Calcitonin, eel TFA

Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes

Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10(-8) to 10(-7) M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10(-9) M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.

Roles of peripheral terminals of transient receptor potential vanilloid-1 containing sensory fibers in spinal cord stimulation-induced peripheral vasodilation

Background: Spinal cord stimulation (SCS) is used to relieve ischemic pain and improve peripheral blood flow in selected patients with peripheral arterial diseases. Our previous studies show that antidromic activation of transient receptor potential vanilloid-1 (TRPV1) containing sensory fibers importantly contributes to SCS-induced vasodilation.
Objectives: To determine whether peripheral terminals of TRPV1 containing sensory fibers produces vasodilation that depends upon the release of calcitonin gene-related peptide (CGRP) and nitric oxide (NO) during SCS.
Methods: A unipolar ball electrode was placed on the left dorsal column at lumbar spinal cord segments 2-3 in sodium pentobarbital anesthetized, paralyzed and ventilated rats. Cutaneous blood flow from left and right hindpaws was recorded with laser Doppler flow perfusion monitors. SCS was applied through a ball electrode at 30%, 60%, 90% and 300% of motor threshold. Resiniferatoxin (RTX; 2 microg/ml, 100 microl), an ultra potent analog of capsaicin, was injected locally into the left hindpaw to functionally inactivate TRPV-1 containing sensory terminals. In another set of experiments, CGRP(8-37), an antagonist of the CGRP-1 receptor, was injected at 0.06, 0.12 or 0.6 mg/100 microl into the left hindpaw to block CGRP responses; N-omega-nitro-l-arginine methyl ester (L-NAME), a nonselective nitric-oxide synthase (NOS) inhibitor, was injected at 0.02 or 0.2 mg/100 microl into the left hindpaw to block nitric oxide synthesis; (4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine, TFA, a neuronal NOS inhibitor, was injected at 0.02 or 0.1 mg/100 microl into the left hindpaw to block neuronal nitric oxide synthesis.
Results: SCS at all intensities produced vasodilation in the left hindpaw, but not in the right. RTX administration attenuated SCS-induced vasodilation at all intensities in the left hindpaw (P<0.05, n=7) compared with responses before RTX. CGRP(8-37) administration attenuated SCS-induced vasodilation in the left hindpaw in a dose dependent manner (linear regression, P<0.05) compared with responses before CGRP(8-37). In addition, L-NAME at a high dose, but not (4S)-N-(4-Amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine, TFA, decreased SCS-induced vasodilation (P<0.05, n=5).
Conclusion: While TRPV1, CGRP and NO are known to be localized in the same nerve terminals, our data indicate that SCS-induced vasodilation depends on CGRP release, but not NO release. NO, released from endothelial cells, may be associated with vascular smooth muscle relaxation and peripheral blood flow increase in response to SCS.

Identification, characterization and quantification of specific neuropeptides in rat spinal cord by liquid chromatography electrospray quadrupole ion trap mass spectrometry

Substance P and CGRP play a central role in neuropathic pain development and maintenance. Additionally, dynorphin A is an endogenous ligand of opioid receptors implicated in the modulation of neurotransmitters including neuropeptides, such as substance P and CGRP. This manuscript proposes a method to characterize, identify and quantify substance P, CGRP and dynorphin A in rat spinal cord by HPLC-ESI/MS/MS. Rat spinal cords were collected and homogenized into a TFA solution. Samples were chromatographed using a microbore C(8) 100 x 1 mm column and a 19 min linear gradient (0:100 --> 40:60; ACN:0.2% formic acid in water) at a flow rate of 75 microL/min for a total run time of 32 min. The peptides were identified in rat spinal cord based on full-scan MS/MS spectra. Substance P, CGRP and dynorphin A were predominantly identified by the presence of specific b CID fragments. Extracted ion chromatogram (XIC) suggested selected mass transitions of 674 --> [600 + 254], 952 --> [1215 + 963] and 717 --> [944 + 630] for substance P, CGRP and dynorphin A can be used for isolation and quantitative analysis. A linear regression (weighted 1/x) was used and coefficients of correlations (r) ranging from 0.990 to 0.999 were observed. The precision (%CV) and accuracy (%NOM) observed were 10.9-14.4% and 8.9-14.2%, 8.8-13.0% and 91.0-110.2% and 97.2-107.3% and 91.8-97.3% for substance P, CGRP and dynorphin A respectively. Following the analysis of rat spinal cords, the mean endogenous concentrations were 110.7, 2541 and 779.4 pmol/g for substance P, CGRP and dynorphin A respectively. The results obtained show that the method provides adequate figures of merit to support targeted peptidomic studies aimed to determine neuropeptide regulation in animal neuropathic and chronic pain models.

A validated HPLC assay for simultaneous analysis of salmon calcitonin and duck ovomucoid

A highly sensitive and selective analytical HPLC method is reported for the simultaneous measurement of salmon calcitonin (sCT) and its enzyme inhibitor, duck ovomucoid (dOVM). The method used a reversed phase C-18 column (4.6 x 250 mm, 5 microm) at room temperature. The elution was achieved using a gradient technique (20-35% B for 10 min, 35-37% B from 10th to 20th min and 37-20% B from 20th to 25th min). The mobile phase used was 0.05% v/v trifluoroacetic acid (TFA) in water and 0.05% v/v TFA in acetonitrile with a flow rate of 1 ml/min. Detection was carried out by UV spectrophotometry at 210 nm. sCT and dOVM were eluted at 7.8 and 15.4 min respectively, free from any interfering endogenous peaks during a run time of 25 min. Linear relationships were observed between the detector response and the concentrations of the analytes (10-100 microg/ml for CT (r2 = 0.996) and 10-100 microg/ml for the dOVM (r2 = 0.999)). The assay was found to be highly selective and sensitive due to the absence of any interfering peaks. The lower C.V. and % error values of the assay indicates that the assay could accurately and precisely quantitate both sCT and dOVM in the examined concentration range. This method can be usedfor the simultaneous quantitative analysis of sCT and dOVM.