Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Gastroenterology and Hepatology
Quantitation of Colonic Luminal Synthesis of Butyric Acid in Piglets
Kien, C. Lawrence*†; Chang, J. C.*; Cooper, James R.*
*Children's Research Institute and †the Department of Pediatrics, The Ohio State University, Columbus, Ohio, U.S.A.
Received October 18, 2001; accepted April 11, 2002.
Address correspondence and reprint requests to C. Lawrence Kien, MD, PhD, Department of Pediatrics, Gastroenterology and Nutrition Division,
University of Texas Medical Branch, Children's Hospital, 301 University Blvd. Galveston, TX 77555–0352, U.S.A. (e-mail: email@example.com).
This study was supported by grants from the Crohn's and Colitis Foundation of America, Inc., New York, New York, and the Children's Research Institute, Columbus, Ohio. Dr. Kien was supported in part by DK61775 during the editorial process.
Objectives: Butyric acid, synthesized via bacterial fermentation in colonic lumen, may play an important role in the nutrition of the colonic mucosa. Since disaccharides, especially lactose, are the principal dietary carbohydrates during infancy, it is important to determine if their fermentation is associated with butyric acid synthesis. The objective of this paper is to describe a newly developed stable isotope method for quantifying butyric acid synthesis in the colonic lumen and to demonstrate its application during cecal infusions of lactose and lactulose in piglets.
Methods: Nine piglets aged 21 to 30 days were studied during acute anesthesia. The 13C enrichment of butyric acid was monitored in the portal vein before and during a 120 minutes cecal infusion of [1–13C]-butyric acid and either unlabeled lactose (N = 4) or lactulose (N = 5).
Results: The luminal synthesis of BA (μmol x kg−1 x min−1) (Mean ± S.D.) was respectively 1.5 ± 0.9 and 1.2 ± 0.6 during lactulose and lactose infusion.
Conclusions: This study provides new quantitative data on in vivo butyric acid production in the mammalian colon.
Butyric acid (BA) is a product of bacterial fermentation of carbohydrate in the rumen of multigastric animals and in the colon of omnivores such as swine or man (1). In cultured, colonic neoplastic cells, BA causes arrest of the cell cycle and stimulates terminal cellular differentiation (2–5), and it induces apoptosis (5,6). However, there is controversy about how these cellular and molecular effects of BA in cultured cells relate to in vivo effects of BA in the colon lumen. Indeed, the literature suggests that BA within the colonic lumen causes decreased apoptosis and increased cell proliferation (7,8), the opposite effect of BA in cultured cells (2–6).
A high colonic luminal concentration of lactate, acetate, BA, or other short chain fatty acids (SCFA) may cause mucosal injury (9,10). On the other hand, BA is a preferential nutrient for energy production by the colonocyte (11–13), and it has been suggested that deficient supply or metabolism of BA may contribute to the etiology of diversion colitis (14), starvation colitis (13), and even ulcerative colitis (15–17). We have hypothesized that the level of uptake of BA by colonocytes may determine its effects on the cell cycle of colonocytes and may determine whether BA serves a potentially vital nutritional role versus causing toxicity when present in excess (8,18). Since BA is readily absorbed by the colonic mucosa (19), the rate of colonic mucosal uptake of BA may directly relate to its level of synthesis in the lumen (8,18). To explore this hypothesis, it is important to be able to quantify the rate of luminal synthesis of BA, which, under steady-state conditions, will equal its removal from the lumen by mucosal absorption (uptake), bacterial metabolism, or fecal excretion.
Since BA is almost completely absorbed by the colon under most conditions and the colonic oxidation of BA is very efficient, assessment of luminal BA concentration or arterial-venous concentration differences across the colon may not be reliable indices of BA production, under all conditions (20). In vitro fermentation studies of colonic fluid or feces also may not be physiologically relevant because of the effects of end-product inhibition (20), and indeed, such studies have provided contradictory information concerning whether disaccharides can be fermented to BA (21,22). Therefore, we have developed stable isotope methodology to measure the rate of fermentation of carbohydrates to short chain fatty acids and to measure the luminal production of BA (20,23).
Starch or fiber fermentation probably serves to provide BA in the adult human, but in the young infant, malabsorption and then fermentation of disaccharides, principally lactose, may be the major mechanism for BA formation (8,24,25). To study carbohydrate malabsorption in infants, we have fed piglets graded intakes of lactulose, a disaccharide of fructose and galactose, which is not digested by mammalian enzymes (8).
This paper describes a new stable isotope method for quantifying colonic luminal bacterial synthesis of BA and its application during simulated disaccharide malabsorption using cecal lactose or lactulose infusions. We used lactose because it is the disaccharide present in mammalian milks and could be a substrate for colonic fermentation under normal conditions in infants as well as during malabsorption states (24,25). Lactulose was studied primarily because it is used experimentally to produce controlled, graded degrees of disaccharide malabsorption (8), but it also is recommended as a stool-softening agent (osmotic laxative) in infants and children (26). We hypothesized that BA production would be quantifiable during `malabsorption` of either lactose or lactulose, in contrast to some data from in vitro incubations (21). We also hypothesized that the luminal production of BA would be similar from either lactose or lactulose.
MATERIALS AND METHODS
The study was approved by our institution's animal use and care committee. Yorkshire/Hampshire pigs (aged 21–30 d) were studied in the fed state. The piglets were either completely sow-reared until the day of study or were fed sow milk replacement formula (Ross Products Division of Abbott Laboratories Inc., Columbus, OH) from about the age of 10 days. Each of the tracer studies described here was conducted while the piglets were anesthetized with isoflurane, after sedation was attained using a combination of tiletamine HCL and zolazepam HCl (7.5 mg/kg i.m., Telazol, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (5 mg/kg, i.m., Rompun, Bayer Corp., Shawnee Mission, KS). A catheter for tracer infusion was inserted into the cecum, and a catheter for blood removal was inserted into the portal vein (PV) (27,28).
Experimental Design and Calculations
In 9 piglets, over a 120 minute period, we conducted cecal infusions of [1–13C]-BA and unlabeled lactose (N = 4) or Lactulose (N = 5) (2.52 g/h or 123 μmol/min). The isotope was infused at an average rate of 2.32 μmol x kg−1 x min−1 (Prime/Minute Infusion Rate = 20/1). Blood was sampled from the PV before the isotope infusion and at 90, 100, 110, and 120 minutes after the beginning of the infusion.
We used a single isotope dilution model for assessing the rate of synthesis (production) of BA in the colonic lumen. The rate of dilution of the isotope is equated to the rate of production of BA (Ra BA) according to the following equation:EQUATION
where d equals the measured molar isotope infusion rate and IE BA is the plateau isotopic enrichment of BA in the PV during the quasi steady state from 90 to 120 minutes (29). IE BA, expressed as moles fraction excess in the equation, is equal to moles percent excess (MPE) divided by 100. MPE was measured using a previously described assay (30).
Theoretical Aspects of the Stable Isotope Model for Assessing Colonic Luminal Production of BA (Colonic Luminal Synthesis)
In contrast to studies involving quantitation of acetate production via fermentation (20), BA synthesis in the colon can be quantified using a single isotope dilution model. Our previous study of endogenous butyric acid synthesis showed that BA was detected in the peripheral arterial circulation and was produced by tissues that were not drained by the PV (23). However, this paper also showed that BA was not detected in the PV when the colon was resected or its complete venous drainage resected. In experiments where 99% enriched BA was infused into an intestinal mesenteric vein, the 13C enrichment of BA in the PV was 96% when the colon vasculature was ligated or resected (23) and 94%, when the colonic vasculature was intact but the piglet was fasted (unpublished pilot studies). Thus, the very small amount of BA that enters the peripheral arterial circulation from endogenous sources does not reach the PV. These previous data indicate that that any net unlabeled BA detected in the PV is derived from the colon lumen, where it is synthesized by bacterial fermentation of carbohydrate. This means that when labeled BA is infused into the cecum, as in the present study, the enrichment of BA in the PV will equal the enrichment of the BA in the colonic lumen at the site of synthesis of BA by bacteria. While undoubtedly much of the BA produced in this way via bacterial fermentation will be metabolized by the colonic mucosa, this will not affect the isotopic enrichment of BA in the PV, only the concentration. Thus, if one infuses BA tracer into the cecum or colon and samples the isotopic enrichment in the PV, one can measure the production of BA within the colon (luminal synthesis)(23).
Table 1 shows the individual data relating to the age, cecal carbohydrate infusion rate, and the rate of BA synthesis expressed both as a rate and as a fraction of the cecal carbohydrate infusion rate.
Molar enrichment of BA was high in this study, ranging from 57.2 to 81.7 MPE in the lactose studies and 46.8 to 78.5 MPE in the lactulose studies. The coefficient of variation in MPE during the quasi steady state ranged from 2.1 to 4.0% for the lactulose studies and 0.1 to 6.1% for the lactose studies. The luminal synthesis of BA (μmol x kg−1 x min−1) (Mean ± S.D.) was respectively 1.5 ± 0.9 and 1.2 ± 0.6 during cecal infusion of lactulose and lactose (P > 0.05). Expressed as the molar percentage of the cecal lactose or lactulose infusion rate, BA synthesis was 8.5 ± 5.2% in the lactulose group and 6.6 ± 2.7% in the lactose group (P > 0.05).
This study provides the first quantitative data on in vivo BA synthesis within the lumen of the mammalian colon. These results also indicate the feasibility of conducting “steady state”, isotopic tracer studies of colonic luminal synthesis of BA. In a previous study (23), we found evidence for endogenous (mammalian) synthesis of BA, and, in the course of that study, we also found indirect, qualitative evidence for BA synthesis in the colonic lumen during cecal infusions of disaccharide. In this study, we applied our isotopic technique to a model of colonic fermentation of lactulose and lactose because we are interested in using lactulose to simulate small intestinal malabsorption of lactose or other disaccharides (8). The present data show that during cecal infusion of lactose or lactulose, production of BA via bacterial fermentation does occur in the young piglet. The rate of production of BA was very small during the co-infusion into the colon of either disaccharide. Expressed as a percentage of the cecal lactose infusion rate, the rate of BA synthesis averaged 8.5% during the lactulose infusion and 6.6% during the lactose infusion. In preliminary studies using a dual stable isotope technique, we found that 17 to 20% of lactose or glucose was fermented to acetate (20). The difference between lactose and lactulose in the mean rate of production of BA was 0.3 μmol x kg−1 x min−1. At present, we cannot determine precisely the biologic significance of this difference; therefore, there is no inherent relevance to assessing the power of this comparison. However, studies by our laboratory in two piglets suggest that when BA is infused into the cecum at approximately the mean rate of production described in this study, neutrophil infiltration of the cecal crypts occurred within 120 minutes, during which there was no associated change in cecal pH. Therefore, this level of BA production or even the small mean difference between the values observed with the two disaccharides could have biologic relevance. However, future studies of chronically cannulated, awake piglets are necessary, in our view to further delineate biologically relevant rates of colonic luminal BA production (20).
The isotopic tracer infusion we used in this study was patterned after that used in previous studies of acetate production in the colon lumen (20). However, since the luminal production rate of butyric acid is lower than that of acetate, the molar enrichment of BA approximated or exceeded 50%, and in all but one tracer infusion study reported here, the average tracer infusion rate exceeded the mean rate of production of BA in the lumen. In mammals, 13C, even at 60 to 70% enrichment, is unlikely to have an isotopic effect of biologic significance (29). There also is no reason to believe, a priori, that the 120-minute BA infusion (labeled or unlabeled) would affect production of BA by the bacteria or the composition of bacterial flora, which is an important potential determinant of overall BA production in the lumen (10). Nevertheless, future applications of this technique under more physiological conditions (e.g., chronically cannulated, awake condition) should also involve true tracer doses, achieving molar enrichment of BA less than 10%. Since the circulating concentration of BA, even in the PV, is quite low, achievement of this goal could require the use of less enriched tracer. If under such conditions, the measured rate of BA production (corrected for the exogenous infusion of tracer ± tracee) approximated the rate of exogenous infusion of BA, then dose response studies would be required to formally exclude any effect of exogenous BA on luminal production of BA.
Studies using this technique eventually could be used to assess whether the rate of production of BA in the colon is affected by the amount of fermentable carbohydrate reaching the colon (disaccharides, fiber, prebiotics, etc.) (8). A previous study involving incubations of feces from adult humans suggested that lactose, lactulose, and some other disaccharides and monosaccharides are primarily fermented to acetate with no evidence that net BA production can occur (21). However, another more recent human fecal incubation study suggested that lactulose and cornstarch produced more BA than rhamnose, or guar (22). By conducting controlled studies in experimental animals of the relationship between the rate of appearance of fermentable carbohydrate into the colon and the rate of synthesis of BA in the colonic lumen (“dose-response studies”), it may be possible to derive an accurate indication of how different carbohydrates (and perhaps different types of bacteria) affect luminal synthesis of BA. Such studies could greatly enhance our fundamental knowledge about the role of BA in gut biology.
As explained in the Introduction, either under- or over-production of BA could have detrimental effects on `colonic health` (8). Our technique could be used in future research to further our understanding of how the colonic luminal supply of BA affects the colonic mucosa. Ultimately, such studies will be necessary to interpret the biologic significance of the rates of luminal synthesis of BA measured in this study. For example, perhaps there is a minimal rate of BA production (and colonic mucosal uptake) that constitutes the “requirement” by colonic mucosa for BA, but a higher rate might lead to toxicity.
In conclusion, this paper provides evidence that malabsorption of lactose and perhaps other carbohydrates may be associated with colonic luminal synthesis of BA, which may provide a source of BA to the colonocyte (24,25,31).
This study was supported by grants from the Crohn's and Colitis Foundation of America, Inc., New York, NY and the Children's Research Institute, Columbus, OH. Dr. Kien was supported in part by DK61775 during the editorial process. The first author is grateful to his colleague, R.R. Wolfe, PhD, for reviewing the manuscript before publication. We are grateful to Henri Brunengraber, MD, PhD, and his laboratory for performing the mass spectrometry analyses for us. We also acknowledge the technical assistance of Jonathan Lash, and the editorial assistance of Rita Porter.
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© 2002 Lippincott Williams & Wilkins, Inc.
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