The median (range) urinary sulphate/creatinine ratio of the control group was found to be significantly higher than that of the patients investigated the values being
6.18 (range; 1.59-14.10) and 1.50 (interquartile range; 0.96-3.02) respectively, p<0.05. Calculation o f the reference values were performed as previously described (see 5.5.2) giving a critical range for the control group o f 4.78-6.81. The median (interquartile range) lactulose/mannitol ratio of the 35 patients whose urine sulphate excretions were
investigated was 0.133 (0.056-0.393) which was higher than the "normal" value of 0.051-0.055 (see 5.5.2). A scatterplot of the permeability data with respect to the urine sulphate/creatinine results (Fig 5.8) showed that a negative relationship existed between these two variables (r = -0.11, p=0.53). Table 5.10 shows sulphate/creatinine values for these subjects separated with respect to small bowel biopsy data. No significant difference was seen between these two groups for excretion o f sulphate.
Table 5.10. The relationship between urine sulphate excretion and small intestinal mucosal status.
median (range) Normal Biopsy (n=16) Abnormal Biopsy (n=14)
Sulphate/Creatinine ratio 1.79* 1.91*
range (0.37-15.08) (0.75-5.26)
Lactulose/Mannitol ratio 0.061 0.189
range (0.013-3.60) (0.014-21.58)
biopsy results were available for 30/35 subjects, hence the median values for children are seen to be higher than the overall median value.
3 . 5
2.5
1.5
0.5
Subject points within this area = abnormal L/M and urine sulphate/creatinine ratios.
5.6 Discussion
The use of SEM GC-MS for the assessment o f urine lactulose and mannitol excretion provided a simple and rapid method for the simultaneous analysis o f urinary mannitol and lactulose.
Although several authors (Ford et al 1985, Hodges et al 1989) have shown that an hyper-osmolar load exerts no detrimental effects on children and enhances the distinction between abnormal and normal children with respect to intestinal permeability, here it was considered that the osmotic effect that may occur with the administration o f such a load was unethical for children who already had failure to thrive and/or diarrhoea.
The oral administration o f an iso-osmolar oral load generally allowed the distinction between patients who had received treatment and had normal jejunal biopsies. Mannitol and to a lesser extent lactulose are normal constituents of the everyday diet. Many of the patients had traces of mannitol and lactulose in their preload urine sample despite an overnight fast. The concentrations calculated were subtracted from the final values, but were mainly insignificant. Any patients who were found not to have complied with the requested overnight fast were not included in study.
When comparing the permeability results of patients with respect to their small bowel biopsies discrepancies may occur ( Ford et al 1985). This was reflected in the poor
specificity value of 38.71%. This may have been due to the fact that the jejunal biopsy is a static procedure that demonstrates the mucosal damage in a particular region o f the small intestine, whilst the differential permeability test provides an overall dynamic evaluation of the small bowel. Thus in the latter case despite focal abnormalities the entire permeability result may still be normal.
Numerous studies have been performed which demonstrate the potential o f the sugar permeability test in the follow-up assessment o f patients receiving treatment for cow's milk protein intolerance and coeliac disease (Nathavitharana et al 1988, Pearson et al 1982). Histological results for these two groups ranged fi-om the mild partial patchy lesions of cow's milk protein intolerance to the totally flat subtotal villous atrophy o f coeliac disease. Here, for patients with coeliac disease a clear distinction was seen between patients who had received a restrictive glûtènfi-ee diet and those children who were untreated. The distinction was not as definite for patients with cow's milk protein intolerance.
This suggested that the coeliac disease patients were more responsive to treatment, leading to a more complete recovery as reflected by the intestinal permeability ratio. When these data were investigated with regards small bowel biopsies, it was shown that patients with coeliac disease and abnormal biopsies had significantly high percent urine recoveries of lactulose. This suggested that for the more severe lesions associated with coeliac disease, the absorptive surface area of the entire mucosal region including the intercellular tight junctions was affected. Comparisons of urinary sugar ratios between the different diagnostic groups showed a significant difference between patients with cystic fibrosis and those with other gastrointestinal disorders. The finding that the urine recovery of both mannitol and lactulose were increased in children with cystic fibrosis, leading to an increased permeability ratio, is a little understood phenomenon. The villi and microvilli architecture o f patients with cystic fibrosis is believed to be essentially normal, with some cases o f increased cellularity and oedema of the lamina propria (Park and Grand 1981). In patients with cystic fibrosis, the mucous lining of the small intestine becomes more
viscous. This viscosity may exert an added osmotic effect during solute absorption, explaining the enhanced movement of both lactulose and mannitol from the intestinal lumen (Murphy et al 1989).
There is much documentation of the damage caused to the small intestine during
micro-organism colonisation with regard to the various degrees of mucosal abnormality, ranging from minor/partial to severe villus atrophy. Villi shortening, inflammatory cell infiltration and irregularity of the surface epithelium may also be seen (Berry and Keeling 1989). Here the biopsy results of the children with intestinal colonisation were varied. However all children in this category (despite treatment), had abnormal permeability ratios. Again this suggested that the dynamic permeability test was more indicative of intestinal mucosal damage. As previously stated the group categorised as failure to thrive of unexplained or secondary aetiology consisted o f patients with a variety o f pathological conditions. Here the biopsy results showed good correlation with the intestinal
An association was seen between the morphometric data and the degree o f small bowel mucosal damage, although the relationships found were not significant (table 5.8) as previously reported, (Ford et al 1985). Percent recovery of lactulose was seen to increase with reduced villus height. This was to be expected, as the excretion o f lactulose in the urine is believed to increase with reduced mucosal integrity and loss o f microvillus absorptive enterocyte cells characterised by increased mucosal atrophy. This was further supported by the existence of a weak negative relationship between crypt depth and urinary mannitol recovery which suggested that as the secretory crypt increased in depth the absorptive surface area was reduced and thus transcellular transportation o f mannitol was limited.
Similarly no significant relationship was seen between disaccharidase activity and intestinal permeability. This would suggest that activity o f these enzymes is only affected in cases of severe chronic mucosal damage.
A relationship between mucosal permeability and growth faltering has been demonstrated (Lunn et al 1991, Sullivan et al 1992). This thesis demonstrated that a low inorganic sulphate excretion was associated with an increase in small bowel permeability, as assessed by the lactulose/mannitol intestinal sugar permeability test (Michie et al 1991).
The main source of inorganic sulphate is dietary in the form of sulphur containing amino acids (SCAA). SCAAs, such as cysteine, are required at the cartilaginous groAVth plates, which have been shown to be sensitive to available sulphur (Van der Kraan et al 1990). Glutathione (GSH), is a tripeptide thiol containing glycine, cysteine and glutamate, present in most cell types. This compound activates thiol requiring enzymes, acts as a co-factor, regulates microtubule formation and protects cells from oxidative damage. Lymphocyte proliferation is dependent on GSH availability as shown by the fact that a cysteine rich dietary protein will have a significant immuno enhancing effect (Boxer et al
1979). It can be hypothesised that a disease process which restricts SCAA availability at the growth plate, either by restricting absorption or by increasing the demand for SCAA for other biological processes, would lead to a decrease in urine excretion o f sulphate. There is no evidence for the former hypothesis, but there is for the latter;
Firstly, the administration o f paracetamol to rats led to a severe reduction in growth velocity unless extra SCAA was included in the diet (McLean et al 1989). Paracetamol detoxification requires sulphation, thus limiting the availability of SCAA to other physiological process such as growth.
Also, a mode of treatment for paracetamol overdose in humans involves N-acetyl cystine administration to facilitate the sulphation pathway and maintain GSH concentrations. Secondly it has been demonstrated that rats injected with alpha Tumour Necrosis Factor (aXNF), a cytokine readily produced in the event o f systemic endotoxaemia and/or bacteraemia, excreted reduced amounts o f inorganic sulphates (Liang et al 1989). Further to this, in patients with failure to thrive and/or diarrhoea, urine concentrations of aTNF, were significantly raised (Michie et al 1990). This is thought to indicate SCAA are being diverted into the synthesis o f acute-phase proteins (which have a high content of SCAA) and GSH production.
Thus with further investigation it would be possible to determine whether intestinal disorders, which lead to an increased permeability o f the small bowel, cause absorption of intestinal toxins which in turn stimulate cytokine production causing diversion of SCAA into GSH synthesis and suppression o f bone growth.
In conclusion, the differential sugar permeability test was a rapid and simple procedure for the assessment o f intestinal mucosal damage. The use o f SIM, GC-MS method of analysis provided a more detailed assessment o f the child presenting with failure to thrive and/or abnormal stools. The discrepancies encountered between the biopsy results and
permeability ratios demonstrated the importance o f dynamic fimctional assessment. The indicated differences in lactulose/mannitol ratios for the aetiologic groups may, on further investigation, serve as criteria for differential diagnoses, particularly in cases of toddler's diarrhoea where jejunal biopsy o f an otherwise normal child could essentially be avoided.