N B Roberts

Accumulation of the endogenous L‐arginine analogue NG‐monomethyl‐L‐arginine in human end‐stage renal failure patients on regular haemodialysis

We measured plasma levels of L‐arginine, its analogue NG‐monomethyl‐L‐arginine (L‐NMMA), and related amino acids in normal subjects and uraemic patients (n = 31), before and after haemodialysis. Plasma levels of L‐arginine were reduced to less than half of normal values in uraemic subjects compared with controls, and were not affected by haemodialysis. L‐NMMA was not detectable in non‐uraemic subjects, but markedly elevated in uraemic patients. In parallel, we used human red blood cells as a model to study the effect of L‐NMMA upon the transport of L‐arginine. L‐NMMA trans‐stimulated L‐arginine transport significantly, confirming that L‐arginine and L‐NMMA share common transport pathways. Our results suggest altered L‐arginine metabolism and the presence of an increased concentration of a NO synthase inhibitor in uraemia. We propose that alterations in plasma L‐arginine levels and increased production of L‐arginine analogues will alter NO synthesis and may help to explain some pathological changes seen in uraemia.

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 mmol/L per cell per h) and CAPD patients (539 61 mmol/L per cell per h) compared with controls (251 39 mmol/L per cell per h). Hihg‐pressure liquid chromatography measurements demonstrated low levels of plasma l‐arginine in uraemic patients both on CAPD (54 3 mmol/L) and not yet on dialysis (80 6 mmol/L) compared with control subjects (146 14 mmol/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.

Resistance to recombinant human erythropoietin due to aluminium overload and its reversal by low dose desferrioxamine therapy.

Seventeen severely anaemic and transfusion-dependent haemodialysis patients with a haemoglobin less than 7 g/dl were treated with recombinant human erythropoietin (r-Hu-EPO). Aluminium toxicity was diagnosed by a positive desferrioxamine (DFO) test and bone biopsy. Seven out of eight patients without aluminium toxicity responded to r-Hu-EPO therapy. Similarly all patients with aluminium toxicity (n = 4) but pre-treated with standard dose of DFO prior to r-Hu-EPO therapy responded but none of the patients with untreated aluminium toxicity (n = 5) responded to r-Hu-EPO therapy. In order to achieve adequate response in these patients, r-Hu-EPO and DFO had to be given in combination. The dose of desferrioxamine used to reverse r-Hu-EPO resistance was less and also used for a short time. We therefore confirm r-Hu-EPO resistance owing to aluminium overload and report its successful and safe reversal with low dose DFO therapy.

Measurement of Copper, Zinc and Magnesium in Serum and Urine by DC Plasma Emission Spectrometry

The application of a DC plasma emission spectrometer to the measurement of copper, zinc and magnesium in serum and urine is reported. Each assay requires only a simple dilution in 1% nitric acid. Comparisons with standard atomic absorption techniques showed good analytical agreement, especially for copper and magnesium. Analysis of quality control preparations for copper and zinc gave results ranging from −1·5% to +3·6% of the stated values for five copper and four zinc experiments, with an unexplained result of +7·6% for one zinc experiment. Reference ranges were compiled for serum zinc and urinary copper which agreed closely with those established by atomic absorption and neutron activation analysis. Assays by DC plasma emission are thus precise and reproducible, and simple enough to authenticate the method for use in a clinical laboratory.

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 μmol/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.

Plasma Emission Spectrometry: Measurement of Calcium, Phosphorus and Chromium in Metabolic Balance Studies

Direct-current plasma emission spectrometry determines food calcium and phosphorus, faecal calcium and chromium, and urinary calcium and phosphorus accurately, precisely and rapidly without acid digestion or dry ashing. Food and faeces are homogenised, diluted and aspirated directly into the plasma. The method makes metabolic balances swifter and less labour-intensive; it enables the use of chromium sesquioxide as an inert marker, for perchlorate digestion is avoided. Both plasma emission (–9.9%) and a dry ashing procedure (–13.5%) give lower values for food phosphorus than those derived from food tables.

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.