RESUMO
An improved straight-phase HPLC method for the separation and quantification of lipid classes is described. Two binary gradient solvent systems were used, one for polar and one for neutral lipids, and detection was performed with a light-scattering detector. The developed HPLC methods were highly reproducible and allowed base-line separation of all investigated polar lipid classes (phosphatidic acid, diphosphatidylglycerol. phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine) and neutral lipid classes (triacylglycerol, free fatty acid, diacylglycerol, cholesterol and monoacylglycerol) except of cholesterol ester and wax ester. Application of the chromatographic systems demonstrated that the methods are suitable for quantitative analysis of the major lipid classes present in lipid extracts from livers and eggs of Atlantic salmon (Salmo salar).
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/análise , Fígado/química , Óvulo/química , Salmão/fisiologia , Animais , Calibragem , Feminino , Modelos Lineares , Lipídeos/classificação , Reprodutibilidade dos TestesRESUMO
The induction of metallothionein and vitellogenin synthesis in rainbow trout liver was studied after injection of oestradiol-17 beta alone or in combination with cadmium or zinc. Intraperitoneal injection of oestradiol-17 beta increased the liver somatic index, with subsequent induction of vitellogenin synthesis. Oestradiol-17 beta did not induce metallothionein synthesis. Injection of cadmium induced the synthesis of metallothionein mRNA and metallothionein. Injection of oestradiol-17 beta in combination with cadmium resulted in inhibition of transcription and translation of both vitellogenin and metallothionein. Chromatography of liver cytosols revealed that cadmium, when co-injected with oestradiol-17 beta, did not bind to metallothionein but would initially bind to high-molecular-mass (HMr) cytosolic proteins. In fish injected with cadmium in combination with oestradiol-17 beta, cadmium was gradually redistributed from HMr proteins to metallothionein. This resulted in induction of metallothionein synthesis and in binding of most of the cadmium to metallothionein. Induction of vitellogenin mRNA was observed 15 days after injection, as cadmium was being redistributed to newly synthesized metallothionein. These findings indicate that cadmium inhibits the transcription of vitellogenin. The binding of cadmium to these non-metallothionein proteins represses the induction of metallothionein and results in increased toxicity of the metal. Preinduction of metallothionein by zinc injections resulted in decreased cadmium sensitivity of the fish and a decrease in the repression of vitellogenin mRNA. Furthermore, a role for metallothionein in the detoxification of cadmium is indicated by the induction of vitellogenin synthesis that occurs once metallothionein has begun sequestering cadmium.
Assuntos
Cádmio/farmacologia , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Metalotioneína/biossíntese , Oncorhynchus mykiss/metabolismo , Vitelogeninas/biossíntese , Animais , Ligação Competitiva , Cádmio/metabolismo , Cádmio/toxicidade , Cobre/análise , Citosol/metabolismo , Interações Medicamentosas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metalotioneína/genética , Tamanho do Órgão/efeitos dos fármacos , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Vitelogeninas/genética , Zinco/farmacologiaRESUMO
In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. Immunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 microns. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17 beta increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 microns) present just inside the oolemma, when the oocytes reached a diameter of 600 microns. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol-17 beta levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 microns had an immunoreactive vitelline envelope with a thickness of about 3 microns. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17 beta observed in females are of physiological significance.
Assuntos
Oócitos/fisiologia , Ovulação/fisiologia , Vitelogeninas/metabolismo , Animais , Transporte Biológico Ativo , Western Blotting , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Estradiol/sangue , Feminino , Imunofluorescência , Soros Imunes , Imuno-Histoquímica , Oncorhynchus mykiss , Oócitos/citologia , Estações do Ano , Vitelogeninas/análiseRESUMO
The use of high-performance anion-exchange chromatography on a Mono Q column for isolation of a glycolipophosphoprotein, vitellogenin, from turbot plasma has been evaluated. The method is an effective, rapid one-step procedure, which gives a pure preparation of vitellogenin as assessed by electrophoresis, [32P]orthophosphate incorporation and amino acid composition.
