Determination of protein synthesis in human ileum in situ by continuous [1-13C]leucine infusion

Peter Rittler1, Hans Demmelmair2, Berthold Koletzko2, Friedrich W. Schildberg1, and Wolfgang H. Hartl1

1 Department of Surgery, Klinikum Grosshadern, Marchioninistr. 15, D-81377 Munich, Germany; and 2 Department of Pediatrics, Dr. v. Haunersches Kinderspital, Ludwig-Maximilian University, 80337 Munich, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Efficient protein synthesis plays an important role in the physiology and pathophysiology of the human gastrointestinal tract. Because of methodological restrictions, no studies on ileal protein synthesis in situ are available in humans. We used advanced mass spectrometry techniques (capillary gas chromatography/combustion isotope ratio mass spectrometry) to determine directly the incorporation rate of [1-13C]leucine into ileal mucosal protein in control subjects and postoperative patients. All subjects had an ileostomy, which allowed easy access to the ileal mucosa. To examine changes in ileal protein synthesis during prolonged isotope infusion (0.16 µmol · kg-1 · min-1, 9.6 µmol/kg prime), studies were performed over a 10-h period. Mucosal biopsies were performed after 3, 6, and 10 h of infusion. Protein synthesis was calculated separately between hour 3 and hour 6 (period 1) and hour 6 and hour 10 (period 2). Control subjects demonstrated an ileal protein fractional synthetic rate of 0.62 ± 0.06%/h in period 1 and of 0.52 ± 0.08%/h in period 2 (not significant). In postsurgical subjects, ileal protein synthesis was significantly higher (1.11 ± 0.14%/h in period 1, P < 0.01 vs. controls in period 1) but declined markedly in period 2 (0.39 ± 0.13, P < 0.01 vs. period 1 after surgery). The rate of protein synthesis in the small bowel of control subjects is, thus far, among the lowest measured in mammals and reflects the comparably slow turnover of human ileal mucosa. Postoperative disturbances of gut integrity lead to an accelerated anabolic response. During prolonged isotope infusion, stimulated protein synthesis declines because of diurnal variations or is erroneously reduced by tracer loss due to an accelerated cell turnover.

mass spectrometry; stable isotopes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EFFICIENT PROTEIN SYNTHESIS is a key process in small bowel metabolism and a requirement for forming new proteins during rapid cell renewal and adaptive responses after disturbances of gut integrity (4). Thus far, in vivo studies on intestinal protein synthesis were almost exclusively restricted to animals, but those studies have only limited applicability to human intestinal protein synthesis.

These limitations result from species differences and refer to enterocyte life span, which, in rodents, amounts to about one-half of that of the human (4). This increased intestinal cell turnover results from a shortened cell cycle time (15) and is associated with a variety of anatomical, histological, and ultrastructural qualities that differ markedly from those in humans (4). Furthermore, rodent small bowel mucosa exhibits several physiological peculiarities uncommon to humans, such as a rapid loss of mucosal protein mass during short-term artificial nutrition (14).

Few studies have addressed human small bowel protein synthesis in situ. Because of methodological restrictions, such studies have thus far been performed only in the duodenum (5, 19-22), a portion of the small bowel that can be reached easily via endoscopy. Accordingly, despite their importance for intestinal physiology and pathophysiology, jejunal or ileal protein synthesis studies are lacking in humans.

We have recently developed a minimally invasive and stable isotope technique to obtain values for fractional protein synthetic rate (FSR) (8). The technique, which requires minimal samples, is based on continuous tracer infusion and multiple tissue biopsies. We sought in the present study to examine ileal protein synthesis in control subjects and patients after major abdominal surgery. All subjects had an ileostomy, which allows easy access to the mucosa of this portion of the bowel. Furthermore, we wanted to study changes in ileal protein synthesis during prolonged isotope infusion, because in the past, several authors (4, 12, 17) had questioned the validity of the continuous tracer infusion technique for measurement of small bowel protein synthesis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Two groups (control and postsurgery) of seven subjects each with cured rectal carcinoma and with ileostomies were carefully screened through their medical histories, physical examinations, and routine blood tests. The groups were comparable with respect to age (control, 68.3 ± 4.0 yr old; postsurgery, 60.3 ± 3.1 yr old), body weight (control, 64.3 ± 2.1 kg; postsurgery, 65.1 ± 3.8 kg), height (control, 168 ± 2 cm; postsurgery, 166 ± 4 cm) and body mass index (control, 22.9 ± 1.0 kg/cm2; postsurgery, 23.4 ± 0.7 kg/cm2).

Control subjects were studied ~6 mo after bowel surgery, which had included construction of a temporary ileostomy. Indication for earlier surgery had been a limited colorectal carcinoma. At the time of study, all control subjects were in good health, had regained their preoperative body weight, and were free from recurrent disease, as indicated by follow- up examinations (lab tests, abdominal CT scan, colonoscopy, and chest X ray). At the time of the study, these subjects were awaiting reconstruction of their normal intestinal passage.

Postoperative patients had been suffering from limited colorectal cancer and had undergone curative elective abdominal surgery that had also included construction of an ileostomy. Only those patients were included whose ileostomy did not show any signs of physical trauma or hypoperfusion at the time of the study. The patients were studied between day 8 and day 10 after surgery, when the loss of body protein and the depression of muscle protein synthesis are maximal (9, 24), and the patients were free from signs of local or systemic infection at the time of the study. No control subject and no patient (before surgery) had a history of previous weight loss or of clinical or laboratory signs of malnutrition or metabolic diseases. Informed consent was obtained after the experimental protocol had been explained in detail. The study was approved by the local institutional review board (protocol no. 134/97).

Experimental protocol. All subjects were inpatients of the general surgical service. Control subjects were scheduled for reconstruction of the normal intestinal passage. Before the study, control subjects consumed their regular hospital diet (~30 kcal · kg-1 · day-1). Postoperative patients received a mixed diet (~25 kcal · kg-1 · day-1), of which one-third was administered enterally (liquids) and the other two-thirds parenterally. After 10 PM, the subjects remained postabsorptive except for consumption of mineral water. A primed-constant infusion of [1-13C]leucine (Tracer Technologies, Sommerville, MA, 99.3 atom % enrichment) was started at 7 AM the next day and continued for 10 h. The infusion rate was 0.16 µmol · kg-1 · min-1 (prime = 9.6 µmol/kg). A blood sample was collected before isotope infusion to determine the background enrichment of protein-bound and free plasma leucine. Plasma background enrichments were used as an indicator of intracellular protein-bound and free leucine background enrichments (8). The first ileal mucosa biopsy was performed after 180 min of isotope infusion, the second after 360 min, and the third after 600 min. Period 1 ranged from minute 180 to minute 360, period 2 from minute 360 to minute 600. All biopsies were taken from the distal portion of the ileum, a portion located in the abdominal wall. The minimum distance between biopsy sites was 2 cm. The average biopsy size was 15 mg wet weight.

Procedures. The principles of the method and the generation of the data are presented and discussed in detail in a previous publication (8). The free and protein-bound amino acids in tissue biopsies were separated by precipitation of the proteins. After hydrolysis of proteins, we separated amino acids from the accompanying impurities by cation-exchange chromatography. For capillary gas chromatography (GC)/combustion isotope ratio mass spectrometry (IRMS) analysis, amino acids from proteins (on average 6-8 ng) were then converted to the N-acetyl-n-propyl (NAP) ester. For gas chromatography/quadrupole mass spectrometry (GC-MS) analysis, we prepared the tert-butyldimethylsilyl (t-BDMS) derivative from free intracellular amino acids. NAP-amino acid derivatives were analyzed in a capillary GC/combustion IRMS system that consisted of a Hewlett-Packard 5890 Series II gas chromatograph that was interfaced to a mass spectrometer Delta S (Finnigan, Bremen, Germany). t-BDMS derivatives were analyzed by a GC-MS system (MSD 5971D, Hewlett-Packard). Isotopomer ratios of the sample were obtained by electron impact ionization and selected ion monitoring at mass-to-charge ratios (m/e) 303 and 302. Data were expressed as tracer-to-tracee ratios.

Calculations. Tissue fractional synthetic rate was calculated by dividing the increment in protein-bound [1-13C]leucine tracer-to-tracee ratio by the enrichment of the precursor, the free homogenate [1-13C]leucine tracer-to-tracee ratio (29). Delta increments of protein-bound [1-13C]leucine enrichment between biopsies were calculated from isotope ratios [13C]/[12C] using a correction factor that takes into account dilution of the label at the carboxyl position by the other carbon atoms in the derivatized leucine (8). FSR was then calculated as follows
FSR = <FR><NU>R<SUB>L</SUB>(<IT>t</IT><SUB>i+1</SUB>) − R<SUB>L</SUB>(<IT>t</IT><SUB>i</SUB>)</NU><DE>{R<SUB>L</SUB>′(<IT>t</IT><SUB>i</SUB>) + R<SUB>L</SUB>′(<IT>t</IT><SUB>i+1</SUB>)}/2</DE></FR> × <FR><NU>1</NU><DE>&Dgr;<IT>t</IT></DE></FR> × 60 × 100 (%/h)
RL(ti) and RL (ti+1) correspond to the tracer-to-tracee ratio of ileal protein-bound leucine in two subsequent samples (i and i+1), separated by the time interval × t (min). RL'(ti) and RL'(ti+1) indicate the tracer-to-tracee ratios of ileal free leucine in two subsequent samples. Average values between RL'(ti) and RL'(ti+1) were used as precursor enrichments for ileal protein synthesis. The factors 60 (min) and 100 are needed to express the FSR in %/h.

To analyze the speed of tracer incorporation separately in period 1 and period 2, we calculated the absolute increase of the tracer-to-tracee ratio/h by the formula
&Dgr;R<SUB>L</SUB>/h = [{R<SUB>L</SUB>(<IT>t</IT><SUB>i+1</SUB>) − R<SUB>L</SUB>(<IT>t</IT><SUB>i</SUB>)}/&Dgr;<IT>t</IT>] × 60

Statistics. Data are expressed as means ± SE. Results obtained at different time points in the same subjects were compared with the paired t-test. Comparisons with controls were made by the unpaired t-test. A P value of <= 0.05 was taken as indicating a significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Analysis of tracer data from the 10-h experiments showed that in both groups (control and postsurgery) isotope infusion led to a stable plateau of the tracer-to-tracee ratio in the mucosa-free homogenate leucine pool (= precursor pool for protein synthesis) that did not change significantly during the study (Table 1). The values of protein-bound leucine enrichment (tracer-to-tracee ratio) at the different sampling points (after 3, 6, and 10 h of isotope infusion) are presented in Table 2. Figures 1, A and B show the values of protein-bound leucine enrichment and the time curve of tracer incorporation into ileal mucosa in control and postsurgical subjects during the 10-h study period. In controls, tracer incorporation was almost linear. On average, the tracer-to-tracee ratio rose by 0.041 ± 0.004%/h in period 1 (hour 3 to hour 6) and by 0.035 ± 0.007%/h in period 2 (hour 6 to hour 10) (not significant). In contrast, incorporation slowed down over time after surgery (rise/h: 0.057 ± 0.005% in period 1, 0.019 ± 0.005% in period 2, P < 0.001). In one postsurgical subject (subject 3), the protein-bound leucine enrichment after 10 h of isotope infusion was actually 10% lower than the corresponding ratio after 6 h of isotope infusion.

                              
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Table 1.   Tracer-to-tracee ratios (%) of [1-13C]leucine to free intracellular leucine from ileal mucosa in control subjects and in postoperative patients


                              
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Table 2.   Individual tracer-to-tracee ratios (%) of [1-13C]leucine in ileum mucosa protein in control subjects and postoperative patients



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Fig. 1.   Time curve of tracer incorporation into ileal mucosa in control (A) and postsurgical subjects (B) during 10-h isotope infusion.

In controls, ileal protein synthesis amounted to 0.62 ± 0.06%/h in period 1 and to 0.52 ± 0.08 in period 2 (not significant). In postsurgical subjects, we observed significantly higher rates of ileal fractional protein synthesis in period 1 (1.11 ± 0.14%/h, P < 0.02 vs. 0.62 ± 0.06 in controls in period 1). During prolonged isotope infusion, the ileal FSR declined in the postsurgery group significantly over time (0.39 ± 0.13%/h in period 2, P < 0.01 vs. 1.11 ± 0.14 in period 1). Ileal protein synthesis in period 2 did not differ significantly between groups (Fig. 2).


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Fig. 2.   Effect of surgery and of prolonged isotope infusion on ileal protein fractional synthetic rate (%/h, mean ± SE). Period 1 ranged from hour 3 to hour 6, period 2 from hour 6 to hour 10. § P < 0.01 vs. control; * P < 0.01 vs. period 1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Choice of the precursor pool. We have used the tissue-free intracellular amino acid enrichment as a substitute for the true precursor pool enrichment (tRNA-bound amino acid enrichment). Arterial plasma ketoisocaproate (KIC) or leucine enrichments were not used for several reasons. First, it was recently shown in human skeletal muscle from postabsorptive subjects that tissue-free leucine enrichment, but not arterial KIC or leucine enrichment, corresponds closely to leucyl-tRNA enrichment, irrespective of the nutritional status or the type of nutrient supply (16). Second, when gut proteolysis is stimulated [as has been found during exercise (6), when counterregulatory hormones are increased (11), or after surgery (18)], the arterial KIC or leucine enrichment is different from the corresponding portal-venous enrichment. The latter cannot be obtained in humans but would have to be known to obtain accurate information on the intracellular enrichment in the intestinal tract (6). Third, arterial indicators of gut precursor pool enrichment do not account for the heterogeneity of protein and precursor pool enrichments that is observed in the intestinal mucosa during intravenous tracer infusion (4). This heterogeneity results from a variable arterial tracer uptake that decreases from the crypt to the upper villus. The impact of these variations on the calculation of protein synthesis will be minimized if the regional protein enrichment (in the tissue from the biopsy) is referred to the corresponding regional precursor pool enrichment. Finally, there is indirect evidence from multiple tracer infusion studies that the tissue-free amino acid enrichment represents a valid indicator of the true precursor pool enrichment also in small bowel. Thus the ratio between the enrichments of differently labeled leucine tracers in the free jejunum amino acid pool was the same as the corresponding ratio in jejunum protein (10).

Validity of the continuous tracer infusion technique. There has been substantial concern about the validity of continuous tracer infusion for measuring gut protein synthesis (4, 12, 17). Based on experiments in growing rats, it has been argued that measurements made by constant infusion of labeled amino acids over several hours are subject to substantial error due to labeled protein loss by turnover, cell loss via exfoliation, or protein secretion over time. According to our data, the arguments against the use of the continuous infusion technique in rodents are not relevant for healthy postabsorptive humans. Thus, as indicated in Fig. 1A, continuous tracer infusion in control subjects was associated with a linear rise of tracer incorporation into ileal mucosa over 3-10 h. Correspondingly, the hourly increase of the tracer-to-tracee ratio was comparable between hour 3 and hour 6, and between hour 6 and hour 10 of the study. The main reason for this difference from animal experiments is presumably the fact that human intestinal cells turn over half as fast as cells from rodents, and cell loss in the human small bowel amounts to only ~0.8 to 2%/h (4).

Notably, the above considerations allow no conclusion regarding the validity of the continuous infusion technique during enteral feeding, wherein the amount of secreted protein may increase rapidly (3), leading to significant label loss and presumably to underestimation of the true synthesis rate. Such a phenomenon may also explain why a recent human study did not detect a significant effect of feeding on duodenal protein synthesis (5). In that study, the tracer amino acid was infused intravenously over 9 h followed by duodenal mucosa biopsy. During isotope infusion, subjects in the fed group received large amounts of enteral calories (30 kcal/kg), whereas control subjects continued to fast. Because of the study's design, the opposing effects on tissue protein enrichment of feeding-induced mucosal protein synthesis and label loss via accelerated protein secretion may have balanced each other out, making the observed rates of protein synthesis during feeding seem comparable with those in subjects who had fasted.

Ileal protein synthesis in control subjects. Mucosal fractional protein synthesis rate in the postabsorptive state amounted to ~0.6%/h. This rate is lower than that obtained from human duodenum in the postabsorptive state (2.6%/h) (20) or after a 36-h fast (1.7%/h) (5). To date, no other studies are available that directly address mucosal FSRs of human distal small bowel; however, several authors have examined small bowel protein synthesis in animal experiments and found rates between 1.5 and 4.5%/h in adult pigs and rats (26, 28). These data suggest that human small bowel FSRs are among the lowest in mammals, and in line with older observations demonstrating a longer cell cycle time and a slower cell turnover in human small bowel than in that of rodents (4, 15).

Effect of surgery on ileal protein synthesis. In the ileum, elective surgery almost doubled FSRs, which were 0.6%/h in control subjects and 1.1%/h in postsurgical subjects. To our knowledge, no comparable data from humans are available for our findings on postoperative intestinal protein synthesis. Ahlman et al. (1) found an increased total amino acid concentration in the duodenum after elective surgery. This finding indicates an increased availability of intracellular amino acids for protein synthesis and would be compatible with our observation of an increased ileal protein synthetic rate.

The higher postoperative ileal protein synthesis may have resulted from earlier intraoperative manipulations causing a minor temporary hyperperfusion. Temporary ischemia or trauma in the small bowel induces a strong compensatory response upon restoration of circulation and markedly stimulates crypt cell proliferation in the small intestine (25). In recent studies on human intestinal epithelial cells in vitro, a small drop in local pH (as can be expected during surgery) significantly accelerated enterocyte proliferation (23). Finally, one cannot completely exclude the possibility that the postsurgical rate of protein synthesis was actually normal and that the rate in controls was depressed due to the presence of the ileostomy for 6 mo.

Changes in postoperative ileal protein synthesis over time. In contrast to controls, we observed a decrease in ileal protein synthesis of nearly 70% during the 10-h study period (Fig. 2) due to deceleration of tracer incorporation into mucosal proteins. During the last 4 h of the study, the tracer-to-tracee ratio in mucosal protein rose by only 0.019%/h, one-third of that observed between hour 3 and hour 6 of the study.

Several mechanisms may explain the declining ileal protein synthesis. A role of fasting is unlikely because, by use of published sources (5, 20), one can calculate that human duodenal protein synthesis declines by only 1.3%/h during a 36-h fasting period. A second explanation would be circadian rhythms in enterocyte cell division. It has not been substantiated that such rhythms exist in humans; however, global intestinal protein synthesis and brush border enzyme synthesis did not exhibit circadian rhythms in growing rats (2, 13). In contrast, there is ample evidence for the existence of such rhythms in mice (for review see Ref. 27). Finally, one cannot ignore method-related reasons to explain declining ileal protein synthesis. As a result of increased postoperative enterocyte proliferation and enzyme secretion, ileal protein turnover might have reached a level on which a significant label loss could occur during prolonged continuous isotope infusion. A nonlinear tracer incorporation into protein would be compatible with a secretory system. It is possible that mucosal constitutive protein synthesis was strongly suppressed after surgery while secretion was either continuing or even increasing. The loss of labeled protein (via cell or enzyme loss or via degradation of rapidly turning over proteins) may have been so extensive that isotope incorporation measurement during constant infusion would yield erroneously low enrichments and consequently seemingly reduced rates of mucosal protein synthesis. A similar problem arose earlier in the intestinal tract of growing rats (12, 17).

It should be noted that in one postsurgical subject (subject 3) the protein-bound leucine enrichment actually decreased by 10% between hour 6 and hour 10 of the study (Table 2). For method-related reasons such a decline is impossible. This decline is, however, unlikely to be exclusively due to analytical variations because we found a coefficient of variation of only 5% during repeated analysis of protein-bound leucine enrichment (interassay variation of ion exchange chromatography, amino acid derivatization, and GC-IRMS analysis) (8). It is more likely that the observed decline can be explained, besides the mechanisms discussed above, by a variation of protein enrichment in the mucosa during intravenous tracer infusion. With this technique, protein enrichment in the intestinal mucosa is heterogenous (4), and the enrichment in a sample will also depend on the percentage of crypt cells and cells from the lower or upper villus in the biopsy. Therefore, a protein enrichment in a biopsy that contains more cells from the upper villus will actually be lower than that in a biopsy that includes more cells from the crypt. In colorectal tissues, the variation between different simultaneous biopsies from the same tissue may, therefore, amount to almost 10% (7).

In conclusion, more work is needed to address the issue outlined above with respect to ileal protein synthesis. However, our data on postoperative ileal mucosa indicate that experiments designed to examine the potential stimulation of intestinal protein metabolism must take into account the observed changes in tracer incorporation during prolonged isotope infusion.


    ACKNOWLEDGEMENTS

This study was supported by grants from the Deutsche Forschungsgemeinschaft (Ha-1439/4-1).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: W. H. Hartl, Chirurgische Klinik, Klinikum Grosshadern, Marchioninistr. 15, D-81377 Munich, Germany (W.HARTL{at}gch.med.uni-muenchen.de).

Received 9 April 1999; accepted in final form 10 November 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 278(4):E634-E638
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