Detection of Nine WADA Prohibited GHRPs and GHS Using the HPLC-MS/MS Method and Their Stability in Human Urine / 中国运动医学杂志

Objective To introduce a practical high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)method for the detection of seven growth hormone-releasing peptides (GHRPs)including GHRP-1,GHRP-2,GHRP-4,GHRP-5,GHRP-6,Hexarelin and Alexamorelin and two growth hormone secretagogues(GHS)including anamorelin and ipamorelin,and study the stability of these nine substances in the human urine.Method The urine samples were purified and extracted by a solid phase extraction procedure using Oasis(R) WCX column.The urine was first centrifuged and taken out 1 mL into a small column,cleaned by 5％ NH4OH and 20％ CH3CN respectively,eluated using the mixture of water and acetomitrile(1/3)with 2％ formic acid,blow-dried in the nitrogen at 35℃ and finally redissolved to be injected into the LC-MS/MS.Result The limits of detection were between 0.01～0.5 ng/mL accordingly.The spiked recoveries at the low concentration(1 ng/mL),medium concentration(2 ng/mL)and high concentration(10 ng/mL)ranged between 40％ and 76％.The intra-and interday precisions of the target substances at these three concentrations were all less than 15％.The indoor temperature,refrigeration condition and multigelation were observed to have significant impact on the anamorelin,GHRP-2,GHRP-4 and GHRP-5.Conclusion The method established in this study is simple,and its specificity and sensitivity meets the international standard and technical documents for laboratories set up by the Wworld Anti-Doping Agency.It has been applied in our routine work.Multigelation should be avoided in the transport,detection and long-term laboratory storage of urine samples.

Effect of Electroacupuncture on Hypothalamic and Hippocampal Expressions of Ghrelin and Ghrelin Receptor mRNA in Functional Dyspepsia Rats / 上海针灸杂志

Objective To investigate the effect of electroacupuncture on hypothalamic and hippocampal expressions of ghrelin and ghrelin receptor (GHS-R) mRNA in functional dyspepsia (FD) rats. Methods Eighty SD rats were randomly allocated to normal control, model control, medication and electroacupuncture groups, 20 rats each. A rat model of FD was made by tail clamp modeling method. The medication group received an oral gavage of cisapride solution and the electroacupuncture group, electroacupuncture treatment. The expressions of ghrelin protein and GHS-R mRNA in the hypothalamus and hippocampus were examined by Western blot and real-time PCR, respectively, in all the four groups. Results Compared with the model control group of rats, the expression of ghrelin protein in the hypothalamus and the expression of GHS-R mRNA in the hypothalamus and hippocampus increased in the medication group (P<0.05) and the expressions of ghrelin protein and GHS-R mRNA in the hypothalamus and hippocampus were up-regulated in the electroacupuncture group (P<0.05). Compared with the medication group of rats, the expressions of ghrelin protein and GHS-R mRNA in the hippocampus were up-regulated in the electroacupuncture group (P<0.05). Conclusions Electroacupuncture and medication can improve gastrointestinal functions and correct an imbalance in the brain-gut axis through the hippocampus-hypothalamic pathway by regulating hypothalamic and hippocampal ghrelin protein and GHS-R mRNA contents and activating hypothalamic neurons and hippocampal excitatory synapses.

Effects of peptidic growth hormone secretagogue receptor (GHS-R) antagonist [D-Lys3] on some of serum hormonal and biochemical parameters in Wistar rat model / Efeitos do antagonista [D-Lys3] do receptor do peptídeo secretagogo do hormônio do crescimento (GHS-R) sobre alguns parâmetros bioquímicos e hormonais séricos em um modelo em ratos Wistar

Objective : The present study investigated the effects of different dosages of a GHS-R antagonist [D-Lys3] on some serum hormonal (cortisol, T3 and T4) and biochemical parameters in a rat.Materials and methods : Thirty-six 60-day-old male rats were assigned to four treatments. [D-Lys3]-GHRP-6 solutions were infused via intraperitoneal injections. Blood was collected and analyzed.Results : The large dosages of a GHS-R antagonist (200 ng/kg BW) caused increases in cortisol, whereas no significant changes occurred when low dosages were injected. There were no significant changes in T3 and T4 following the administration of the GHS-R antagonist, but a considerable increase was observed in blood glucose levels of the groups (G50, G100, and G200 ng/kg BW). There was a significant increase in total protein when the greatest dose was administrated (G200 ng/kg BW). However, total cholesterol, triglycerides, and albumin showed no significant changes.Conclusions : Exogenous GHS-R antagonist can cause an increase in glucose and moderate increases in cortisol and total protein, yet it has no significant effect on T3 and T4 levels or on the concentrations of serum lipids. The effect of GHS-R antagonist is not completely adverse to the effects of ghrelin. Further molecular studies are necessary to identify the physiological effects of the peptidic GHS-R antagonist. Arq Bras Endocrinol Metab. 2014;58(3):288-91.

Objetivo : O presente estudo investigou os efeitos de diferentes doses do antagonista do GHS-R [D-Lys3] sobre alguns parâmetros hormonais (cortisol, T3 e T4) e bioquímicos em ratos.Materiais e métodos : Trinta e seis ratos machos com 60 dias de idade foram alocados para quatro tratamentos. Soluções de [D-Lys3]-GHRP-6 foram administradas por meio de injeções intraperitoneais e foram coletadas e analisadas amostras.Resultados : Doses altas de antagonista de GHS-R (200 ng/kg PC) levaram a aumento do cortisol, enquanto não houve diferença significativa quando foram injetadas doses baixas. Não houve alterações significativas em T3 e T4 depois da administração do antagonista do GHS-R, mas foi observado aumento considerável nos níveis de glicose sanguínea dos grupos (G50, G100 e G200 ng/kg PC). Houve aumento significativo na proteína total quando foi administrada a maior dose (G200 ng/kg PC), entretanto, não foram observadas alterações no colesterol total, nos triglicérides e na albumina.Conclusões : O antagonista do GHS-R exógeno pode causar aumento da glicose e aumento moderado do cortisol e proteína total, embora não haja efeitos significativos nos níveis de T3 e T4 ou na concentração de lipídios séricos. O efeito do antagonista de GHS-R não é completamente adverso aos efeitos da grelina. Devem ser feitos outros estudos moleculares para se identificar os efeitos fisiológicos do peptídeo antagonista do GHS-R. Arq Bras Endocrinol Metab. 2014;58(3):288-91.

Hazard Classification of Household Chemical Products in Korea according to the Globally Harmonized System of Classification and labeling of Chemicals

OBJECTIVES: This study was conducted to review the validity of the need for the application of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) to household chemical products in Korea. The study also aimed to assess the severity of health and environmental hazards of household chemical products using the GHS. METHODS: 135 products were classified as 'cleaning agents and polishing agents' and 98 products were classified as 'bleaches, disinfectants, and germicides.' The current status of carcinogenic classification of GHS and carcinogenicity was examined for 272 chemical substances contained in household chemical products by selecting the top 11 products for each of the product categories. In addition, the degree of toxicity was assessed through analysis of whether the standard of the Republic of Korea's regulations on household chemical products had been exceeded or not. RESULTS: According to GHS health and environmental hazards, "acute toxicity (oral)" was found to be the highest for two product groups, 'cleaning agents and polishing agents', and 'bleaches, disinfectants, and germicides' (result of classification of 233 household chemical products) at 37.8% and 52.0% respectively. In an analysis of carcinogenicity assuming a threshold of IARC 2B for the substances in household chemical products, we found 'cleaning agents and polishing agents' to contain 12 chemical substances and 'bleaches, disinfectants, and germicides' 11 chemical substances. CONCLUSION: Some of the household chemical products were found to have a high hazard level including acute toxicity and germ cell mutagenicity, carcinogenicity, and reproductive toxicity. Establishing a hazard information delivery system including the application of GHS to household chemical products in Korea is urgent as well.

Acute and Subchronic Inhalation Toxicity of n-Octane in Rats

OBJECTIVES: We have investigated the toxic effects of the inhalation of subchronic and acute levels of n-octane. METHODS: The rats were exposed to n-octane of 0, 2.34, 11.68 and 23.36 mg/L (n = 5 rats/group/gender) in an acute inhalation test (Organization for Economic Co-operation and Development (OECD) TG 403), or to 0, 0.93, 2.62 and 7.48 mg/L (n = 10 rats/group/gender) for a subchronic inhalation test (OECE TG 413), to establish a national chemical management system consistent with the Globally Harmonized Classification System (GHS). RESULTS: Acutely-exposed rats became lethargic but recovered following discontinuation of inhalation. Other clinical symptoms such as change of body weight and autopsy finds were absent. The LC50 for the acute inhalation toxicity of n-octane was determined to exceed 23.36 mg/L and the GHS category was 'not grouping'. Subchronically-treated rats displayed no significant clinical and histopathological differences from untreated controls; also, target organs were affected hematologically, biochemically and pathologically. Therefore, the no observable adverse effect level was indicated as exceeding 7.48 mg/L and the GHS category was 'not grouping' for the specific target organ toxicity upon repeated exposure. CONCLUSION: However, n-octane exposure should be controlled to be below the American Conference of Industrial Hygienists recommendation (300 ppm) to prevent inhalation-related adverse health effects of workers.

Expression of ghrelin and its receptor GHS-R 1A in pancreatic endocrine tumors and the plasma levels of ghrelin and leptin / 中华胰腺病杂志

Objective To study the plasma and intratumoral levels of ghrelin, leptin and their relationship and clinical significance in patients with pancreatic endocrine tumor.Methods Preoperative plasma levels of ghrelin and leptin were detected by ELISA in 11 patients with pancreatic endocrine tumors and 28 normal controls.Expressions of ghrelin and its receptor GHS-R 1A were tested in 11 tumors and 27 paired control tissues by immunohistochemistry staining, and they were correlated with the clinicopathological characteristics.Results The plasma levels of ghrelin was ( 16.0 ± 5.0) pg/ml, which was significantly lower than that in normal controls [ (21.0 ± 2.0) pg/ml, P = 0.047 ].The plasma levels of leptin was (0.34 ±0.03 ) ng/ml, which was not significantly different with that in normal controls [ 0.38 ± 0.04) ng/ml ].There was positive association between plasma levels of leptin and ghrelin (P =0.015 ), but was not associated with clinicopathological parameters.The plasma levels of leptin in control group was positively associated with BMI (P = 0.002), but they were not associated in patients with tumor.The expression rate of ghrelin in tumor tissue was significantly lower than that in control group (64% vs 100%, P = 0.004 ).But the expression rate of GHS-R I A was not significantly different between the two groups.The expression of ghrelin and GHS-R1A in tumor was not significantly associated with clinicopathological parameters.Conclusions The ghrelin and its receptor GHS-R 1A were extensively expressed in pancreatic endocrine tumors, and the serum levels of ghrelin and leptin was changed.

Clinical Observation on Effect of Prevention of Glenohumeral Subluxation with Electro-acupuncture Therapy in Super-early Period of Cerebral Stroke in 76 Cases / 国际中医中药杂志

Objective To observe the preventive effects of electroacupuncture therapy on glenohumeral subluxation(GHS)in patients of cerebral stroke of super-early period. Methods A total of 160 cases with cerebral stroke were randomly recruited into a control group of 80 cases and a treatment group of 80 cases. The control group was treated with conventional medicine and rehabilitation training, while the treatment group was given electro-acupuncture therapy based on conventional medicine and rehabilitation training. All treatments were performed once in each day, and one treatment course lasted for ten days. The preventive effect was evaluated after the successive three courses. Results 4 cases in treatment group and 2 cases in control group quitted in therapeutic process. The GHS incidence rate in the third course of treatment group was significantly lower than control group (P<0.05). The severe GHS incidence rate of treatment group was significantly lower than control group in the second course (P<0.05)and the third course (P<0.01). The GHS scale of the treatment group showed no significant difference in all three courses (P>0.05), while the control group showed significant difference in one course of all three(P<0.05). Conclusion Electro-acupuncture therapy can significantly decrease GHS incidence rate, relieve pathogenetic condition, control its progress, and so thus prevent GHS of stroke patients in super-early period.

Harmonização e classificação toxicológica de agrotóxicos em 1992 no Brasil e a necessidade de prever os impactos da futura implantação do GHS / Harmonization and toxicological classification of pesticides in 1992 in Brazil and the need to foresee the impacts from the forthcoming introduction of GHS

Em 1992, o Brasil modificou seus critérios de classificação toxicológica de agrotóxicos adequando-os à recomendação de classificação de periculosidade da Organização Mundial da Saúde (OMS). Em 2002, o Sistema Globalmente Harmonizado de Classificação e Rotulagem de Produtos Químicos (GHS) foi adotado pela Organização das Nações Unidas. Em decorrência, a OMS está adequando ao GHS sua recomendação de classificação de agrotóxicos, o que também deverá ser feito pelo Brasil. Considerou-se oportuno estimar o impacto da alteração de critérios, ocorrida em 1992, na reclassificação toxicológica dos produtos comerciais que se encontravam registrados na ocasião. Encontrou-se que 58,6 por cento do total dos agrotóxicos então registrados (74,9 por cento das formulações líquidas e 31,0 por cento das sólidas) podem ter sofrido reclassificação para classes toxicológicas consideradas de me-nor periculosidade, sofrendo mudanças na comunicação de riscos expressa na rotulagem. Isto pode ter ocasionado conseqüências negativas devido a confusões de interpretação pelos agricultores. Nos países que já dispõem de sistemas de classificação de periculosidade de agrotóxicos, como o Brasil, recomenda-se estimar, antes da implantação, os impactos das mudanças que poderão decorrer da adoção do GHS.

In 1992, Brazil modified its toxicological classification criteria for pesticides. This modification aimed to follow the guidelines of the World Health Organization (WHO) hazard classification of pesticides. In 2002, the United Nations adopted the "Globally Harmonized System for the Classification and Labelling of Chemicals" (GHS). As a result, the WHO is reviewing its classification and Brazil will probably follow the example. Therefore it seems timely to estimate the impact of the changes in the criteria adopted in 1992 in the toxicological reclassification and labelling of the commercially available products registered at that time. It is estimated that 58.6 percent of the total of registered pesticides (74.9 percent liquid and 31.0 percent solid formulations) might have been reclassified to less hazardous classes. The hazard warnings on the labels may have caused negative consequences due to misinterpretations by farmers or rural workers. For countries already having a hazard classification system, it is therefore recommendable to estimate the possible impact of the changes before implanting the GHS.

Role of Glucocorticoids in Fasting-induced Changes in Hypothalamic and Pituitary Components of the Growth Hormone (GH)-axis

To directly test if elevated glucocorticoids are required for fasting-induced regulation of growth hormone (GH)-releasing hormone (GHRH), GHRH receptors (GHRH-R) and ghrelin receptors (GHS-R) expression, male rats were bilaterally adrenalectomized or sham operated. After 7 days, animals were fed ad libitum or fasted for 48 h. Bilateral adrenalectomy increased hypothalamic GHRH to 146% and decreased neuropeptide Y (NPY) mRNA to 54% of SHAM controls. Pituitary GHRH-R and GHS-R mRNA levels were decreased by adrenalectomy to 30% and 80% of sham-operated controls. In sham- operated rats, fasting suppressed hypothalamic GHRH (49%) and stimulated NPY (166%) mRNA levels, while fasting increased pituitary GHRH-R (391%) and GHS-R (218%) mRNA levels. However, in adrenalectomized rats, fasting failed to alter pituitary GHRH-R mRNA levels, while the fasting-induced suppression of GHRH and elevation of NPY and GHS-R mRNA levels remained intact. In fasted adrenalectomized rats, corticosterone replacement increased GHRH-R mRNA levels and intensified the fasting-induced decrease in GHRH, but did not alter NPY or GHS-R response. These data suggest that elevated glucocorticoids mediate the effects of fasting on hypothalamic GHRH and pituitary GHRH-R expression, while glucocorticoids are likely not the major determinant in fasting-induced increases in hypothalamic NPY and pituitary GHS-R expression.

International Comparison of Criteria for Evaluating Sensitization of PRTR-Designated Chemical Substances

In this study, we aim to compare the criteria for sensitizers among national organizations in various countries and international organizations, and to specify whether each Pollutant Release and Transfer Register (PRTR)-designated chemical substance is a sensitizer by each organization. The definition of sensitizing chemicals and the designation of respective sensitizers according to the PRTR law, Japan Society for Occupational Health (JSOH), American Conference of Governmental Industrial Hygienists (ACGIH), European Union (EU), and Deutsche Forschungsgemeinshaft (DFG) were studied. Of the 435 PRTR-designated chemical substances, 15 are listed as sensitizers according to the PRTR law, 16 as sensitizers of the airway and 21 as sensitizers of the skin by JSOH, 12 as sensitizers (no discrimination) by ACGIH, 19 (airway) and 85 (skin) by EU, and 15 (airway) and 43 (skin) by DFG. Only 9 substances were designated as sensitizers by all these organizations. The variation in the designation of sensitizers is accounted for by the differences in the classification criteria and grouping of chemical substances. JSOH limits the definition of sensitizers to substances that induce allergic reactions in humans and uses only human data. Other organizations utilize not only human evidence but also appropriate animal tests. In addition, EU designates an isocyanate as a sensitizer except those for which there is evidence showing that they do not cause respiratory sensitivity. The worldwide enforcement of the globally harmonized system (GHS) of classification and labeling of chemicals could promote not only the consistent designation of sensitizers among national and international organizations, but also the development of testing guidelines and classification criteria for mixtures.

International comparison of criteria for evaluating sensitization of PRTR-designated chemical substances

In this study, we aim to compare the criteria for sensitizers among national organizations in various countries and international organizations, and to specify whether each Pollutant Release and Transfer Register (PRTR)-designated chemical substance is a sensitizer by each organization. The definition of sensitizing chemicals and the designation of respective sensitizers according to the PRTR law, Japan Society for Occupational Health (JSOH), American Conference of Governmental Industrial Hygienists (ACGIH), European Union (EU), and Deutsche Forschungsgemeinshaft (DFG) were studied. Of the 435 PRTR-designated chemical substances, 15 are listed as sensitizers according to the PRTR law, 16 as sensitizers of the airway and 21 as sensitizers of the skin by JSOH, 12 as sensitizers (no discrimination) by ACGIH, 19 (airway) and 85 (skin) by EU, and 15 (airway) and 43 (skin) by DFG. Only 9 substances were designated as sensitizers by all these organizations. The variation in the designation of sensitizers is accounted for by the differences in the classification criteria and grouping of chemical substances. JSOH limits the definition of sensitizers to substances that induce allergic reactions in humans and uses only human data. Other organizations utilize not only human evidence but also appropriate animal tests. In addition, EU designates an isocyanate as a sensitizer except those for which there is evidence showing that they do not cause respiratory sensitivity. The worldwide enforcement of the globally harmonized system (GHS) of classification and labeling of chemicals could promote not only the consistent designation of sensitizers among national and international organizations, but also the development of testing guidelines and classification criteria for mixtures.