Diagnostic strategy for mucolipidosis II/III.

Overlapping clinical phenotypes are a diagnostic challenge to the clinician, especially in the cases of mucolipidosis (ML) and mucopolysaccharide disorders (MPS), due to overlapping phenotypes. Present study was carried out in 147 children suspected to have ML or MPS and 100 controls. They were screened for ML II/III by colorimetric method using substrate pNCS. Six children were found screen positive for ML II/III and further confirmatory study showed significantly raised activity in plasma confirming high specificity of the ML screening test. Forty-two (28.5%) children out of remaining 141 children that were screen negative, were found to have various MPS disorders, while rest 99 had normal enzyme activity in plasma and leucocytes. Present study demonstrates prompt and specific chemical method that can be used as a tool for estimating ML II/III, with high specificity.

Erythrocyte zinc in hospital patients.

BACKGROUND: Abnormal thyroid function tests (serum thyrotropin [TSH], free thyroxine [T(4)] and free triiodothyronine [T(3)]) are frequently seen in hospitalized patients. Assessment of thyroid function in these patients is difficult. It has been suggested that acutely ill patients may be hypothyroid at the tissue level. Erythrocyte zinc (EZn) has been shown to be increased in hypothyroidism. The aim of this study was to examine EZn as an index of thyroid status of hospital patients. METHODS: In order to assess the thyroid status at tissue level, we measured EZn in 26 healthy subjects, 39 critically ill patients and 19 hospitalized geriatric patients. EZn was measured in young cells, as the effect of illness is likely to be seen in the newly formed cells. RESULT: TSH and free T(3) were lower in critically ill patients and serum free T(3) was lower in geriatric patients. EZn in young cells was higher in both patient groups (by 13% and 23%, respectively). EZn in old cells was also higher in the geriatric group. CONCLUSION: We conclude that EZn is higher in hospitalized patients, suggesting that these patients may be hypothyroid at the tissue level.

Low circulating estradiol and adrenal androgens concentrations in men on glucocorticoids: a potential contributory factor in steroid-induced osteoporosis.

Reductions in circulating estradiol concentrations could be implicated in the pathogenesis of steroid-induced osteoporosis (SIOP) in men. We assessed serum estradiol and adrenal androgens (dehydroepiandrosterone sulfate [DHEAS] and androstenedione) in 77 men (group A: idiopathic osteoporosis [IOP], n = 38, aged [mean +/- SD] 57.7 +/- 12.1 years; group B: SIOP, n = 39, aged 55.3 +/- 13.1 years). We also studied the relationship between bone mineral density (BMD) and serum estradiol in the group of men with SIOP. In group B, we observed a higher prevalence of low serum testosterone concentrations (<9.0 nmol/L) (P =.0052), which was significantly correlated with steroid dosage (r = -0.42, P =.0089) and estradiol concentrations (r = 0.42, P =.012). There was a significant positive association between BMD at the lumbar spine and serum estradiol (P =.004) in the men with SIOP (group B). A high proportion of subjects had low serum estradiol concentrations (<48 pmol/L) in both groups (group A: 44.7 %, group B: 36 %). Serum adrenal androgens concentrations were also significantly suppressed in group B (serum androstenedione-group A: 4.99 +/- 1.8; Group B: 2.1 +/- 1.6 nmol/L; P =.0001). Serum DHEAS was undetectable in 59% of patients in group B versus 6% in group A (P =.001). Reductions in androstenedione also correlated with steroid dosage (r = -0.35, P =.01). In conclusion, the data show that adrenal androgens synthesis is markedly suppressed in men with SIOP. The clinical relevance of this finding remains to be determined. This study also shows a positive association between serum estradiol and BMD and a high prevalence of low serum estradiol in men with SIOP. Low serum estradiol may contribute to bone loss in men with SIOP.

Comparison of the humoral markers of bone turnover and bone mineral density in patients on haemodialysis and continuous ambulatory peritoneal dialysis.

Renal osteodystrophy is an important complication in patients with end-stage renal disease on maintenance dialysis. The aim of this study was to compare the biochemical markers of bone formation (serum collagen type I C-terminal propeptide) and resorption (serum deoxypyridinoline - DPD - and pyridinoline - PYR) with the bone mineral density (BMD) at lumbar spine, femoral neck, and forearm in patients with end-stage renal disease on haemodialysis (HD) versus continuous ambulatory peritoneal dialysis (CAPD). Fifty-nine adult patients, 45 on CAPD (18 females, 27 males) and 14 on HD (2 females, 12 males), were studied. The mean age was 44 +/- SEM 1.6 and 54.4 +/- 4.8 years, respectively. No significant differences in serum calcium, phosphorus, creatinine, and parathyroid hormone were found between patients on HD and CAPD in predialysis samples. Serum urea was significantly lower (p = 0.02) in the CAPD group. Serum PYR (nmol/l) and DPD (nmol/l) were significantly higher in patients on HD as compared with those on CAPD: 105 +/- 23.3 versus 43.7 +/- 3.47 (p = 0.007) and 31.0 +/- 2.4 versus 24.4 +/- 1.4 (p = 0.027), respectively. The results were still significantly higher in the HD patients following correction for serum creatinine and body mass index. There was a close correlation between dialysate DPD and creatinine in both dialysis modalities (HD r = 0.9, CAPD r = 0.76). The clearance of DPD did not differ significantly between the CAPD membrane and the HD membrane (p = 0.22). Serum collagen type I C-terminal propeptide was not significantly different between the HD and CAPD patients. The results were unaffected following correction for age and gender. The BMD was measured in 38 (65%) of the patients (HD n = 8, CAPD n = 30) by dual-energy X-ray absorptiometry and expressed as 'Z' scores. This was reduced at all sites in the patients with end-stage renal disease. The BMD was significantly lower at the ultradistal forearm (mostly trabecular bone) in HD patients as compared with CAPD patients (n = 0.02). A similar trend was observed at the lumbar spine, although the results failed to reach significance. In the whole population (n = 38), linear regression analysis revealed a significant negative correlation between BMD at the ultradistal forearm and serum PYR (r = -0.35, p = 0.04) and DPD (r = -0.33, p = 0.049). Combined measurements of BMD and biochemical markers of bone resorption may have potential in the identification of patients at high risk of bone loss who may require further evaluation of bone remodeling by bone histomorphometry.

Size at birth, adult intestinal calcium absorption and 1,25(OH)(2) vitamin D.

BACKGROUND: Adult bone mineral status is modified by early environmental influences, but the mechanism of this phenomenon is unknown. Intestinal calcium absorption and vitamin D metabolism are integrally involved in bone metabolism and may be programmed during early life. AIM: To examine the early-life influences on calcium absorption and its control in 322 post-menopausal female twins. METHODS: Intestinal calcium absorption was assessed by the stable strontium (Sr) method. Serum PTH, 25(OH) and 1,25(OH)(2) vitamin D were measured and recalled birth weight recorded. RESULTS: Fractional intestinal Sr absorption (alpha Sr) was correlated with serum 1,25(OH)(2) vitamin D (p<0.001), but not with 25(OH) vitamin D. Birth weight was inversely associated with serum 1,25(OH)(2) vitamin D (p=0.04), the association being independent of serum calcium, phosphate, creatinine and PTH. Birth weight was inversely correlated with alpha Sr (p=0.03), this association being independent of age, season, customary calcium intake and serum 25(OH) vitamin D; however, when serum 1,25(OH)(2) vitamin D was added into the model, the association became non-significant, suggesting that the association was partially mediated via serum 1,25(OH)(2) vitamin D. DISCUSSION: We found a significant inverse association between birth weight and intestinal calcium absorption that is partially explained by an association between serum 1,25(OH)(2) vitamin D and birth weight. This suggests a mechanism whereby the intra-uterine environment might affect adult skeletal status.

Effects of prednisolone on bone turnover in patients with corticosteroid resistant asthma.

BACKGROUND: Although corticosteroid resistant (CR) bronchial asthma is associated with impaired in vitro an din vivo glucocorticoid (GC) responsiveness in mononuclear cell function, it is not known whether this is a cell specific phenomenon or whether these patients are at risk of glucocorticoid side-effects as corticosteroid-sensitive (CS) subjects. In order to address this question we have examined the effects of GCs on biochemical indices of bone turnover in vivo. METHODS: Six CS and six CR subjects received prednisolone 40 mg orally at 09.00 daily for 5 days. At 08.30 on day 1 and day 6 a fasting blood was taken for estimation of serum osteocalcin, tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP; total and bone isoenzyme), and a urine sample taken for estimation of free deoxy-pyridinoline crosslinks. TRAP and ALP were measured by a colorimetric method and osteocalcin and deoxy-pyridinoline crosslinks by ELISA. RESULTS: Serum osteocalcin (nmol/L) decreased from 1.39 +/- 0.09 (mean +/- SEM) to 1.14 +/- 0.07 (P = 0.023) and from 1.19 +/- 0.05 to 0.96 +/- 0.1 (P = 0.037) in the CS and CR groups respectively. There was no difference between baseline and post-treatment levels in either group. Serum total ALP (units/L) decreased from 178 +/- 7 to 168 +/- 4 (P = 0.02) and from 195 +/- 22 to 175 +/- 16 (P = 0.04) in the CS and CR groups, respectively. There was no difference between baseline and post-treatment levels in either group. There was no significant elevation of deoxy-pyridinoline crosslinks or TRAP and no suppression of bone ALP in either group. CONCLUSION: We suggest that CR asthmatics may be equally at risk from the metabolic side-effects of glucocorticoids.

Gastric carcinogenesis in the rat induced by duodenogastric reflux without carcinogens: morphology, mucin histochemistry, polyamine metabolism, and labelling index.

Chronic duodenogastric reflux induces gastric adenocarcinomas in the rat without the use of carcinogens. Altogether, 186 male Wistar rats were randomised to undergo either a simple gastrojejunostomy or a gastrotomy and sacrificed at eight weekly intervals for 56 weeks. No control animals developed dysplasia or carcinoma. All rats subjected to a gastrojejunostomy showed hyperplasia of the proliferative neck zone, with increased sulphomucin production adjacent to the scar. Low grade dysplasia was found at 16 weeks, and carcinoma was first seen at 32 weeks. Most carcinomas were well differentiated mucin secreting adenocarcinomas of the expanding type, which secreted a mixture of sialomucins and sulphomucins. Duodenogastric reflux was associated with a 100% increase in labelling index (assessed autoradiographically with tritiated thymidine) in the gastric mucosa when compared with corresponding tissue adjacent to a gastrotomy scar. This increase was significant at eight weeks and persisted for 56 weeks after surgery. This study supports the theory that, in this model, hyperplasia precedes the development of carcinoma.

Postheparin plasma diamine oxidase in health and intestinal disease.

In animals, the distribution of the enzyme diamine oxidase is confined, almost exclusively, to the small bowel mucosa. In humans, plasma diamine oxidase is at or below assay detection limits but can be liberated into the circulation from binding sites in the intestine by i.v. heparin. Therefore, the authors wished to see if diamine oxidase could be released by a low and safe dose of heparin (5000 U) and if the resultant area under the concentration-time curve would provide a noninvasive marker of segmental intestinal disease. In 17 control subjects, the mean area under the curve (following administration of 5000 U i.v. heparin) was 35.9 +/- 5.0 (SEM) mU.L-1.2 h-1; in 6 individuals studied on two separate occasions, postheparin plasma diamine oxidase profiles were reproducible (r = 0.98; p less than 0.001). The longitudinal distribution of diamine oxidase in the gastrointestinal tract, measured in 12 gastric, 16 jejunal, 6 ileal, and 18 colonic biopsies, was similar in humans to that found in animals. In patients with normal peroral biopsies, there was a linear relationship between jejunal mucosal and postheparin plasma diamine oxidase activities (r = 0.84; p less than 0.01). The areas under the curve in controls were then compared with those in patients with segmental intestinal diseases: 21 with ileal disease with or without colonic Crohn's disease (10 unoperated and 11 with ileal resection), 7 with non-Crohn's ileal resection, 8 with ulcerative colitis, 10 with untreated and 7 with treated celiac disease (6 studied before and after a gluten-free diet), and 5 studied during total parenteral nutrition and again after resumption of oral feeding. The results in the 18 ileectomized patients were subdivided into those with major (arbitrarily greater than 75 cm) and minor (less than 75 cm) resections. Areas under the curve were markedly reduced in nonresected Crohn's patients (6.0 +/- 1.79 mU.L-1.2 h-1; p less than 0.001 vs. controls), correlating inversely, in a first-order relationship, with disease activity (r = 0.82; p less than 0.001) and returning toward normal in 4 patients achieving disease remission. Low areas under the curve in total parenteral nutrition patients (4.5 +/- 0.9; p less than 0.001) were also reversible on resumption of oral feeding. However, areas under the curve were not significantly lower in patients with limited ileal resection (less than or equal to 75 cm), with celiac disease (untreated and treated), or ulcerative colitis than in controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of intestinal diamine oxidase (DAO) depletion by heparin on mucosal polyamine metabolism.

The aim of this study was to examine the relative importance of the enzymes ornithine decarboxylase (ODC) and diamine oxidase (DAO) in intestinal polyamine metabolism in normal rats. To study this we measured jejunal and ileal DAO and ODC activities and polyamine levels 2 h after injecting, in non-fasting male Wistar rats, either normal saline (controls) or a large dose of i.v. heparin to deplete intestinal mucosal DAO stores. We found that heparin significantly reduced mean DAO activity (p less than 0.001). As a result putrescine levels increased significantly in both jejunum (p less than 0.02) and ileum (p less than 0.01). Heparin-induced DAO depletion had no significant effect on spermine and spermidine levels at either site. The increased putrescine was associated with reduced ODC activity in both jejunum (p less than 0.01) and ileum (p less than 0.05). ODC activity correlated negatively with putrescine (p less than 0.001). These results suggest that (i) in normal rats, DAO is more important than ODC in regulating mucosal putrescine levels and (ii) putrescine exerts feedback inhibition of ODC.

Role of polyamines in the early adaptive response to jejunectomy in the rat: effect of DFMO on the ileal villus:crypt axis.

To study the role of the polyamines putrescine, spermidine and spermine and of the enzymes controlling their synthesis (ornithine decarboxylase; ODC) and degradation (diamine oxidase; DAO) along the villus:crypt axis at the crucial early stage of the ileal adaptive response to jejunectomy, we measured polyamine concentrations and the activities of ODC, DAO and alkaline phosphatase (a marker of enterocyte maturity) in epithelial cells isolated by the Weiser technique from villus tips, mid villi, lower villi and crypts 4 days after surgery in transected control (TRC) and jejunectomised rats untreated or given the specific ODC blocker, alpha-difluoromethyl ornithine (DFMO, 2% in drinking water beginning 3 days before surgery). In the TRCs, there was a diminishing villus tip-to-crypt gradient not only in alkaline phosphatase but also in ODC and DAO activities. After jejunectomy, there were up to 93% increases in mean enterocyte ODC activity when compared with the corresponding cell fractions from the TRCs, but in both the control and jejunectomised rats, DFMO treatment markedly inhibited ODC activity (p less than 0.05-0.01) and reduced spermidine and particularly putrescine concentrations (p less than 0.005-0.001) in all four cell fractions. Only 4 days post-operation, jejunectomy stimulated a significant increase in ileal wet weight but DFMO treatment completely prevented this adaptive response and significantly reduced segmental intestinal weight (mg/cm) in the TRCs. These results (i) extend our knowledge of polyamines and related enzymes along the villus:crypt gradient in the normal intestine, (ii) provides the first data on these variables after resection, and (iii) lend further support to the hypothesis that changes in enterocyte ODC activity and in putrescine and spermidine concentrations play an important role in initiating the ileal adaptive response to proximal small bowel resection in the rat.

Effect of fasting and feeding on polyamines and related enzymes along the villus: crypt axis.

Fasting and feeding have profound effects on crypt cell production and small bowel mucosal growth but the mechanism whereby food stimulates villus tip enterocytes to influence crypt cell production is unknown. We therefore measured the activities of ornithine decarboxylase (ODC), diamine oxidase (DAO) and alkaline phosphatase (ALP--a marker of enterocyte maturity) and polyamine concentrations in epithelial cells from villus tips, mid villi, lower villi and crypts of small intestine in non-fasted controls and 18-24 h fasted rats. Fasting reduced crypt cell production and caused villus hyperplasia, DAO activity (mU/g) increased in control villus tips from 9.6 +/- (SEM) 0.8 to 12.3 +/- 1.5 after fasting (NS), from 7.6 +/- 0.4 to 13.9 +/- 3.0 in mid villi (p less than 0.01), from 5.7 +/- 1.0 to 10.4 +/- 7.4 in lower villi (p less than 0.01) and from 5.4 +/- 0.9 to 12.8 +/- 1.5 in the crypts (p less than 0.001). ALP showed a similar pattern of results. In contrast, fasting lowered ODC activity (pmol/mg protein/h) dramatically from 319 +/- 82 in control villus tips to 16.7 +/- 3.0 during fasting, from 297 +/- 59 to 10.7 +/- 3.6 in mid villi, from 224 +/- 45 to 6.3 +/- 2.8 in lower villi and from 150 +/- 31 to 5.8 +/- 3.3 in the crypts. Fasting reduced putrescine concentrations in all fractions but particularly in the crypts and in general was associated with increases in spermidine and spermine concentrations. The role of DAO in the maintenance of low putrescine concentrations during fasting is unclear.

Is the intestine the sole source of heparin-stimulated plasma diamine oxidase? Acute effects of jejunectomy, ileectomy and total enterectomy.

The assumption that the intestine is the sole/major source of post-heparin diamine oxidase (DAO) has been tested only once by Kobayashi et al. in 1969. In this study we tested whether the intestine is indeed the sole/major source of post-heparin plasma DAO and furthermore we studied the relative contribution of jejunum and ileum to post-heparin plasma DAO. We measured non-fasting plasma DAO before and every 15 min for 2 h after i.v. heparin (4,000 U/kg BW) and compared the resultant areas under the concentration-time curves (AUCs: mU/l X 2 h) in unoperated controls, jejunectomised, ileectomised and totally enterectomised (from gastro-oesophageal to colorectal junctions) male Wistar rats. Total enterectomy virtually abolished the post-heparin rise in plasma DAO, the mean AUC falling from 55.3 +/- (SEM) 10.2 in controls to 2.1 +/- 1.1 after enterectomy (p less than 0.001). The mean AUCs after jejunectomy and ileectomy were 25.6 +/- 5.5 and 10.75 +/- 2.42, respectively. Therefore in acute jejunal and ileal resection the AUCs were 2.1 and 5.4 times, respectively, lower than those seen in the controls (p less than 0.02 and p less than 0.001). The difference in mean AUC values between jejunectomy and ileectomy groups was also statistically significant (p less than 0.05). We conclude that total enterectomy abolishes the post-heparin rise in circulating DAO activity, confirming that the intestine is the major source of post-heparin plasma DAO. The ileum contributes more than the jejunum to this post-heparin rise in plasma enzyme activity.

Aminoguanidine blocks intestinal diamine oxidase (DAO) activity and enhances the intestinal adaptive response to resection in the rat.

The aim of this study was to assess the effect inhibiting diamine oxidase (DAO) activity on intestinal adaptation after 80% proximal small bowel resection in the rat. Aminoguanidine (AG), a DAO inhibitor, was administered subcutaneously (25 mg kg-1 day-1) to rats for 11 days after small bowel transection (n = 6) or resection (n = 6). Two additional groups of animals (n = 6) served as transection or resection controls and received normal saline subcutaneously for the same period. On day 12 after operation, the animals were sacrificed, mucosal homogenates prepared from the ileal remnants (or from the control ileum), analysed for DAO and ornithine decarboxylase (ODC) activities, and the concentrations of the polyamines putrescine, spermidine, spermine, and the indices of mucosal mass (wet weight, protein and DNA) measured. The results were expressed both per unit length intestine and per milligram mucosal DNA. AG treatment completely inhibited DAO activity in both the transection control and resected rats. In the AG-treated resection group, inhibition of DAO activity was accompanied by increases (p less than 0.005) in all three indices of mucosal mass (wet weight, protein and DNA per centimetre intestine), putrescine concentrations (p less than 0.05) and ODC activity per centimetre intestine (p less than 0.001) when compared with the saline-treated resection controls. The results of this study suggest that inhibition of the putrescine-degrading enzyme, DAO, enhances the adaptive response to intestinal resection. Therefore, DAO may play a major role in regulating adaptive intestinal mucosal growth. Furthermore, inhibition of DAO activity could be important therapeutically in patients with the short bowel syndrome.

Cholestasis of total parenteral nutrition: bile acid and bile lipid metabolism in parenterally nourished rats.

As food in the intestine "drives" the enterohepatic circulation and bile acids influence bile flow, we postulated that the cholestasis of total parenteral nutrition might be due to bile acid changes, and the cholelithiasis and biliary sludge of total parenteral nutrition to bile lipid changes. We therefore studied bile acid and bile lipid metabolism in the following groups of rats, with and without bile fistula: (a) nonfasted, orally fed controls, (b) orally fed controls fasted for 20 h, and (c) after 7 days of total parenteral nutrition. Biliary bile acid concentration (35.4 +/- 2.5 mM) and secretion (253 +/- 20.0 mumol/100 g body wt.24 h) increased significantly in the rats on TPN and the rats fasted for 20 h (38.8 +/- 2.5 and 243 +/- 23.4 mM, respectively) when compared with the orally fed controls (26.5 +/- 2.5 and 178 +/- 23.5 mM, respectively). Bile flow, however, was unchanged. Bile acid pool size (Eriksson washout technique) also increased from 43.4 +/- 3.0 mumol/100 g body wt in the controls to 50.5 +/- 4.8 in the group fasted for 20 h and 65.6 +/- 5.3 in the TPN group (p less than 0.05-0.01). Similar bile acid pool sizes (carcass extraction method) were found in the nonfistulated animals. Biliary cholesterol secretion and saturation were significantly less in the TPN rats than in the other two groups. Liver microscopy indicated only minimal fatty change, but serum bile acid and alkaline phosphatase levels were increased in the TPN group (p less than 0.05). Thus, during TPN bile acids stagnate within the enterohepatic circulation, increasing biliary bile acid concentration and secretion rates and expanding the pool size. However, the absence of an associated choleresis, together with abnormal liver function tests, suggest that alterations in bile acid metabolism cause a relative cholestasis in this model.

Longterm pancreaticobiliary diversion stimulates hyperplastic and adenomatous nodules in the rat pancreas: a new model for spontaneous tumour formation.

A model for spontaneous tumour formation in the rat pancreas is described that requires neither cocarcinogens nor dietary manipulation. Short term hypercholecystokininaemia, when induced by raw soya flour feeding, induces benign and malignant tumours of the rat pancreas. Pancreaticobiliary diversion (PBD) results in hypercholecystokininaemia and in the short term, pancreatic hyperplasia. Longterm PBD was done to establish whether hypercholecystokininaemia thus produced would also lead to pancreatic neoplasia. After a period of 16-21 months hyperplastic and adenomatous nodules, one of the latter showing carcinoma in situ, were found in PBD rats but not in sham operated control rats.

Mucosal polyamine profile in normal and adapting (hypo and hyperplastic) intestine: effects of DFMO treatment.

The polyamines, putrescine, spermidine, and spermine, are believed to play an important role in modulating normal and adaptive intestinal mucosal growth. Polyamine synthesis is rate limited by ornithine decarboxylase (ODC) and ODC activity is specifically inhibited by -difluoromethyl ornithine (DFMO). To assess the importance of polyamines in adaptive growth we first measured mucosal polyamine profiles at different sites in the normal rat intestine and compared the results with those obtained in adaptive hypoplasia (seven days parental nutrition, TPN), in the adaptive hyperplasia of two weeks after 90% small bowel resection (SBR) or pancreatico biliary diversion (PBD). We then examined the effects of DFMO (2% in drinking water, daily from two days before surgery) on the polyamine concentrations and the adaptive response to PBD. The hyperplasia of SBR and PBD was associated with increases in all the polyamine concentrations particularly putrescine. TPN induced a modest degree of hypoplasia and little change in polyamine synthesis resulting in subnormal polyamine concentrations and significantly inhibited the mucosal adaptive response. Changes in polyamine metabolism are important in intestinal mucosal adaptation and by controlling these changes adaptive growth can be controlled.