Changes, if any, in intestinal permeability to cellobiose and mannitol and in the CE/MA ratio are presented as mean ± SE and/or median (range) of the percentage recoveries of each probe molecule in the 5-hour urine collection. The normal ranges of the two control groups were calculated in the same way, and the two values were considered separately in the calculations. To define the normal upper limit of CE/MA, the mean + 2 standard deviations (SD) of cumulative control groups was calculated. Differences between mean values of control (pediatric and adult) and IDDM patient groups were assessed using the nonparametric Mann-Whitney test.
Correlation of NMR in contrast with the Corcoran and Page (25) methods for mannitol analysis was achieved by means of the linear regression analysis (r correlation factor). The r correlation factor was also applied in the comparison of percentage of CE, percentage of MA, and CE/MA with duration of disease, glycosylated hemoglobin, and amount of insulin therapy.
Informed consent was obtained from the patients or children's parents before submitting to the CE/MA test. The test is noninvasive and was well tolerated, as expected, by all patients and control subjects.
All values are express as mean ± standard error. The orally administered sugars were well tolerated by patients and control subjects; there were no symptoms suggestive of lactose intolerance. All urine specimens were free of aldehydes and ketone bodies.
All the enrolled subjects completed the test, and the individual percentages of recovered molecular probes are shown in Table 1. In 12 healthy children and 20 adult healthy volunteers the CE/MA ratio was 0.016 ± 0.010 and 0.009 ± 0.002, respectively. To define an upper limit of normal range for the data, the mean ± SE for our 32 controls was calculated (0.008 ± 0.010), and values higher than 0.028 (i.e., mean + 2 SD) were considered significantly changed.
In 11 patients with IDDM, glycosuria was present in a range of 0.25-0.50 g/l; therefore, urinary cellobiose was measured by the enzymatic assay in the remaining 20 patients. The median (range) percentages of cellobiose recovered in these 20 patients was 0.0% (range, 0-3.6%) and did not differ significantly from the levels in either group of control subjects.
The mean percentage of mannitol recovered in all 31 children with IDDM was far higher (50.15 ± 4.4%) than in control groups (children, 17.5 ± 2.2%; adults, 31.9 ± 2.5%). The difference in results between the study and the control children's groups was statistically significant (p = 0.003) and also between the study group and the adult control group (p = 0.03). There were higher concentrations than normal upper limit in 22 of 31 patients.
In 16 of these 22 patients and in 4 out of 9 patients with a normal percentage of mannitol excretion, cellobiose was measured. Individual distribution of the percentage of mannitol recovery is shown in Figure 3. A high percentage of mannitol recovery in urine was confirmed by NMR (Figs. 1 and 2; see also the NMR results section).
The median (range) value of the CE/MA ratio in 20 patients with IDDM was 0.0% (range, 0-0.06%), which was not statistically significant when compared with ratios in both control groups. Two patients (Table 1, CA and ML) had values beyond the upper normal limit (mean + 2 SD). In both patients no clinical or laboratory signs of inflammatory or malabsorptive intestinal disease were found, even after 1 year of follow-up. None of the investigated parameters correlated, except percentage of mannitol versus length of disease (r = 0.914).
Nuclear Magnetic Resonance
In Figure 1 in spectrum for urine of patients with IDDM after mannitol administration is compared with that of standard mannitol in water, under the same pH conditions, as described in Materials and Methods. The shape of the spectra in the illustrated region unambiguously demonstrates the presence of mannitol in high concentration in the urine sample. The NMR values for five points strictly correlated with the values obtained according to the Corcoran and Page method (r = 0.993; Fig. 2).
Intestinal permeability in patients with diabetes mellitus has seldom been reported (9-10). In one of these studies (9) lactulose and rhamnose were administered as a hyperosmolar solution to 48 men with diabetes mellitus, of whom 8 had IDDM. The overall data showed a significantly augmented permeability to lactulose and a similar trend in rhamnose, with a ratio of the two probes that was not statistically different from ratios in 13 healthy subjects and 40 patients with type II diabetes mellitus. The increase in permeability was the highest in the 8 patients with IDDM. Other specific data of interest were not presented in the article, such as the differential effect of insulin treatment in the two groups of patients with diabetes mellitus, which on the whole caused an increase in permeability to both sugars, and the effect, if any, of neuropathy or angiopathy, which were reported by most patients.
In the other study reported by Cooper et al. (10), intestinal permeability in 12 patients with diabetes mellitus who had diarrhea, neuropathy, and retinopathy, and in 10 patients with uncomplicated diabetes and 25 healthy control subjects, was investigated by a lactulose-mannitol test. We do not know how many if any patients with IDDM were enrolled in the study. The investigators found that the diarrhea was associated with an augmented excretion ratio of both probes, significant because of a lowered permeability to mannitol, in comparison with the same ratio in the patients with uncomplicated diabetes mellitus.
In both studies the ratios between the probes used in patients with diabetes mellitus and control subjects largely overlapped. No correlations with metabolic parameters (blood and urinary glucose, glycosylated hemoglobin) or with renal function parameters (creatinemia, urinary proteins) were found (9,10). Also, in the patients in our study, none of these parameters, or any associated pathologic conditions, such as thyroiditis, rheumatoid arthritis, multiple sclerosis, or secondary amenorrhea, was correlated to intestinal permeability values. The data reported from the literature (9,10) show an increase of permeability to both probes, with only the renal excretion of the larger one significantly higher in certain patients with diabetes mellitus than in control subjects. Although the ratios were in the normal range, the results seem to be not specific and similar to those reported in bacterial overgrowth of the small intestine (29).
We studied a homogeneous series of young patients with uncomplicated IDDM, and our data differ from those in the reported literature in that a significantly higher renal excretion of only the small probe was shown in most cases.
The CE/MA test procedure has been proposed by Strobel et al. (13) as a relatively convenient tool in the study of intestinal permeability and has since been used by other investigators (14-18). In our laboratory it was reliable with no substantial flaws in healthy children and adults; in diseases such as scleroderma (21), radiation enteritis (19-20), cancer chemotherapy (23), postcolectomy pouch (24), and in type II diabetes mellitus (30). The increase of the ratio, which has been shown in different pathologic conditions such as celiac disease (14,16,17), malnutrition (31), iron deficiency (32), idiopathic and infectious diarrhea (33), food allergy (18), cystic fibrosis (15), and Crohn's disease (34-35), was not reproduced in our patients with IDDM. Although we measured percentages of recovered CE in 20 of 31 children, our results may be limited by the 11 (35%) undetectable cases with no data on cellobiose urinary excretion. We cannot exclude that all these cases had a higher than normal CE/MA ratio. The increased mannitol excretion in 6 of 11 such as cases makes that possibility unlikely.
We cannot explain the increased intestinal permeability to mannitol. The endogenous production of mannitol can reasonably be excluded because we administered 2 g sugar, an amount far above the eventual endogenously produced quantity (36). An increase of the intestinal flow rate, although within the normal range, was shown to decrease the estimated pore size of the jejunal diffusion pathway from 13 to 8 angstrom (37) in the normal subject. That could imply a preferential route for mannitol compared with that of cellobiose. In symptom-free patients with diabetes, however, jejunal cecal transmit time seems to be longer than that in healthy subjects (38). Another possibility is that the selective increase in permeability to mannitol is secondary to an increased rate of the mechanism experimentally investigated and proposed by Bijlsma et al. (39) but not confirmed however by others (40) (i.e., a paracellular solvent drag caused by the intravillous hyperosmolarity produced by local countercurrent movements).
The prevalence of celiac disease associated with IDDM has been shown recently to be higher than expected. Patients with celiac disease determined by screening with anti-gliadin (3) or anti-endomysial antibody tests (11) often have few or no symptoms and an earlier age of onset of IDDM (41) and are predominantly female. Some with IDDM who are antibody negative, may later test positive (3), even in the presence of persistently negative results in analyses of small bowel biopsy specimens. From our series we report on two patients (6.2%) who at the 1-year follow-up, showed seroconversion to positive for anti-endomysial antibodies and a typical histologic pattern in a small bowel specimen.
In conclusion, our study showed an increase in intestinal permeability to mannitol not previously reported, clear-cut and unassociated with that of the larger probe, in at least two thirds of 31 young patients, all with uncomplicated IDDM and free from clinical and serologic signs of celiac disease at the time of their recruitment. The high intestinal permeability to mannitol represents a functional alteration apparently opposite to those reported in overt celiac disease, in which the reduced mannitol urinary recovery is caused by the loss of absorptive villous surface which produced an increase in CE/MA ratio. The intestinal permeability to mannitol was normal in our healthy children, young adult controls, and adult patients with type II diabetes mellitus (30). It is difficult to define the impact of this finding, whether it is a primary feature in patients with IDDM or an initial alteration of the villi's function, preceding their atrophy in patients with IDDM, in whom celiac disease could develop later.
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Keywords:© 1999 Lippincott Williams & Wilkins, Inc.
Cellobiose; Diabetes mellitus type I; Intestinal permeability; Mannitol