L-citrulline Levels Research Articles

L-arginine plasma levels and severity of idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (iPAH) is a rare disease of unknown aetiology characterized by a poor prognosis. Impairment of nitric oxide (NO) synthesis or NO-induced vasorelaxation has been suspected to play a role in the development of iPAH. This study was performed to investigate possible correlations between the plasma levels of the NO-related aminoacids L-arginine, L-citrulline and N-hydroxy-L-arginine (L-NHA) and the severity of iPAH. In twelve iPAH patients hemodynamics were measured by right heart catheterization, and plasma levels of L-arginine, L-citrulline and L-NHA were determined in blood samples from the pulmonary artery, peripheral artery and peripheral vein by high-performance liquid chromatography analysis. In eight of twelve patients a six minute walk test was performed. Plasma levels of L-arginine strongly correlated to right atrial pressure, cardiac output, cardiac index, mixed-venous oxygen saturation, six minute walk data and NYHA functional class at all sites of blood sampling (p < 0.05). The results suggest a possible role of the NO precursor L-arginine in the pathogenesis of iPAH.

Effect of agmatine on brain l-citrulline production during morphine withdrawal in rats: A microdialysis study in nucleus accumbens

Agmatine, an endogenous nitric oxide (NO) synthase inhibitor and ligand for imidazoline receptors, has been previously shown to prevent morphine dependence in rats. The present study was designed to investigate NO formation in nucleus accumbens core region (NAcc) during naloxone (NL)-precipitated morphine withdrawal in rats treated with agmatine or l-NAME by using intracerebral microdialysis in freely moving rats, through measuring extracellular l-citrulline concentrations, an indirect sign of NO production since equal amounts of l-citrulline and NO are produced from l-arginine. l-Citrulline levels in the NAcc core did not change following administration of agmatine (40 mg/kg i.p.) or l-NAME (100 mg/kg i.p.) in control rats. Both agmatine and l-NAME attenuated withdrawal symptoms of morphine in NL (2 mg/kg i.p.)-precipitated withdrawal. l-Citrulline levels showing the release of NO increased in morphine-dependent rats during NL-precipitated withdrawal. Agmatine and l-NAME treatments significantly suppressed the increase in l-citrulline levels compared to physiological saline-treated rats in this setting. The results suggest that the release of l-citrulline in NAcc may be involved in the processes of morphine withdrawal and agmatine as an endogenous inhibitor of NO synthase may be one of the factors involved in the changes in the physiology and behavioral state during opioid withdrawal and may have pharmacological importance.

Effect of agmatine on mophine dependence in comparison with L-NAME: a microdialysis study in nucleus accumbens

Background Agmatine is an endogenous amine synthesized from Larginine. It has several biological actions including NMDA receptor blockade and nitric oxide inhibition. It potantiates morphine analgesia, prevents tolerance and inhibits all semptoms of withdrawal. The aim of present study was to investigate effect of agmatine on L-citrulline levels during morphine dependence and withdrawal in nucleus accumbens.

Effect of dietary conjugated linoleic acid on serum levels of N 2O 5 and l-citrulline in goat kids

Forty new born male kids were allotted into two groups for the evaluation of the effects of dietary conjugated linoleic acid (CLA) on nitric oxide (NO) and l-citrulline levels in serum. The control group received no supplement CLA and the CLA group received 20 g/kg milk DM of CLA from birth to 1-month old. The kids were fed colostrum for 2 days and milk replacer from days 3 to 29. Blood samples ( n = 40) were taken at 1, 8, 15, 22, and 29 days of age. N 2O 5 (nitrite + nitrate) concentration in serum ranged from 21.93 to 26.15 and 34.90 to 40.59 μmol in control and CLA kids. The l-citrulline values ranged from 0.30 to 0.40 and 15.54 to 19.81 μmol in control and CLA kids.

Effects of prolonged oral supplementation with l‐arginine on blood pressure and nitric oxide synthesis in preeclampsia

Several lines of evidence point to the dysfunction of the endothelial l-arginine-NO system in preeclampsia. We investigated the influence of dietary supplementation with l-arginine on blood pressure and biochemical measures of NO production in women with preeclampsia in prospective, randomized, placebo-controlled study. The 61 preeclamptic women on a standardized low nitrate diet received orally 3 g of l-arginine (n = 30) or placebo (n = 31) daily for 3 weeks as a supplement to standard therapy. The differences between the two groups in systolic (SBP), diastolic (DBP) and mean arterial blood pressures (MAP) as well as in plasma levels of selected aminoacids, plasma concentrations of nitrates/nitrites (NOx) and in 24-h urine NOx excretion were determined. After 3 weeks of treatment, values of SBP, DPB and MAP were significantly lower in the group taking l-arginine as compared with the placebo group (SBP: 134.2 +/- 2.9 vs. 143.1 +/- 2.8; DBP: 81.6 +/- 1.7 vs. 86.5 +/- 0.9; MAP: 101.8 +/- 1.5 vs. 108.0 +/- 1.2 mmHg, P < 0.01). Importantly, treatment with exogenous l-arginine significantly elevated 24-h urinary excretion of NOx and mean plasma levels of l-citrulline. Exogenous l-arginine did not influence plasma concentrations of l-arginine, l-ornithine and methylated arginines (ADMA, SDMA, L-NMMA). We conclude that in women with preeclampsia, prolonged dietary supplementation with l-arginine significantly decreased blood pressure through increased endothelial synthesis and/or bioavailability of NO. It is tempting to speculate that the supplementary treatment with l-arginine may represent a new, safe and efficient strategy to improve the function of the endothelium in preeclampsia.

The Influence of a Load of l-Arginine on Serum Amino Acids and Pancreatic Apoptosis/Proliferation and ATP Levels in the Rat

Administration of high doses of amino acids like ethionine, methionine, and arginine causes pancreatic tissue damage. The initial mechanism behind these effects is not known. The aim of this study was to show the early effects of a load of L-arginine on programed cell death/proliferation and ATP levels in the pancreas. We analyzed in rats the effects of intraperitoneal administration of L-arginine on serum amino acids, pancreatic cell apoptosis/proliferation, and ATP levels at 8, 16, and 24 hours. Serum amino acid concentrations were measured with HPLC, tissue ATP was measured fluorometrically, apoptosis was studied with caspase-3 activity and histone-associated DNA-fragments, and proliferation was studied with thymidine autoradiography. After a load of l-arginine, there were initially increased serum levels of L-arginine and L-citrulline, but these fell below control levels after 24 hours as well as amino acids in the glutamate family (ornithine, proline, histidine, and glutamine). Initially, increased ATP levels in the pancreatic tissue returned to control levels at 24 hours. The acinar cells proliferation was suppressed and the apoptosis rate strongly increased at 16 and 24 hours. Pancreatic histology showed vacuole formation in the acinar cells at 8 hours. At 16 hours, there was less vacuolization, but apoptotic bodies were seen, and at 24 hours there was cell degeneration but no necrosis. After a load of l-arginine, amino acid metabolism causes a high ATP production in the pancreatic tissue that may cause mitochondrial initiation of cell death.

Role of adenosine transport in gestational diabetes-induced L-arginine transport and nitric oxide synthesis in human umbilical vein endothelium.

Gestational diabetes is associated with increased L-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial L-arginine/NO pathway via A(2) purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 microM), and intracellular L-arginine (1.9 mM) and L-citrulline (0.7 mM) levels compared with normal cells (0.05 microM, 0.89 mM, 0.35 mM, respectively). Incubation of HUVEC from normal pregnancies with 1 microM nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44(mapk) activation, and were blocked by the A(2a) purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the L-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A(2a) purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of L-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus.

Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells.

Insulin induces vasodilatation in human subjects and increases L-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. L-arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca2+, L-arginine and L-citrulline levels, L-[3H]citrulline formation from L-[(3)H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased L-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca2+, L-[3H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the L-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.

Upregulation of endothelial nitric oxide synthase in rat aorta after ingestion of fish oil-rich diet.

A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (.NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affects the vascular activity per se and the point of the.NO-cGMP pathway at which fish oil acts. For this purpose, two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids, either corn oil (CO) or menhaden oil (MO), for 8 wk. We studied the mRNA and protein levels of endothelial NO synthase (eNOS) and NOS activity. The bioavailability of vascular.NO was assessed directly by electron spin resonance spectroscopy. The levels of cGMP, l-arginine, and l-citrulline were also evaluated in homogenates. Superoxide anion (O(2)(-).) production and related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% relative to the rats fed the CO-rich diet) and NOS activity (102%);.NO production increased by 90%, cGMP levels increased by 100%, and l-arginine decreased by 30%. No change in aortic O(2)(-). production was caused by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.

Effects of ACE inhibition and AT1-receptor blockade on haemodynamic responses to L-arginine in Type 1 diabetes

Angiotensin-converting enzyme (ACE) inhibitors have been shown to improve endothelial function in Type 1 diabetes. However, the potential of ACE inhibitors (ACE-I) to enhance the haemodynamic effects of L-arginine (L-arg), the precursor of nitric oxide (NO), has not been evaluated. Furthermore, angiotensin receptor blockers (ARBs), another group of inhibitors of the renin-angiotensin system (RAS), have not been studied in this context. Using a randomised, crossover design, the acute effects of L-arg (200 mg/kg) on blood pressure (BP) and renal haemodynamics were determined in uncomplicated Type 1 diabetic patients before and after three weeks of treatment with the ACE-I ramipril (5 mg/day) or the ARB losartan (50 mg/day). L-arg alone did not influence BP or renal haemodynamics. BP responses to L-arg were not modulated by ACE-I or ARB. In contrast to the systemic responses, L-arg induced significant renal vasodilation after treatment with ramipril (p<0.05). Unlike ramipril, losartan did not modulate renal haemodynamic responses to L-arg. L-arg administration was paralleled by increments in plasma L-citrulline levels, determined as a measure of L-arg-induced systemic NO production. These responses were not influenced by RAS inhibitors. No changes in other indicators of the systemic and renal NO production, such as plasma and urinary nitrates/nitrites, were detected in response to L-arg alone or after treatment with RAS inhibitors. ACE-Is have greater potential than ARBs to enhance L-arg effects in the kidney in uncomplicated Type 1 diabetes. Neither RAS inhibitor influenced the systemic effects of L-arg. The lack of changes in renal NO indicators parallelling the haemodynamic responses, suggests that the effects of ACE-I on L-arg-induced renal haemodynamic changes could be also attributable to NO-independent mechanisms.

Interaction between Nitric Oxide and Cyclooxygenase Pathways in Endothelial Cells

Nitric oxide (NO) and cyclooxygenase (COX) derived prostaglandins (PGs) are involved in vascular homeostasis. Contradictory results have been obtained in previous studies on the putative ‘cross-talk’ between these two pathways. Our aim was to evaluate the interaction between NO and PG release in human microvascular endothelial cells (HMEC-1) and in umbilical vein endothelial cells (HUVEC). Methods: Medium samples were assayed for nitrite/nitrate (NO_x) and L-citrulline levels while lysed cells were assayed for endothelial NO synthase (eNOS), as markers of NO production. Prostaglandin E₂ (PGE₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were assessed as indicators of COX activity. Results: The NO donor sodium nitroprusside and L-arginine increased PGs levels in both cell types. N^G-monomethyl-L-arginine (L-NMMA) significantly inhibited 6-keto-PGF_1α release without significantly reducing PGE₂levels. Indomethacin increased both NO_x, eNOS and L-citrulline levels. PGE₂ treatment did not modify NO_x values. Conclusion: The stimulation of PGs by NO may represent an additional pathway used by exogenous nitrovasodilators to elicit vasodilation. Reduction of PGs by inhibition of COX was compensated by enhanced NO. Conversely, PGsdid not compensatedecreased NO following L-NMMA treatment. Treatment with the vasodilatator prostaglandin E₂did not modifyNO_x levels.

Plasma nitric oxide (nitrate/nitrite) levels in acute stroke and their relationship with severity and outcome

Endothelium-derived nitric oxide is beneficial in experimental stroke. We assessed plasma NO levels in patients with acute stroke and their association with both severity and outcome. Plasma nitric oxide (NO), assessed as nitrate/nitrite (NOx), cyclic guanosine monophosphate (cGMP, second messenger to NO), L-arginine (substrate for NO) and L-citrulline (co-product with NO) levels were measured in 228 patients with acute ischemic stroke, 49 patients with acute hemorrhagic stroke, and 38 age and gender-matched normal volunteers. Stroke severity was assessed using the Glasgow Coma Scale, and the outcome was judged by discharge destination (home, institution, or dead). In our study, stroke patients had low levels of NOx (μmol/l): ischemic 49.9 (26.1); hemorrhage 41.7 (19.5); and control 64.0 (36.3) (P <.001); L-arginine (μmol/l): ischemic 85.1 (32.3); hemorrhage 69.9 (24.4); and control 104.0 (30.0) (P <.001); and L-citrulline (μmol/l): ischemic 30.5 (12.3); hemorrhage 26.7 (12.1); and control 39.4 (13.5) (P <.001). Cyclic GMP levels were elevated in stroke: ischemic 21.2 (16.1); hemorrhage 24.7 (17.5); and control 15.8 (9.2) (P =.024). Patients who died or became institutionalized had lower NOx, L-arginine, and L-citrulline levels, and higher cyclic GMP levels, than patients who were discharged home. NOx levels were not associated with feeding status in patients. Low levels of NOx are present in stroke and are associated with severity and outcome. Because endothelium-derived NO is beneficial in acute stroke, administering NO might be beneficial in acute stroke. Copyright © 2003 by National Stroke Association

Cerebrospinal fluid and plasma concentrations of nitric oxide metabolites are increased in dementia with Lewy bodies

We compared cerebrospinal fluid (CSF) and plasma concentrations of nitric oxide metabolites (NO − x) in 22 patients with dementia with Lewy bodies (DLB) and 13 matched controls. We found a pronounced increase in NO − x CSF and plasma levels in DLB patients. No changes were found in l-arginine and l-citrulline levels in CSF or plasma. There was no correlation between CSF and plasma levels and age, age of onset, duration of the disease or scores of the MiniMental State Examination. These findings reveal that excessive nitric oxide production may be related to the pathogenesis of DLB.

Investigation of extracellular L-citrulline concentration in the striatum during alcohol withdrawal in rats.

In this study, changes in striatal extracellular L-citrulline concentrations were investigated hourly for 5 h following alcohol withdrawal in chronic alcohol feeding Wistar rats. Alcohol (7.2% ethyl alcohol, v/v) was given to rats as modified liquid diet for 20 days. Signs of alcohol withdrawal appeared from the 1st h of alcohol withdrawal and the total alcohol withdrawal scores remained higher during the course of experiments. The mean of basal levels of L-citrulline in the microdialysis samples collected in conscious rat model from the striatum of control and alcoholized rats were found to be 1.28 +/- 0.48 microM and 0.35 +/- 0.08 microM, respectively. L-citrulline levels in the striatum of alcoholized rats increased by 4 folds significantly within 1 h following alcohol withdrawal. The increased striatal L-citrulline concentration was blocked by NG-nitro-L-arginine methyl ester (L-NAME; 60 mg/kg), a nitric oxide synthase inhibitor, pretreatment. Our results indicate an increased L-citrulline level in the rat striatum during early alcohol withdrawal and this situation may be related to an increased nitric oxide production.

Increased l-citrullinel-arginine plasma ratio in severe preeclampsia

Objective: To evaluate nitric oxide (NO) production in patients with pregnancy-induced hypertension or preeclampsia and in controls. Methods: Four groups of pregnant women were included: 17 patients with pregnancy-induced hypertension, ten with mild or moderate preeclampsia, 17 with severe preeclampsia, and 44 normotensive women matched for weeks of gestation at blood sampling with the cases. Plasma levels of l-citrulline and l-arginine were measured by using high-performance liquid chromatography. Results: The mean plasma levels of l-citrulline and the ratio of l-citrulline to l-arginine, which reflects NO production, were higher in women with severe preeclampsia than in controls, patients with pregnancy-induced hypertension, and patients with mild or moderate preeclampsia. Conclusion: Nitric oxide production is enhanced in severe preeclampsia, possibly as a compensatory phenomenon for the increased synthesis and release of vasoconstrictors and platelet-aggregating agents.

Regulation of ICAM-1/CD54 expression on human endothelial cells by hydrogen peroxide involves inducible NO synthase.

Expression of the inducible isoform of nitric oxide synthase (iNOS) is stimulated by cytokines in human epithelial cells. This work indicates that incubation of human umbilical cord endothelial cells with combinations of interleukin-1beta, tumor necrosis factor alpha, and interferon-gamma stimulated the synthesis of iNOS mRNA, as detected by reverse transcriptase-polymerase chain reaction. It is important to note that 50, 100, and 200 microM hydrogen peroxide was able to stimulate iNOS directly. Furthermore, 100 microM H2O2 enhanced synthesis of the oxidation products, nitrite (NO2-) and nitrate (NO3-) at 12 and 36 h. iNOS protein, detected by Western blot analysis, as well as L-citrulline levels, were also increased. When endothelial cell monolayers were incubated for 1 h with 100 microM H2O2 and subsequently with cytokines, iNOS mRNA was further augmented. Under the same conditions, we regularly observed an inhibition (25%) of intercellular adhesion molecule-1 (ICAM-1/CD54) expression. The latter was reversed when the NOS inhibitor N(G)-monomethyl-L-arginine was added, as shown by flow cytometry. These data suggest a specific effect of endogenous hydroperoxides on the biosynthesis and processing of the human endothelial iNOS isoform. We propose that H2O2 induces a temporary NO-dependent modulation of adhesion molecule expression to limit the tissue destruction that accompanies the vascular recruitment of leukocytes.

L-glutamate, L-arginine and L-citrulline levels in cerebrospinal fluid of Parkinson's disease, multiple system atrophy, and Alzheimer's disease patients.

Alterations in neuronal nitric oxide (NO) production may play a role in the pathophysiology of Parkinson's disease (PD) Alzheimer's disease (AD), and multiple system atrophy (MSA). The biosynthesis of NO is dependent on the availability of L-arginine, the substrate for NO-synthase (NOS), and on L-glutamate, which stimulates NO synthesis via the NMDA receptor. In this process L-citrulline is formed. We measured the levels of these amino acids in cerebrospinal fluid (CSF) of 108 PD patients, 12 AD patients, 15 MSA patients and 21 healthy subjects. A slight but statistically significant elevation of CSF L-citrulline was found in MSA patients, while CSF L-glutamate was found to be significantly decreased in AD patients. We found no significant changes in L-arginine levels. Although the relation between the CSF levels of these amino acids and neuronal NO production is still unclear, our findings suggest that AD is associated with a decrease in NO synthesis.

Nitric oxide biomarkers increase during exercise-induced vasodilation in the forearm.

The purpose of the study was to determine if exercise-induced vasodilation was associated with an increase in forearm plasma levels of nitric oxide (NO) biomarkers (NO2- + NO3- and L-citrulline). Twelve healthy subjects (27+/-6 yrs) performed incremental rhythmic forearm exercise with the nondominant hand for 6 min each at 15, 30 and 45% of maximal voluntary contraction (MVC). Forearm blood flow (FBF) was determined in the exercise arm using venous occlusion plethysmography. Blood samples were obtained from the antecubital vein of the exercise and nonexercise arms for the measurement of NO biomarkers. In the exercise arm, FBF increased by a mean of 150%, 335% and 585% above baseline at 15, 30 and 45% of MVC, respectively. (ANOVA, P= 0.0001). Venous plasma NO2- + NO3- levels increased from 24+/-4 micromol/L at baseline, to 29+/-5, 32+/-4 and 3+/-4 micromol/L (ANOVA, P = 0.0001). Venous plasma L-citrulline levels increased from 31+/-5 micromol/L at baseline to 58+/-10, 87+/-7 and 141+/-15 micromol/L (ANOVA, P = 0.0001). There was a linear relationship between FBF and venous plasma NO2- + NO3- (slope= 0.38+/-0.10, P=0.0007) and between L-citrulline, (slope= 5.1 +/-1.3, P = 0.0004). Venous plasma levels of NO2- + NO3- and L-citrulline in the nonexercise arm were unchanged. These results demonstrate that exercise-induced vasodilation in the forearm is associated with forearm plasma levels of NO2- + NO3- and L-citrulline, in vivo markers of NO production.

Identification of coumarins from lemon fruit (Citrus limon) as inhibitors of in vitro tumor promotion and superoxide and nitric oxide generation.

Three coumarins were isolated as siginificant inhibitors of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation in Raji cells from the peel of lemon fruit. They were identified as 8-geranyloxypsolaren (LE-1), 5-geranyloxypsolaren (bergamottin, LE-2), and 5-geranyloxy-7-methoxycoumarin (LE-3), respectively, by spectroscopic analysis. Three isolates had no potential O(2)-scavenging and markedly suppressed TPA-induced superoxide (O(2)(-)) generation in differentiated human promyelocytic HL-60 cells. Furthermore, LE-1 and LE-3 reduced both lypopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Similarly, they were found to be NO generation inhibitors rather than scavengers by measuring the extracellular L-citrulline levels. The occurrence of these coumarins in a lemon fruit was abundant in the flavedo of the peel based on quantitative analysis using high-performance liquid chromatography (HPLC). The present study suggests that the coumarins in lemon fruit are promising chemopreventive agents by inhibiting radical generation.

The L-arginine-nitric oxide system regulates platelet aggregation in pregnancy.

To investigate the ex vivo platelet aggregation in response to a substrate (L-arginine [L-Arg]) and a donor (sodium nitroprusside [SN]) of nitric oxide (NO) in nonpregnant women and in normotensive pregnancies. Platelet aggregation was studied with a dual-channel aggregometer and expressed as a percentage of light transmission. Measurements were done at baseline and after preincubation with scalar doses of L-Arg and SN. The intraplatelet L-citrulline (L-Cit) levels were measured by high-performance liquid chromatography (HPLC). In both groups, the baseline aggregation values were similar for adenosine diphosphate (ADP) and collagen stimuli. Wilcoxon rank-sum testing demonstrated that in both groups the addition of L-Arg had a significant effect on aggregation: Doses of 50 to 5000 mumol decreased ADP-induced aggregation. Conversely, collagen-induced aggregation was affected only by the highest dose of L-Arg. Sodium nitroprusside administered in doses of 2.5 to 250.0 nmol decreased ADP- and collagen-induced platelet aggregation equally. ADP-induced aggregation values obtained after (SN) incubation were positively correlated with gestational age. Intraplatelet L-Cit levels showed a significant rise after the incubation with L-Arg, and this effect was negatively correlated with gestational age. The L-Arg-NO system regulates platelet aggregation during pregnancy. Moreover, a physiologic reduction of platelet sensitivity to the antithrombotic effect of NO occurs.