Interference of hydroxyphenylpyruvic acid, hydroxyphenyllactic acid and tyrosine on routine serum and urine clinical chemistry assays; implications for biochemical monitoring of patients with alkaptonuria treated with nitisinone.

OBJECTIVES: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1). MATERIALS AND METHODS: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter. RESULTS: No significant effect (p > 0.1) was observed up to 225 µmol/L of HPPA and HPLA, and up to 5000 µmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked. CONCLUSIONS: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory.

Development of a universal double-digest RAD sequencing approach for a group of nonmodel, ecologically and economically important insect and fish taxa.

The generation of genome-scale data is critical for a wide range of questions in basic biology using model organisms, but also in questions of applied biology in nonmodel organisms (agriculture, natural resources, conservation and public health biology). Using a genome-scale approach on a diverse group of nonmodel organisms and with the goal of lowering costs of the method, we modified a multiplexed, high-throughput genomic scan technique utilizing two restriction enzymes. We analysed several pairs of restriction enzymes and completed double-digestion RAD sequencing libraries for nine different species and five genera of insects and fish. We found one particular enzyme pair produced consistently higher number of sequence-able fragments across all nine species. Building libraries off this enzyme pair, we found a range of usable SNPs between 4000 and 37 000 SNPS per species and we found a greater number of usable SNPs using reference genomes than de novo pipelines in STACKS. We also found fewer reads in the Read 2 fragments from the paired-end Illumina Hiseq run. Overall, the results of this study provide empirical evidence of the utility of this method for producing consistent data for diverse nonmodel species and suggest specific considerations for sequencing analysis strategies.

The Pigment in Alkaptonuria Relationship to Melanin and Other Coloured Substances: A Review of Metabolism, Composition and Chemical Analysis.

The pigments found in plants, animals and humic substances are well described and classified. In humans considerable progress has been made with the main pigment melanin in defining its biochemistry, the different types and function. However, analytical techniques to show these differences in vivo are still not readily available. NMR and IR spectroscopy are relatively insensitive and reveal only major structural differences. Techniques utilising MS are useful in determining elemental content but require further studies to optimise conditions for accurate mass analysis. How the components may be structurally organised seems to be the most problematic with scanning TEM and the improved FTIR of use in this respect. As regards understanding the nature of the pigment related to HGA seen in patients with Alkaptonuria (AKU), it is still thought of as a melaninlike pigment simply because of its colour and likewise thought to be a polymer of undetermined size. It is important that detailed analysis be carried out to define more accurately this pigment. However, observations suggest it to be the same as the HGA-derived pigment, pyomelanin, produced by bacteria and containing both quinone and phenolic groups. The interesting developments in alkaptonuria will be to understand how such a polymer can cause such profound collagen and connective tissue damage and how best to reverse this process.

Interferences of homogentisic acid (HGA) on routine clinical chemistry assays in serum and urine and the implications for biochemical monitoring of patients with alkaptonuria.

OBJECTIVES: We have assessed the effect of elevated concentrations of homogentisic acid (HGA) as in alkaptonuria (AKU), on a range of routine chemistry tests in serum and urine. DESIGN AND METHODS: HGA was added to pooled serum and a range of assays was analysed with Roche Modular chemistries. Effects on urine were assessed by diluting normal urine with urine from a patient with AKU, adding HGA to urine and after lowering output of urinary HGA with nitisinone treatment. RESULTS: Serum enzymatic creatinine showed 30% negative interference with 100µmol/L HGA and >50% at 400µmol/L. Serum urate 100 to 480µmol/L was reduced up to 20% at 100 and to 50% with 400µmol/L HGA. Serum cholesterol between 3 and 11mmol/L was reduced by 0.5mmol/L with 400µmol/L HGA. Urine enzymatic creatinine and urate with >2mmol/L HGA showed concentration dependent negative interference up to 80%. A positive interference in urine total protein by benzethonium turbidometric assay was observed, with 10mmol/L HGA equivalent to 1g/L protein. Jaffe creatinine, Na, K, Cl, Mg, Ca, phosphate, ALT, GGT, ALP activities and urea in serum and or urine were not affected by increases in HGA. CONCLUSIONS: To avoid interferences by HGA in alkaptonuria concentration of HGA should be established before samples are assayed with peroxidase assays and benzethonium urine protein.

An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases.

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

Diabetes in pregnancy: Effect on glycation and acetylation of the different chains of fetal and maternal hemoglobin.

BACKGROUND: Measurement of glycated fetal hemoglobin to assess maternal glycemic control is unreliable. Electrospray ionization-mass spectrometry (ESI-MS) has been suggested as a definitive procedure. OBJECTIVE: This study aimed to evaluate glycation and acetylation by ESI-MS of the separate chains of neonatal fetal hemoglobin in comparison with glycation of maternal hemoglobin, to assess the impact of maternal diabetes. METHODS: Twenty-nine non-diabetic (31 neonates) and 15 diabetic women (15 neonates) were recruited. Whole blood was collected at delivery from the mothers and cord blood from their respective neonate. The blood samples were diluted in acetonitrile:water (50:50) and treated with a cation exchange resin to remove sodium and potassium adducts on the hemoglobin. The α- and ß-chain glycated maternal hemoglobin and α- and γ-chain glycated and acetylated hemoglobin of the neonate were measured by ESI-MS. HbA1c was measured on the Menarini 8160. RESULTS: Mean α- and γ-chain and overall glycated hemoglobin and acetylated fetal hemoglobin were 1.23±0.41%, 1.62±0.47%, 1.24±0.37%, and 8.56±0.84% in the infant of the non-diabetic mother (INDM) and 1.39±0.21%, 1.92±0.61%, 1.64±0.59%, and 8.44±0.63% in the infant of the diabetic mother (IDM). Mean maternal α- and ß-chain, overall glycated hemoglobin and HbA1c were 1.98±0.38%, 4.28±0.76%, 3.13±0.51%, 5.39±0.39% (non-diabetic) and 2.40±0.83%, 4.71±0.90%, 3.58±0.83%, 6.15±0.71% (diabetic). Overall glycated hemoglobin levels were significantly higher in the IDM (p=0.006) compared to the INDM. Maternal glycated hemoglobin was significantly higher than neonatal glycated hemoglobin in the control (p<0.0001) and diabetic group (p<0.0001). A significant correlation was observed between maternal glucose concentration and overall glycated fetal hemoglobin in the IDM (p=0.02). Glucose concentrations in the diabetic mother and the IDM were not significantly different (p=0.5) and were significantly correlated (p<0.0001). HbA1c was not significantly different in the two maternal groups. No significant difference was observed between AcHbF in IDM or INDM (p=0.61). CONCLUSIONS: Measurement of overall glycated fetal hemoglobin, incorporating α- and γ-chain glycations, seems best to assess abnormal glucose homeostasis during pregnancy. Accurate assay of this parameter is critical for stratification of risk of possible post birth developmental complications.

Acidification and urine calcium: is it a preanalytical necessity?

BACKGROUND: It has been suggested that for the accurate measurement of calcium in urine, samples must be collected into bottles containing acid. Acidification poses risks to both patients and laboratory staff. Here we reappraise whether acidification is a preanalytical necessity. METHODS: Twenty-four-hour urine samples were collected from 133 patients into bottles without acid or preservatives. In a subset of 29 patients, 10 mL aliquots were prepared to test the effect on urine calcium of 0.1, 1.0 and 5.0 mol/L hydrochloric acid (HCl). Calcium was then measured immediately after acidification, after 12 h and seven days storage at 4 degrees C. In a separate study, urine calcium concentrations in paired control (non-acidified) and acidified (with 5 mol/L HCl) samples were compared in 133 patients. When available, we recorded the time from start of urine collection to time of analysis. Calcium was measured using the cresolphthalein complexone colorimetric endpoint assay on the Roche Modular system. RESULTS: There was no significant difference in the calcium concentration in the 29 cases studied between the varying acid concentrations tested compared with non-acidified urine (P = 0.987). Overall, in 133 patients there was no difference between control and acidified samples (P = 0.888). We found no correlation between basal urine pH and urine calcium at all time points studied. CONCLUSIONS: Our results suggest that the acidification of urine samples is not a preanalytical necessity for the measurement of urine calcium.

Bacterial killing in gastric juice--effect of pH and pepsin on Escherichia coli and Helicobacter pylori.

The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5-7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1x10(6) E. coli ml-1 and 1x10(8) H. pylori ml-1) were pre-incubated with test solutions at 37 degrees C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml-1 at pH 3.5 reduced viable counts by 100% for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50% of the control after 20 min incubation in 1 mg pepsin ml-1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

Characterization of cationic amino acid transport systems in rat erythrocytes: lack of effect of uraemia on L-arginine influx.

1. Chronic renal failure (CRF) is associated with the abnormal regulation of nitric oxide (NO) synthesis at the systemic level. The transport of L-arginine, upregulated in blood cells from uraemic patients, modulates NO synthesis in this pathological condition. The model of partial nephrectomy in rats is widely accepted as a valid model of uraemia. Because there are no reports of L-arginine transport in blood cells from uraemic rats, the aim of the present study was to investigate L-arginine transport in red blood cells (RBCs) from these rats. 2. The kinetics of L-arginine transport in RBC and plasma and the amino acid profiles of RBC were investigated in control, sham-operated and subtotally nephrectomized rats. 3. L-Arginine transport was mediated via the cationic amino acid transport system y+ and a transport system with kinetics resembling the human system y+L. In control RBC, the apparent Ki for L-leucine inhibition of L-arginine transport via system y+L was 0.16 +/- 0.02 and 4.8 +/- 2 mmol/L in the presence of Li+ and Na+, respectively. 4. The Vmax values for L-arginine transport via system y+L and system y+ were similar in RBC from control sham-operated and uraemic rats. Moreover, L-arginine concentrations in plasma and RBC were not affected by uraemia. 5. The findings of the present study provide the first evidence that L-arginine transport in rat erythrocytes is mediated by two distinct cationic transport systems with characteristics of systems y+ and y+L, which accept neutral amino acids only in the presence of Li+. In contrast with previous studies in uraemic patients, plasma levels and maximal transport rates of L-arginine were not altered in this rat model of CRF.

Review article: human pepsins - their multiplicity, function and role in reflux disease.

Human gastric juice contains a multiplicity of proteinases. These are classified as aspartic proteinases because of enzymic activity dependent on two oppositely placed aspartic acids in the active site region. At least seven zones of activity can be visualized by agar gel electrophoresis and a similar number of separate proteins resolved by high performance ion exchange chromatography. The major enzyme secreted (up to 70% of the total) pepsin 3b is sensitive to the selective inhibitor pepstatin whereas gastricsin or pepsin 5 (20% of the total) is not. Minor enzymes including pepsin 1, which has an associated proteincarbohydrate complex attached is variable and can be < 5% in normal and up to 20% of the total as in peptic ulcer patients. The activity of these enzymes depends on the substrate and pH with significant digestion occurring up to pH 4.5. It has also been shown that these enzymes can bind to substrates like collagen up to pH 5.5. In gastric secretion studies of patients with reflux oesophagitis the amount of pepsin and the profile of the enzymes in basal secretions, and that after pentagastrin stimulation, was found to be not different from healthy non-refluxers. Thus the problem with reflux is that gastric juice appears in the oesophagus, an area without any natural protection from proteolytic damage. The ability to reduce gastric secretion is therefore important in effective treatment. However, being able also to inhibit enzymic activity or protect substrates from damage using alginates offers considerable scope for future therapies.

Activation of L-arginine transport in undialysed chronic renal failure and continuous ambulatory peritoneal dialysis patients.

1. Treatment with haemodialysis and continuous ambulatory peritoneal dialysis (CAPD) presents different pathophysiological profiles and it has been suggested that clinical outcome in chronic renal failure may depend on the mode of dialysis. The transport of L-arginine, a precursor of nitric oxide, into blood cells is increased in uraemic patients on haemodialysis. The present study was designed to investigate L-arginine transport into red blood cells (RBC) in uraemic patients not yet on dialysis and on CAPD therapy. 2. Eleven uraemic patients not yet on dialysis and 17 on CAPD were included in the study. L-Arginine transport into RBC and plasma and RBC amino acid profiles were analysed in these sets of patients. 3. L-Arginine transport via system y(+), but not y(+)L, into RBC, was significantly increased in undialysed uraemic patients (459 +/- 40 micromol/L per cell per h) and CAPD patients (539 +/- 61 micromol/L per cell per h) compared with controls (251 +/- 39 micromol/L per cell per h). High-pressure liquid chromatography measurements demonstrated low levels of plasma L-arginine in uraemic patients both on CAPD (54 +/- 3 micromol/L) and not yet on dialysis (80 +/- 6 micromol/L) compared with control subjects (146 +/- 14 micromol/L). 4. Our findings provide the first evidence that uraemic patients not yet on dialysis and on CAPD present with an activation of L-arginine transport via system y(+) into RBC associated with reduced plasma levels of L-arginine.

A rare ganglioneuroblastoma secreting dopamine and the value of its measurement in diagnosis and prognosis.

A case is described of a patient with a ganglioneuroblastoma, initially located in the right adrenal, which produced an excess of dopamine (7646 and 7959 nmol/24 h), approximately two and a half times the upper limit of the normal daily urine output. The urinary excretion of noradrenaline, adrenaline and methylated derivatives was always within the normal reference ranges. The patient was generally well, with normal blood pressure and only mild flushes. Two years after surgical resection, recurrence was indicated by an increase in urinary dopamine (8507 nmol/24 h); it was located in the tumour bed and left side of the neck by CT and (123)I MIBG scans. The patient was treated with a high dose of (131)I MIBG, with subsequent reduction in dopamine production. This was repeated on four other occasions, the latest being in January 2005. The output of dopamine was thus used as a marker of tumour diagnosis and progression and it is recommended that the assay of dopamine be included in the screening of catecholamine-secreting tumours to avoid possible misdiagnosis.

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A.

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.

Activation of L-arginine transport in peripheral blood mononuclear cells in chronic renal failure.

Transport of LL-arginine, the precursor for nitric oxide (NO) synthesis, has been investigated in human peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers and chronic renal failure patients. Chronic renal failure patients were either on treatment by haemodialysis or continuous ambulatory peritoneal dialysis (CAPD). Saturable influx of L-arginine in PBMCs was mediated by the cationic amino acid transport systems y(+) and y(+)L. Initial rates of L-arginine transport (2 microM) via system y(+) were significantly increased in chronic renal failure patients, whereas transport via system y(+)L was unaffected. The increase in L-arginine transport via system y(+) was: 1.7-fold in uraemic patients on CAPD, 4.3-fold in uraemic patients pre-haemodialysis and 2.6-fold post-haemodialysis. When the intracellular PBMCs amino acid profile was analysed in chronic renal failure patients and control subjects, L-lysine and L-arginine concentrations were significantly increased in pre-haemodialysis uraemic patients and restored to normal values by haemodialysis and CAPD. The present study provides the first evidence that system y(+) mediates the increased transport of L-arginine in PBMCs from patients with chronic renal failure. The increased activity of system y(+) may provide the necessary supply of L-arginine to sustain NO synthesis in PBMCs exposed to increased levels of circulating cytokines in chronic renal failure.

Different Mg to Fe ratios in the mixed metal MgFe hydroxy-carbonate compounds and the effect on phosphate binding compared with established phosphate binders.

Due to the side effects of the current oral phosphate binders, there is a need for effective alternatives. A number of mixed metal hydroxy-carbonate compounds (MMHCs) based on Mg and Fe have recently been established as effective phosphate binders. We have now carried out further studies on the MMHCs with different ratios of Mg(2+):Fe(3+) in different forms to assess for phosphate binding efficacy and ionic release in aqueous solution and food slurries. The compounds that provide the most promise are those with Mg(2+):Fe(3+) ratios of 2:1 and 4:1 in the unaged/dry form. Their phosphate binding efficacy was compared with a wide range of established phosphate binders, such as aluminum hydroxide [Al(OH)(3)], calcium carbonate (CaCO(3)), calcium acetate (CaAc(2)), magnesium hydroxide [Mg(OH)(2)], and lanthanum carbonate [La(2)(CO(3))(3)] in various food slurries. The results showed that the MgFe compounds were much more effective (on a weight for weight basis) than the established binders, and their properties were relatively pH independent. Calcium compounds (CaCO(3) and CaAc(2)) were ineffective under the experimental conditions. Mg(OH)(2) was effective at low pH but not at pHs greater than 5.0, and also released two- to threefold more magnesium than the MgFe compounds. Al(OH)(3) showed some degree of efficacy, but the binding capacity was, at best, less than 50% of the MMHCs. La(2)(CO(3))(3) required at least a 10-fold increase in weight to give comparable binding to the MMHCs. In conclusion, MgFe hydroxy-carbonate compounds are effective phosphate binders and may provide a better alternative to both existing and emerging binders for combating hyperphosphataemia.

In-vitro studies of aluminium-induced toxicity on kidney proximal tubular cells.

The toxicity of aluminium (Al) to various cells is well described. However, little is known about its effect on kidney cells, which can be exposed to relatively high concentrations. In this study, the effect of aluminium as the citrate complex in concentrations up to 100 microM/l was investigated using a monolayer cell culture of kidney proximal tubular cells (PTC). Aluminium was found to be slightly toxic; at 100 microM/l the PTCs lost viability by 15, 20 and 24% after incubation for 24, 48 and 72 h, respectively. Viability was significantly reduced (P<0.001) after 48 h incubation with aluminium concentrations of 25, 50, 75 and 100 microM/l compared with controls. Lactate dehydrogenase (LDH) release was significantly increased (P<0.001) with 100 microM/l Al to 44.67+/-1.76 and 50.33+/-0.88 compared with controls 24+/-1.00 and 28.33 2.34 U/l after 24 and 48 h incubation, respectively, indicating damage to the plasma membrane. However, N-acetyl-beta-D-glucosaminidase (NAG) release in the medium of cells exposed to aluminium showed no difference from control values (P>0.1). Glucose consumption in aluminium-exposed cells at 100 microM/l was slightly, but not significantly (P=0.14), increased during 48 h incubation. Electron micrographs of cells exposed to aluminium at 100 microM/l showed a slight reduction in microvilli density and the cell tight junctions were not as clear compared with the control cells. Pretreatment with protective agents glutathione and tiopronin partly restored the viability of kidney proximal tubular cells to control values, whereas vitamin C and/or cysteine showed no effect. This study indicates that aluminium may show toxicity to kidney cells in culture. Several sites on the cell, i.e. microvilli, membrane and the cell junction, seem to be affected, however the mechanism(s) of damage remain unclear.

The evaluation of novel mixed metal hydroxy-carbonates as phosphate binders: an in-vivo study in the rat.

A number of novel phosphate binders based on mixed metal hydroxide structures incorporating Fe and Ca, or Fe and Mg (classified as CT, Crosfield test compounds), were compared with the established phosphate binders Mg(OH)2, Al(OH)3, CaCO3 and a commercial hydrotalcite (Al- and Mg-based) using a rat model. The changes in urine and soluble faecal phosphate were used to evaluate efficacy of phosphate binding. The binders were mixed into a standard rat maintenance food at a concentration of 1% (w/w). Four rats were used for each binder study group and fed over 7 days. Urine and faeces were collected (in a metabolic cage) over the last 24-h study period and the phosphate content measured. The urinary phosphate was significantly reduced (P < 0.001) with CTFeCa (72+/-44 microm), CTFeMg (13+/-4 microm), CT100 (26+/-11 microm), and Mg(OH)2 (65+/-53 microm), compared with control (766+/-188 microm), Al(OH)3 (1,256+/-279 microm), and CaCO3 (857+/-25 microm). The soluble phosphate content of the faeces was significantly reduced (P < 0.05) by up to 60 % with CTFeCa, CTFeMg and Mg(OH)2, and up to 40% with CT100 and Al(OH)3, compared with 30% in controls and 10% with CaCO3. The new mixed metal hydroxy-carbonate compounds based on FeCa or FeMg are effective phosphate binders in-vivo and warrant further testing in patients.

The development and in-vitro evaluation of novel mixed metal hydroxy-carbonate compounds as phosphate binders.

The currently available phosphate binders are relatively inefficient and suffer from clinical side-effects of increased absorption of calcium and aluminium and the diarrhoea-inducing effects of magnesium. A new class of compounds based on mixed metal hydroxides has been developed and evaluated for their potential as phosphate binders. The mixed metal hydroxides were prepared using a standard procedure for hydrotalcite (Al2Mg6(OH)16.CO3.4H2O) by substituting Fe3+ for Al3+, with Mg2+ or Ca2+ as the divalent metal ion. Phosphate precipitation (binding) was examined at different pH values in aqueous solution and in various food mixtures in comparison with hydrotalcite, Al(OH)3, CaCO3 and Mg(OH)2 on the same weight-to-weight basis. A series of compounds with differing ratios of metal ions (Fe:Mg/Ca 1:2 or 1:3) gave analytically similar ratios to those predicted from the initial amounts added. CTFeCa bound > 90% phosphate in aqueous solution compared with 65% binding with CTFeMg, 85% binding with Mg(OH)2, and less than 30% binding for CaCO3 and Al(OH)3. The mixed metal compounds also bound up to 80% phosphate in various food matrices, which was relatively independent of changes in pH, compared with Mg(OH)2, where binding decreased from 85% at pH 3.0 to 25% at pH 8.0. Al(OH)3 and CaCO3 were relatively ineffective phosphate binders under all the conditions tested. The mixed metal hydroxides compounds show considerable promise as phosphate binders over those currently available and warrant further patient-based in-vivo testing.

Mucosal protective effects of ecabet sodium: pepsin inhibition and interaction with mucus.

Pepsin, acid and Helicobacter pylori are major factors in the pathophysiology of peptic ulcer disease and reflux oesophagitis. Ecabet sodium reduces the survival of H. pylori in the stomach and inhibits pepsin activity in the gastric juice of experimental animals. Here we have investigated the effects of ecabet sodium on some of the factors involved in the dynamics of the mucosal barrier, i.e. pepsins and mucins. This study used gastric juice obtained from 12 non-symptomatic volunteers and nine patients with reflux oesophagitis. Ecabet sodium significantly inhibited pepsin activity in human gastric juice, with a maximum inhibition of 78%. Pepsin 1, the ulcer-associated pepsin, was inhibited to the greatest extent. The ability of gastric juice to digest mucin was significantly inhibited by ecabet. As with gastric juice proteolytic activity, the inhibitory effect of ecabet on mucolysis was greater in gastric juice from patients with reflux oesophagitis than in that from controls. Ecabet sodium showed a positive interaction with gastric mucin, as assessed by an increase in viscosity. Thus ecabet sodium may reduce the aggressive potential of gastric juice towards the mucosa, which may be relevant in the treatment of reflux oesophagitis and peptic ulcer disease. In addition, it may strengthen the mucus barrier in peptic ulcer disease and gastritis.