
814
718
209
100
291
C
124
96
772
541
309
159
571
D
119
110
924
400
247
74
618
E
133
92
692
677
313
169
463
Mean
110
88
807
574
273
135
496
10 + SE Controls (n = 10)
8
38
57
20
20
57
Mean
119
89
772
547
344
193
620
-c SE P*
7 NS
4 NS
46 NS
10 NS
12
14
20
< .02
< .05
< .05
NOTE. Values are means -+ SE. *Patients
Y controls:
comparison
by nonparametric
Mann-Whitney
test.
‘1
44
DARMAUN
a lower de novo synthetic rate (Table 2). Glutamine itself exerts feedback inhibition on the glutamine synthetase of cultured myocytes.14 It is tempting to speculate that the same mechanism regulates glutamine production in vivo. Thus, a reduced intestinal mass might at first suppress
ET AL
glutamine consumption, inducing an initial increase in the plasma glutamine level; the latter could inhibit glutamine production, so that in the long run basal levels of plasma glutamine might be reestablished through a lower rate of glutamine synthesis.
REFERENCES
1. Windmueller HG, Spaeth AE: Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine. J Biol Chem 253:69-76,1978 2. Hanson PJ, Parsons DS: Metabolism and transport of glutamine and glucose in vascularly perfused rat small intestine. Biochem J 166:509-519,1977 3. Watford M, Lund P, Krebs HA: Isolation and metabolic characteristics of rat and chicken enterocytes. Biochem J 178:589596,1979 4. Souba WW, Smith RJ, Wilmore DW: Glutamine metabolism by the intestinal tract. JPEN 9:608-617,1985 5. Darmaun D, Manary MJ, Matthews DE: A method for measuring both glutamine and glutamate levels and stable isotope enrichment. Anal Biochem 147:92-102,1985 6. Darmaun D, Matthews DE, Bier DM: Glutamine and glutamate kinetics in humans. Am J Physiol251:E117-126,1986 7. Bulus N, Cersosimo E, Hishan F, et al: Physiological importance of glutamine. Metabolism 38:Sl-5, 1989 8. Haubrich WS: Anatomy and physiology of the small intestine,
in Bockus HL (ed): Gastroenterology, vol 2. Philadelphia, PA, Saunders, 1966, pp 3-9 9. Roediger WEW: Utilization of metabolic fuels by the colonic mucosa. Gastroenterology 83:423-429,1982 10. Feldman EJ, Dowling RH, MC Naughton J, et al: Effects of oral versus intravenous nutrition on intestinal adaptation after small bowel resection in the dog. Gastroenterology 70~712-719, 1976 11. Hughes CA, Ducker DA: Adaptation of the small intestine; does it occur in man? Stand J Gastroenterol17:149-158,1982 12. Ashy AA, Ardawi MSM: Glucose, glutamine, and ketonebody metabolism in human enterocytes. Metabolism 37:602-609, 1988 13. Felig P, Wahren J, Karl I, et al: Glutamine and glutamate metabolism in normal and diabetic subjects. Diabetes 22573-578, 1973 14. Smith RJ, Larson S, Stred SE, et al: Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells. J Cell Physiol120:197-203,1984
Metabolism
After Small Intestinal
Resection
in Humans
Dominique Darmaun, Bernard Messing, Bernard Just, Monique Rongier, and Jehan-Fransois
Desjeux
Glutamine and leucine kinetics were measured using stable isotopes in five enterectomized patients (residual small bowel, 80 * 25 cm [mean f SE]) who were in a near normal nutritional status at distance from surgery. While parameters of leucine metabolism were normal, rates of whole body glutamine utilization were reduced by 20% in the patients. The data suggest that the small intestine plavs a prominent role in glutamine utilization in vivo in humans.
A
LTHOUGH THE prominent role of the small intestine in glutamine utilization has been demonstrated in animals,‘-4 it remains to be established in humans. If glutamine is indeed a major fuel and a nitrogen donor for the human small intestine, extensive resection of this organ may alter whole body glutamine metabolism in vivo; therefore, the present study assessed glutamine kinetics using stable isotope methodology. METHODS
Informed consent was obtained from five patients and 10 control subjects. Extensive bowel resection had been performed because of abdominal trauma (patient A), intestinal infarction (patients B and C), benign mesenteric fibroma (D), and radiation-induced enteritis (E). Residual small bowel length was estimated from x-rays obtained at least 6 months after resection. Three patients (A, B, and C) received parenteral nutrition (PN) in the postoperative period for 3, 4, and 60 months, respectively. All patients were weaned from PN for more than 3 months before inclusion in the study. Patients were free of inflammation, sepsis (C-reactive protein, .4 f .l mg/dL), or cancer. Although intestinal adaptation had probably occurred (patients were weaned from PN) the patients had short bowel syndrome, ie, moderate diarrhea and malabsorption; only 78% of ingested energy and nitrogen was absorbed (Table 1). Patients were instructed to maintain their usual levels of activity and dietary intake at home and to collect their stools for the 3-day period preceding the test. Absorbed dietary intake (Table 1) was calculated by subtracting measured nitrogen and caloric fecal losses from the intake, as estimated using a 3-day dietary history. Fat mass was estimated from four skinfold thickness measurements with a Harpenden calliper. Controls were 10 healthy adults studied after an overnight fast under a self-selected diet. The night before the study patients were admitted to the Nutrition Unit where they ate dinner at 7:00 PM and then remained fasting until completion of the test at 11:OOAM on the next day. The next morning, primed continuous infusions of sterile, pyrogen-free solutions of L-[l-*3C]leucine (90% ‘jC) and L-[2-“N]glutamine (97% 15N;CEA, Gif-sur-Yvette, France) providing 6 and 2 kmol kg-’ he’, respectively, were administered intravenously in the postabsorptive state as described.” Amino acid levels and stable isotope enrichments were determined in plasma by gas chromatography mass spectromet$ on a
From INSERMu-290, H&pita1St. Lazare, Paris, France. Supported in part by a Grant No. 8860121 from the Minist&e de la Recherche et de la Technologie France. Address reprint requests to Dominique Darmaun, MD, INSERM u-290 H6pital St. Lazare, 107Rue Fbg-St-Denis, 75010 Paris, France. Copyright 0 1991 by W.B. Saunders Company 0026-0495l9ll4001-0009$03.00/0
42
Nermag RlOlOT instrument (Nermag, Argenteuil, France) with a precision of approximately l%, as defined by the coefficient of variation (CV) = 100 x SD/mean of three measurements of same sample. Amino acid appearance rates (Ra) were calculated using equation 1: Ra = i [Ei/Ep - 11,
(I)
where i and Ra are the tracer infusion rate and the amino acid appearance rate, respectively (pmol kg-’ . h-l), and Ei and Ep are the tracer enrichments in the infusate and plasma at steady-state (mole % excess), respectively. Isotopic steady-state was defined by a CV < 10% in tracer enrichment over the last 2 hours of infusion (n = 5 to 7 arterialized plasma samples). Because leucine is an essential amino acid, leucine Ra (Ra,,) is entirely derived from protein breakdown in the postabsorptive state. In contrast, the nonessential amino acid glutamine has two inflow components to its flux? release from protein breakdown (BGln)and glutamine de novo synthesis (DGln);the former is estimated as follows: I&,, = k x RaLcu,
(2)
where k is the assumed ratio of glutamine to leucine content of body protein (8.0 and 13.9 g amino acid per 100 g protein, respectively); therefore, D,,, = Ra,,, - B,,,.
(3)
RESULTS
As expected from inclusion criteria, the patients were in a fair nutritional status (Table 1); besides, when corrected for fecal losses, their nutritional intake was close to the controls’ intake. Parameters of leucine metabolism, ie, plasma leucine level ([Leu]), leucine appearance rate, and leucine metabolic clearance rate (MCR,, = Ra,,/[Leu]) were similar in patients and controls (Table 2). While plasma glutamine level was normal, both glutamine turnover and metabolic clearance rate (MCR,,, = Ra,,,/[Gln]) were significantly depressed in the patients; the reduction was entirely accounted for by a smaller rate of glutamine de novo synthesis (Table 2). DISCUSSION
Based on infusion of stable isotope-labeled glutamine, this study demonstrates that whole body glutamine utilization is reduced in enterectomized patients. This reduction in glutamine turnover is unlikely to result from malnutrition since (1) the patients’ nutritional status was as close to normal as possible (Table 1); and (2) leucine appearance rate, an index of whole body protein turnover, was unaltered (Table 2). Similarly, neither surgical stress
Metabolism, Vol 40, No 1 (January), 1991: pp 42-44
GLUTAMINE
METABOLISM
43
AFTER ENTERECTOMY
Table 1. Clinical and Nutritional Characteristics
of the Patients
Nutrient Intake* Sexi
Residual Small
Delay Post
Age
Bowel
Surgery
WI
lcm)
(mol
A
Ml26
120
15
61
54
B
F/58
15
42
45
33
kcal in
Weight
g protein in
abs
% IBW
Fat Mass
Alb
Prealb
Hb
(% BWI
(g/dL)
lmg/dL)
(g/dL)
abs
kg
1.5
1.1
60
92
14
3.4
23
12.7
1.8
1.3
51
94
30
3.3
33
13.3
-
Patients
C
Ml49
30
108
54
38
2.1
1.7
54
81
10
4.0
25
12.2
D
Ml30
80
13
55
42
2.5
2.1
61
95
16
4.3
29
13.9
E
Ml34
150
148
56
43
1.8
1.4
60
95
nd
4.1
45
14.0
39
80
65
54
42
1.9
1.5
57
91
18
3.8
31
13.2
25 + SE 6 Controls (n = 10; 8M, 2F)
27
3
0.3
0.2
2
2
4
0.2
4
0.4
Mean
-
6
Mean
28
38
1.5
66
97
&SE
7
2
0.4
2
2
Abbreviations:
IBW. ideal body weight;/
BW. body weight;
“Daily nutrient intake per kg of body weight; intake minus fecal loss.
kcal,total
Alb, albumin;
Prealb, prealbumin;
energy (fat + carbohydrate
nor artificial nutrition can account for this finding, since patients were investigated several months after surgery and/or weaning from PN. Besides the small intestine, several organs such as liver, kidney, and lymphoid tissue contribute to glutamine consumption in animals.’ Because glutamine metabolism was assessed by means of whole body kinetics rather than through measurement of glutamine balance across the gut, the organ site of the reduction in glutamine utilization in the patients cannot be ascertained from our results. Since the short bowel patients studied were free of liver, kidney, or immune disease, the present findings nevertheless suggest that the reduction in glutamine turnover is related to enterectomy per se, ie, to a reduced intestinal cell mass. The fact that the depression in glutamine utilization reached only 10% to 25% despite a more than 5O%8 reduction in small bowel length suggests that additional factors can influence glutamine utilization: (1) the patients retained an intact duodenum and/or colon, and both tissues are known to utilize glutamine in animals’; (2) intestinal adaptation’“,”
Hb, blood hemoglobin.
+ protein);
in, dietary intake; abs, absorbed dietary intake =
had obviously occurred in our patients and may be associated with a compensatory increase in intestinal cell number or activity,‘” and, consequently, in glutamine requirement. The present findings point to the human small intestine as a “target organ” for glutamine metabolism in vivo and are in keeping with data obtained in preparations of isolated perfused intestine or whole animals.‘~’ As for the human species, although the capacity of human enterocytes to oxidize glutamine has been documented in vitro,” the only evidence previously obtained in vivo was the positive arterioportal venous gradient for glutamine concentrations (533 v 460 umol/L) reported in four patients studied peroperatively during cholecystectomy.” The present study is the first to document a reduced rate of glutamine turnover in short bowel patients and thus to provide quantitative evidence for the prominent role of the small intestine in glutamine utilization in vivo. Finally. as glutamine kinetics were assessed at steady state, the depressed turnover measured in our patients also reflects a depression in glutamine production, as a result of
Table 2. Leucine and Glutamine Metabolism in Enterectomized
Patients
Leucine
ILeul (~mol/U
-
Glutamlne
MCR (irmaI’
1-l
h-‘j
(mL. kg-‘.
IW (pmoNLl
h-‘1
(pmol
Ra kg-‘. h-‘1
MCR (pmol
i ‘. h
‘)
(mL.kg.‘-h
Patients
-
A
85
72
847
535
288
174
538
B
86
70
814
718
209
100
291
C
124
96
772
541
309
159
571
D
119
110
924
400
247
74
618
E
133
92
692
677
313
169
463
Mean
110
88
807
574
273
135
496
10 + SE Controls (n = 10)
8
38
57
20
20
57
Mean
119
89
772
547
344
193
620
-c SE P*
7 NS
4 NS
46 NS
10 NS
12
14
20
< .02
< .05
< .05
NOTE. Values are means -+ SE. *Patients
Y controls:
comparison
by nonparametric
Mann-Whitney
test.
‘1
44
DARMAUN
a lower de novo synthetic rate (Table 2). Glutamine itself exerts feedback inhibition on the glutamine synthetase of cultured myocytes.14 It is tempting to speculate that the same mechanism regulates glutamine production in vivo. Thus, a reduced intestinal mass might at first suppress
ET AL
glutamine consumption, inducing an initial increase in the plasma glutamine level; the latter could inhibit glutamine production, so that in the long run basal levels of plasma glutamine might be reestablished through a lower rate of glutamine synthesis.
REFERENCES
1. Windmueller HG, Spaeth AE: Identification of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine. J Biol Chem 253:69-76,1978 2. Hanson PJ, Parsons DS: Metabolism and transport of glutamine and glucose in vascularly perfused rat small intestine. Biochem J 166:509-519,1977 3. Watford M, Lund P, Krebs HA: Isolation and metabolic characteristics of rat and chicken enterocytes. Biochem J 178:589596,1979 4. Souba WW, Smith RJ, Wilmore DW: Glutamine metabolism by the intestinal tract. JPEN 9:608-617,1985 5. Darmaun D, Manary MJ, Matthews DE: A method for measuring both glutamine and glutamate levels and stable isotope enrichment. Anal Biochem 147:92-102,1985 6. Darmaun D, Matthews DE, Bier DM: Glutamine and glutamate kinetics in humans. Am J Physiol251:E117-126,1986 7. Bulus N, Cersosimo E, Hishan F, et al: Physiological importance of glutamine. Metabolism 38:Sl-5, 1989 8. Haubrich WS: Anatomy and physiology of the small intestine,
in Bockus HL (ed): Gastroenterology, vol 2. Philadelphia, PA, Saunders, 1966, pp 3-9 9. Roediger WEW: Utilization of metabolic fuels by the colonic mucosa. Gastroenterology 83:423-429,1982 10. Feldman EJ, Dowling RH, MC Naughton J, et al: Effects of oral versus intravenous nutrition on intestinal adaptation after small bowel resection in the dog. Gastroenterology 70~712-719, 1976 11. Hughes CA, Ducker DA: Adaptation of the small intestine; does it occur in man? Stand J Gastroenterol17:149-158,1982 12. Ashy AA, Ardawi MSM: Glucose, glutamine, and ketonebody metabolism in human enterocytes. Metabolism 37:602-609, 1988 13. Felig P, Wahren J, Karl I, et al: Glutamine and glutamate metabolism in normal and diabetic subjects. Diabetes 22573-578, 1973 14. Smith RJ, Larson S, Stred SE, et al: Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells. J Cell Physiol120:197-203,1984
