Dual sugar gut-permeability testing on blood drop in animal models

doi:10.1016/j.cccn.2004.09.023

Clinica Chimica Acta

Volume 352, Issues 1–2, February 2005, Pages 191-197

https://doi.org/10.1016/j.cccn.2004.09.023 Get rights and content

Abstract

Background

Intestinal permeability is determined by measuring nonmetabolized sugars. In animals, intestinal permeability is determined in urine, using cumbersome and expensive metabolic cages. We developed an HPLC method for determining concentrations of lactulose (L) and l-rhamnose (R) in blood-drop of rabbits and mice, and we compared these results with the procedure based on sugars excreted in urine. We measured the intestinal permeability induced by a fragment (ΔG) of the zonula occludens toxin which opens the paracellular pathway.

Methods

The animals received sugar solution and later received the same solution+ΔG. Five-hour urine collection and timed blood tests were performed after ingestion of sugars. Sugars were measured with HPLC, and the percentage of recovered sugars was expressed as L/R ratio.

Results

At 60 min after administration of sugars, the mean L/R ratio for rabbits and mice was 0.026 and 0.052, respectively. At 60 min after administration of sugars+ΔG, the mean L/R ratio for rabbits and mice was 0.22 and 0.53. The mean L/R ratio in the urine was 0.023 at basal condition and 0.25 after ΔG ingestion.

Conclusions

Testing small serum samples for sugar permeability is effective for monitoring changes in permeability of the gut in animals. This cheap simple method allows us to measure in vivo the biological activity of other molecules which modulate the paracellular pathway.

Introduction

Dual-sugar tests of small intestinal permeability are widely used as a noninvasive means of assessing intestinal damage in humans and animals [1], [2]. This useful screening procedure can also contribute to understanding the modulation of the paracellular pathway of the intestinal epithelium in experimental animal models [3]. Lactulose (L) and l-rhamnose (R) are the most common probe saccharide molecule probes, and the L/R ratio in a 5-h urine collection is a well-established method of assessing small intestine permeability both in humans and animals [4], [5]. However, the collection of 5-h urine samples is not always easy (i.e., in very young children) [4] and is both expensive and invasive in animals, involving metabolic cages or bladder catheterization [3], [5]. Two previous studies in sick infants [6] and dogs suffering from inflammatory bowel disease [5] have shown that the dual sugar test can be simplified using timed blood instead of urine samples with equally good results. In the present study, we investigated the performance of the dual sugar test using very small blood samples to assess small intestine permeability in rabbits and mice at basal condition and after oral administration of a Vibrio cholerae zonula occludens toxin-derived molecule (the ΔG fragment) that acts on the tight junction of the small intestine, causing a reversible increase in intestinal permeability [7]. We also compared the blood results with the standard procedure based on urine collection.

Section snippets

Chemicals

The lactulose, l-rhamnose, FMOC-Cl (9-flourenylmethyl chloroformate), hydrazine hydrate, ethanol, acetic acid, and HPLC-grade acetonitrile were purchased from Sigma.

Synthesis of FMOC-hydrazine

The FMOC-hydrazine was prepared following the method of Rooyakkers et al. [8]. Briefly, the procedure was as follows: 1 g FMOC was dissolved in 100 ml ethanol. This solution was added dropwise to 10 ml of hydrazine hydrate, and the mixture was stirred for 30 min at room temperature (RT). The resulting FMOC-hydrazine precipitate was

Calibration and limit of detection

The linearity of the detector response was assessed using decreasing concentrations of l-rhamnose and lactulose in aqueous solutions. The method was found to be linear over the range of 1–1250 pmol. The detection limit was 0.08 pmol for lactulose and 0.02 pmol for l-rhamnose.

Analytical recovery and precision

Recovery was determined with known quantities of sugars added to the serum. Recovery rates were between 98% and 102% for both sugars. The coefficient of variation (CV) was determined by injecting 10 samples each of aqueous

Discussion

This study shows that an intestinal permeability test can be performed in an animal model on extremely small quantities of serum, thereby making it possible to do without expensive and invasive metabolic cages. Moreover, the single blood samples (corresponding to the 3.3% of total blood volume for mice and to 0.2% for rabbits) are in line with recommendations for the withdrawal of blood in animals, according to which samples below 10% of total blood volume have no significant haemodynamic

Acknowledgement

This work was supported by grant R.F.149/03 from I.R.C.C.S. “Burlo Garofolo” Trieste, Italy.

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