Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects

Cornelia C. Metges1,2, Antoine E. El-Khoury1, Lidewij Henneman3, Klaus J. Petzke2, Ian Grant3, Shahinaze Bedri1, Paulo P. Pereira1, Alfred M. Ajami4, Malcolm F. Fuller3, and Vernon R. Young1

1 Laboratory of Human Nutrition, School of Science and Clinical Research Center, Massachusetts Institute of Technology, Cambridge 02139; 4 MassTrace, Woburn, Massachusetts 01801; 2 Unit of Protein Metabolism, Department of Biochemistry and Physiology of Nutrition, German Institute of Human Nutrition, 14558 Bergholz-Rehbrücke, Germany; and 3 Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration of L-[1-13C]lysine and L-[6,6-2H2]lysine, respectively. [15N2]urea or 15NH4Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with 15NH4Cl. [15N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [13C]- and [2H2]lysine enrichments during the 12-h fed period were observed between the two 15N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after 15NH4Cl and [15N2]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg · kg-1 · day-1, depending on the [15N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.

lysine kinetics; amino acid requirement; tracer balance; lysine-15-nitrogen; gas chromatography-combustion-isotope ratio mass spectrometry


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CURRENT INTERNATIONAL RECOMMENDATIONS for indispensable amino acid requirements of adult humans (12) are derived from nitrogen balance studies in men and women (22, 36). Earlier studies by us and others, using approaches based on 13C-labeled amino acid oxidation, suggested that adult lysine requirements are considerably higher than previously thought (23, 44, 45). Careful 24-h tracer studies have provided general support for the minimal physiological requirement as estimated in short-term experiments for phenylalanine, leucine, and lysine, and possibly for other indispensable amino acids, and they have established that the tracer-balance concept is valid (3, 11, 20). However, there remains controversy about the quantitative values for the amino acid requirements in adult humans, as reviewed by Waterlow (42).

Among the reasons suggested for the discrepancy between nitrogen balance and tracer-derived amino acid requirement estimates is the possibility that the metabolic requirement, i.e., the irreversible loss of indispensable amino acids (of which oxidation is the major component), is met not only by the diet but also by amino acids synthesized de novo by the gastrointestinal microflora, which are then absorbed. On the basis of the interpretation of urinary 15N urea excretion after labeled urea administration, it has been claimed that urea nitrogen can be salvaged by urea hydrolysis in the colon and that this nitrogen can be incorporated by the intestinal microflora into amino acids that are subsequently absorbed by the host (18). Although it has been shown in the pig that colonic absorption of amino acids is possible (30, 31), experimental evidence in nonruminant animals does not indicate a quantitatively important amino acid absorption from the colon (7, 17, 19). Nevertheless, results in the pig demonstrated that amino acids synthesized by the microflora can be absorbed supposedly in the small intestine and used for host protein synthesis (25, 40). Tracer studies in animals and humans have shown a transfer of nonspecific nitrogen into dispensable and indispensable amino acids (1, 25, 32). For a majority of amino acids, this may reflect nitrogen exchange or reversible transamination. However, lysine does not undergo transamination in mammalian tissue (6). Therefore, appearance of 15N-labeled lysine in body proteins and plasma amino acids after administration of a 15N-labeled nitrogen source, such as ammonium or urea, must reflect the de novo synthesis of lysine and its subsequent absorption from the gastrointestinal tract. Comparative experiments with germ-free and conventional rats (39) have confirmed that lysine de novo synthesis is due to the activity of the indigenous microflora in the gastrointestinal tract.

Although in uremic patients and human subjects consuming a low-protein diet microbial lysine can be made available to the human host (15, 38), there has been no previous attempt to quantify the significance of this source of lysine in host tissue metabolism. The purpose of the present investigation, therefore, was to quantify the contribution of microbial lysine to whole body lysine turnover in healthy adult human subjects. In a pilot study in pigs (25), we found that the degree of [15N]lysine labeling is dependent on the 15N source ([15N2]urea or 15NH4Cl) used. This article describes the appearance of 15N-labeled lysine in plasma after oral administration of isonitrogenous amounts of [15N2]urea and15NH4Cl, respectively, in normal young adults. In addition, lysine turnover, lysine oxidation, and lysine splanchnic uptake were measured during a 24-h oral/intravenous tracer protocol. Because the intestinal site of synthesis and subsequent absorption of microbial lysine is uncertain, we studied, additionally, the microbial synthesis of lysine in otherwise healthy subjects with ileostomies after administration of 15NH4Cl by use of the same protocol. This enabled us to compare estimates of the microbial lysine contribution to host tissue lysine homeostasis by using fecal and ileal microbial protein as putative precursor pools for the [15N]lysine absorption.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects and diets. Six male subjects aged 21.7 ± 1.6 yr (weight 81.7 ± 13.3 kg; height 1.82 ± 0.04 m) were recruited within the student population at the Massachusetts Institute of Technology (MIT) and the community of the Boston-Cambridge area. The study was performed at the Clinical Research Center (CRC) of the MIT. The subjects were healthy according to medical history, physical examination, vital signs, blood parameters, and urinalysis. They had no recent weight loss, unusual dietary practice, or pharmacological treatment. Subjects who smoked and consumed more than five or more alcoholic drinks per week were excluded from the study. Subjects were allowed to engage in their usual daily activities but not to participate in competitive sports and perform strenuous exercise. Body weight was monitored daily and did not change throughout the experimental period. The purpose of the study and the potential risks involved were fully explained to each of the subjects. Written informed consent was obtained, and the subjects received financial compensation for their participation in the study. The study was approved by the MIT Committee on the Use of Humans as Experimental Subjects and the MIT-CRC Advisory Committee.

A second group of seven otherwise healthy subjects were ileostomates who were studied at the Human Nutrition Unit of the Rowett Research Institute, Aberdeen, Scotland. The group comprised three men and four women with terminal ileum ileostomies following ulcerative colitis or cancer of the colon (1 subject). Their mean age, weight, and height were 59.7 ± 12.3 yr, 71.6 ± 15.0 kg, and 1.68 ± 1.27 m, respectively. Body weight did not change during the experimental period. Clinical examination and routine clinical chemistry parameters were in a normal range for their age. The intestinal resection was performed >= 6 mo before the study. They did not take any medications and had no known diseases of the upper gastrointestinal tract. Each subject gave informed written consent to participate in this study, which was approved by the Joint Ethical Committee of Grampian Health Board and University of Aberdeen.

All subjects received a protein (N) adequate, semi-synthetic, diet based on a crystalline L-amino acid mixture (Ajinomoto USA, Teaneck, NJ; 160 mg N · kg-1 · day-1) patterned essentially as in a hen's egg except for the moderate, but presumably adequate, lysine and leucine content (total lysine and leucine intake: 45 and 40 mg · kg-1 · day-1, respectively; Table 1). To adjust for the somewhat lower nitrogen contribution of leucine and lysine, glycine and alanine were added. The level of energy intake was based on estimates of resting energy expenditure and subjects' activity level. Average diet composition for the MIT subjects is given in Table 2. The diet composition for the subjects studied in Aberdeen was the same but adjusted for their different energy requirements. No other foods or beverages were allowed except tap water, decaffeinated tea or coffee with or without artificial sweetener, and bouillon.

                              
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Table 1.   Composition of crystalline L-amino acid mixture


                              
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Table 2.   Diet composition

Total nitrogen intake for the MIT subjects was 167 mg · kg-1 · day-1, due to the additional urea and ammonia tracer intake, supplied in gelatine capsules, and a lysine supplement to account for the lysine tracer given during the 24-h tracer protocol (see 24-h Tracer protocol). The ileostomates received a total nitrogen intake of 163 mg · kg-1 · day-1. The energy intake of the MIT subjects was 186 kJ · kg-1 · day-1, whereas the ileostomates consumed 134 kJ · kg-1 · day-1 because of a lower level of physical activity. Two out of three meals per day were consumed under the supervision of the dietary staff.

The MIT subjects received the experimental diet during each of two randomly assigned 11-day periods with a break of 4-8 wk in between, when subjects consumed their usual diet. During the two periods, isonitrogenous amounts of either [15N2]urea or 15NH4Cl (3.45 mg 15N · kg-1 · day-1; 2.1% of total N intake) were given. The 15N tracer was started on day 5, in the morning. Therefore, subjects received with each of the three daily meals one gelatine capsule containing either [15N2]urea [580 mg/day; 99.1 atom percent (AP); Isotec, Miamisburg, OH] or 15NH4Cl (1,000 mg/day; 99.8 AP; Isotec), respectively, during days 5-10. The ileostomy subjects were residents in the Human Nutrition Unit of the Rowett Research Institute and consumed the same experimental diet for 10 days. Beginning on the morning of day 5, the 1,000-mg amount of 15NH4Cl was given in three gelatine capsules per day, and this continued until the end of day 10. Hence, 3.9 mg 15N · kg-1 · day-1 (2.5% of total N intake) were ingested. This subject group did not receive [15N2]urea.

24-h Tracer protocol. At 1600 on day 10, the subjects consumed their last meal in the CRC and were then admitted as inpatients to the MIT Medical Department. Two small catheters were placed into veins of the nondominant arm by use of aseptic sterile procedures. One catheter was introduced into an antecubital vein for tracer infusion, and one catheter was placed into a superficial dorsal vein of the hand for blood collection (10). Beginning at 1800 on day 10 of the diet, oral doses (1/24 of daily dose) of the respective 15N tracer ([15N2]urea or 15NH4Cl) were given hourly. Hourly portions of 15N tracers were weighed individually and dissolved in ~25 ml of tap water immediately before ingestion. Simultaneuosly, a 24-h infusion protocol was performed in which an L-[1-13C]lysine · HCl tracer (99 AP; MassTrace, Woburn, MA) was administered intravenously and L-[6,6-2H2]lysine · HCl (99 AP; MassTrace) was given by mouth in hourly intervals to assess lysine kinetics. Because the analytic data of two of the six subjects were questionable, they were not included in data evaluation. The primed, constant intravenous infusion of [13C]lysine was administered at a known rate of 3.5 µmol · kg-1 · h-1 (prime 5.25 µmol · kg-1 · h-1 ). The bicarbonate pool was primed (0.6 µmol/kg) with 13C sodium bicarbonate (99 AP; Cambridge Isotope Laboratories, Andover, MA). The oral tracer [2H2]lysine was given at a rate of 2.5 µmol · kg-1 · h-1 (prime 3.75 µmol/kg). All tracers were tested to be sterile and pyrogen free by an independent laboratory. The intravenous tracers were prepared in physiological saline under sterile conditions and were infused with a screw-driven pump at a rate of 8 ml/h (model 22; Harvard Apparatus, Millis, MA). The oral tracer was dissolved in distilled water and given in hourly doses of 8 ml.

The feeding regimen on the tracer day 11 consisted of 10 equal hourly meals (each supplying 1/10 of total 24-h intake), which were started at 0600. Because the subjects remained in bed during the 24-h tracer protocol period, energy but not nitrogen supply was decreased to ~83% of that for the prior 10 days. The 24-h tracer protocol was not performed in the ileostomates.

Breath 13CO2 background enrichment. To correct the breath 13CO2 baseline for food-derived changes, a tracer-free study was performed in three additional young MIT subjects (20.7 ± 0.9 yr; 61.6 ± 15.0 kg; 1.65 ± 0.1 m). They received the standard experimental diet plus unlabeled ammonium chloride, as described in Subjects and diets, and 30-min breath samples were taken. To account for the lysine intake by the tracers, unlabeled lysine was added as hourly oral doses. The mean value for each 30-min interval of the 24-h protocol was used to correct the 13C enrichment in breath during [13C]lysine tracer infusion.

Sample collection. Urine and feces from MIT subjects were collected over 24 h starting on day 3 of the dietary period and continued throughout the remainder of the experimental period (day 3 0800 to day 11 1800). The subjects were instructed to collect feces directly in clean plastic bags and then to freeze the sample promptly at -20°C. During the 24-h tracer protocol, urine was collected at consecutive 3-h intervals. Fasted blood samples were drawn into heparinized tubes from an antecubital arm vein by venipuncture on days 3 or 4 (baseline samples for [15N]lysine enrichments) and day 8. Throughout the 24-h tracer protocol, hourly blood samples were taken via the catheter in the hand vein. Ten minutes before blood sampling, the hand was placed into a customized warming box (65°C air temperature) to obtain arterialized blood.

Breath samples were taken every 30 min for measurement of breath 13CO2. Between 360 and 720 min, no breath samples were obtained so that subjects could maintain the normal sleeping pattern. Instead, blood samples (2 ml) were used to liberate 13CO2 from blood bicarbonate, as previously described (10). Total carbon dioxide production (VCO2) was determined by indirect calorimetry, also as described previously (10).

Starting on day 5, 24-h ileostomy fluid was collected continuously and pooled over 24-h periods. Subjects emptied their bags whenever fluid was present and froze the fluid immediately. One fasted blood sample was obtained in the morning of day 10. Because no baseline samples of ileal fluids and plasma were taken, total urinary nitrogen was used as a baseline for calculation of 15N enrichments (atom percent excess, APE).

Sample analysis. Ileal digesta and feces were pooled daily, and the microbial fraction was obtained by differential centrifugation as recently described (39). The microbial pellet was precipitated, and the protein was hydrolyzed and purified by filtration. Nitrogen content in ileal digesta was determined by a micro-Kjeldahl method. Lysine content in ileal microbial protein was determined after acid hydrolysis by ion exchange chromatography (33). Plasma was separated in a refrigerated centrifuge at 1,000 g and stored frozen at -20°C until analysis. The isolation of free amino acids was performed as described by Metges and Petzke (24).

Because 15N enrichment in lysine in the plasma samples was extremely low, a highly sensitive methodology was used. [15N]lysine and the other amino acids of plasma and microbial protein were derivatized to form N-pivaloyl-i-propyl esters and measured by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Briefly, amino acid esters are separated on a capillary column (Ultra-2, Hewlett-Packard, Waldbronn, Germany) by gas chromatography (HP 5890, Hewlett-Packard). The effluent is directed on-line into a combustion interface. Amino acids are combusted in an oxidation furnace at 980°C to CO2, N2, NOx, and H2O. Combustion gases are reduced in a reduction oven at 600°C, CO2 and H2O are eliminated, and N2 is directed into the ion source of an isotope ratio mass spectrometer (Finnigan delta S, Finnigan MAT, Bremen, Germany). Details on this methodology have been reported elsewhere (24, 25). Samples were measured in duplicate.

Urinary 15N baseline enrichment was measured by gas IRMS (Europa Scientific, Crewe, UK) (39).

Plasma free [13C]- and [2H2]lysine enrichment was measured in trifluoracetyl-n-propanol ester by GC-MS. Plasma (100 µl) was subjected to cation exchange (Bio-Rad AG 50W-X8, 100-200 mesh, H+ form). After evaporation of the eluate, 0.5 ml of freshly prepared propanol-HCl (1:5 acetyl chloride-propanol, vol/vol) was added, and the mixture was heated at 90°C for 30 min. Propyl esters were dried under N2. Then 100 µl acetonitrile and 100 µl trifluoroacetic anhydride were added, and the mixture was heated at 60°C for 30 min. After evaporation of excess reagent under N2 at room temperature, the residue was redissolved in 0.5 ml ethyl acetate and transferred to autosampler microvials. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) and were of analytical grade.

Samples (1.5 µl) were injected splitless into a Varian (Walnut Creek, CA) Saturn 2000 quadrupole ion trap GC-MS equipped with a model 3400 GC and then onto a DB-WAX capillary column (0.25 mm × 30 m, 0.25 µm film thickness; J&W Scientific, Folsom, CA). Helium was the carrier gas, set to a head pressure of 55 kPa and adjusted so as to deliver 1 ml/min at 250°C. The injector temperature was programmed to rise from 140 to 190°C at 15°C/min, whereas the oven temperature was programmed to rise from 180 to 250°C at 35°C/min after a 2-min isothermal phase. Sample retention times under these conditions averaged 6 min.

Isotopic enrichments were monitored in EI/MS/MS mode under default settings to accommodate a filament emission of 90 µA, with a multiplier gain of autotune + 200 V and an automatic gain control target of 5,000. For the prescan, the parent ions at or above mass-to-charge ratio (m/z) 380 (C13H18N2O4F6) were isolated in an 8-amu parent mass window (m/z 377-384). During the subsequent analytic scan, the same parent window was isolated and subjected to nonresonant excitation so as to produce a predominant (product) ion centered at m/z 380. These were sampled using a narrow rF scan, and the ion current ratios at m/z 381/380 and 382/380, corresponding to the two lysine tracer/tracee ratios of interest, were computed postrun from the narrow-range product ion scan data. Plasma samples were analyzed in duplicate.

For purposes of calibration, a training data set was created by compiling the mass spectral response of graded mixtures of [13C]- and [2H2]lysine, together with unlabeled lysine, over a 0- to 10-mole fractional range for each tracer. Multilinear regression was then used to generate a prediction equation, correlating the two ion pair ratios obtained spectrometrically on standards as the explanatory variable for tracer-to-tracee (tracer/tracee) mole ratios. Thus, for [13C]lysine in the presence of [2H2]lysine plus tracee, the regression relationship between tracer/tracee mole ratio (MR1) was described by MR1 = 1.070 × RAE1 - 0.261 × RAE2; and for [2H2]lysine in the presence of [13C]lysine plus tracee, the tracer/tracee mole ratio (MR2) relationship was described by MR2 = -0.261 × RAE1 + 1.070 × RAE2.

In these equations, the paired ion current ratios RAE1 and RAE2 represent the baseline corrected relative abundances at m/z 381/380 and 382/380, respectively. The equation was then applied in determining ion pair mole ratios from the corresponding spectral information afforded by the plasma samples, and the resulting mole ratios were converted algebraically into tracer mole fractional excess (MFE) for each tracer, after subtraction of the corresponding baseline plasma values for each set of samples. Analysis of replicate standards (n = 5) under these conditions showed a coefficient of variation ranging from 2 to 7%, the latter values being those for the detection of 0.5 MFE of either lysine isotopolog in the presence of a 5 MFE of the other. Also, by way of validation, the accuracy of estimates for this methodological approach was found to fall within 5% of expected values on average, based on analysis of sham mixtures prepared gravimetrically and with known tracer(s)-to-tracee composition.

Evaluation of primary data. Lysine oxidation (Lys Ox) was computed for each half-hour interval during the 24-h tracer protocol, except during the first 2 h of the study, when it was taken to be equal to that measured at the end of the 2-h period to reduce any possible effect of the lysine and bicarbonate prime
Lys Ox (&mgr;mol · kg<SUP>−1</SUP> · 30 min<SUP>−1</SUP>) 
= <FR><NU><SUP> 13</SUP>CO<SUB>2</SUB> production (&mgr;mol · kg<SUP>−1</SUP> · 30 min<SUP>−1</SUP>)</NU><DE><SUP> 13</SUP>C plasma lysine enrichment (MFE %/100)</DE></FR>
where 13C plasma lysine enrichment is the average of the two enrichments determining the specific half-hour interval. Mean lysine oxidation for the fasted and fed states was calculated during 480-720 min and 1,020-1,320 min of the tracer period, respectively.
<SUP> 13</SUP>CO<SUB>2</SUB> production (&mgr;mol ⋅ kg<SUP>−1</SUP> ⋅ 30 min<SUP>−1</SUP>) = <A><AC>V</AC><AC>˙</AC></A><SC>co</SC><SUB>2</SUB> (&mgr;mol · kg<SUP>−1</SUP> · 30 min<SUP>−1</SUP>) 
×<SUP> 13</SUP>CO<SUB>2</SUB> breath enrichment<SUP>#</SUP> × 1/R<SUP>##</SUP>
and # = APE/100
and ## = recovery of 13CO2 [as reported by El-Khoury et al. (9); fasted state: 76.6%; fed state: 85.1%].

Lysine turnover (rate of appearance, Lys Ra) was calculated as follows
Lys R<SUB>a</SUB> = i × (E<SUB>i</SUB>/E<SUB>p</SUB> − 1)
where i is the tracer infusion rate (µmol · kg-1 · 30 min-1), Ei is the enrichment in AP of the administered isotope ([13C]- or [2H2]lysine), and Ep is the enrichment in plasma at isotopic steady state during fasting (480-720 min) and feeding (1,020-1,320 min).

Lysine splanchnic uptake (Lys Spl Upt) was computed for the fasted and fed states from plasma enrichments of orally and intravenously administered lysine (E[2H2]Lys and E[13C]Lys) at isotopic steady state, normalized for infusion rate (ir) of tracers, as follows
Lys Spl Upt 
= {1 − [(E<SUB>[<SUP>2</SUP>H<SUB>2</SUB>]Lys</SUB>/ir<SUB>[<SUP>2</SUP>H<SUB>2</SUB>]Lys</SUB>)/(E<SUB>[<SUP>13</SUP>C]Lys</SUB>/ir<SUB>[<SUP>13</SUP>C]Lys</SUB>)]} × 100
This fraction represents the fraction of lysine taken up by the gut and the liver during its first pass.

The contribution of microbial [15N]lysine (Contr Lysmicr) to total lysine appearance in the peripheral circulation was estimated from the fraction of lysine that was of microbial origin and the lysine turnover. Thus the former was calculated from the ratio of the mean enrichment of [15N]lysine in plasma (Ep [15N]Lys) for the fast and fed states to the enrichment of [15N]lysine in either fecal or ileal microbial lysine (Em [15N]Lys). These values were then multiplied by the appropriate lysine turnover (Lys Ra) value, derived from the intravenously administered [1-13C]lysine tracer. Thus
Contr Lys<SUB>micr</SUB> (&mgr;mol · kg<SUP>−1</SUP> · 30 min<SUP>−1</SUP>) 
= Lys R<SUB>a</SUB> × (E<SUB>p [<SUP>15</SUP>N]Lys</SUB>/E<SUB>m [<SUP>15</SUP>N]Lys</SUB>)

Statistics. All results were expressed as means ± SD. The hypotheses to be tested were 1) that [13C]lysine kinetics after intake of isonitrogenous amounts of 15NH4Cl and [15N2]urea, respectively, would not differ from each other, 2) microbial lysine contribution to host metabolism would not differ when 15NH4Cl and [15N2]urea, respectively, were given as 15N tracers to the MIT subjects, and 3) that 15N enrichment of ileal microbial lysine (in ileostomates) would be lower than fecal microbial lysine (in MIT subjects) after the intake of 15NH4Cl. Hence we used a two-sided, paired t-test to evaluate group means for hypotheses 1 and 2 and a one-sided, unpaired t-test to compare group means of ileostomates and MIT subjects (hypothesis 3). To compare the fasted and fed states during the 24-h tracer protocol in the MIT subjects, the mean values were compared using a two-sided, paired t-test. A P value of <0.05 was considered to be significant. Nonsignificantly different means were labeled NS.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[15N]lysine labeling. The plasma lysine 15N enrichments in the MIT subjects on days 3 and 4, before the intake of the two 15N tracers, did not differ from each other, and they corresponded to the 15N natural abundance ([15N2]urea period: 0.3671 ± 0.0003; 15NH4Cl period: 0.3668 ± 0.0004 15N AP). Because a baseline plasma sample was not available for the ileostomates, urinary total nitrogen was measured. The result (0.3658 ± 0.0003 15N AP) was similar to plasma lysine of the MIT subjects.

The plasma [15N]lysine enrichment (APE) after 6 days of 15NH4Cl tracer intake was threefold greater than after [15N2]urea administration in MIT subjects in the fasted state (Table 3; Fig. 1). This difference between the two 15N tracers was already apparent on day 8 ([15N2]urea: 0.0028 ± 0.0011; 15NH4Cl: 0.0067 ± 0.0014; NS). Mean enrichments on day 8 after 15NH4Cl and [15N2]urea, respectively, were significantly different from zero (one-sided t-test; P < 0.01; P < 0.05).

                              
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Table 3.   15N enrichment of plasma lysine and microbial protein (ileal and fecal) lysine, respectively, in MIT subjects and ileostomates during administration of [15N2]urea and 15NH4Cl



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Fig. 1.   Daily pattern of plasma lysine 15N enrichment on days 10-11 throughout 24-h hourly oral intake of 0.14 mg 15N · kg-1 · h-1, either as 15NH4Cl (black-triangle) or [15N2]urea () after 6 days of intake of the respective 15N tracer. Values are means ± SD for 6 subjects. Results are expressed as difference in enrichment [15N atom percent excess (APE)] from initial value on experimental days 3 and 4 (considered zero enrichment baseline). Feeding began with small meals at 0600 (720 min) and was continued until 1500 (1,260 min), providing in total (tracer included) 45 mg dietary lysine · kg-1 · day-1.

During the 24-h tracer protocol on days 10 to 11, the mean plateau enrichments of plasma [15N]lysine after 15NH4Cl intake differed slightly between the fasted and fed states (0.0176 ± 0.0087 vs. 0.0118 ± 0.0034 APE; NS; Fig. 1; Table 3). After [15N2]urea consumption, fasted and fed state plasma [15N]lysine enrichments were not different (0.0056 ± 0.0035 vs. 0.0059 ± 0.0053 APE; NS; Fig. 1; Table 3).

Comparison of fasted-state plateau enrichments (during the 24-h tracer protocol), after [15N2]urea and 15NH4Cl tracer intake, respectively, showed significantly higher values with the 15NH4Cl tracer, although variability (25-100%) was high (P < 0.05; Table 3). During the fed state, values after 15NH4Cl tracer intake also tended to be higher than after [15N2]urea, but this difference did not reach statistical significance.

Plasma [15N]lysine enrichment of ileostomates on day 10 was somewhat but not significantly lower than that in MIT subjects (Table 3). However, it should be noted that the ileostomates had consumed the 15NH4Cl tracer for only 5 days and had not received tracer during the night before the final blood sample was drawn for analysis.

The 15N enrichment of microbial lysine from feces and ileal fluid increased steadily after initiation of the tracer (Fig. 2), and after 3 days following the start of 15N intake an apparent plateau was reached (mean slope 0.02; NS; Fig. 2). The [15N]lysine enrichment with 15NH4Cl as tracer was higher than that with [15N2]urea, but the difference did not reach statistical significance (Table 3). In comparison, the enrichment of ileal microbial lysine was significantly lower than that for fecal microbial lysine in MIT subjects after administration of 15NH4Cl (P < 0.001; Fig. 2; Table 3). At the end of the experimental period, after 15NH4Cl and [15N2]urea intake, respectively, fecal microbial lysine was on average 15 and 25 times more enriched than plasma lysine (Table 3). In contrast, ileal microbial lysine enrichment was 4.5 times higher than plasma lysine enrichment in the ileostomates after 15NH4Cl administration.


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Fig. 2.   Microbial fecal (15NH4Cl: black-triangle; [15N2]urea: ) and ileal (15NH4Cl: open circle ) lysine enrichments throughout experimental time frame. Daily 15N intake started on day 5. Results are expressed as difference in enrichment (15N APE) from initial value on experimental days 3 and 4 (considered zero enrichment baseline). Samples were not obtained on day 11. Note that on some days (days 5, 7, and 9), fecal samples of only 4 MIT subjects could be obtained and that ileostomates ingested 13% more 15N · kg-1 · day-1 than MIT subjects because of their lower body weight.

Daily total nitrogen output via ileal fluid (621 ± 310 g/day) was 1.51 ± 0.45 (SD) g on day 9. The lysine content in ileal microbial protein was 4.75 g/16 g N. Hence, assuming that, as in the pig, 50% of total ileal nitrogen was of microbial origin (43) and that the lysine concentration in endogenous protein is similar to mixed body protein (~8%) (27), ~0.22 g lysine of microbial origin and ~0.38 g lysine of endogenous origin were lost daily under the current experimental conditions. This corresponds to an approximate ileal total loss of 8.5 mg lysine · kg-1 · day-1.

The 15N ratio of plasma free lysine to microbial lysine did not differ between the two 15N tracers ingested (Table 4). However, if the plasma lysine enrichment of the MIT subjects given the 15NH4Cl tracer was related to ileal microbial lysine enrichment, the ratio was higher (Table 4; fasted state: P < 0.01; fed state: P < 0.001). Computed for the fasted state only, in ileostomates, when ileal microbial lysine enrichment is used as a precursor, the apparent fractional contribution of microbial lysine to plasma lysine was lower than in MIT subjects (0.21 vs. 0.44; Table 4).

                              
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Table 4.   Ratio of 15N enrichment in plasma free lysine to microbial protein lysine after [15N2]urea and 15NH4Cl intake, calculated either from fecal or ileal microbial lysine enrichment

Lysine kinetics. As expected, the course of 13CO2 background enrichment in three subjects in the tracer-free study after consumption of the experimental diet for 10 days changed only minimally with the experimental diet (typically -0.0008 APE fasted state; -0.0003 APE fed state). The course of 13CO2 enrichment and 13CO2 production throughout the 24-h tracer protocol for the 15NH4Cl and [15N2]urea intakes was apparently different, although this did not reach statistical significance at any given interval (Fig. 3, A and B).




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Fig. 3.   Pattern of change during a 24-h primed continuous intravenous infusion of [1-13C]lysine (infusion rate 3.5 µmol · kg-1 · h-1). Values are 13C enrichment of expired carbon dioxide (A), µmol 13CO2 expired (B), and lysine oxidation (C) for each 30-min interval throughout 24-h tracer protocol (means ± SD of 4 subjects). Feeding began with small meals at 0600 (720 min) and was continued until 1500 (1,260 min), providing in total (tracer included) 45 mg dietary lysine · kg-1 · day-1. black-triangle, After 15NH4Cl intake; , after [15N2]urea intake.

The lysine oxidation rates, however, were significantly higher with 15NH4Cl tracer intake for the intervals 540-720 min (fasted state) and 1,110-1,200 min (fed state), respectively (P < 0.05; Fig. 3C). Total lysine oxidation measured over 24 h was 34.2 mg · kg-1 · day-1 during 15NH4Cl tracer intake and 19.2 when [15N2]urea was given (Table 5). Lysine turnover, estimated with the intravenous [13C]lysine tracer, was higher when the 15NH4Cl tracer was given than it was with [15N2]urea, but this was only significant for the fed state (Table 5). When lysine turnover was estimated with the orally administered [2H2]lysine, the same difference between the 15N tracers was seen in the fed but not the fasted state. Lysine turnover estimated with the oral tracer was higher (Table 5). Lysine splanchnic uptake was estimated not to differ between 15N tracers.

                              
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Table 5.   24-h Lysine kinetics in MIT subjects receiving 15NH4Cl and [15N2]urea, respectively, in small oral doses during continuous intravenous infusion of [1-13C]lysine and hourly oral administration of [6,6-2H2]lysine

If the ratio of plasma [15N]lysine to microbial [15N]lysine is used as a direct measure of the fraction of circulating lysine that is derived from the intestinal microflora, then the estimate of the contribution of fecal microbial lysine to lysine turnover, measured by [1-13C]lysine per day with the 15NH4Cl data, was 28.9 ± 29.7 mg · kg-1 · day-1, and with [15N2]urea it was 11.7 ± 7.5 mg · kg-1 · day-1 (NS; 4 subjects). However, when the microbial contribution was calculated on the basis of ileal microbial lysine enrichment with the 15NH4Cl data, it was 129.6 ± 85.6 mg · kg-1 · day-1 for the MIT subjects. If a similar lysine turnover in ileostomates is assumed, a possible 67.9 ± 13.7 mg · kg-1 · day-1 lysine was derived from microbial sources. The quantitative, nutritional meaning of the estimates will be presented in DISCUSSION.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although the present data confirm the less extensive, earlier investigations by others on body [15N]lysine labeling after administration of 15N-labeled nitrogen precursors (15, 38), a major question posed at the outset of this investigation was the quantitative extent to which microbially synthesized lysine was made available for metabolism in host tissues and its net contribution to the lysine economy of the individual subject. This question turns out to be far more difficult to answer from the present data than we had anticipated. After methodological and design aspects are addressed, the various reasons for this will be discussed.

We have measured plasma and microbial [15N]lysine without consideration of its enantiomeric form. A study investigating D-amino acid contents in cultures of several bacteria revealed that D-lysine was not detectable, whereas, for example, D-alanine and D-aspartic acid ranged between 8 and 80% and 2 and 60% of total amino acids, respectively (5). In serum of adult rats ingesting standard food, no D-lysine was detected (37). Also, to the best of our knowledge, D-lysine in human plasma has not been reported in the literature, although other plasma amino acids (serine, alanine, aspartic acid, phenylalanine, tyrosine) have been shown to be present in D form (2, 28). Therefore, we do not consider that a significant contribution of D-lysine is likely here. Nevertheless, if this were not the case, then we would have overestimated the availability of microbially derived lysine for host utilization.

The reason for including ileostomates in this study is that, although microbial activity is associated principally with the large intestine, it is by no means confined solely to that part of the gastrointestinal tract (8, 13), and the flora of the lower ileum is qualitatively similar to that of feces (8). Given that the major sites of amino acid absorption are in the small intestine, it was important to determine the 15N enrichment in the microbial protein of digesta as close as possible to those sites and where there was no functional large intestine to complicate interpretation of the results. Nevertheless, we recognize that ileal fluid of ileostomates may not be the same as the contents of an intact ileum (4, 16, 29, 41); however, our assessment, for the present purpose, was that conventional ileostomy resembles the environment in the intact ileum more than it does the colon, and this gave us the rationale for comparing and contrasting the data from the two groups of subjects studied here.

To help readers understand our results and the discussion that follows, we present a scheme in Fig. 4 showing that 15N from either 15NH4Cl or [15N2]urea, after breakdown by microbial urease, is incorporated either directly or indirectly into microbial lysine (and other microbial amino acids) in the small intestine. This depicts the complexity of the pathways that lysine might take within the intestine and between the intestine and host tissues, which introduces difficulties in readily interpreting the quantitative significance of our findings.


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Fig. 4.   Schematic outline of metabolism of microbial lysine in gastrointestinal tract.

The 15N from nonspecific nitrogen sources is returned to the intestinal tract in the 15N-labeled amino acids in endogenous secretions (pancreatic, biliary, mucosal) and as 15N urea (Fig. 4). Intravenous infusion of 15N-labeled amino acids is followed by labeled plasma amino acids appearing rapidly in the jejunal fluids and pancreatic secretions (14, 21). The quantity of endogenous protein that is recycled in the intestine makes it a potentially significant source of nitrogen for microbial growth. In pigs, 90% of all endogenous nitrogen secreted into the gut was found to be reabsorbed (19). Although these experiments do not explain the involvement of the enteric flora in this process, it is probable that microbial proteolytic activity is involved because mucins and some other digestive secretions are resistant to mammalian digestive enzymes.

The differences we observed between ileal and fecal microbial protein in [15N]lysine enrichment are in accord with investigations in the pig (40) showing that, after the pigs were fed 15NH4Cl for 10 days, [15N]lysine enrichment increased dramatically from the ileum to the caecum. Also, the difference in fecal microbial and plasma lysine 15N enrichment seen in this study between 15NH4Cl and [15N2]urea confirms earlier results in the pig (25) and is presumably due, in part, to the greater retention of 15N label from ammonium chloride (24a). However, because of high interindividual variation the difference did not always reach statistical significance. If allowance is made for the different 15N retention, then the microbial enrichments would not appear to be different, although an additional reason for the different enrichments might be differences in ammonia nitrogen and urea nitrogen metabolism by the microflora (35, 26).

Using the 15N enrichment of fecal microbial lysine with 15NH4Cl as the label, we estimated an uptake of ~29 mg · kg-1 · day-1; with [15N2]urea, the estimate was 12 mg · kg-1 · day-1, although this was not statistically different. It is obvious, however, that the digesta undergo many changes during passage from the duodenum to the rectum, and it is unlikely that the [15N]lysine in microbial protein from feces is truly representative of that occurring at sites of amino acid absorption in the intestine. It was for that reason that we sampled ileal digesta from ileostomates as being closer to the sites of absorption. The corresponding estimate of microbial lysine absorption for the ileostomates (when the same rate of lysine turnover is assumed as in the MIT subjects) is ~68 mg · kg-1 · day-1. If the 15N enrichment of lysine in ileal digesta were assumed to represent the 15N enrichment of absorbed lysine in the MIT subjects given 15NH4Cl, their uptake of microbial lysine would be calculated to be ~130 mg · kg-1 · day-1, a value that exceeds by ~10-fold both the apparent dietary requirement and, therefore, the irreversible loss of body lysine (12).

In considering the plausibility of these different estimates of microbial lysine uptake, one must consider that they refer to the gross uptake of amino acid synthesized by the microflora and do not necessarily represent a net increase of that magnitude in the total lysine available for metabolism. To the extent that 1) the growth of the microbes that give rise to the labeled lysine is supported by the degradation of endogenous protein (Fig. 4) and 2) luminal amino acids of endogenous origin are oxidized by intestinal tissues, then the microbially derived lysine in plasma can be seen as part of the normal mechanism by which endogenous nitrogen and amino acids are recycled, rather than microbial amino acids serving as a net source of amino acids, which are additional to those supplied in the diet. Support for this view comes from previous [1-13C]leucine oxidation studies (10, 11), where there is an excellent concordance between both predicted and measured protein oxidation when estimated from leucine oxidation and N excretion values, which would not have been the case if there had been a significant net uptake of leucine from the gut microflora. There seems little reason to expect, if there were a significant entry of microbially derived lysine into body tissues, that this would not also apply to the other nutritionally indispensable amino acids, including leucine.

Although the true 15N enrichment of the labeled lysine that is actually absorbed might be closer to that of the ileal digesta protein than to that of the feces, it could quite conceivably be higher (30). Furthermore, if 15N-labeled substrates entering the gut lumen were used preferentially by microbes in juxtaposition to the intestinal wall, with turnover and release of their constituent proteins and amino acids in that spatial domain, then the 15N enrichment of lysine being absorbed from the gastrointestinal tract would not be accurately reflected by either the ileal or fecal microbial protein-bound lysine.

The loss of lysine at the terminal ileum was estimated in the present study to be ~8.5 mg · kg-1 · day-1. Presumably there is also loss of endogenous lysine due to bacterial and tissue intestinal oxidative metabolism (Fig. 4), as indicated by the measured first-pass lysine extraction by splanchnic tissues (and microflora) of ~25% of the dietary lysine (Table 5). It could be, therefore, that any entry of lysine of microbial origin helps to counteract the endogenous lysine losses, rather than adding in a net way to the total exogenous (diet) input of lysine.

The lysine oxidation rates measured in this study are considerably lower than the ~45 mg · kg-1 · day-1 level of lysine intake, indicating that we have significantly underestimated the true whole body lysine oxidation rate. This is in accord with our previous oral/intravenous [1-13C]lysine tracer studies (9). Lysine turnover rates were in the same range as reported earlier and were somewhat higher for the orally adminstered lysine tracer (9). It was surprising, however, that there were differences between various measures of lysine kinetics between the two different studies in which either [15N2]urea or 15NH4Cl was used as a tracer (Table 5). There are a number of possible explanations. First, it could be due to the 15N tracers per se. However, this seems unlikely, because the amount of nitrogen supplied with the two 15N tracers was equal. If account is taken of the 1.6-fold higher 15N retention with 15NH4Cl compared with [15N2]urea (24a), and it is assumed that all of the additional nitrogen retained was used to produce microbial lysine, this would amount to a difference between the two tracers of only 3 mg · kg-1 · day-1. This does not explain the observed difference in lysine kinetics between the two tracers (Table 5). Second, although administration of NH4Cl causes acidosis and increases protein breakdown and amino acid oxidation (34), this occurs at doses that are severalfold higher than the total 1 g/day dose used in this study. Third, and on the basis of the foregoing comments, the observed differences would appear to be due to experimental and biological factors that cannot be identified at this time.

In conclusion, to reliably estimate the quantitative contribution made by lysine (or other indispensable amino acids) of bacterial origin to host metabolism, use of the present paradigm seems doubtful. Although our data do suggest a potentially significant nutritional uptake into body tissues of amino acids of microbial origin, the quantification of this uptake, and its net contribution to the amino acid economy, require a better understanding of nitrogen and carbon transactions of amino acids and related compounds between the intestinal tissues and the microflora within the gastrointestinal tract.


    ACKNOWLEDGEMENTS

We are grateful to the staff of the MIT Clinical Research Center and the Unit Protein Metabolism of the German Institute of Nutrition for skillful technical assistance.


    FOOTNOTES

This research was supported by National Institutes of Health Grants DK-42101, P-30-DK-40561, and RR-88, and a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany.

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 correspondence and reprint requests: C. C. Metges, German Institute of Human Nutrition, Arthur-Scheunert-Allee 114-116, 14558 Bergholz-Rehbrücke, Germany (E-mail: metges{at}www.dife.de).

Received 18 November 1998; accepted in final form 24 May 1999.


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