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.

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.

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.

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.

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.

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.

Five human gastric aspartic proteinases: N-terminal amino acid sequences and amino acid composition.

Human pepsin A consists of 4 or more isoenzymes (designated 1, 3a, 3b and 3c) one of which, pepsin 1, contains up to 50% carbohydrate moieties. The amino-acid composition and N-terminal sequence of pepsin 1 and the other isoforms have been determined and compared with data obtained for pepsin 3b and gastricsin (pepsin C or pepsin 5). Pepsins were isolated from penta-gastrin stimulated gastric juice using repetitive chromatography on DEAE-cellulose, or high performance ion-exchange chromatography. Sequencing was performed using automated solid-phase Edman degradation with a microsequence facility. The amino-acid compositions were similar for pepsins 1, 3a, b and c and the N-terminal sequences of pepsins 1, 3a and c, reported for the first time, were shown to be identical with that for pepsin 3b (the main component of pepsin A) although residue 28 was unassigned in pepsin 1. Residue 30 in all four isoenzymes is valine and we cannot confirm reports of major pepsins with leucine in this position. For gastricsin the sequence differed from the pepsin isoenzymes and in position 24 we find pro rather than ala as was first described. These observations suggest that pepsin 1 is identical to 3b or a mixture of 3a, 3b and 3c but not gastricsin. This data supports the hypotheses that the four pepsin isoenzymes are products of the same gene(s) but have undergone varying levels of post translational modification.

Elemental analysis of femoral bone from patients with fractured neck of femur or osteoarthrosis.

The elemental composition of bone has been determined by inductively coupled atomic emission and mass spectrometry to test the hypothesis that changes in major or minor elemental concentrations may contribute to the risk of fracture. Femoral bone was obtained from patients at operation for the treatment of fracture and compared with that of patients with osteoarthrosis and a necropsy control group. The data suggest that there are no major differences in bone elemental composition in patients with fractures compared with the control group. Bone adjacent to joints with osteoarthrosis tends to be less mineralized (per unit trabecular bone volume) than control bone and bone from fracture patients, and has significantly lower concentrations of boron, lead and, zinc. These observations may reflect the more rapid turnover of bone close to the arthritic joint.

Hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, codon 143 CAC-->TAC--a variant with altered oxygen affinity that compromises measurement of glycated hemoglobin in diabetes mellitus: structure, function, and DNA sequence.

OBJECTIVE: To determine the nature and characteristics of a unique hemoglobin variant that causes a spurious increase in glycated hemoglobin (HbA1c). MATERIAL AND METHODS: Blood specimens from four unrelated persons with this hemoglobin variant were examined by conventional laboratory methods, including electrophoresis, high-performance ion-exchange chromatography, and isoelectric focusing; by amino acid sequence analysis, polymerase chain reaction-based DNA sequence analysis, and electrospray ionization mass spectrometry, to establish the molecular structure; and by studies of oxygen affinity under varied conditions, to define the functional characteristics of the hemoglobin variant. RESULTS: The unique hemoglobin variant observed in these four cases is due to the mutation CAC-->TAC, at beta-globin gene codon 143, corresponding to beta 143 (H21) His-->Tyr. This amino acid substitution affects an important 2,3-diphosphoglycerate binding site and slightly increases the oxygen affinity of the hemoglobin variant. CONCLUSION: A hitherto unrecognized hemoglobin variant, encountered in four unrelated persons of Irish or Scots-Irish ancestry, hemoglobin Old Dominion/Burton-upon-Trent, beta 143 (H21) His-->Tyr, has now been characterized at the molecular, structural, and functional levels. Although it is associated with a slight increase in oxygen affinity, it is without hematologic effect, and its only clinical significance is that it coelutes with HbA1c on ion-exchange chromatography and thereby causes a spurious increase in HbA1c and compromises the use of this analyte to monitor the treatment of diabetes mellitus.

Necropsy study of the association between sudden cardiac death, cardiac isoenzymes and contraction band necrosis.

AIMS: To assess whether a quantitative analysis of myocardial contraction bands could aid the postmortem identification of early myocardial infarction, especially if used in conjunction with cardiac isoenzyme activities. METHODS: Sixty four coroner's necropsies were grouped by gross and histological findings into 26 cases of definite non-cardiac death, 12 cases of definite myocardial infarction and 26 cases in which there was occlusive coronary artery atheroma, but no demonstrable evidence of infarction. Using multiple sections of left ventricular myocardium stained with Heidenhain's iron haematoxylin, the number of myocardial cells containing contraction bands per unit area was quantified. The results were analysed statistically using logistic regression, and were then compared and combined with results from the statistical analysis of postmortem cardiac isoenzymes that had recently been undertaken on the same cases. RESULTS: The number of cells containing contraction bands per unit area was higher in cases of definite myocardial infarction compared with those of non-cardiac deaths. In addition, cases of occlusive coronary artery atheroma only could be identified, indicating the presence of early myocardial infarction. The accuracy of this identification could be improved by combining these results with the results from the statistical analysis of postmortem cardiac isoenzymes. CONCLUSION: The quantitative assessment of myocardial contraction band necrosis can provide useful additional information in cases of sudden death, where myocardial infarction is suspected but not identified on routine histological staining. The value of the information obtained is increased when used in conjunction with the postmortem measurement of cardiac isoenzyme activities.

Necropsy study of association between sudden death and cardiac enzymes.

AIMS: To determine if cardiac enzymes measured at necropsy could be used to predict early myocardial infarction. METHODS: Cardiac enzyme activities were measured in body fluids at necropsy. Coroners' necropsies were grouped by gross and microscopic findings into cases of definite myocardial infarction, cases with occlusive coronary artery atheroma but no identifiable myocardial infarction, and non-cardiac cases. Pericardial fluid, peripheral venous blood, and blood from the right atrium were collected. Total creatine phosphokinase, creatine phosphokinase isoenzymes, aspartate aminotransferase and hydroxybutarate dehydrogenase activities were measured and the results analysed by logistic regression. RESULTS: The values of creatine phosphokinase and its isoenzymes were raised in those who had died of cardiac disease and were most discriminatory. Cases of early myocardial infarction without evidence of infarction on routine histological examination could be identified from enzyme activities. CONCLUSIONS: Measurement of cardiac enzymes in blood and pericardial fluid at necropsy can provide valuable additional information in cases of sudden death as a result of myocardial ischaemia which have occurred before macroscopic or microscopic evidence of myocardial infarction.

Assay of gastricsin and individual pepsins in human gastric juice.

AIMS: To develop and validate an analytical procedure for the quantitation of pepsins and gastricsin in human gastric juice and to assess its potential in a controlled gastric secretory study. METHODS: High performance ion-exchange chromatography was used to separate human pepsin 1, 3a, 3b, 3c and gastricsin from gastric juice. Computed chromatographic areas for each enzyme were quantified by relation to a known amount of a secondary standard porcine pepsin. The assay procedure was validated by recovery and analytical precision studies. Gastric secretions after pentagastrin and insulin stimulation from 10 patients with portal hypertension were used to assess the potential of the analytical procedure. RESULTS: The assay precision varied from 1.5 to 9.0% within batch and 7.5 to 18.1% between batch, with about 100% recoveries of porcine pepsin A from human gastric juice over the assay range 0.025-0.5 mg/ml. A fourfold increase in combined pepsin and gastricsin concentration was observed following pentagastrin and insulin stimulation. The mean percentage content of pepsins 3a, 3b, 3c, and 1 in non-stimulated gastric juice were 4%, 72%, 12% and 1.4%, respectively, and did not change significantly after gastric stimulation. An approximate doubling of the percentage of gastricsin (10% to 20%) relative to the pepsins was observed, however, after both insulin and pentagastrin stimulation. CONCLUSIONS: This procedure for quantifying individual human pepsins and gastricsin in gastric juice is simple and reliable. It may be of considerable importance in determining the mechanisms involved in the control and secretion of these digestive enzymes in man, including the effect of anti-ulcer drugs and our understanding of the pathophysiology of peptic ulcer disease.

Age does not influence levels of HbA1c in normal subject.

To resolve whether haemoglobin A1c(HbA1c) levels in normal subjects increase with age, we measured HbA1c in 399 patients undergoing routine oral glucose tolerance test (OGTT). The OGTT results categorized the patients into 127 normal, 94 impaired glucose tolerance (IGT) and 178 diabetic. None of these groups showed a significant correlation between HbA1c and age and we cannot, therefore, see a need for age-specific reference ranges for HbA1c. Some of the confusion in the literature may have arisen from less rigorous categorization of subjects than we used, resulting in the inclusion of some individuals with IGT or diabetes in the 'normal' groups of other studies. The prevalence of such abnormality would be expected to be greater amongst older subjects, falsely suggesting a correlation between HbA1c and age, and we were able to demonstrate this with our own data when insufficiently rigorous criteria were applied for the selection of normal subjects.

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.

Increased absorption of aluminium from a normal dietary intake in dementia.

Serum aluminium was significantly raised (p < 0.01) up to 2-3-fold, in patients with dementia including Alzheimers Disease (AD) 0.66 +/- 0.2 (mumol/l mean +/- 1 s.d.) and patients on regular aluminium hydroxide therapy 0.54 +/- 0.17, compared with healthy volunteers 0.21 +/- 0.13, although not as high as in patients with end stage renal failure on regular dialysis 0.88 +/- 0.42. The urine outputs (mumol/l mean +/- 1 s.d.) of aluminium and silicon, respectively, were also significantly increased up to 5-fold in dementia 2.89 +/- 1.78 (n = 23) and 1587 +/- 645 (n = 22) and patients on regular aluminium hydroxide therapy 5.03 +/- 2.08 (n = 8) and 998 +/- 364 (n = 21) compared with healthy volunteers 0.95 +/- 0.82 (n = 84) and 471 +/- 332 (n = 114). The increase in urine aluminium was thus associated with a similarly marked increase in the output of silicon. The increased absorption of aluminium in dementia patients is equivalent to the intestinal loading in Aludrox therapy. Also silicon appears to be important in the renal excretion of the absorbed aluminium. Whether this is a phenomenon related to the elderly or the process of dementia warrants further study.

Human erythrocyte and plasma amino acid concentrations during exercise.

PURPOSE: This investigation examined the effects of exercise and maltodextrin (Md) or placebo (Pl) ingestion on plasma and erythrocyte concentrations of amino acids. METHODS: The erythrocyte and plasma concentrations of 17 amino acids, as well as plasma glucose and insulin, were analyzed in eight healthy trained male subjects before, during, and 25 min after 90-min cycle ergometer exercise at 65% peak oxygen uptake. The two treatments involved ingestion of orange-flavored water (Pl) or orange-flavored 10% maltodextrin solution (Md). RESULTS: Two-way ANOVA revealed 1) that plasma concentrations of alanine and tyrosine changed significantly during the treatments, 2) that the plasma concentrations were significantly different between treatments for glycine and threonine, 3) that all erythrocyte concentrations increased significantly throughout the treatments except for arginine and tyrosine, and 4) that there were no significant differences in erythrocyte concentrations between the treatments. Three-way ANOVA highlighted the significant differences in the time responses between plasma and erythrocyte concentrations; the changes in erythrocyte levels from rest being significantly different from plasma for all amino acids except aspartic acid, glycine, and ornithine. Plasma glucose concentrations became elevated and remained above rest values in Md but fell below rest values in Pl: the differences in concentration between treatments were significant. Correspondingly, plasma insulin was significantly higher in Md during exercise. CONCLUSION: These results highlight that far from being slow in the uptake of amino acids, the erythrocyte in fact sequesters amino acids at an appreciable rate during exercise without a corresponding elevation in the plasma amino acids. For a greater understanding of amino acid changes during exercise, the analysis of both plasma and erythrocytes is recommended.

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.

An improved automated method for the analysis of inorganic phosphate without dialysis.

A procedure is described for automating the analysis of inorganic phosphate in physiological media without dialysis. Sodium dodecyl sulphate is used to maintain protein solubility during the analysis. Sample analysis was measured at 60 and 90 samples an hour. The inorganic phosphate results obtained for a series of human plasma and for various internal control sera by non-dialysis and dialysis showed good agreement.

Serum aluminium measurement by DC plasma emission spectrometry.

A serum aluminium assay has been established using a DC plasma emission spectrometer and calibration with serum based standards. The assay is linear between 0.20 and 18.0 mumol/L aluminium. The procedure gives good analytical recovery and precision. The assay was validated by comparison(s) with an electrothermal atomic absorption (ETAAS) procedure which showed good agreement. Serum aluminium concentrations have been compared in normal individuals, in undialysed chronic renal failure patients with or without aluminium hydroxide treatment, and in dialysed patients treated with oral aluminium hydroxide. Non-dialysed patients had higher serum aluminium concentrations if treated with aluminium hydroxide. Haemodialysis patients had the highest serum aluminium concentrations whilst those on peritoneal dialysis had levels similar to those of the non-dialysed chronic renal failure group.