L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.

Summary 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.

BACKGROUND Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS A total of 619 standards (molecular weight, 45–1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods—2 reversed phase and 1 normal phase—coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD−/− mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU.

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