Enhancement of fracture healing after citrulline supplementation in mice.

Around 10 % of long bone fractures show inadequate bone healing resulting in non-union development. A deregulated arginine-citrulline-nitric oxide metabolism caused by a poor nutritional status of the patients is a risk factor for non-unions. Additionally, previous research in mice with a disrupted arginine to citrulline conversion showed delayed healing. The study hypothesis was that stimulating said metabolism could positively influence the healing process through promotion of collagen synthesis and angiogenesis. Adult wild-type mice underwent a femur osteotomy and plate-screw osteosynthesis. Mice were randomly divided into three groups and received daily oral supplementation of arginine, citrulline or 0.9 % saline (control). Body weight and food intake were measured daily. After 14 d, the mice were euthanised and femora collected. Callus formation was assessed by micro-computed tomography and concentrations of amino acids and enzymes in the femora were measured. Only citrulline-treated mice showed significantly increased bridging of the fracture gap when compared to control mice. Femur citrulline and ornithine concentrations were increased in citrulline-treated animals. qPCR showed significantly decreased expression of inflammatory markers, whereas increased expression of angiogenic and collagen-producing factors was observed in citrulline-treated mice. Although food intake did not show any difference between the three groups, animals treated with citrulline showed a weight gain of 0.3 g, compared with a 0.1 g decline in the control group. Daily oral citrulline supplementation stimulated callus formation and improved the inflammatory response, positively contributing to the enhanced healing response. Finally, the increased weight gain pointed toward a better post-operative recovery.

Visceral hypersensitivity in irritable bowel syndrome: evidence for involvement of serotonin metabolism--a preliminary study.

BACKGROUND: Altered serotonergic (5-HT) metabolism and visceral perception have been associated with the pathogenesis of irritable bowel syndrome (IBS). Aim of this preliminary study was to assess the effect of the direct precursor of 5-HT, 5-hydroxytryptophan (5-HTP), on systemic 5-HT metabolites and visceral perception and to assess potential differential responses between IBS and controls. METHODS: 15 IBS patients and 15 healthy volunteers participated in this randomized double-blind placebo controlled study. Visceroperception was measured by rectal barostat. The 100 mg 5-HTP or placebo was ingested orally. Serotonergic metabolites were assessed in platelet poor plasma. KEY RESULTS: 5-HTP induces rectal allodynia in a significant number of healthy controls; IBS patients exhibit lowered pain thresholds in both placebo and 5-HTP conditions. 5-HTP induces rectal hyperalgesia in hypersensitive but not in non-hypersensitive IBS patients. Administration of 5-HTP significantly increased plasma 5-HTP levels (p < 0.001), did not affect 5-HT levels (p > 0.05), while levels of the main metabolite of 5-HT, 5-hydroxyindoleacetic acid, increased significantly (p < 0.05) in both groups. The magnitude of these changes observed in 5-HT metabolites was significantly greater in IBS patients. CONCLUSIONS & INFERENCES: Oral administration of 5-HTP induced significant alterations in systemic 5-HT metabolites that were accompanied by increased visceroperception of pain in controls and hypersensitive IBS patients. Changes in 5-HT metabolism appear to be important factors involved in visceral hypersensitivity as the 5-HTP-induced pro-nociceptive response was observed in all hypersensitive IBS patients and to a lesser magnitude in a significant number of healthy controls but in none of the non-hypersensitive IBS patients.

Serotonergic reinforcement of intestinal barrier function is impaired in irritable bowel syndrome.

BACKGROUND: Alterations in serotonergic (5-HT) metabolism and/or intestinal integrity have been associated with irritable bowel syndrome (IBS). AIMS: To assess the effects of the precursor of 5-HT, 5-hydroxytryptophan (5-HTP), on mucosal 5-HT availability and intestinal integrity, and to assess potential differences between healthy controls and IBS patients. METHODS: Fifteen IBS patients and 15 healthy volunteers participated in this randomised double-blind placebo-controlled study. Intestinal integrity was assessed by dual-sugar test and by determining the mucosal expression of tight junction proteins after ingestion of an oral bolus of 100 mg 5-HTP or placebo. Mucosal serotonergic metabolism was assessed in duodenal biopsy samples. RESULTS: 5-HTP administration significantly increased mucosal levels of 5-HIAA, the main metabolite of 5-HT, in both healthy controls (7.1 ± 1.7 vs. 2.5 ± 0.7 pmol/mg, 5-HTP vs. placebo, P = 0.02) and IBS patients (20.0 ± 4.8 vs. 8.1 ± 1.3 pmol/mg, 5-HTP vs. placebo, P = 0.02), with the latter group showing a significantly larger increase. Lactulose/L-rhamnose ratios were significantly lower after administration of 5-HTP (P < 0.05) in healthy controls and were accompanied by redistribution of zonula occludens-1 (ZO-1), pointing to reinforcement of the barrier. In IBS, expression of the tight junction proteins was significantly lower compared to healthy controls, and 5-HTP resulted in a further decrease in occludin expression. CONCLUSIONS: Oral 5-HTP induced alterations in mucosal 5-HT metabolism. In healthy controls, a reinforcement of the intestinal barrier was seen whereas such reaction was absent in IBS patients. This could indicate the presence of a serotonin-mediated mechanism aimed to reinforce intestinal barrier function, which seems to dysfunction in IBS patients.

Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions.

A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 microM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 microM (butyric acid) to 150 microM (propionic acid) to 440 microM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the L-arginine/NO pathway in health and disease.

Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid L-arginine by the action of nitric oxide synthases (NOS) in what is called the L-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, L-arginine and its simultaneously generated product, L-citrulline. Considering only a fraction of the endogenous L-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of L-arginine and/or L-citrulline alone. Instead, to estimate NO-related changes in the L-arginine and/or L-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of L-arginine to NO and L-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.

Plasma protein synthesis measurements using a proteomics strategy.

The analysis of the synthesis of proteins has been the subject of many studies in animals and humans. Plasma proteins can be used as an easy accessible source of specific proteins. In this paper, an innovative method to study the synthetic rate of plasma proteins is described. This methodology, based on the proteomics approach, enables the direct observation of the effects of posttranslational modifications of protein synthesis and/or degradation. The methodology is based on 1D or 2D electrophoresis and subsequent electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS). Protein synthesis is measured in isotopically labeled peptides of the identified proteins. This innovative method can be used to assess amino acid adequacy and safety by studying protein synthesis and posttranslational modification of plasma proteins in more detail.

Effects of pre- and post-absorptive factors on the lactulose/rhamnose gut permeability test.

It is assumed that the outcome of the lactulose/rhamnose gut permeability test is not influenced by pre- or post-absorptive factors. The aim of our study was to investigate the role of a pre-absorptive factor, i.e. small-intestinal transit, and a post-absorptive factor, i.e. renal clearance. Ten healthy male subjects were studied. Urinary lactulose and rhamnose excretion was measured after intraduodenal administration of lactulose and rhamnose following induction of increased intestinal permeability using chenodeoxycholic acid (chenodiol), in the absence and in the presence of accelerated intestinal transit. Urinary sugar excretion was measured after intravenous administration of either a regular dose (50 mg/50 mg) or a high dose (250 mg/250 mg) of lactulose/rhamnose. The intraduodenal experiments showed that a combination of accelerated small-bowel transit and increased permeability did not lead to significant differences in the recovery of lactulose (P=0.647) or rhamnose (P=0.889), or in the lactulose/rhamnose ratio, compared with those under conditions of increased permeability alone (P=0.68). However, lactulose recovery was significantly lower (P=0.025) after intravenous administration of a high dose of the sugars. There was no significant difference in urinary rhamnose recovery (P=0.575) between the high and the regular doses. This resulted in a significantly lower lactulose/rhamnose ratio (P=0.021) after intravenous administration of a high dose, compared with a regular dose, of the sugars. In conclusion, the assumption that post-absorptive processes do not influence the outcome of the lactulose/rhamnose permeability test appears not to be valid.

Automated isolation of high-purity plasma albumin for isotope ratio measurements.

Measurement of the incorporation of labeled amino acids in plasma albumin, isolated from plasma sampled at different time points after infusion start is a well-known technique to study human albumin synthesis. Unfortunately, no chromatographic method has been described yet, enabling the automated isolation of high-purity albumin from large numbers of plasma samples as is required to study the kinetics of this process. Therefore, we developed a fast protein liquid chromatographic method, capable of processing 200 microliters amounts of plasma in 74 min (injection to injection). The system can run unattended as the FPLC system is connected to a sample processor equipped with a polyether ether ketone (PEEK) sample loop and a cooled sample tray. Albumin isolation was divided into three steps. First, plasma samples were injected onto a 1-ml Blue Sepharose HiTrap affinity column, equilibrated with 50 mmol/l phosphate buffer (pH 7.0). After elution of non-binding protein, switching the solvent to phosphate buffer with 1.5 mol/l sodium chloride eluted albumin. The resulting albumin fraction was desalted on-line by directing it through two consecutive HiTrap 5-ml desalting columns, whereafter it was retained in the system within a 5-ml PTFE loop, connected to a motor value. After switching this valve, thus bypassing the sample loop, the phosphate buffers were changed automatically to Tris buffers. Final purification involved elution of the captured fraction over a 1-ml ion-exchange Resource Q column, using a sodium chloride gradient, ranging from 0 to 0.5 mol/l in Tris buffer (20 mmol/l, pH 7.5). A more than 99% purity of the final albumin fraction was confirmed by capillary electrophoresis.

Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry.

The change in amino acid enrichment, an indicator of a change in protein synthesis and/or degradation, is usually measured using gas chromatography-mass spectrometry and/or (GC-combustion) isotope ratio mass spectrometry. Unfortunately, often a complex and sensitive derivatization procedure and/or a large amount of sample is required. Also, these techniques are less suited to study intermediary metabolism, in which the simultaneous application (and thus measurement) of multiple amino acid tracers is preferred. Alternatively, in this study the possibilities of the coupling of liquid chromatography and mass spectrometry were explored, resulting in the measurement of both the concentration and isotope enrichment of o-phthaldialdehyde (OPA)-derivatizated plasma amino acids in one run. This was achieved by the injection of OPA-derivatizated amino acids into an automated HPLC system. After the elution of buffer salts and reagent excess to drain using column switching, the column effluent was directed via a fluorescence detector into a Thermoquest Model LCQ benchtop LC-MS. Mass spectrometric measurements were performed in "zoom-scan" mode, employing multiple scan events if the target components were not baseline separated. Best signal-to-noise ratio's were obtained using the LCQ's electrospray probe in the negative mode. Still, when working under standard conditions the total ion current of OPA-amino acid derivatives eluting at the beginning of the chromatogram (e.g., citrulline, arginine and glycine) was by a factor of 5 lower, compared to components eluting in the last part of the chromatogram (leucine, valine, and ornithine). These differences could be minimized by increasing the temperature of the heated capillary to 260 degrees C and by applying 5% collision energy (between the skimmer and the first octapole) to the first eluting components. A further improvement could not be obtained by the addition of makeup liquids like ammonia, acetic acid, methanol, or acetonitrile (up to 25% of column effluent flow). Considering these results and the fact that the first eluting amino acid derivatives are the most polar ones, we hypothesized that hydration of these components interferes with the ionization process. A linear calibration curve was obtained for both fluorescent response and total ion current (TIC) for all amino acids in the range from 5 to 1000 pmol per injection. The coefficient of variation of the fluorescent response was typically on the order of 1-4%, for the TIC this was between 4 and 9%. However, measurement of isotope ratios requires not only the determination of the area of the base peak, but also of the area of the (enriched) isotopomeric peak(s), having a much lower abundance. Therefore, isotope ratio measurements require the injection of at least 25 pmol of the amino acid derivative of interest (except for ARG 50 pmol) to obtain true ratio's. The accuracy of the isotope enrichment measurement was determined by the injection of a standard containing all major physiological amino acids (400 pmol each) and a standard at physiological concentrations (ranging from 50 pmol (CIT) to 350 pmol (VAL). Standard deviation of the isotopic ratios ranged from 0.1 to 0. 5% for the high (400 pmol) standards and from 0.2 to 0.8% for the low (physiological) standard, which is comparable with GC-MS. A plot of the results against the theoretical values gave a linear curve for all isotopes studied (R2 ranged from 0.9984 to 0.9997). However, the [1-13C]-enriched amino acids measured (LEU, GLY, and VAL) gave a closer agreement to the expected values as was found for [ureido-13C-5,5-2H2]-enriched citrulline and [guanidino-15N2]-enriched arginine. We could not determine whether this was due to the measurement procedure itself or resulting from an instability of the tracers in solution. Nevertheless, the results were reproducible and the theoretical value could be calculated using the tangent of the enrichment curves. (ABSTRACT TRUNCATED)