Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor

We have found that phosphorylation of a G-protein-coupled receptor by protein kinase C (PKC) disrupts modulation of ion channels by the receptor. In AtT-20 cells transfected with rat cannabinoid receptor (CB1), the activation of an inwardly rectifying potassium current (Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by 100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected, and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop of CB1. Mutating this serine to alanine did not affect the ability of CB1 to modulate currents, but it eliminated disruption by PMA, demonstrating that PKC can disrupt ion channel modulation by receptor phosphorylation.     

Cannabinoid-induced hypotension and bradycardia in rats mediated by CB1-like cannabinoid receptors

Previous studies indicate that the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide HCl (SR141716A), inhibits the anandamide- and delta9-tetrahydrocannabinol- (THC) induced hypotension and bradycardia in anesthetized rats with a potency similar to that observed for SR141716A antagonism of THC-induced neurobehavioral effects. To further test the role of CB1 receptors in the cardiovascular effects of cannabinoids, we examined two additional criteria for receptor-specific interactions: the rank order of potency of agonists and stereoselectivity. A series of cannabinoid analogs including the enantiomeric pair (-)-11-OH-delta9-THC dimethylheptyl (+)-11-OH-delta9-THC dimethylheptyl were evaluated for their effects on arterial blood pressure and heart rate in urethane anesthetized rats. Six analogs elicited pronounced and long lasting hypotension and bradycardia that were blocked by 3 mg/kg of SR141716A. The rank order of potency was (-)-11-OH-delta9-THC dimethylheptyl > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > THC > anandamide > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol, which correlated well with CB1 receptor affinity or analgesic potency (r = 0.96-0.99). There was no hypotension or bradycardia after palmitoylethanolamine or (+)-11-OH-delta9-THC dimethylheptyl. An initial pressor response was also observed with THC and anandamide, which was not antagonized by SR141716A. We conclude that the similar rank orders of potency, stereoselectivity and sensitivity to blockade by SR141716A indicate the involvement of CB1-like receptors in the hypotensive and bradycardic actions of cannabinoids, whereas the mechanism of the pressor effect of THC and anandamide remains unclear.     

CB1 receptor antagonist precipitates withdrawal in mice exposed to Delta9-tetrahydrocannabinol

Although tolerance to cannabinoids has been well established, the question of cannabinoid dependence had been very controversial until the discovery of a cannabinoid antagonist, SR141716A. The objective of this study was to develop and characterize a mouse model of precipitated withdrawal indicative of cannabinoid dependence. Using a dosing regimen known to produce pharmacological and behavioral tolerance, mice were treated with Delta9-tetrahydrocannabinol (Delta9-THC) twice a day for 1 wk. SR141716A administration after the last Delta9-THC injection promptly precipitated a profound withdrawal syndrome. Typical withdrawal behavior was an increase in paw tremors and head shakes that was accompanied with a decrease in normal behavior such as grooming and scratching. Of the three Delta9-THC regimens tested, daily Delta9-THC injections of 10 and 30 mg/kg produced the greatest number of paw tremors and head shakes and the least number of grooms after challenge with SR141716A. Precipitated withdrawal was apparent after 2, 3, 7 and 14 days of treatment based on an increase in paw tremors in Delta9-THC-treated mice as compared with vehicle-treated mice. These findings are consistent with SR141716A-precipitated withdrawal in rats. Moreover, these results suggest that mice are a viable model for investigating dependence to cannabinoids.     

Alteration in the brain content of substance P (1-7) during withdrawal in morphine-dependent rats

OBJECTIVE: To evaluate hoof size, shape, and balance as risk factors for catastrophic musculoskeletal injuries (CMI), including suspensory apparatus failure (SAF) and cannon bone condylar fracture (CDY) in Thoroughbred racehorses. ANIMALS: 95 Thoroughbred racehorses that died between 1994 and 1996. PROCEDURE: 38 quantitative measures of hoof size, shape, and balance were obtained from orthogonal digital images of the hoof and were compared between case horses with forelimb CMI (70), SAF (43), and CDY (10) injuries and control horses whose death was unrelated to the musculoskeletal system (non-CMI, 25). Comparison of group means between cases and controls was done using ANOVA, and multivariable logistic regression was used to estimate odds ratios. RESULTS: Odds of CMI were 0.62 times lower for a 5mm increase in ground surface width difference and 0.49 times lower for a 100-mm2 increase in sole area difference. Odds of SAF were 6.75 times greater with a 10 degrees increase in toe-heel angle difference and 0.58 times lower with a 100-mm2 increase in sole area difference. Odds of CDY were 0.26 times lower with a 3 degrees increase in toe angle, 0.15 times lower with a 5-mm increase in lateral ground surface width, and 0.35 times lower with a 100-mm2 increase in sole area difference. CLINICAL RELEVANCE: Decreasing the difference between toe and heel angles should decrease risk of SAF for Thoroughbred racehorses and should be considered in addition to increasing toe angle alone to help prevent catastrophic injury. Trimming the hoof to perfect mediolateral symmetry may not be a sound approach to avoiding injury.     

CB1 cannabinoid receptor: cellular regulation and distribution in N18TG2 neuroblastoma cells

In order to characterize cellular regulation of CB1 cannabinoid receptors, synthesis and turnover studies were performed. Metabolic labeling of N18TG2 cells with 35S-labeled amino acids was followed by immunoprecipitation from cell lysates using an affinity-purified antibody generated to the N-terminal 14-amino-acid segment of the CB1 receptor. During a 2 h labeling period, CB1 receptors were rapidly and constitutively synthesized (rate: 0.86%/min). The majority of newly synthesized CB1 cannabinoid receptors (70%) was degraded rapidly by a first-order process (t1/2=4.8 h). The remaining nascent receptors, which were degraded slowly (t1/2>24 h), may represent the pool of potentially functional receptors. Trypsin treatment of intact confluent cells, designed to cleave the extracellular antibody recognition site, did not alter the recovery of metabolically labeled immunoprecipitated CB1 receptors. This suggests that a large percentage of newly synthesized receptors was inaccessible to the protease and is probably intracellular. Immunocytochemistry revealed CB1 cannabinoid immunoreactivity both intracellularly and on the cell surface. Subcellular membrane fractions exhibited receptor binding activity on plasma membranes and nuclear-associated membranes. Only low-affinity binding was seen in the chromatin fraction. An hypothesis has been developed to explain these results and form the basis for future studies.     

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.     

Opioid withdrawal activates MAP kinase in locus coeruleus neurons in morphine-dependent rats in vivo

Opioid dependence is widely believed to result from neuroadaptations in specific brain regions. However, the precise molecular mechanisms underlying these adaptations are not yet clear. Our aim was to explore the role of mitogen-activated protein kinase (MAPK) in mu opioid receptor signalling in vivo. Using anti-phospho MAPK antibodies, activated MAPK was detected in cortical neurons (layers II/III), median eminence, amygdaloid and hypothalamic nuclei in untreated animals. Dense nuclear and cytoplasmic staining was observed resulting in full visualization of processes in these cells. Chronic, but not acute, administration of morphine greatly diminished this staining pattern while mu opioid receptor levels and levels of MAP kinase as detected with a phosphorylation state-independent antibody were unchanged. When opioid withdrawal was precipitated with naloxone a dramatic increase in MAP kinase phosphorylation was observed in somata and fibres of locus coeruleus, solitary tract and hypothalamic neurons. Thus, the differential activation state of MAPK could have important implications for understanding the mechanisms underlying opioid tolerance and dependence.     

Forced opiate withdrawal under anaesthesia augments and prolongs the occurrence of withdrawal signs in rats

This study examined the impact of the introduction of lecturer-practitioner (LP) roles in four Nuffield Hospitals over a period of two years. A series of interviews was conducted with the practitioners themselves, the staff and the managers. LPs also maintained structured diaries. Nursing practice in the independent sector is influenced by a number of factors, including caring for patients in individual rooms and working directly with consultants because there are no junior medical staff. Nurses encountered patients with very diverse problems, and accordingly needed help to maintain a wide professional knowledge base. Nursing staff benefited from the presence of an LP in terms of linking theory to practice, role modelling, obtaining career advice and introducing change. Non-nursing staff also benefited from the LPs' adoption of an interdisciplinary approach to certain care situations. LPs were valued as a resource for staff and seen as important in enabling the company to maintain its image as a provider of high-quality care. The LP was also well placed to raise the profile of nursing in the independent sector through research activities. LPs themselves benefited from their experiences and were able to develop their own skills.     

Clonidine blocks acquisition but not expression of conditioned opiate withdrawal in rats

Previous studies in rodents have reported that clonidine, an alpha 2-adrenergic receptor agonist, attenuated conditioned aversions to naloxone-precipitated opiate withdrawal when administered prior to each withdrawal conditioning episode. The current study was designed to determine whether clonidine could modify the expression of previously established conditioned place aversions and conditioned suppression of operant responding. Dose- and time-dependent effects of clonidine on activity and suppression of operant responding for food identified appropriate treatment parameters for subsequent studies in which rats rendered dependent on opiates through implantation of morphine pellets were tested for: (1) conditioned place aversion; and (2) conditioned suppression of operant responding for food (fixed ratio-15 schedule), in a paradigm wherein rats received four pairings of naloxone with a distinct tone and odor stimulus. Clonidine dose-dependently blocked the acquisition of both conditioned behaviors when administered prior to naloxone on each conditioning trial, but was ineffective in blocking the expression of these conditioned withdrawal signs when administered prior to the test session.     

Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands

Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is located exclusively in the periphery (CB2). The purposes of this study were to explore further the binding requirements of the CB2 receptor and to search for compounds displaying distinct affinities for either cannabinoid receptor. The binding affinities of a series of cannabinoids tested previously at the CB1 receptor were determined at cloned human CB1 and CB2 receptors using a filtration assay. In addition, possible allosteric regulation of the CB2 receptor was examined. Sodium and a GTP analog elicited a concentration-dependent decrease in specific binding to the CB2 receptor. The affinity of cannabinol for CB2 receptors (Ki = 96.3 +/- 14 nM) was confirmed to be in approximately the same range as that of delta 9-THC (Ki = 36.4 +/- 10 nM). Affinities at cloned CB1 and CB2 receptors were compared with affinities determined in the brain. Although most of the chosen compounds did not discriminate between CB1 and CB2, several ligands were identified that showed selectivity. Affinity ratios demonstrated that two 2'-fluoro analogs of anandamide were over 23-fold selective for the CB1 receptor and confirmed the CB1 selectivity of SR141716A {N- (piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride}. In addition, WIN-55, 212-2 {(R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl) methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone} and a closely related propyl indole analog were shown to be 6.75- and 27.5- fold selective, respectively, for the CB2 receptor. These ligands can now serve as a basis for the design of compounds with even greater selectivity.     

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or coincubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC50 73 nM), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction. These results provide functional evidence of the existence of prejunctional cannabinoid CB1-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.     

Clonidine antagonizes the aversive effects of opiate withdrawal and the rewarding effects of morphine only in opiate withdrawn rats.

The researchers asked whether clonidine, an α?-noradrenergic agonist, would block selectively the motivational effects of opiate withdrawal and whether clonidine's effects would respect the boundary between nondeprived and deprived motivational states. In a place conditioning paradigm, clonidine (0.05 mg/kg ip) blocked the rewarding effects of morphine in opiate-withdrawn rats (as well as the aversive properties of withdrawal itself), but did not affect morphine place preferences (2 and 20 mg/kg) in drug-naive rats. Furthermore, clonidine blocked the acquisition of morphine (15 mg/kg), but not LiCI (15 mg/kg), conditioned taste aversions in water-deprived rats. The results suggest that the motivational system activated in deprived animals includes dopaminergic and noradrenergic components that are in series with each other. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors

OBJECTIVES: We studied the clinical spectrum, histology, and malignant potential of colonic polyps in Indian children (< or =12 yr). METHODS: Two hundred thirty-six children with colonic polyps were studied from January 1991 to October 1996. They were evaluated clinically and colonoscopic polypectomy was done. Children with five or more juvenile polyps were labeled as having juvenile polyposis and serial colonoscopic polypectomies were done every 3 wk. Colectomy was performed when there were intractable symptoms or clearing of the polyps by colonoscopy was not possible. Histological examination of the polyps was done. Follow-up colonoscopy was done in children with juvenile polyposis only. RESULTS: The mean age of these children was 6.12 +/- 2.7 yr, with a male preponderance (3.5:1). Rectal bleeding of a mean duration of 14 +/- 16 months was the presenting symptom in 98.7%. Solitary polyps were seen in 76%, multiple polyps in 16.5%, and juvenile polyposis in 7% (n = 17) of the children. A majority (93%) of the polyps were juvenile and 85% were rectosigmoid in location. Adenomatous changes, seen in 11%, were more common in juvenile polyposis (59%) than in juvenile polyps (5%). Among those with juvenile polyposis, colon clearance was achieved in eight, six required colectomy for intractable symptoms, and three were still on the polypectomy program. Polyps recurred in 5% of children with juvenile polyps and 37.5% of those with juvenile polyposis. CONCLUSIONS: Juvenile polyps remain the most common colonic polyps in children. A significant number of cases of polyps are multiple and proximally located, which emphasizes the need for total colonoscopy in all. Juvenile polyps should be removed even if asymptomatic because of their neoplastic potential. Colonoscopic polypectomy is effective even in juvenile polyposis. Surveillance colonoscopy is required in juvenile polyposis only.     

SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor

Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.     

Further evidence for the presence of cannabinoid CB1 receptors in mouse vas deferens

Basophil granulocytes and their mediators are involved in the pathogenesis of allergic inflammation. We evaluated basophil count, blood histamine content, eosinophil count and serum total IgE levels in one hundred-thirteen healthy newborns at birth. 108 children were prospectively studied with a follow-up to 18 months of age for development of topic disorders. No difference was found in newborns with biparental family history of atopy (FHA) in comparison with newborns with monoparental FHA and with newborns without FHA. Children who developed atopic disorders had neonatal basophil count higher than those who did not develop atopic symptoms (p = 0.03). No significant correlation was found between basophil and eosinophil counts (r = 0.013), between basophil count and serum total IgE levels (r = 0.012) and between basophil count and blood histamine content. Positive predictive value and sensitivity of basophil count for allergy up to 18 months of age was respectively only 33% and 27%. Our data indicate that an increased basophil count at birth is not associated with FHA and is not a good predictive marker of atopy.     

A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain

CB1-type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn-2 arachidonylglycerol (2-AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2-AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1-type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAH-immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complementary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.     

Neurobiology of withdrawal motivation: Evidence for two separate aversive effects produced in morphine-naive versus morphine-dependent rats by both naloxone and spontaneous withdrawal.

In drug-naive rats, the rewarding effects of morphine are blocked by lesions of the tegmental pedunculopontine nucleus (TPP), but not by neuroleptics. In dependent rats (chronically treated with morphine), morphine reward is blocked by neuroleptics, but not by TPP lesions. Just as this activation of opiate receptors in naive vs dependent rats produces different mechanisms of reward, this study concludes that reduced opioid activity on these opiate receptors produces different mechanisms of aversion. Neuroleptics blocked the conditioned place aversions produced by naloxone and spontaneous withdrawal in morphine dependent, but not naive, rats, without attenuating the somatic withdrawal syndrome induced by naloxone in dependent rats. The researchers suggest that the aversive effects of endogenous opioid withdrawal in naive rats are mediated by different neural substrates than the aversive effects of exogenous opioid withdrawal in dependent rats. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence that the cannabinoid CB1 receptor is a 2-arachidonoylglycerol receptor. Structure-activity relationship of 2-arachidonoylglycerol, ether-linked analogues, and related compounds

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.     

Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.