Arginine Availability in Reamed Intramedullary Aspirate as Predictor of Outcome in Nonunion Healing.

Fracture healing and nonunion development are influenced by a range of biological factors. Adequate amino acid concentrations, especially arginine, are known to be important during normal bone healing. We hypothesize that bone arginine availability in autologous bone marrow grafting, when using the reamer-irrigator-aspirator (RIA) procedure, is a marker of bone healing capacity in patients treated for nonunion. Seventeen patients treated for atrophic long bone nonunion by autologous bone grafting by the RIA procedure were included and divided into two groups, successful treatment of nonunion and unsuccessful, and were compared with control patients after normal fracture healing. Reamed bone marrow aspirate from a site distant to the nonunion was obtained and the amino acids and enzymes relevant to arginine metabolism were measured. Arginine and ornithine concentrations were higher in patients with successful bone healing after RIA in comparison with unsuccessful healing. Ornithine concentrations and arginase-1 expression were lower in all nonunion patients compared to control patients, while citrulline concentrations were increased. Nitric oxide synthase 2 (Nos2) expression was significantly increased in all RIA-treated patients, and higher in patients with a successful outcome when compared with an unsuccessful outcome. The results indicate an influence of the arginine-nitric oxide metabolism in collected bone marrow, on the outcome of nonunion treatment, with indications for a prolonged inflammatory response in patients with unsuccessful bone grafting therapy. The determination of arginine concentrations and Nos2 expression could be used as a predictor for the successful treatment of autologous bone grafting in nonunion treatment.

Intestinal permeability before and after albendazole treatment in low and high socioeconomic status schoolchildren in Makassar, Indonesia.

Intestinal helminths are highly prevalent in low-SES children and could contribute to poor health outcomes either directly or via alteration of the gut microbiome and gut barrier function. We analysed parasitic infections and gut microbiota composition in 325 children attending high- and low-SES schools in Makassar, Indonesia before and after albendazole treatment. Lactulose/Mannitol Ratio (LMR, a marker of gut permeability); I-FABP (a surrogate marker of intestinal damage) as well as inflammatory markers (LBP) were measured. Helminth infections were highly prevalent (65.6%) in low-SES children. LMR and I-FABP levels were higher in low-SES children (geomean (95%CI): 4.03 (3.67-4.42) vs. 3.22 (2.91-3.57); p. adj < 0.001; and 1.57 (1.42-1.74) vs. 1.25 (1.13-1.38); p. adj = 0.02, respectively) while LBP levels were lower compared to the high-SES (19.39 (17.09-22.01) vs. 22.74 (20.07-26.12); p.adj = 0.01). Albendazole reduced helminth infections in low-SES and also decreased LMR with 11% reduction but only in helminth-uninfected children (estimated treatment effect: 0.89; p.adj = 0.01). Following treatment, I-FABP decreased in high- (0.91, p.adj < 0.001) but increased (1.12, p.adj = 0.004) in low-SES children. Albendazole did not alter the levels of LBP. Microbiota analysis showed no contribution from specific bacterial-taxa to the changes observed. Intestinal permeability and epithelial damage are higher while peripheral blood inflammatory marker is lower in children of low-SES in Indonesia. Furthermore, treatment decreased LMR in helminth-uninfected only.

Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Mass spectrometry imaging of L-[ring-13C6]-labeled phenylalanine and tyrosine kinetics in non-small cell lung carcinoma.

BACKGROUND: Metabolic reprogramming is a common phenomenon in tumorigenesis and tumor progression. Amino acids are important mediators in cancer metabolism, and their kinetics in tumor tissue are far from being understood completely. Mass spectrometry imaging is capable to spatiotemporally trace important endogenous metabolites in biological tissue specimens. In this research, we studied L-[ring-13C6]-labeled phenylalanine and tyrosine kinetics in a human non-small cell lung carcinoma (NSCLC) xenografted mouse model using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI). METHODS: We investigated the L-[ring-13C6]-Phenylalanine (13C6-Phe) and L-[ring-13C6]-Tyrosine (13C6-Tyr) kinetics at 10 min (n = 4), 30 min (n = 3), and 60 min (n = 4) after tracer injection and sham-treated group (n = 3) at 10 min in mouse-xenograft lung tumor tissues by MALDI-FTICR-MSI. RESULTS: The dynamic changes in the spatial distributions of 19 out of 20 standard amino acids are observed in the tumor tissue. The highest abundance of 13C6-Phe was detected in tumor tissue at 10 min after tracer injection and decreased progressively over time. The overall enrichment of 13C6-Tyr showed a delayed temporal trend compared to 13C6-Phe in tumor caused by the Phe-to-Tyr conversion process. Specifically, 13C6-Phe and 13C6-Tyr showed higher abundances in viable tumor regions compared to non-viable regions. CONCLUSIONS: We demonstrated the spatiotemporal intra-tumoral distribution of the essential aromatic amino acid 13C6-Phe and its de-novo synthesized metabolite 13C6-Tyr by MALDI-FTICR-MSI. Our results explore for the first time local phenylalanine metabolism in the context of cancer tissue morphology. This opens a new way to understand amino acid metabolism within the tumor and its microenvironment.

Acute haemodynamic changes during haemodialysis do not exacerbate gut hyperpermeability.

INTRODUCTION: The gastrointestinal tract is a potential source of inflammation in dialysis patients. In vitro studies suggest breakdown of the gut barrier in uraemia leading to increased intestinal permeability and it is hypothesised that haemodialysis exacerbates this problem due to mesenteric ischaemia induced by blood volume changes during treatment. METHOD: The effect of haemodialysis on intestinal permeability was studied in ten haemodialysis patients and compared with five controls. Intestinal permeability was assessed by measuring the differential absorption of four orally administered sugar probes which provides an index of small and whole bowel permeability. A multi-sugar solution (containing lactulose, rhamnose, sucralose and erythritol) was orally administered after an overnight fast. Plasma levels of all sugar probes were measured hourly for 10 h post-administration. In haemodialysis patients, the procedure was carried out twice - once on a non-dialysis day and once immediately after haemodialysis. RESULTS: Area under curve (AUC) for lactulose:rhamnose (L:R) ratio and sucralose:erythritol (S:E) ratio was similar post-dialysis and on non-dialysis days. AUC for L:R was higher in haemodialysis patients compared with controls (0.071 vs. 0.034, P=0.001), AUC for S:E ratio was not significantly different. Levels of lactulose, sucralose and erythritol were elevated and retained longer in haemodialysis patients compared with controls due to dependence of sugars on kidney function for clearance. CONCLUSION: We found no significant acute changes in intestinal permeability in relation to the haemodialysis procedure. Valid comparison of intestinal permeability between controls and haemodialysis patients was not possible due to the strong influence of kidney function on sugar levels.

Citrulline Supplementation Improves Organ Perfusion and Arginine Availability under Conditions with Enhanced Arginase Activity.

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with L-arginine supplementation exhibited less consistent results; however, L-citrulline, the precursor of L-arginine, may be a promising alternative. In this study, we determined the effects of L-citrulline compared to L-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with L-citrulline or L-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. L-arginine and L-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that L-citrulline, and not L-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.

Novel multi-sugar assay for site-specific gastrointestinal permeability analysis: a randomized controlled crossover trial.

BACKGROUND & AIMS: Increased gastrointestinal (GI) permeability is an important hallmark of many conditions, potentially leading to antigen exposure and sepsis. Current permeability tests are hampered by analytical limitations. This study aims to compare the accuracy of our multi-sugar (MS) and the classical dual sugar (DS) test for detection of increased GI permeability. METHODS: Ten volunteers received permeability analysis using MS (1 g sucrose, lactulose, sucralose, erythritol, 0.5 g rhamnose in water) or DS (5 g lactulose, 0.5 g rhamnose), after indomethacin or placebo. Blood and urine were analyzed by isocratic LC-MS. RESULTS: MS testing revealed significantly elevated urinary lactulose/rhamnose (L/R) ratios after indomethacin, due to enhanced lactulose excretion (P < .01) and unaltered rhamnose excretion. The DS test showed increased L/R ratios, due to increased lactulose excretion and decreased rhamnose excretion (both P < .05). After indomethacin, plasma L/R increased in both assays (P < .05 and P < .01). Urinary and plasma L/R ratios correlated significantly. Indomethacin increased sucrose excretion and 0-1 h sucrose/rhamnose. Colon permeability was unchanged. CONCLUSIONS: Sensitive permeability analysis is feasible in plasma and urine using MS or DS test. In contrast to the DS test, monosaccharide excretion is not decreased by the MS test. In short, the MS test provides accurate, site-specific information on gastroduodenal, small, and large intestinal permeability. Registered at US National Library of Medicine (http://www.clinicaltrials.gov, NCT00943345).

Systematic review of ophthalmate as a novel biomarker of hepatic glutathione depletion.

BACKGROUND: Sustainability of hepatic glutathione (GSH) homeostasis is an important cellular defense against oxidative stress. Therefore, knowledge of liver GSH status is important. However, measurement of plasma GSH and tissue is difficult due to its instability. Alternatively, ophthalmate (OPH), an endogenous tripeptide analog of GSH, has been suggested as a potential indicator to assess GSH depletion. AIM: To provide an overview of present knowledge with respect to the usefulness of OPH as a biomarker for oxidative stress and hepatic GSH homeostasis. METHODS: A systematic, computerized search combined with a cross-reference search of the literature described in PubMed (January 1975 to January 2012) was conducted, key words: 'ophthalmate' and 'ophthalmic acid'. RESULTS: Twenty-two articles were included. Hepatic OPH levels increase inversely proportional to a drop in hepatic GSH in mice with paracetamol (PCM) induced hepatotoxicity. Little is known about the stability of OPH in human plasma. To measure the very low physiological concentrations of plasma OPH, liquid chromatography-mass spectrometry techniques can be employed. OPH synthesis can be measured in humans, using stable isotope labeling with a deuterated water ((2)H2O) load. CONCLUSION: OPH may be a promising biomarker to indicate hepatic glutathione depletion, but the suggested biological pathways need further unraveling.

High sensitivity measurement of amino acid isotope enrichment using liquid chromatography-mass spectrometry.

Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC-MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5 µmol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline.

Ophthalmate detection in human plasma with LC-MS-MS.

Based on animal experimentations, ophthalmate (OPH) has recently been suggested as a potential plasma biomarker to probe hepatic GSH homeostasis. Up until now, the inability to accurately determine OPH concentrations in human plasma prohibited further studies of OPH metabolism in humans. This study therefore aimed to study the influence of delayed sample preparation on OPH concentrations using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Venous plasma samples from 5 healthy human volunteers were incubated for varying times (5, 30, 60 and 120 min) at temperatures of 4 °C and 37 °C to investigate potential enzymatic degradation. At 37 °C, the decrease in OPH reached significance after 120 min (74.6% (range: 56.2-100.0%; p<0.0001)). At 4 °C, the same trend was observed but did not reach significance. These findings indicate ongoing enzymatic activity, stressing the need for immediate sample deproteinization to obtain reliable plasma concentrations. To investigate the feasibility of the here developed method, baseline arterial plasma values of 21 patients scheduled for partial liver resection were determined to be 0.06±0.03 µmol/l (mean±s.d.). In addition, in pooled samples from 3 patients, an OPH calibration curve was spiked to arterial plasma, arterial whole blood and liver biopsy material, resulting in a linear calibration curve in all cases. Individual measurements of baseline samples revealed that both arterial whole blood and liver biopsy material contained significant levels of endogenous OPH, namely 16.1 (11.8-16.4) µmol/l and 80.0 (191.8-349.2) µmol/kg, respectively. In conclusion, the present LC-MS/MS assay enables the accurate measurement of OPH in human plasma, whole blood and liver biopsies. Freshly prepared samples and immediate deproteinization are mandatory to block enzymatic degradation.

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure.

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.

Novel analytical approach to a multi-sugar whole gut permeability assay.

Many pathophysiological conditions are associated with increased gastrointestinal permeability, reflecting an elevated risk of endotoxaemia, inflammation, and sepsis. Permeability tests are increasingly used in clinical practice to obtain information on gastrointestinal functioning, but tests are often restricted to the small intestine, and require large oral sugar doses. Therefore, a novel multi-sugar assay was developed, allowing assessment of whole gut permeability changes in urinary and plasma samples collected at regular intervals from 10 healthy volunteers at baseline and after intake of monosaccharides (rhamnose and erythritol) and disaccharides (sucrose, lactulose, and sucralose). Samples were analyzed by isocratic cation-exchange LC-MS. Sample preparation and detection conditions were optimized. After centrifugation, chromatographic separation was achieved on an IOA-1000 column set at 30°C. Column effluent was mixed with ammonia for sugar-ammonium adduct formation. The lower limit of detection was 0.05 µmol/L for disaccharides and 0.1 µmol/L for monosaccharides. Linearity for each probe was between 1 and 1000 µmol/L (R(2): 0.9987-0.9999). Coefficients of variation were <5% in urine, and <9% in plasma. Recovery data were within the 90% to 110% range at all spiked concentrations. This highly sensitive novel LC-MS approach resulted in a significant decrease of the detection limit for all sugar probes, allowing a 5-fold reduction of the commonly used lactulose dose and the addition of sugar probes to also assess the gastroduodenal and colon permeability. In combination with its extended application in plasma, these features make the novel assay a promising tool in the assessment of site-specific changes in gastrointestinal permeability in clinical practice.

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

Application of liquid chromatography-mass spectrometry to measure short chain fatty acids in blood.

A new liquid chromatography-mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 micromol/l (R(2) ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 micromol/l for propionic and butyric acid and 0.1 micromol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.

Measurement of amino acid isotope enrichment by liquid chromatography mass spectroscopy after derivatization with 9-fluorenylmethylchloroformate.

An automated method is described to measure tracer-tracee-ratios (TTR) of plasma amino acids after their separation as 9-fluorenylmethylchloroformate derivatives. In a 45 min cycle, 5 microl plasma aliquots were derivatized, HPLC separated and subjected to electrospray ionization. By applying source collision induced dissociation, derivatives were dissociated at the peptide bond releasing the originating amino acids into the mass spectrometer. This approach enabled the determination of plasma amino acid TTRs with a standard deviation between 0.15 and 0.36%, which is sufficient to study the fate of infused tracers and their conversion products in an in vivo experiment in humans.

Increased glutamate levels in the vitreous of patients with retinal detachment.

Experimental models have implicated glutamate in the irreversible damage to retinal cells following retinal detachment. In this retrospective study we investigated a possible role for glutamate and other amino acid neurotransmitters during clinical rhegmatogenous retinal detachment (RRD). Undiluted vitreous samples were obtained from 176 patients undergoing pars plana vitrectomy. The study group consisted of 114 patients (114 eyes) with a rhegmatogenous retinal detachment. Controls included 52 eyes with an idiopathic macular hole or idiopathic epiretinal membrane and 10 eyes with a traction retinal detachment due to proliferative diabetic retinopathy. Vitreous concentrations of glutamate, gamma-aminobutyric acid (GABA), taurine, glycine, and aspartate were determined by high-pressure liquid chromatography (HPLC). Multivariate analysis was used to examine a possible association between amino acid neurotransmitter levels and several clinical variables including visual acuity. The mean vitreous concentration of glutamate in eyes with a rhegmatogenous retinal detachment (16.6 +/- 5.6 microM) was significantly higher as compared to the controls (13.1 +/- 5.2 microM) (P = 0.001). Taurine levels were also increased in RRD, whereas no significant difference could be observed in glycine, aspartate and GABA levels when comparing RRD with controls. A correlation was found between increased vitreous glutamate and a lower pre-operative visual acuity. No association was, however, observed between post-operative visual acuity and the level of any of the five amino acid neurotransmitters. RRD was associated with a significantly increased vitreous glutamate concentration. Using visual acuity as a functional parameter in this study, we could not demonstrate a correlation between vitreous glutamate, or any of the other tested amino acid neurotransmitters and visual outcome.

Increased nitric oxide (NO) pathway metabolites in the vitreous fluid of patients with rhegmatogenous retinal detachment or diabetic traction retinal detachment.

BACKGROUND: Nitric oxide (NO) plays a significant role in physiological and pathological processes in the retina. In the L-arginine-NO pathway, NO synthase (NOS) converts L-arginine to NO and L-citrulline. Increased NO production, mediated by inducible NOS has been implicated in the pathogenesis of various vitreoretinal diseases. In the present study it is hypothesized that in rhegmatogenous retinal detachment (RRD), the production of NO pathway metabolites might be upregulated. METHODS: Using high-pressure liquid chromatography citrulline, arginine and nitrite were measured in vitreous fluid of 93 eyes with RRD, nine eyes with a traction retinal detachment due to proliferative diabetic retinopathy (PDR), and in 49 control samples of vitreous fluid from eyes without retinal detachment. RESULTS: The mean vitreous concentrations of citrulline and arginine were significantly increased in eyes with RRD (9.6+/-4.3 and 97.3+/-29.2; respectively) or in eyes with a traction retinal detachment (25.8+/-10.3 and 130.7+/-23.7; respectively) as compared to control eyes (7.1+/-3.2 and 75.9+/-18.1; respectively). The mean level of nitrite was also higher in vitreous fluid of patients with RRD (2.24+/-1.4) or patients with a traction retinal detachment (2.21+/-0.72) than in the controls (2.01+/-0.72), although not significantly so. CONCLUSIONS: We found increased levels of NO pathway metabolites in the vitreous fluid of eyes with retinal detachment, which may reflect a possible role of NO in the pathogenesis of this disease.

The liquid chromatography mass spectrometry approach to measure amino acid isotope ratios.

PURPOSE OF REVIEW: Although the application of liquid chromatography mass spectrometry has seen a rapid development in the world of clinical science since the introduction of atmospheric pressure ionization, its potential is still hardly recognized in the field of stable isotope biomedical research, as indicated by the small number of publications on this topic. Nevertheless, considering the polar nature of many biological substrates of interest as well as the non-destructive nature of electrospray ionization, a liquid chromatography mass spectrometry approach simplifies mass spectrometric spectra, allows the application of multiple labelled tracers, and provides an easy on-line sample processing, thus minimizing labour while at the same time providing concentration data and good reproducibility in low quantities of sample. RECENT FINDINGS: These features make it an excellent tool to perform both clinical and animal experiments that usually generate large numbers of samples, which when processed through the classic approach employing gas chromatography combustion isotope ratio mass spectrometry systems would require much more work and time and thus analytical costs. SUMMARY: The present paper is intended to introduce the functionality, limitations and options of representatives from the present family of modern liquid chromatography mass spectrometry systems into the field of stable isotope biomedical research, with a focus on amino acid metabolism.

Quantitative in vivo assessment of arginine utilization and nitric oxide production in endotoxemia.

BACKGROUND: Until recently no methods were available to quantitate nitric oxide (NO) production in vivo. The advent of stable isotope techniques has allowed quantitation of NO production in different animal models and human disease states. METHODS: In vivo NO production was assessed with the use of stable isotope labeled arginine. Enrichments of metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Knock-out mice were used to assess the influence of knocking out inducible NOS (iNOS) or constitutively expressed NOS (cNOS) on arginine-NO metabolism. Pig models were used to assess the role of individual organs on arginine-NO fluxes. RESULTS: In mice under basal conditions cNOS mediates half of the NO production. After endotoxin challenge NO production doubles as a result of iNOS induction and cNOS-mediated NO production is downregulated. In larger animal models (pig) whole body NO production is augmented after endotoxin challenge, largely resulting from NO production in liver, intestine and kidney. Arginine supplementation increases NO production in pigs in liver, intestine and kidney both in the basal state and after endotoxin challenge. CONCLUSIONS: Stable isotope techniques employing LC-MS allow in vivo assessment of NO production in small and large animal models and in patients. This allows definition of the role that iNOS and cNOS-mediated NO production play in several disease states.