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The influence of starvation and refeeding on skeletal muscle protein phosphorylation
P.15
Proteinase
following
abdominal
in skeletal
activity
Patients and methods: Metabolically healthy patients (n = 9) undergoing elective cholecystectomy, by laparotomy, participated in the study. The protein synthesis rate was determined by using the flooding dose technique f?rpploying intravenous injection of L-[d,]phenylalanine (45 mg/kg, 7.5 and 1.5 atoms-% excess). The incorporation into muscle protein was measured by taking percutaneous biopsies from the quadriceps femoris muscle. The rate of muscle protein synthesis was calculated from the increase in enrichment of labeled phenylalanine in protein by using gas chromatography mass spectrometry. The protein synthesis rate was initially determined in the postabsorptive state before surgery. Postoperatively the patients received total parenteral nutrition (TPN), consisting of equal energy contents of carbohydrate and fat (135 kJ/kg bw) and an amino acid nitrogen supply of 0.2 g N/kg bw/24 h or saline only. The second assessment was performed 24 h postoperatively. Results: Protein synthesis rate in quadriceps muscle was 1.61 + 0.12%/24 h in the basal state preoperatively and decreased by 28% to 1 .I 6 k 0.09%/24 h, 24 hours postoperatively (p < 0.02). The patients receiving TPN decreased by 15%. while those who got saline decreased by 38%. Conclusion: These preliminary results suggest that total parenteral nutrition, when given continuously postoperatively, did not counteract the decrease in muscle protein synthesis rate observed after surgery. However, the provision of TPN may give a less pronounced decrease when compared to saline controls.
muscle
surgery
F. Hammarqvist, A. YOU der Decken, E. Vinnars and J. Wernerman The Metabolic Research Laboratory, St Goran’s Hospital, Stockholm, Sweden Surgical trauma elicits changes in skeletal muscle metabolism leading to muscle protein catabolism mirrored by a decrease in protein synthesis and an altered pattern of the muscle free amino acids. The aim of this study was to determine the activity of proteindegradating enzyme systems, acid and neutral proteinases, in muscle tissue obtained during the immediate postoperative period. Methods: Metabolically healthy patients (n = 10) undergoing elective cholecystectomy were studied. Percutaneous muscle specimens were obtained from the vastus lateralis muscle preoperatively, 24, and 72 hours postoperatively. The patients received a standardised energy supply consisting of 2 g glucose/kg bw during the day of operation and thereafter 3 g glucose/kg bw/d. The acid and neutral proteinase activity was determined by measuring the rate of tyrosine release from added substrates at acid and neutral pH. Also the autolytic degradation of intrinsic proteins without an added substrate was measured. The activity was expressed as pg tyrosin released/mg wet weight muscle/h and per pg DNA/h respectively. Results: Preoperatively the acid proteinase activity was 1.72 f 0.12 pg tyrosine released/mg wet weight muscle/h. At 24 and 72 hours following surgery the activity decreased to 88.3 f 6.7% (p < 0.05) and 92.7 k 4.6% (p = 0.065) of the initial values. The activity expressed per pg DNA/h was preoperatively 3.64 i 0.21 and 24 and 72 hours postouerativelv 85.9 + 3.3% (D i 0.05) and 101.4 + 2.3% of t(e initial’ value.-The act’ivity of t’he neutral proteinases remained unchanged. The autolytic degradation decreased to 78.9 f 9.1% of the initial values after 24 hours (p =z 0.05) but was not different from preoperative values 72 h following surgery. Conclusion: Assessment of the activities of the proteinase systems may be a useful tool with which it is possible to study protein degradation in human tissues. In this study the acid proteinase activity decreased following abdominal surgery of moderate size indicating a decrease in the degradation of muscle proteins. This decrease is concomitant with the known postoperative decrease of muscle protein synthesis, but will probably still not outbalance the change in protein synthesis since substrates are mobilised during the postoperative period from peripheral tissues.
P.16
Protein
synthesis
rate
in human
P.17 The influence of starvation and refeeding on skeletal muscle protein phosphorylation Elisabeth Svanberg-Larsson, Helen Zachrisson, PerAnders Larsson, Yoshikazu Nogochi and Kent Lundholm Department of Surgery, Sahlgrenska Hospital, GCiteborg. Sweden Protein balance in skeletal muscles is controlled by alterations in protein synthesis and degradation. This tuned regulation is essentially controlled by phosphorylation/dephosphorylation mechanisms. Very little information is available on protein phosphorylation in relationship to feeding and malnutrition. Therefore, the aim of this study was to evaluate whether phosphorylation kinetics is possible to evaluate and quantify in skeletal muscles in relation to refeeding experiments. Methods: Adult weight-stable mice were starved for 18 h and thereafter refed for 3 h with a standard rodent chow. The hind limb muscles were excised, homogenised and ultracentrifuged whereafter the supernatant was dialysed. The dialysate containing soluble proteins and co-factors were used for phosphorylation experiments in vitro in the presence of 3ZP-ATP. The proteins were then separated by electrophoresis according to pH and the molecular size. The degree of the phosphorylation among various proteins was visualized by autoradiography and subsequently quantified by electrophoretic gel densitometry. Human muscle tissue specimens were also prepared accordingly and the phosphorylations were conducted in the same way as for mouse muscles. Results: Refeeding for 3 h stimulated phosphorylation of proteins preferentially of high molecular size and in the range of acidic iso-electric points, whereas starvation induced phosphorylation of lower-sized proteins in the range with higher iso-electric points. Thus, both starvation and refeeding induced changes in phosphorylation compared to freely-fed animals although different proteins were affected.
skeletal
muscle decreases 24 hours after abdominal gery irrespective of intravenous nutrition
sur-
I. Tjiider,
P. EssBn, M.A. McNurlan. P.J. Garlick and J. Wernerman Depts. of Anesthesiology and Intensive Care at Huddinge University Hospital, St Gorant Hospital, Stockholm, Sweden, and Rowett Research Institute, Aberdeen, UK Previous studies have shown that the protein synthesis rate in human skeletal muscle decreases by 30% immediately after surgery. On the third postoperative day the decline in protein synthesis rate is 50%, irrespective of total parenteral nutrition, but after one nights fast. The aim of this study was to determine the protein synthesis rate before and 24 hours following surgery, with total parenteral nutrition given continuously throughout the study period. 49
Conclusion: The rapid stimulation of protein synthesis in a starvation-refeeding situation includes a complicated network of regulatory activities. This cascade is predominantly controlled by phosphorylation/dephosphorylation of different proteins as visualized by autoradiograms in the present study. This technique represents a new approach to study protein synthesis regulation in both animal and human muscle biopsies. This may open up a wide field of nutritional research aspects.
P.18 The effect and degradation
of insulin on protein
After killing them, liver total RNAs were purified, denaturized and fixed on nylon membranes by dot and Northern blot procedures. Hybridizations were performed using a 1.1 Kb human cathepsin D cDNA probe labelled with 32P-dCTP by random priming. Autoradiographies were used to quantify hybridization signals. Cathepsin D activity was measured by a modified Anson’s method using bovine haemoglobin as substrate. Results: We observed a continuous increase in gene expression throughout the fasting period. Enzyme activity doubled at 24 hours of fasting and maintained a similar value until the 72 hours.
synthesis
in man
Ann-Charlotte Miiller-Loswick, Anders Hyltander, U/la Kbrner, Dwight E. Matthews and Kent Lundholm Departments of Anaesthesia I & Surgery I & II, Sahlgrenska Hospital, Gliteborg. Sweden and Cornell Medical Center, The New York Hospital, New York, USA
Control 24 hours 48 hours 72 hours (‘I p less than
0.01
blot units)
Activitv (u,g,Ty;{h.w
wt)
12.611.0’ 9.5k1.7’ 11.a*2.2*
(Student
T test) with regard
to
chol
Conclusions: Cathepsin D gene expression is activated with regard to the fasting time. Moreover, there is no correlation with enzyme activity. This different behaviour could be due either to a lack in the efficiency of mRNA translation and/or to cellular enzyme regulators.
The role of insulin as a physiological regulator of protein balance is unclear. Our previous work has demonstrated that insulin in itself did not influence on either protein synthesis or degradation in the presence of fasting levels of plasma amino acids. The present study has evaluated whether insulin regulates protein balance in human skeletal muscles in the presence of elevated plasma levels of amino acids. A combined and primed constant infusion of L-[U-‘4C]-tyrosine and D5-L-phenylalanine were used to measure both disposal (protein synthesis) of tyrosine and phenylalanine across the leg and the arm before and appearing (protein degradation) during 3.5 hrs with hyperinsulinization (glucose clamp) in the presence of increased levels of plasma amino acids. The disposal (synthesis) of tyrosine and phenylalanine (56 f 10, 68 + 11 nmol/min/g leg tissue) was not increased by insulin at high post-feeding levels (100-I 20 mu/I). The appearance (protein degradation) of tyrosine and phenylalanine in the fasted state (61 + 10, 73 f 7) was decreased by hyperinsulinemia to 37 k 6 and 338 + 6 nmol/min/g for both tyrosine and phenylalanine respectively. Similar results were obtained from measurements across the forearm. Insulin stimulated glucose uptake across both leg and arm muscles as expected during hyperinsulinization and the whole body glucose uptake as 7.23 + 0.68 mg/min/kg at loo-120 mU/I plasma. The results demonstrate that tyrosine and phenylalanine gave the same information on amino acid flux during steady state measurements across leg and arm muscle tissue in man. Hyperinsulinemia did not stimulate protein synthesis but depressed protein degradation by 50% in the presence of elevated plasma concentrations of amino acids.
P.19 Cathepsin D gene expression mitted to long-term fasting
Northern (relative 100 190 272 345
P.20 Protein turn-over assessed by leucine glutamine fluxes in adult caeliac patients
and
S. Dutra. F. Thuillier, D. Darmaun, B. Messing, M. Rongier and J. F. Desjeux HBpital Saint-Lazare and lnserm U.290. Paris, France
in rats sub-
A.L. Andre& S. Schwartz, M. Crespo. E. Garcia, M.A. Arbbds and J. Ldpez Metabolic Unit Research 3. Grisolia: Department of Biochemistry, Hospital ‘Vail de Hebrdn’, Barcelona, Spain Cathepsin D is one of the most important proteolytic enzymes and plays an important role in protein breakdown level, but its control mechanisms are poorly understood. The aim of this work was to study both activity and gene expression of cathepsin D in rat liver submitted to long-term fasting in order to demonstrate that the enzyme participates in the increase of liver protein breakdown observed in fasting and that this increase is mainly produced through the stimulation of gene expression. Material and methods: 20 male Sprague-Dawley rats (120-I 30 g) were divided into four groups and fasted at different periods (24, 48, 72 hours and a control group).
Glutamine (Gln) is an important substrate for rapidly proliferating cells including epithelial cells of the intestinal mucosa. Caeliac disease is characterized by a marked increase of the renewal of intestinal crypt cells aimed at compensating the villous atrophy. Since it was demonstrated a 20% reduction of Gln utilisation associated with enterectomy in human being (Darmaun et al. Metabolism 1991, 40, 42-4) we postulated an opposite increase in Gln utilisation in active caeliac disease. Therefore the present study assessed Gln and Leucine (Leu) kinetics using stable isotope methodology. Whole body Leu and Gln kinetics were measured in 6 healthy adults and 4 active caeliac using a 4 h i.v. infusion of 4 pmol L-[1 -‘3C] Leu and 7 pmol kg-’ h-’ of L-[2-‘5N] Gln in the post absorptive state. Steady state 13C-enrichment in plasma Leucine was used to calculate the Leu appearance rate (Ra) whereas Gln Ra was derived from plasma 15N enrichment in the last 2 h of the stable isotope infusion. Gln protein breakdown (B) was derived from Ra Leu: B Gln = 1.7375 x Ra Leu, permitting to estimate the Gln de novo synthesis: D Gln = Ra Gln - B Gln. Caeliac patients (weight: 91 f 6% of IBW, albumine: 28 + 7 g/l) had steatorrhea and total villous atrophy documented by endoscopic upper jejunal biopsies; they were active caeliac either resistants (n = 2) or following errors (n = 2) in gluten exclusion. Results in pmol kg-’ h-’ (mean + SD) were as follows: Patients Controls
Ra Leu 113+10 96kll
B Gin 196+18 167120
D Gin 378k32 168k23
Ra Gin 378f45i 335f16
Mann- Whitney test: ‘p = 0.066. tp = 0.038. ’NS
Conclusion: Compared with controls there is a slight but significant increase in Leu (18%) and Gln (15%) kinetics in patients with active caeliac diease. Whether these increased fluxes are dependent upon protein malnutrition or enhanced cells renewal in the small mucosa cannot be precised from the present study. 50
Proteinase
following
abdominal
in skeletal
activity
Patients and methods: Metabolically healthy patients (n = 9) undergoing elective cholecystectomy, by laparotomy, participated in the study. The protein synthesis rate was determined by using the flooding dose technique f?rpploying intravenous injection of L-[d,]phenylalanine (45 mg/kg, 7.5 and 1.5 atoms-% excess). The incorporation into muscle protein was measured by taking percutaneous biopsies from the quadriceps femoris muscle. The rate of muscle protein synthesis was calculated from the increase in enrichment of labeled phenylalanine in protein by using gas chromatography mass spectrometry. The protein synthesis rate was initially determined in the postabsorptive state before surgery. Postoperatively the patients received total parenteral nutrition (TPN), consisting of equal energy contents of carbohydrate and fat (135 kJ/kg bw) and an amino acid nitrogen supply of 0.2 g N/kg bw/24 h or saline only. The second assessment was performed 24 h postoperatively. Results: Protein synthesis rate in quadriceps muscle was 1.61 + 0.12%/24 h in the basal state preoperatively and decreased by 28% to 1 .I 6 k 0.09%/24 h, 24 hours postoperatively (p < 0.02). The patients receiving TPN decreased by 15%. while those who got saline decreased by 38%. Conclusion: These preliminary results suggest that total parenteral nutrition, when given continuously postoperatively, did not counteract the decrease in muscle protein synthesis rate observed after surgery. However, the provision of TPN may give a less pronounced decrease when compared to saline controls.
muscle
surgery
F. Hammarqvist, A. YOU der Decken, E. Vinnars and J. Wernerman The Metabolic Research Laboratory, St Goran’s Hospital, Stockholm, Sweden Surgical trauma elicits changes in skeletal muscle metabolism leading to muscle protein catabolism mirrored by a decrease in protein synthesis and an altered pattern of the muscle free amino acids. The aim of this study was to determine the activity of proteindegradating enzyme systems, acid and neutral proteinases, in muscle tissue obtained during the immediate postoperative period. Methods: Metabolically healthy patients (n = 10) undergoing elective cholecystectomy were studied. Percutaneous muscle specimens were obtained from the vastus lateralis muscle preoperatively, 24, and 72 hours postoperatively. The patients received a standardised energy supply consisting of 2 g glucose/kg bw during the day of operation and thereafter 3 g glucose/kg bw/d. The acid and neutral proteinase activity was determined by measuring the rate of tyrosine release from added substrates at acid and neutral pH. Also the autolytic degradation of intrinsic proteins without an added substrate was measured. The activity was expressed as pg tyrosin released/mg wet weight muscle/h and per pg DNA/h respectively. Results: Preoperatively the acid proteinase activity was 1.72 f 0.12 pg tyrosine released/mg wet weight muscle/h. At 24 and 72 hours following surgery the activity decreased to 88.3 f 6.7% (p < 0.05) and 92.7 k 4.6% (p = 0.065) of the initial values. The activity expressed per pg DNA/h was preoperatively 3.64 i 0.21 and 24 and 72 hours postouerativelv 85.9 + 3.3% (D i 0.05) and 101.4 + 2.3% of t(e initial’ value.-The act’ivity of t’he neutral proteinases remained unchanged. The autolytic degradation decreased to 78.9 f 9.1% of the initial values after 24 hours (p =z 0.05) but was not different from preoperative values 72 h following surgery. Conclusion: Assessment of the activities of the proteinase systems may be a useful tool with which it is possible to study protein degradation in human tissues. In this study the acid proteinase activity decreased following abdominal surgery of moderate size indicating a decrease in the degradation of muscle proteins. This decrease is concomitant with the known postoperative decrease of muscle protein synthesis, but will probably still not outbalance the change in protein synthesis since substrates are mobilised during the postoperative period from peripheral tissues.
P.16
Protein
synthesis
rate
in human
P.17 The influence of starvation and refeeding on skeletal muscle protein phosphorylation Elisabeth Svanberg-Larsson, Helen Zachrisson, PerAnders Larsson, Yoshikazu Nogochi and Kent Lundholm Department of Surgery, Sahlgrenska Hospital, GCiteborg. Sweden Protein balance in skeletal muscles is controlled by alterations in protein synthesis and degradation. This tuned regulation is essentially controlled by phosphorylation/dephosphorylation mechanisms. Very little information is available on protein phosphorylation in relationship to feeding and malnutrition. Therefore, the aim of this study was to evaluate whether phosphorylation kinetics is possible to evaluate and quantify in skeletal muscles in relation to refeeding experiments. Methods: Adult weight-stable mice were starved for 18 h and thereafter refed for 3 h with a standard rodent chow. The hind limb muscles were excised, homogenised and ultracentrifuged whereafter the supernatant was dialysed. The dialysate containing soluble proteins and co-factors were used for phosphorylation experiments in vitro in the presence of 3ZP-ATP. The proteins were then separated by electrophoresis according to pH and the molecular size. The degree of the phosphorylation among various proteins was visualized by autoradiography and subsequently quantified by electrophoretic gel densitometry. Human muscle tissue specimens were also prepared accordingly and the phosphorylations were conducted in the same way as for mouse muscles. Results: Refeeding for 3 h stimulated phosphorylation of proteins preferentially of high molecular size and in the range of acidic iso-electric points, whereas starvation induced phosphorylation of lower-sized proteins in the range with higher iso-electric points. Thus, both starvation and refeeding induced changes in phosphorylation compared to freely-fed animals although different proteins were affected.
skeletal
muscle decreases 24 hours after abdominal gery irrespective of intravenous nutrition
sur-
I. Tjiider,
P. EssBn, M.A. McNurlan. P.J. Garlick and J. Wernerman Depts. of Anesthesiology and Intensive Care at Huddinge University Hospital, St Gorant Hospital, Stockholm, Sweden, and Rowett Research Institute, Aberdeen, UK Previous studies have shown that the protein synthesis rate in human skeletal muscle decreases by 30% immediately after surgery. On the third postoperative day the decline in protein synthesis rate is 50%, irrespective of total parenteral nutrition, but after one nights fast. The aim of this study was to determine the protein synthesis rate before and 24 hours following surgery, with total parenteral nutrition given continuously throughout the study period. 49
Conclusion: The rapid stimulation of protein synthesis in a starvation-refeeding situation includes a complicated network of regulatory activities. This cascade is predominantly controlled by phosphorylation/dephosphorylation of different proteins as visualized by autoradiograms in the present study. This technique represents a new approach to study protein synthesis regulation in both animal and human muscle biopsies. This may open up a wide field of nutritional research aspects.
P.18 The effect and degradation
of insulin on protein
After killing them, liver total RNAs were purified, denaturized and fixed on nylon membranes by dot and Northern blot procedures. Hybridizations were performed using a 1.1 Kb human cathepsin D cDNA probe labelled with 32P-dCTP by random priming. Autoradiographies were used to quantify hybridization signals. Cathepsin D activity was measured by a modified Anson’s method using bovine haemoglobin as substrate. Results: We observed a continuous increase in gene expression throughout the fasting period. Enzyme activity doubled at 24 hours of fasting and maintained a similar value until the 72 hours.
synthesis
in man
Ann-Charlotte Miiller-Loswick, Anders Hyltander, U/la Kbrner, Dwight E. Matthews and Kent Lundholm Departments of Anaesthesia I & Surgery I & II, Sahlgrenska Hospital, Gliteborg. Sweden and Cornell Medical Center, The New York Hospital, New York, USA
Control 24 hours 48 hours 72 hours (‘I p less than
0.01
blot units)
Activitv (u,g,Ty;{h.w
wt)
12.611.0’ 9.5k1.7’ 11.a*2.2*
(Student
T test) with regard
to
chol
Conclusions: Cathepsin D gene expression is activated with regard to the fasting time. Moreover, there is no correlation with enzyme activity. This different behaviour could be due either to a lack in the efficiency of mRNA translation and/or to cellular enzyme regulators.
The role of insulin as a physiological regulator of protein balance is unclear. Our previous work has demonstrated that insulin in itself did not influence on either protein synthesis or degradation in the presence of fasting levels of plasma amino acids. The present study has evaluated whether insulin regulates protein balance in human skeletal muscles in the presence of elevated plasma levels of amino acids. A combined and primed constant infusion of L-[U-‘4C]-tyrosine and D5-L-phenylalanine were used to measure both disposal (protein synthesis) of tyrosine and phenylalanine across the leg and the arm before and appearing (protein degradation) during 3.5 hrs with hyperinsulinization (glucose clamp) in the presence of increased levels of plasma amino acids. The disposal (synthesis) of tyrosine and phenylalanine (56 f 10, 68 + 11 nmol/min/g leg tissue) was not increased by insulin at high post-feeding levels (100-I 20 mu/I). The appearance (protein degradation) of tyrosine and phenylalanine in the fasted state (61 + 10, 73 f 7) was decreased by hyperinsulinemia to 37 k 6 and 338 + 6 nmol/min/g for both tyrosine and phenylalanine respectively. Similar results were obtained from measurements across the forearm. Insulin stimulated glucose uptake across both leg and arm muscles as expected during hyperinsulinization and the whole body glucose uptake as 7.23 + 0.68 mg/min/kg at loo-120 mU/I plasma. The results demonstrate that tyrosine and phenylalanine gave the same information on amino acid flux during steady state measurements across leg and arm muscle tissue in man. Hyperinsulinemia did not stimulate protein synthesis but depressed protein degradation by 50% in the presence of elevated plasma concentrations of amino acids.
P.19 Cathepsin D gene expression mitted to long-term fasting
Northern (relative 100 190 272 345
P.20 Protein turn-over assessed by leucine glutamine fluxes in adult caeliac patients
and
S. Dutra. F. Thuillier, D. Darmaun, B. Messing, M. Rongier and J. F. Desjeux HBpital Saint-Lazare and lnserm U.290. Paris, France
in rats sub-
A.L. Andre& S. Schwartz, M. Crespo. E. Garcia, M.A. Arbbds and J. Ldpez Metabolic Unit Research 3. Grisolia: Department of Biochemistry, Hospital ‘Vail de Hebrdn’, Barcelona, Spain Cathepsin D is one of the most important proteolytic enzymes and plays an important role in protein breakdown level, but its control mechanisms are poorly understood. The aim of this work was to study both activity and gene expression of cathepsin D in rat liver submitted to long-term fasting in order to demonstrate that the enzyme participates in the increase of liver protein breakdown observed in fasting and that this increase is mainly produced through the stimulation of gene expression. Material and methods: 20 male Sprague-Dawley rats (120-I 30 g) were divided into four groups and fasted at different periods (24, 48, 72 hours and a control group).
Glutamine (Gln) is an important substrate for rapidly proliferating cells including epithelial cells of the intestinal mucosa. Caeliac disease is characterized by a marked increase of the renewal of intestinal crypt cells aimed at compensating the villous atrophy. Since it was demonstrated a 20% reduction of Gln utilisation associated with enterectomy in human being (Darmaun et al. Metabolism 1991, 40, 42-4) we postulated an opposite increase in Gln utilisation in active caeliac disease. Therefore the present study assessed Gln and Leucine (Leu) kinetics using stable isotope methodology. Whole body Leu and Gln kinetics were measured in 6 healthy adults and 4 active caeliac using a 4 h i.v. infusion of 4 pmol L-[1 -‘3C] Leu and 7 pmol kg-’ h-’ of L-[2-‘5N] Gln in the post absorptive state. Steady state 13C-enrichment in plasma Leucine was used to calculate the Leu appearance rate (Ra) whereas Gln Ra was derived from plasma 15N enrichment in the last 2 h of the stable isotope infusion. Gln protein breakdown (B) was derived from Ra Leu: B Gln = 1.7375 x Ra Leu, permitting to estimate the Gln de novo synthesis: D Gln = Ra Gln - B Gln. Caeliac patients (weight: 91 f 6% of IBW, albumine: 28 + 7 g/l) had steatorrhea and total villous atrophy documented by endoscopic upper jejunal biopsies; they were active caeliac either resistants (n = 2) or following errors (n = 2) in gluten exclusion. Results in pmol kg-’ h-’ (mean + SD) were as follows: Patients Controls
Ra Leu 113+10 96kll
B Gin 196+18 167120
D Gin 378k32 168k23
Ra Gin 378f45i 335f16
Mann- Whitney test: ‘p = 0.066. tp = 0.038. ’NS
Conclusion: Compared with controls there is a slight but significant increase in Leu (18%) and Gln (15%) kinetics in patients with active caeliac diease. Whether these increased fluxes are dependent upon protein malnutrition or enhanced cells renewal in the small mucosa cannot be precised from the present study. 50