Evaluation Of Progesterone And Estrogen Biology Essay

Evaluation Of Progesterone And Estrogen Biology EssaySteroid hormones viz. progesterone, o o estrogen were estimated through tender loving care in a concent dimensionn and m dependent manner i.e. 2.5mg/ml, 5.0mg/ml for 30 45 minutes and, 0.2mg/ml, 0.4mg/ml for 30 45 minutes respectively. Progesterone and estrogen were stained with 50% v/v aq. resolve of Conc. H2SO4 and were estimated through tender loving care in a concentration and time dependent manner.Different steroid hormones travel at different rates cod to the differences in their attraction to the stationary phase and because of differences in solubility in the solvent. These Rf values obtained from both the hormones were then comp atomic frame 18d and it was pitch that there was a conceivable difference. Further, a orbit on the interaction of steroid hormones with rich loony toonss and proteins was nethertaken using a spectrophotometer. Steroid hormones viz. progesterone and estrogen were made to interact with measured amounts of alcohol, stearic point and bovine serum white (BSA) and their absorbance were recorded at the excitation wavelength of 410 nm using a spectrophotometer. Progesterone (conc.2mg/ml) and estrogen (conc. 0.5mg/ml) were each tangled with 0.1 ml, 0.2ml and 0.4ml of stearic blistering (conc. 0.5mg/ml) and 5 mg, 10 mg and 15 mg of BSA separately and their absorbance were noted at 410nm. A s electric arc shift in the absorbance was found on the over only interaction of steroids progesterone and estrogen with alcohol, stearic acid and BSA respectively, when excited to 410 nm. Thus an attempt was made to establish a valid spectrophotometric appendage for the study of interaction of steroid hormones with fatty acids and proteins.Key words Progesterone, estrogen, stearic acid, bovine serum egg white, chromatographyRunning title Evaluation of progesterone and estrogen debutSteroids are a class of compounds that have a cyclopentano-perhydro-phenanthrene skeleton and that occur in nature and in synthetic products. The bile acids, androgens, estrogens, corticosteroids, ecdysteroids, sterols and vitamin D are compounds include in the class of steroids. Steroids and their metabolites are substructurevass by thin- bottom chromatography (TLC) in a variety of samples such as biological samples or plants and pharmaceutical formulations. TLC continues to be an important dust for the determination of steroids because of its ad vanguardguardtages. Many samples can be analyzed simultaneously and quickly at relatively low cost, multiple separation techniques and sleuthing procedures can be applied and the detecting limits are often in the low nanogram range and quantitative densitometric methods are accurate. The white-mediated hepatic uptakes of discharge fatty acids 1, bile acids and Rose Bengal 2 have been reported, but the kinetics are not sufficiently clear. It was appriseed by some experimental results in vitro using a smooth-spoken membrane s ystem (hexane source phase/bovine serum/hexane receiving phase) that the rate of uptake of steroids from blood to intracellular space is controlled not only in the free form but likewise in forms detain with serum proteins such as albumin 3. Nowadays, it is commonly accepted that the fractions of steroids which bind with high affinity to plasm proteins such as globulins are less advantageously available to tissues. Although steroids bind with low affinity to man serum albumin (HSA), most of the circulating steroids were outflow to HSA due to the high concentration of albumin 4. Recent studies have shown that, in addition to free steroids, those bound to albumin in plasma may also be available to tissues 5. The stick to of steroids to albumin is affected by temperature, pH, fatty acid and competing ligands. Plasma levels of FFA vary throughout the day 6 and the availability of steroids to tissues is changed by FFA covert to albumin. Serum albumin is the most abundant protein i n the circulatory system, accounting for 60% of the total serum protein . In mammals, albumin is synthesized by the liver and possesses a half-life in circulation of 19 days. Its principal function is to transport fatty acids, a great variety of metabolites and drugs such as anti-coagulants, tranquilizers and general anesthetics. Serum albumin has been one of the most studied proteins for over 40 years because its primary structure is very well cognise for a long time and its tertiary structure was determined a few years ago by X-ray crystallography.It is known that progesterone and estrogen were bound to the same site on serum albumin (SA) 7 and consequently a competition effect exist probably mingled with these two steroid sex-hormones. Then, even if in vivo, the balance between the progesterone and the estrogen rate was controlled by multiple mechanisms and Gleasons observation could be explained by an enhancement of the progesterone displacement to its SA binding site by estr ogen when Mg2+ cation concentration increased 8. SA is the major plasma protein responsible for the reversible binding of a wide range of drugs 9. Extensive studies on different aspects of drug-SA interactions are quench in progress because of the clinical significance of the process, oddly in the case of tightly bound drugs (K 105M1) 10. Numerous analytical techniques are used for protein binding studies and they are continuously being added to, along with extending knowledge about the complex mechanisms involved in the drug-SA binding process 11. It has recognized that an inverse affinity exists between the number of polar groups in a steroid and the strength of interaction with serum albumin. With the aid of a spectrophotometric procedure (12), the validity of which was established by a semblance with the method of equilibrium dialysis, the influence of structural alterations in sex steroids on the interaction with bovine serum albumin (BSA) was investigated. It was found th at introduction of 0x0 or hydroxyl radical groups in various positions weakens this interaction, and that introduction of m ethyl group groups has the opposite effect. Characteristic differences were noted for CY substituents in their influence on the strength of association. Furthermore, the elution profiles of gel-filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the steroid/BSA complexes and that FFA potentiates the binding of these steroids to BSA. In the case of HSA, the binding affinities of progesterone and testosterone were not greatly affected by bound FFA 13,14.This study was undertaken towards chromatographic rating of progesterone and estrogen using the H2SO4 staining method and spectrophotometric analysis of the interaction of both the sex steroids with BSA and stearic acid.Materials and MethodsDrugs and chemicals Progesterone, Estrogen, Silica gel (ACME Chemicals, India)Sulphuric acid reagent, benzene, ethyl acetate, bovine serum albumin, stearic acid, neutral spirits, propylene glycolTLC Equipment All the equipments used for TLC used were from ACME synthetics, Mumbai, India. Acme applicator, TLC glass plates, Beakers, hairlike pipettes for spotting solutions, Rulers, hot plates, Gloves were used for all chemical usage.Thin layer chromatography Thisis a chromatographytechnique used to separate mixtures.Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer ofadsorbentmaterial, usuallysilica gel, aluminium oxide, orcellulose. This layer of adsorbent is known as thestationary phase.After the sample has been applied on the plate, asolventor solvent mixture (known as themobile phase) is drawn up by the plate viacapillary action. Because differentanalytes ascend the TLC plate at different rates, separation is achieved. In steroid analysis, TLC is the method of choice, especially when many simultaneous analyses have to be carried out , hundreds of analyses can be performed in a short time and with small demands on equipment and space. Samples can be analyzed with minimal clean up, and analyzing a sample by the use of multiple separation steps and static post-chromatographic detection procedure is also possible because all sample components are stored on the layer without the chance of loss. The time required in TLC analysis is about 10-60 min. As little as 0.001 g of steroids/spot can be can be detected by TLC. Using a TLC plate with thicker adsorbent layers (0.5-2 min), several grams of substance can be isolated.Sulphuric acid staining methodAfter plate development, the plate is dried and sprayed with a 50% v/v aqueous solution of sulfuric acid in a fume hood. The hood was exposed to extremely corrosive materials and, thus, all fittings were made of glass or acid skanky plastic. After spraying, and while still in the fume hood, the plate is heated to about 80C for about 45 minutes on a hot plate or in an oven. The solutes are partially oxidized leaving behind a charred deposit of b inadequacy carbon that is easy to distinguish. This method will detect most in volatile organic compounds. Sulfuric acid spray reagent was found to be particularly sui sidestep for differentiating natural estrogens from diethylstilbestrol in both a mixture of natural steroids and in biologic preparations under daylight as well as ultraviolet light.Solvent system Benzene and Ethyl acetate in the ratio of 51Spectrophotometric evaluationSpectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a cuvette placed in the spectrophotometer. Spectrophotometry takes advantage of the dual nature of light. Namely, light has1. A particle nature which gives rise to the photoelectric effect2. A wave nature which gives rise to the visible spectrum of light.The spectrophotometer can measure the amount of light (of certain fr equency) transmitted or adsorbed by the solution. This light that has not been absorbed by the solution in the cuvette, will strike the phototube. The photons of light that strike the phototube will be converted into electrical energy. This current that is produced is very small and must be amplified before it can be efficiently detected. The signal is proportional to the amount of light which originally struck the phototube and is thus an accurate measurement of the amount of light which has passed through (been transmitted by) the sample. Different compounds having dissimilar atomic and molecular interactions have trace absorption phenomena and absorption spectra. Concentration of every component may be found from the spectrophotometer measurements and calibration curve made using the samples of known concentration. In this study the spectrophotometer used was Ultrospec III (Pharmacia). All samples were analysed at an absorbance of 410 nm and optical compactness was recorded.SUL FURIC ACID SPRAY CABINETExperimental Protocols1) interaction OF STEROIDS WITH BOVINE blood serum ALBUMIN (BSA)The sample solution consists of progesterone and estrogen mixed each with stearic acid and BSA in three different concentrations and the absorbance is noted at 410 nm.2ml progesterone (2mg/ml) + 1 ml stearic acid (0.5mg/ml) + 5mg BSA2ml progesterone (2mg/ml) + 1 ml stearic acid (0.5mg/ml) + 10mg BSA2ml progesterone (2mg/ml) + 1 ml stearic acid (0.5mg/ml) + 15mg BSA2ml estrogen (0.5mg/ml) + 1 ml stearic acid (0.5mg/ml) + 5mg BSA2ml estrogen (0.5mg/ml) + 1 ml stearic acid (0.5mg/ml) + 10mg BSA2ml estrogen (0.5mg/ml) + 1 ml stearic acid (0.5mg/ml) + 15mg BSA2) INTERACTION OF STEROIDS WITH ALCOHOLSThe sample solution consists of progesterone and estrogen mixed each with ethanol and propylene glycol in the following concentrations and the absorbance of each sample is noted at 410nm.0.5ml progesterone (2mg/ml) + 2 ml ethanol0.5ml progesterone (2mg/ml) + 2 ml propylene glycol0.5m l estrogen (0.5mg/ml) + 2 ml ethanol0.5ml estrogen (0.5mg/ml) + 2 ml propylene glycol3) INTERACTION OF STEROIDS WITH STEARIC ACIDThe sample solution consists of progesterone and estrogen mixed each with ethanol and stearic acid in three different concentrations and the absorbance is noted at 410 nm.0.5ml progesterone (2mg/ml) + 2 ml ethanol + 0.1 ml stearic acid(0.5mg/ml)0.5ml progesterone (2mg/ml) + 2 ml ethanol + 0.2 ml stearic acid(0.5mg/ml)0.5ml progesterone (2mg/ml) + 2 ml ethanol + 0.4 ml stearic acid(0.5mg/ml)0.5ml estrogen (0.5mg/ml) + 2 ml ethanol + 0.1 ml stearic acid(0.5mg/ml)0.5ml estrogen (0.5mg/ml) + 2 ml ethanol + 0.2 ml stearic acid(0.5mg/ml)0.5ml estrogen (0.5mg/ml) + 2 ml ethanol + 0.4 ml stearic acid(0.5mg/ml)Results The results of experiments conducted with chromatographic and spectrophotometric evaluation of progesterone and estrogen are depicted in Table 1 2 and foreshadows 1 2. The results show appreciable difference in Rf values of progesterone and estroge n depending on the elution time. Similarly the addition of BSA to the reaction mixture of progesterone and estrogen showed appreciable difference in optical density as depicted in Figures 1 2. Stearic acid (0.5mg) was also able to shift the absorbance values of progesterone.TABLE 1 Conc. H2SO4 STAINEDPROGESTERONES.NOPROGESTE-RON CONC. (mg/ml)TIME DURATION (min)DIST. MOVED BY SOLUTE (cm)DIST. MOVED BY SOLVENT (cm) retentivity FACTOR (Rf)12.5306.67.20.91622.5459.39.80.94835.0306.77.20.93045.0459.59.80.9690.1ml progesterone (25mg/ml) + 50% v/v aq. solution of Conc. H2SO40.2ml progesterone (25mg/ml) + 50% v/v aq. solution of Conc. H2SO4SOLVENT SYSTEM Benzene and Ethyl acetate in the ratio of 51TABLE 2 Conc. H2SO4 STAINED ESTROGENS.NOESTROGEN CONC. (mg/ml)TIME DURATION (min)DIST. MOVED BY SOLUTE (cm)DIST. MOVED BY SOLVENT (cm)RETENTION FACTOR (Rf)10.2306.67.80.85820.2456.78.20.80430.4306.87.8087140.4456.88.20.8290.2ml estrogen (1mg/ml) + 50% v/v aq. solution of Conc. H2SO40.4ml estrogen (1mg/ml) + 50% v/v aq. solution of Conc. H2SO4SOLVENT SYSTEM Benzene and Ethyl acetate in the ratio of 51Figure 1Figure 2DiscussionSteroids and their metabolites are analyzed by thin-layer chromatography (TLC) in a variety of samples such as biological samples or plants and pharmaceutical formulations. TLC continues to be an important method for the determination of steroids because of its advantages 15.Many samples can be analyzed simultaneously and quickly at relatively low cost, multiple separation techniques and detection procedures can be applied and the detection limits are often in the low nanogram range and quantitative densitometric methods are accurate. The results of this study suggest the novel mechanism using concentrated H2SO4 to identify the eluted progesterone and estrogen on a thin layer chromatography plate. The results of the TLC obtained were satisfactory and although this technique is comparatively octogenarian but can be applied to find out the Rf values of the steroids in a simple manner. The benzene and ethyl acetate crew proved to be an effective mobile phase. There was some difference in the Rf values based on the time duration of TLC but it was marginal. The results are shown in Table 1 and Table 2.To evaluate the effect of fatty acid and protein, stearic acid and BSA, we conducted studies on the absorbance using the spectrophotometer. Our results suggest that addition of BSA to the reaction mixture caused significant change in optical density in both steroids i.e progesterone and estrogen (Fig.1 2). The cellular effects evoke by estrogen and progesterone 16 covalently conjugated to membrane impermeable BSA have been attributed to non-genomic actions mediated by membrane associated hormone receptors. It has previously been established that for physiological concentrations of progesterone and albumin in the circulating blood, approximately 99% of the steroid is bound to albumin. If more than one molecule of the protein participa ted in the binding of one molecule of progesterone, dimerization or complexing of more than two albumin molecules would result and should be demonstrable by the viscosity and by the sedimentation behavior of the system. Determination of viscosity and sedimentation constants under the conditions outlined in the experimental part failed to reveal any differences between the albumin solutions with and without progesterone. It was concluded from these findings that only one molecule of human serum albumin complexes with one molecule of progesterone. It would seem reasonable to assume that a similar molar relationship exists in the interaction of other steroid molecules with serum albumin. The steric relationship between the two components in the complex was investigated by measuring the strength of interaction between albumin and epimeric steroids. Such a comparison should permit conclusions as to the spatial arrangement of the interacting molecules. Thus there are multiple reasons for the change in absorbance with addition of BSA to the reaction mixture 17.Furthermore, fatty acid tends to minimize or eliminate the well-known differences in affinity between bovine and human albumin for interactions with these two steroids 18. The values for binding affinity in the interaction of testosterone with these batches of human serum albumin are significantly higher than those previously published by some authors and the value for progesterone-bovine albumin interaction is not in treaty with the polarity rule. Studies of these same interactions by ultraviolet difference spectroscopy give further evidence of the augmentation in binding but, in the case of defatted bovine albumin only, the aromatic difference troughs are indicative of tyrosine perturbation whereas refatted bovine albumin, defatted and refatted human albumin manifest tryptophan perturbation. Quantitative correlation of perturbation with level of bound steroid suggests that fatty acid alters the ratio (possib ly hydrogen-bonded to non hydrogen-bonded) of two forms of bound steroid. This concept gains in validity when one considers the nature of the binding forces between steroid and protein. For lack of specific knowledge as to these forces, the binding between neutral steroids and proteins may best be interpreted as mediated by hydrogen bonds and van der Waals forces 19. Common to both of these binding mechanisms is the low energy of the bond, of the order of 5 to 10 kcal per mole, which explains the ready dissociation of the complexes. Furthermore, they are highly dependent upon the distance the van der Waals forces for spherical atoms are inversely proportional to the seventh power of the distance between the atomic centers. For efficient interaction, therefore, a close fit of the steroid and protein surfaces would be required.Whether the presence of stearic acid alone in any way alters chemical configuration and affects the absorbance by spectrophotometer can only be ascertained by s pecific chemical tests The change in stearic acid mediated absorbance with progesterone can be attributed to interplay of chemical forces between the steroid and the fatty acid. Studies with lauric, myristic, and palmitic acid showed that the decrease of binding affinity for progesterone was proportional to the amount of fatty acid added to albumin, and to its chain length. These results confirm and extend other findings of inhibition of progesterone binding to human albumin by saturated fatty acids.In conclusion this study describes a novel H2SO4 staining technique to visualize the sex steroids, progesterone and estrogen and a change in spectrophotometric absorbance of progesterone and estrogen by BSA and stearic acid.Legend for Figures and TablesTable 1 Table 1 describes the thin layer chromatographic evaluation of progesterone (2.5 5 mg) conducted using the concentrated H2SO4 staining technique and eluted for a duration of 30 and 45 minutes. The last column of the table shows th e Rf values obtained.Table 2 Table 2 describes the thin layer chromatographic evaluation of estrogen (0.2 0.4 mg) conducted using the concentrated H2SO4 staining technique and eluted for a duration of 30 and 45 minutes. The last column of the table shows the Rf values obtained.Figure 1 Figure 2 describes the optical density values at absorbance of 410 nm obtained with estrogen (0.25 1 mg), ethanol (2ml), bovine serum albumin (10mg) and stearic acid (0.5 mg) alone or in combination.Figure 2 This figure describes the optical density values at absorbance of 410 nm obtained with progesterone (1 4 mg), ethanol (2ml), bovine serum albumin (10mg) and stearic acid (0.5 mg) alone or in combination.