EXTRACTION AND PURIFICATION OF HORMONES LECTURER IN CHARGE

EXTRACTION AND PURIFICATION OF HORMONES LECTURER IN CHARGE OF ENDOCRINOLOGY II BAMIDELE OLUBAYODE

EXTRACTION Extraction is a way to separate a desired substance when it is mixed with other substances. The mixture is brought into contact with a solvent in which the substance of interest is soluble but the other substances present are insoluble. Extractions use two immiscrble phases to separate the susbtance from one phase into the other.

IMPORTANCE OF EXTRACTIONS To improve the quality of life (extraction of medicines from plants by pharmaceutical industry to meet health needs). Many advances can be directly traced to the development of each new separation method. The first step in understanding the chemical reactions of life is to learn what substances are present in samples obtained from biological sources.

PURIFICATION This is the process of isolating and thus removing substances considered to be contaminants. Pure results of a successful process are termed isolate. Extraction and purification are vital components of almost any protein-specific research effort. But the methods used during these processes will depend on the nature of both the protein and the solution. purification

PURIFICATION CONT’D The extraction and purification of proteins (hormones) techniques may include: Chromatography methods exploit the physical properties of either the target protein or the other elements in the solution. Using various paper, gas or liquid chromatography methods, the protein and its surrounding elements are typically dissolved in a mixture and then passed through a stationary phase prior to analysis. Centrifugation, filtration, sonication and other fractionation techniques can be used to break up and remove the cell parts that surround and contain the target protein, like cell membranes and DNA.

PURIFICATION CONT’D During precipitation techniques, ammonium sulfate is added to solution and protein samples are gathered and concentrated as they precipitate. This method is often used for bulk protein extraction. Gel electrophoresis techniques can be used to identify denatured or non-natured proteins by passing samples through an electrically charged gel matrix. The matrix typically separates the protein molecules by p. H or molecular weight.

EXTRACTION AND PURIFICATION OF HORMONES Hormones are needed to be isolated and purified mainly for: their chemical characterisation, which increases our understanding of their biochemistry and makes their synthesis possible; replacement therapy in which contamination with other hormones is undesirable; the development of specific diagnostic assay.

EXTRACTION AND PURIFICATION OF PITUITARY HORMONES Pituitary hormones were first isolated from tissue by fractionation procedures involving precipitation by various salts and solvents. However, the development of chromatographic materials has lessened the problems of fractionation and purification. The techniques usually depend on the particular pituitary hormone or class of pituitary hormone. Isolation poses a difficult hypothalamic hormones are concentration. problem since present in low

EXTRACTION AND PURIFICATION OF HUMAN GROWTH HORMONE A simple method for the extraction and purification of human growth hormone has been developed. This involves initial alkaline the acetone-dried pituitary powder. Then, the bulk of the impurities is precipitated at p. H 4. 8 and the crude hormone subsequently precipitated with alcohol at 25% v/v concentration. extraction of

EXTRACTION AND PURIFICATION OF HUMAN GROWTH HORMONE The crude hormone is then purified by a single Sephadex G-100 gel filtration, 75% of the activity of the crude being obtained as virtually pure monomer and the remaining 25% in the form of two fractions containing two separate polymers. A simple and rapid paper chromatographic assay method has also been developed for following the extraction and purification stages which gives good agreement with the corresponding radioimmunoassays. (Clin Endocrinol Metab 37: 860, 1973)

EXTRACTION AND PURIFICATION OF PROLACTIN During an electrophoretic investigation of proteins from the pituitary of a pregnant woman a band of protein was identified which had the characteristics of prolactin. This led to the first purification of human prolactin from frozen human pituitary glands by Lewis et al. (1971). Initial extraction with saline removed a large proportion of the growth hormone. Prolactin was extracted from the residue at p. H 9 5 in 60 % ethanol and precipitated with 85 % ethanol at p. H 6.

EXTRACTION AND PURIFICATION OF PROLACTIN Chromatography of the redissolved precipitate was carried out on Sephadex G 100 and DEAE cellulose. Hwang et al. (1972) removed insoluble proteins with ammonium acetate at p. H 5 and extracted the residue at p. H 10. 5 Ethanol fractionation was carried out on the supernatant at p. H 8. 5 and the fraction which precipitated between 25% and 85% ethanol was redissolved in alkali and submitted to chromatography on sophadex G-100 at p. H 9. Final purification was achieved by ion exchange chromatography on DEAE AND CM cellulose.

EXTRACTION AND PURIFICATION OF FOLLICLE STIMULATING HORMONE AND LEUTINIZING HORMONE Roos et al. (1963) harvest their glycoprotein-rich fraction from the supernatant retrieved from the ammonium sulphate precipitation of growth hormone. By increasing the concentration of ammonium sulphate the FSH-LH-rich fraction is precipitated. Some TSH, however, precipitates with the growth hormone. Further purification of LH and FSH is achieved by chromatography on DEAE cellulose, gel filtration on Sephadex G 100, adsorption chromatography on hydroxyapatite, and preparative polyacrylamide electrophoresis.

EXTRACTION AND PURIFICATION OF FOLLICLE STIMULATING HORMONE AND LEUTINIZING HORMONE In the method of Jones et al. (1977) the p. H 4. 9 supernatant is found to be a rich source of glycoprotein hormones (Mc. Lean et al. , 1977). Ammonium sulphate (53 g/l) is added to the supernatant and protein precipitating between 50% and 75% ethanol is harvested. Initial chromatography is carried out on Sephadex G 100, which, surprisingly, separates most of the LH activity from FSH and TSH. The LH is further purified on DEAE and CMcellulose. FSH and TSH, after removal of the final traces of LH by DEAE cellulose chromatography, are separated on CM-cellulose. They are then further purified by rechromatography on CM-cellulose.

EXTRACTION AND PURIFICATION OF ADENOCORTICOTROPIC HORMONE Adenocorticotropic hormone (ACTH) is extremely susceptible to proteolytic degradation and therefore care is required during its isolation. The first preparation of purified human ACTH (MW 4500) was achieved by Lerner et al. (1968). They used the ACTH –rich oxycellulose concentrate from the Raben procedure for growth hormone as the starting material. ACTH is eluted off the oxycellulose with 0. 1 M hydrochloric acid and freeze dried.

EXTRACTION AND PURIFICATION OF ADENOCORTICOTROPIC HORMONE Further purification is achieved by ion exchange chromatography on CM-cellulose and gel filtration on sephadex G 25 and G 50. One major and several minor peptides were purified, all possessing similar amino acid compositions and biological activity.

EXTRACTION AND PURIFICATION OF STEROID HORMONE Steroids consist of an essential lipophilic cyclopentanoperhydrophenanthrene nucleus modified on the periphery of the nucleus or on the side chain by the addition of hydrophilic groups. Although steroid are widely distributed in nature and many thousands have been synthesised in the laboratories of parmaceutical and chemical organisations. Quantification of steroid hormones is used to dectect anabolic steroid metabolites in urine sport of sport person. There are several methods of extraction and

EXTRACTION AND PURIFICATION OF STEROID HORMONE 1. Immunoaffinity extraction 2. Chromatography 3. Mass spectrometry 4. Liquid –liquid phase extraction 5. Solid –liquid phase extraction • The steroid to be extracted is gotten either from the blood or urine. • Chromatographic system whereby the steroids are separated in a passage over celite microcolumns is procedure for accurate, sensitive and highly specific measurement of cortisol and progesterone.

REFERENCES AND MATERIALS FOR FURTHER READING Lowry P. J et al. Purification of anterior pituitary and hypothalamic hormones. J. clin. Path. , 30, Supply. ( Ass. Clin. Path. ), 7, 16 -21. Stroud S. W. et al. A simple Method for the Extraction and Purification Of Human Growth Hormone and Its Assay by Paper Chromatography. J. Clin. Endocrinogy Metab. 37: 860, 1973. Makin H. L. J et al. General Methods for the Extraction, Purification, and Measurement of. Steroids by Chromatography and Mass Spectrometry. Chapter 3, Steroid Analysis, 163 DOI 10. 10 / _3, © Springer

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