Tiletamine

ABSTRACT. Backgound: Intestinal malabsorption of disaccharides or oligosaccharides occurs normally in preterm infants, the majority of the world's adult population when ingesting dairy products, and in sick patients with abnormal intestinal motility or intestinal damage. Although there may be benefit to colonic fermentation, there also could be mucosal damage from excessive fermentation. Moreover, excess fluid in the colon also may impair fermentation. There are few data that address the effects of moderate amounts of carbohydrate, such as 50% of intake, reaching the colon. We addressed the hypothesis that normal weight gain and stool characteristics would occur during disaccharide malabsorption approximating 50% of intake. Methods: Twelve piglets (aged 17 days) were randomized to receive control sow milk replacement formula (CON; lactose 60 g/L) or CON modified so that lactulose replaced 50% of the lactose (LAC). During a 5- to 7-day period, weight gain and fecal characteristics were observed before autopsy for cecal histology and determination of cecal cell proliferation using bromodeoxyuridine. Results: Neither group developed diarrhea. In LAC and CON, formula intakes (g/kg/d) were similar (279 + or - 30 and 280 + or - 35), as were the ratios of weight gain:fofmula intake (g/mL): 0.23 + or - 0.04 and 0.22 + or - 0.04. Mean cecal cell proliferation was not diminished in the group receiving lactulose (p > .05). Conclusion: These data suggest that moderate (50%) disaccharide malabsorption per se does not cause diarrhea and does not impair dietary energy use or colonic cell proliferation. (Journal of Parenteral and Enteral Nutrition 27:323-326, 2003)

Intestinal malabsorption of disaccharides or oligosaccharides occurs normally in preterm infants, the majority of the world's adult population when ingesting dairy products, and in sick patients with abnormal intestinal motility or intestinal damage.1-4 Although there may be benefit to colonic fermentation, there also could be mucosal damage from excessive fermentation.5,6 Moreover, excess fluid in the colon also may impair fermentation.4 There are few data that address the effects of moderate amounts of carbohydrate, such as 50% of intake, reaching the colon. Our studies of lactose digestion and lactose fermentation in preterm infants suggested that > 50% of lactose might reach the colon (ie, malabsorbed) without causing diarrhea or significantly affecting weight gain or N balance.7-10 However, in these studies, the degree of disaccharide maldigestion was assessed using stable isotope tracer techniques or breath H^sub 2^ studies. As a further test of the hypothesis that small intestinal malabsorption of even 50% of dietary disaccharide will not cause diarrhea nor appreciably affect growth, we constructed the experiment described in this paper in which lactulose was substituted for lactose at a level equal to 50% of the lactose concentration. Because in a recent study4 we showed that more severe disaccharide malabsorption caused colitis and decreased cell proliferation in the colonie mucosa, we also assessed the effects of disaccharide malabsorption on cecal mucosal structure and cell proliferation.

MATERIALS AND METHODS

Animals, Feedings, and Design

Twelve standard Yorkshire/Hampshire piglets were used. The Children's Hospital Animal Care and Use Committee approved the research protocol. On day 7 of life, the piglets were transported from the pig farm to the laboratory. The piglets then were fed a carbohydrate-free sow milk replacement formula (Ross Products Division of Abbott Laboratories, Inc, Columbus, OH) supplemented with, lactose (60 g/L) to accustom them to artificial nipple feeding. On day 17 of life, the piglets were randomized to 2 formula study groups: (1) a control group (CON) that continued to receive the sow milk replacement formula with 60 g lactose/L and (2) a lactulose substituted group (LAC) in which the carbohydrates lactulose and lactose were added to achieve a concentration of 30 g/L of each (corrected for dilution by the volume of lactulose syrup added). The piglets were studied in groups of 2 over a 7-day period. Each of the piglets in the CON group was pair-fed to a corresponding piglet in the LAC group.

Lactulose is a disaccharide of fructose and galactose that cannot be digested by mammalian enzymes. Experimentally, it permits one to simulate the effects of sugar maldigestion, both in the colon where it likely will be fermented in a very similar way to lactose11,12 and in the small intestine where fermentation also may occur (theoretically) and where undigested sugar also could exert an osmotic effect and could increase water flux through the bowel.

During the study, body weight, formula intake, and stool characteristics were monitored. Observation of 1 loose or watery stool was counted as diarrhea for that day. We then compared the groups in terms of the number of days of loose stools/diarrhea. At the time of colon biopsy and sacrifice on day 24 of age, breath was collected from the endotracheal tube for determination of H^sub 2^ CH^sub 4^, and CO2 concentrations (Quintron Instrument Corp, Milwaukee, WI) in order to assess the fermentation of carbohydrate, especially lactulose, in the colon.13 At the same time, cecal pH was measured (Model Omega PHH-253; Omega Technologies Co, Stamford, CT), and tissue was removed for determination of cecal cell proliferation and assessment of colonic injury or inflammation.

At aged 24 days, the 12 piglets were anesthetized with xylazine (4 mg/kg intramuscular [IM]; Rompun; Bayer Corp, Shawnee Mission, KS) and a combination of tiletamine HCl and zolazepam HCl (6 mg/kg IM; Telazol; Fort Dodge Laboratories, Inc, Fort Dodge, IA). Bromodeoxyuridine (BrdU) was injected into an ear vein.4 Two hours later, the piglets were reanesthetized with the Telazol/Rompun combination and tissues were obtained for histology before sacrifice.

Histology

The tissues for light microscopy were fixed within a few seconds in 10% neutral buffered formalin, embedded in paraffin, and then sectioned at 5 [mu]m. The slides were stained with hematoxylin-eosin for histologic assessment of injury or inflammation using a previously published scoring system:14 grade 0, no abnormalities; 1, crypt dilatation and mucin retention; 2, cryptitis or 2 crypt abscesses; 3, multiple crypt abscesses; 4, fibrinopurulent debris and mucosal ulceration; 5, transluminal necrosis or perforation. Unstained, paraffin-embedded slides were stained for BrdU labeling, and proliferation index (PI) for the total cecal crypt and for the lower 40% of the crypt was then estimated as previously described and expressed as percentage.4

Statistics

For assessing differences between groups, we used the 2-sample t test. All results are expressed as mean + or - SD. The level of probability selected for statistical significance was .05.

RESULTS

Neither group developed diarrhea or histologic evidence of colitis; the histologic injury score was 0 in all piglets. Breath concentration (ppm/5% CO2) of H^sub 2^ was not significantly different in the LAC and CON groups, but breath concentration of methane was 95% higher in LAC compared with CON (p = .055; Table). Cecal pH was approximately equal in the LAC (5.5 + or - 0.2) and CON (5.6 + or - 0.2) groups (p > .05).

In LAC and CON, formula intakes were similar, as were the ratios of weight gain:formula intake (g/mL; Table).

For the total cecal crypt, the PI (%) was not different in the LAC group (20 + or - 11) compared with the CON group (14 + or - 7; p = .28; Table). For the lower 40% of the crypt, the PI also was not different in the LAC group (40 + or - 18) compared with CON (29 + or - 11; p = .24).

DISCUSSION

These data suggest that in contrast to a more extreme form of experimental disaccharide malabsorption (87% of normal intake),4 a more moderate degree of malabsorption (50%) does not cause diarrhea or decreased cell proliferation in the colon. The present study design more closely approximates the degree of lactose malabsorption observed in the preterm infant.10,15 Our previous data suggest that colonic salvage may compensate for failure of small intestinal digestion of lactose in some infants, but despite the many experimental approaches that can be used to show this,15 none of them can be as direct as the one we could ethically use in piglets, namely, replacing lactose with lactulose to simulate 50% maldigestion of sugar. The present results indicate that, as predicted in the preterm infant, colonic salvage of carbohydrate can play a significant role in compensating for intestinal carbohydrate malabsorption.

In the LAC group, the percentage of the ingested kilocalories from malabsorbed sugar (lactulose) relative to the total energy ingested by the piglet was 12%, or stated another way, these piglets effectively experienced a 12% deficit in dietary energy digestible in the small intestine. This value can be compared with an average of 9% in preterm infants we have studied (maximum 30%) who do not manifest diarrhea10,16 and of 26% and 30%, respectively, in human adults and piglets with severe diarrhea associated with lactulose ingestion.4,17 Fermentation in the colon will salvage some, but not all, of the carbohydrate energy reaching the colon,7 but lack of difference in weight change in the LAC group also could relate to physiologic adjustments (change in basal energy expenditure) or behavioral changes (less physical activity). In addition, one also could argue that there are limitations in using weight change over 7 days to assess energy balance. In a study of piglets fed diets of different energy densities for 1 week at a comparable period of life, Zijlstra et al18 observed a 74% decrease in daily weight gain associated with a feeding regimen that resulted in an 8% decrease in energy density of the feeding but a 61% decrease in total daily energy intake (because of lower feed intake). Although one cannot directly extrapolate these data to a linear model, there was in their study approximately a 1% decrease in weight gain per 1% decrease in metabolizable energy intake.18 Thus, the relative lack of any change in weight gain in our study was a bit surprising, but it illustrates the potentially important role of coloniC salvage that is also seen in humans with short bowel syndrome and intact colons.19,20 At the very least, in the piglet, 50% malabsorption of disaccharide apparently has no major acute detrimental effects on energy balance.

This study and our previous study indicate that weight gain as a function of energy intake is not demonstrably affected by moderate to severe disaccharide malabsorption,4 although as recently pointed out, we have observed nonsignificant increases in the ratio of energy intake to weight gain in preterm infants.21 Thus, in considering potential detrimental effects of carbohydrate malabsorption in infants,15,22 one must realize that "normal" weight gain may not be a sufficient criterion for optimal health of the colon. However, in comparing these 2 studies, there are important differences in how cecal mucosal structure and cell turnover are affected. With severe lactulose malabsorption, cecal cell proliferation was decreased 52% compared with the control,4 whereas during moderate malabsorption (the present study), there was a trend toward markedly higher cell proliferation in the LAC group compared with the control. Because the goal of the entire research project was to study feeding regimens that caused decreased cell proliferation and colitis, we could not study more piglets, but our post hoc power analysis suggested that the trends we observed in cell proliferation would be significant with a power of 0.81 with 11 piglets in each group. Of course, even if we had observed a statistically significant increase in cell proliferation with moderate lactulose malabsorption, at this stage in our investigations, there is no basis for characterizing a stimulation of colonic cell proliferation as an advantageous outcome of production of butyric acid versus an adverse, toxic result of overproduction of unbuffered, short-chain fatty acids.5,6

ACKNOWLEDGMENTS

The study was supported in part by grants from the Crohn's and Colitis Foundation of America and the Children's Research Institute, and the piglet formula was provided by Ross Products Division of Abbott Laboratories, Inc. The authors acknowledge the technical assistance of Jonathan Lash with the animal procedures. We thank Rita Porter for her editorial assistance.

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C. Lawrence Kien, MD, PhD*[dagger][double dagger]; James R. Cooper, DVM, PhD*; and Wendy L. Frankel, MD[sec]

From the * Children's Research Institute, and the [dagger] Department of Pediatrics, The Ohio State University, Columbus, Ohio; the [double dagger] Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas; and [sec] the Department of Pathology, The Ohio State University, Columbus, Ohio

Received for publication December 28, 2002.

Accepted for publication May 21, 2003.

Correspondence: C. Lawrence Kien, MD, PhD, Department of Pediatrics, Gastroenterology Division, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0352. Electronic mail may be sent to clkien@utmb.edu.

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