ABSTRACT. Background: Amino acids are an integral part of parenteral nutrition because of their anabolic action helping to conserve body protein after surgical stress. At the gastrointestinal tract, an adequate supply of amino acids may be particularly important because of the gut's high rate of protein turnover, cell division, and proliferation. However, no information is available about the effects of amino acids on human intestinal protein metabolism after surgery. Methods: Studies were performed in postabsorptive patients 8-10 days after major abdominal surgery. Mass spectrometry techniques (capillary gas chromatography/ combustion isotope ratio mass spectrometry) were used to directly determine the incorporation rate of 1-[^sup 13^C]-leucine into colon mucosal protein. All subjects had a colostomy, which allowed easy access to the colon mucosa, and consecutive sampling from the same tissue was performed during continuous isotope infusion (0.16 µmol/kg min). Isotopic enrichments were determined at baseline and after a 4-hour infusion of amino acids or after infusion of saline (control group). Results: Compared with baseline, infusion of amino acids reduced fractional colon protein synthesis significantly by -29.2 ± 8.3%. This decrease was also significantly different from the corresponding (insignificant) change during saline infusion (+19.4 ± 26.9%, p

Amino acid supply has gained its firm place during parenteral nutrition in a wide range of patients who cannot tolerate enterai feeding. Amino acids are usually considered necessary to reduce protein loss after surgical stress. An optimal conservation of body protein will be obtained with daily administration of 1.2-1.5 g amino acids per kg body weight.1 Potential anabolic actions of amino acids are known to occur in muscle and in splanchnic tissues.2 At the intestinal tract, amino acid metabolism and protein turnover are particularly important because an efficient formation of new protein is essential for maintaining a high rate of cell division and proliferation and for the production of cellular structure compounds and secreted enzymes.3'4 Unfortunately, in humans effects of specific substrates have almost exclusively been studied in the whole splanchnic bed, not allowing a differentiation between hepatic and intestinal changes.5-7 Virtually nothing is known on the effects of amino acids on human intestinal protein metabolism in situ.

We sought in the present study to examine the effects of a standard amino acid infusion on colon protein synthesis using stable isotopes (1-[^sup 13^C]-leucine) and advanced mass spectrometry techniques. Studies were performed in patients after major abdominal surgery whose mucosal function may be altered and who are frequent candidates for parenteral nutrition.

MATERIALS AND METHODS

Subjects

Two groups of postoperative patients (control, n = 5; amino acid infusion, n = 12) with cured rectal carcinoma and colostomies were carefully screened through their medical history, physical examinations, and routine blood tests. The groups were comparable with respect to age (control, 64.8 ± 3.9 years; amino acid infusion, 63.5 ± 2.9) and body mass index (control, 24.9 ± 1.0 kg/cm^sup 2^; amino acid infusion, 27.5 ± 1.3). Patients had had limited colorectal cancer and had undergone curative, elective abdominal surgery, which had also included construction of a colostomy. All patients had a preoperative bowel preparation (orthograde flushing by oral colonoscopy fluids) and received prophylactic IV antibiotics at the time of surgery. Anesthesia consisted of epidural application of analgesic and anesthetic drugs and was continued no longer than the fifth postoperative day. The patients were studied between days 8 and 10 after surgery, when body protein loss peaks,8 had an uneventful postoperative course, and were free from signs of organ malfunction and local or systemic infection. No patient (before surgery) had a history of previous weight loss or clinical and 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 134/97).

Experimental Protocol

All subjects were inpatients of the general surgical service. Before the study, patients received a mixed diet (approximately 25 kcal/kg day, of which 5 kcal/kg day were administered as protein or IV amino acids); two-thirds of the calories were administered enterally (liquids) and the other one-third parenterally. Of the calculated energy demand (25 kcal/kg day), only 60% was provided on day 1 to day 3 after surgery, 80% between day 4 and day 6, and 100% thereafter. In any case, a daily postoperative protein/amino acid intake of 1.2 g /kg was assured. Remaining nonprotein calories were given as carbohydrates between day 1 and day 6. After the sixth postoperative day, half of the nonprotein calories were given as carbohydrates, the other half as fat. Nutrition was started as parenteral nutrition. If gastrointestinal function was considered sufficient, tea per mouth was provided on postoperative days 1 and 2, clear liquids on postoperative days 3 and 4, and regular liquid food on the subsequent days. Daily oral consumption was recorded by the dieticians, and eventual caloric deficits were compensated by adjusting parenteral nutrition.

After 10 PM on the day before surgery, subjects remained postabsorptive, except for consumption of mineral water. A primed-constant infusion of 1-[13C]leucine (Tracer Technologies, Sommerville, MA; 99.3 atom percent enrichment) was started at 7 AM the next day. The isotope infusion rate was 0.16 µmol/kg min (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.9

The first mucosa biopsy was performed after 180 minutes of isotope infusion, the second after 360 minutes, and the third after 600 minutes. The baseline period (period I) ranged from minutes 180-360. Between minutes 360 and 600 (after the second biopsy, period II), patients in the amino acid group (n = 12) received a continuous infusion of commercial amino acid solutions with comparable amino acid composition (Glamin, Baxter, Erlangen, Germany; or Parentamin/Dipeptamin, Serag-Wiessner, Naila, Germany; and Fresenius-Kabi, Bad Homburg, Germany). The total amino acid infusion rate was 67 mg/kg h. Patients in the control group remained in a fasting state (saline infusion, η = 5). Arterialized blood samples for measurement of amino acid concentrations were taken at the same time as mucosa biopsies. The minimum distance between biopsy sites was 2 cm. The average biopsy size was 10 mg wet weight. All biopsies were taken from a portion of the colon that was located in the abdominal wall. No patient was included with any signs of mucosa swelling, edema, necrosis, bleeding, or malfunction. If mucosal function was unclear, we used laser Doppler flowmetry10 to confirm the normality of mucosal flow patterns.

Procedures

Study methods and data analysis were discussed in detail previously.9 The free and protein-bound amino acids in tissue biopsies were separated by protein precipitation. After protein hydrolysis, amino acids were separated from the accompanying impurities by cationexchange chromatography. For capillary gas chromatography and combustion isotope ratio mass spectrometry analysis, amino acids from proteins (on average 7-8 ng) were then converted to the W-acetyl n-propyl ester. For gas chromatography and quadrupole mass spectrometry analysis, we prepared the tert-butyldimethylsilyl derivative from free intracellular amino acids. JV-acetyl n.-propyl-amino acid derivatives were analyzed in a capillary gas chromatography and combustion isotope ratio mass spectrometry system that consisted of a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard) interfaced to a mass spectrometer Delta S (Finnigan MAT, Bremen, Germany). Tert-butyldimethylsilyl derivatives were analyzed by a gas chromatography and quadrupole mass spectrometry system (MSD 5971D, Hewlett-Packard). Isotopomere ratios of the sample were obtained by electron impact ionization and selected ion monitoring at mass-to-charge ratios 303 and 302. Data were expressed as tracer:tracee ratios.

Plasma amino acid levels were measured by an autoanalyzer (Beckman Instruments, Fulton, CA).

Calculations