1. Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography
I H Lee, S Pollack, S H Hsu, J R Miksic J Chromatogr Sci. 1991 Apr;29(4):136-40. doi: 10.1093/chromsci/29.4.136.
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.
2. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes
J Cornish, K E Callon, C Q Lin, C L Xiao, T B Mulvey, G J Cooper, I R Reid Am J Physiol. 1999 Nov;277(5):E779-83. doi: 10.1152/ajpendo.1999.277.5.E779.
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.
3. Roles of peripheral terminals of transient receptor potential vanilloid-1 containing sensory fibers in spinal cord stimulation-induced peripheral vasodilation
Mingyuan Wu, Naoka Komori, Chao Qin, Jay P Farber, Bengt Linderoth, Robert D Foreman Brain Res. 2007 Jul 2;1156:80-92. doi: 10.1016/j.brainres.2007.04.065. Epub 2007 Apr 30.
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.
