Introduction

Intestinal permeability may vary with changing pathological circumstances transiently as well as persistently. The variations can occur due to liver cirrhosis, nonspecific intestinal inflammations, acute pancreatitis, chemotherapy, etc. [1–5].

Many agents were tested to assess their characteristic for measuring intestinal permeability. Currently, two agents are used as optimal markers: mannitol/l-rhamnose to evaluate transcellular absorption and lactulose/cellobiose to evaluate paracellular absorption.

Lactulose and mannitol are convenient and the most frequently used agents in present clinical practice, enabling a test of the intestinal permeability (test L/M). They present insignificant affinity for the monosaccharide transporter system, therefore being passively absorbed and having the advantage of not being metabolized and excreted in urine unchanged [4, 6, 7].

Determination of lactulose and mannitol are performed using the gas chromatography (GC) and high-performance liquid chromatography (HPLC) method, and enzymatic methods [8–11].

Medical literature and clinical practice report different methods for expression and evaluation of measured (obtained) values of lactulose and mannitol in urine: the lactulose/mannitol ratio, the lactulose/creatinine ratio, and lactulose excretion in urine collected during the test duration, which is usually 5 h, measured as a percentage of the orally administered dose. The purpose of our study was to evaluate the diagnostic value (sensitivity, specificity, and AUC), and its separate aspects, provided by the L/M test in a cohort of patients with Crohn’s disease and liver cirrhosis.

Study Cohort and Methods

The study included 21 men with a mean age of 57 (47–67) years and five women with a mean age of 52 (40–61) years, diagnosed with alcohol-related liver cirrhosis, CHILD score B–C. Clinically significant ascites was present in six patients.

The cohort of patients with Crohn’s disease included seven men with a mean age of 31 (20–45) years and 13 women with a mean age of 33 (22–47) years with the active form of Crohn’s disease (A—CDAI of 220–280) affecting terminal ileum and colon. The control group consisted of nine men with a mean age of 35 (24–60) years and 11 women with a mean age of 27 (22–50) years; these subjects were either employees of University Hospital Brno or students of the medical faculty presenting with no symptoms of disease of the digestive tract.

L/M Test Description

In the morning, the examined subject was asked to empty the urinary bladder and consequently drink on an empty stomach a test solution consisting of 5 g lactulose, 10 g mannitol, and 40 g sucrose diluted in 100 mL water.

The patient’s urine was then collected for the next 5 h. The urine of each subject was stored in a vessel containing chlorhexidine as a conservative. The urine was stirred, the volume precisely measured, and an aliquot specimen sent to the laboratory. Until the laboratory analysis, the samples were stored at −20°C.

Evaluation Methods

To detect lactulose and mannitol in urine, we used the set sugar absorption test; lactulose, mannitol in urine-enzymatic, Instruchemie, NL.

Lactulose determination is a fully enzymatic method. In the first stage, lactulose is hydrolysed into fructose and galactose using β-galactosidase. The fructose formed is further quantitatively converted through three consecutive enzymatic reactions, in the presence of hexokinase, glucose phosphate isomerase, and glucose 6-phosphate dehydrogenase, to gluconate-6-phosphate and NADPH. The activity of mannitol dehydrogenase catalyzes the conversion of mannitol to fructose and NADH.

The NADPH and NADH formed in the reactions were estimated by measuring the change in absorbance at 340 nm.

The methods were performed by using a Cobas Mira analyzer. Creatinine in urine was measured on a Modular (Roche) analyzer, with a kinetic blanked compensated Jaffé method. The values obtained allowed the calculation of the lactulose/mannitol ratio, the lactulose/creatinine ratio, and the percentage of urinary lactulose excretion of the ingested dose.

Statistical Methods

The normal (Gaussian) distribution of all the parameters under study was verified with the Kolmogorov–Smirnov test.

For each parameter, in each patient cohort, the data collected were processed with Student’s t-test (significant at P < 0.05), calculating the mean, the standard deviation (SD), and the statistical significance of the differences versus the control group.

The values of the cut-off levels, the sensitivity, the specificity, and the area under curve (AUC) were calculated to determine the tested parameters in both cohorts.

The diagnostic value of the lactulose/mannitol ratio, the lactulose/creatinine ratio, and the percentage of urinary lactulose excretion of the ingested dose were expressed as the comparison of their receiver operating characteristic (ROC) curves.

MedCalc for Windows version 9.3.1 was used for the statistical calculations, receiver operating characteristic (ROC), cut-off levels for maximal sensitivity and specificity, and the graphical representations.

Results

The basic statistical indicators of the tested parameters in the L/M test in the cohort of patients with Crohn’s disease and liver cirrhosis versus the control cohort are presented in Table 1. In both cohorts, the mean value of the three tested parameters was highly significantly different from the control group. The sensitivity, specificity, and AUC of the determined cut-off levels in Crohn’s disease and in liver cirrhosis are presented in Table 2A, B. In the cohort of patients with Crohn’s disease, the calculated lactulose/mannitol ratios demonstrated nearly ideal values. The next two indicators, the lactulose/creatinine ratio and the percentage of lactulose recovery showed nonideal but still very reliable and comparable values. In the cohort of patients with liver cirrhosis, the calculated values of the sensitivity, the specificity, and the AUC were lower then in the foregoing cohort, nonetheless, the priority given to the lactulose/mannitol ratio was preserved.

Table 1 Two urinary ratios (lactulose/mannitol, lactulose/creatinine) and lactulose recovery in a 5-h urinary volume in controls and patients with Crohn's disease and liver cirrhosis
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Table 2 Sensitivity, specificity, and AUC for the lactulose/mannitol ratio, the lactulose/creatinine ratio, and lactulose recovery in patients with Crohn's disease (A) and liver cirrhosis (B)
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The area under the curve (ROC curves area) is a suitable parameter to summarize the overall discriminative or diagnostic value of a model featuring Crohn’s disease (Fig. 1a) and liver cirrhosis (Fig. 1b). For both diseases, the diagnostic value of the tested parameters was assessed as follows (in order of decreasing importance): lactulose/mannitol, lactulose/creatinine, and lactulose recovery.

Fig. 1
figure 1

Comparison of ROC curves for lactulose/mannitol, lactulose/creatinine, and lactulose recovery in patients with Crohn's disease (a) and liver cirrhosis (b)

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Discussion

Morphological and functional integrity of the small intestine underlies two opposite processes: the highly effective mechanism of nutrient absorption that includes specific transport movements and, in the opposite direction, a highly effective and complex defence mechanism that serves as a protective barrier to prevent harmful bacteria, toxins, antigens, and other pathogens from entering the body. When the epithelium of the small intestine become impaired, it can lose the tight cell junctions and allow bacteria and toxins to enter the blood stream, which may cause sepsis and multiple organ failure.

Macromolecules with antigen characteristics may produce local or system response of the immune system, leading to serious allergies. The pathological symptoms and reactions that are a consequence of increased intestinal permeability is a condition called leaky gut syndrome [12, 13].

Schematically, the complex processes of the intestinal mucous membrane can be divided into transcellular and paracellular fluxes. It follows that mucosal intestinal permeability can be assessed on the basis of the quality and functions of paracellular structures of the intestinal mucous membrane.

The assessment of the mucosal intestinal integrity (permeability) is measured by urinary excretion of orally administered test unmetabolizable sugars. The usual scheme of the test lies in oral administration of two sugars of a different molecular size and with a different mechanism of absorption. Monosaccharides, such as l-rhamnose or mannitol, are absorbed through the transcellular pathway and reflect the degree of absorption of small molecules (<0.65 nm). Disaccharides, such as lactulose or cellobiose, are absorbed through the paracellular junction complex, which corresponds to the permeability of larger molecules (>0.93 nm).

The results of our investigation justify the priority given to the L/M ratio used to determine and appraise the degree of intestinal permeability within the L/M test. A further advantage of this ratio is the elimination of errors due to nonmucosal factors, because variables such as rate of gastric emptying, intestinal transit, impairment of renal function, and diuresis should affect both sugars similarly [14–18].