The Ethics Of Enron And Worldcom - 1821 Words

The desire for money, power, and praise can lead some top executives to participate in unethical behavior to satisfy these needs. Some businesses simply fabricate their financials while others invent fake companies to inflate their asset’s value and profitability. Both situations are attempts to increase their earnings as much as possible for top executives own interests. The two most well-known and fairly recent instances of major accounting scandals include Enron and WorldCom. How did Enron, once one of the five largest audit and accountancy partnerships in the world, and WorldCom, once the United States’ second largest long distance telephone company, both end in bankruptcy just one year after the other? By exploring the history, scandal, and aftermath of Enron and WorldCom, comparing the two business’ malpractice, understanding the ethical issues involved, considering the historical context of the scandal which includes the Sarbanes-Oxley Act of 2002, and my opinion regarding the effect, it will become clear how these once successful businesses ended with criminal charges. Enron Corporation was an American energy and service company based in Houston, Texas. In 1985, Kenneth Lay merged two natural gas pipeline companies, InterNorth and Houston Natural Gas to form Enron. This new company eventually had 36,000 miles of pipe stretching across the United States and into Canada, making it the second largest pipeline network in the United States. By 1992, Enron became theShow MoreRelatedEnron and Worldcom Case Study1225 Words   |  5 PagesEnron and WorldCom Case Study This report is based on the demise of Enron Corporation and WorldCom. Both the firms are demised due to the ethical lapses. These ethical lapses come into existence when managements of the firm, uses unethical practices to accomplish the goals of the firm. Maintaining financial and accounting standards in the business practices are necessary. The profession of accounting has become a mockery due to the accounting scandals that took place all over the world in theRead MoreEssay on The Consequences of Bad Business Decisions1304 Words   |  6 Pagespractices. Once respected businesses like Enron, WorldCom, and Arthur Anderson have been found deceiving there customers, stockholders, and employees. C.E.O.s try to achieve the American dream and pursue capitalism to its fullest potential. In doing so, business leaders have lost their values and ethics, and make bad business decisions. The downfalls of a company are the consequence of C.E.O.s bad decisions. According to Marjaana Kopperi business ethics, can simply be defined in terms of socialRead MoreThe Sarbanes Oxley Act Of 20021133 Words   |  5 Pagesinto customers, the Securities and Exchange Committee proposed and implemented a new law. This policy was put in place to regulate the accounting practices and to make them more honest. Titled the Sarbanes-Oxley Act of 2002 (enacted just after the WorldCom scandal), basically set rules and regulations in place that included (but not limited too): a Public Company Accounting Oversight Board to provide independent justification and to oversee the accounting department; Auditor Independence to limit theRead MoreEnron Questionable Transactions Essay765 Words   |  4 PagesEnron Questionable Transactions Question 1 The question which segment of its operations got Enron into difficulties is simple to answer, everything. Almost every all segments of their operation were improper. First of all, they practice unethical and dishonest practices which victimized workers, consumers, taxpayers and stockholders. Enron created partnerships within their own organization which led to them creating new financial instruments, called SPE’s (special purpose entities) which wasRead MoreThe Sarbanes Oxley Act Of 20021015 Words   |  5 PagesThe Sarbanes-Oxley Act of 2002, also known as the SOX Act, is enacted on July 30, 2002 by Congress as a result of some major accounting frauds such as Enron and WorldCom. The main objective of this act is to recover the investors’ trust in the stock market, and to prevent and detect corporate accounting fraud. I will discuss the background of Sarbanes-Oxley Act, and why it became necessary in the first section of this paper. The second section will be the actà ¢â‚¬â„¢s regulations for the management, externalRead MoreFraud : The Perfect Fraud Storm1420 Words   |  6 PagesFinancial Statement Fraud Option #2 The perfect fraud storm occurred between the years 2000 and 2002 involving two of the largest energy and telecom corporations in the United States: Enron and WorldCom. It was determined that both organizations fraudulently overstated assets, created assets from expenses or overstated revenues, costing investors billions of dollars and resulting in both organizations declaring bankruptcy (Albrecht, Albrecht, Albrecht Zimbelman, 2012). Nine factors contributedRead MoreA Case Of Accounting Fraud1555 Words   |  7 PagesAnother major case of accounting fraud driven by the desire to build and protect one’s personal financial condition is the WorldCom debacle. Bernie Ebbers had to show continually growing net worth in order to avoid margin calls on his own WorldCom stock that he had pledged to secure loans. When WorldCom, the telecommunications giant, failed and was put into bankruptcy, the U.S. witnessed the largest accounting frauds in history. Former CEO, Bernie Ebbers, was convicted of orchestrating this accountingRead MoreThe Sarbanes Oxley ( Sox ) Act Of 20021617 Words   |  7 Pagesbrief historical summary of SOX will be presented, including the events leading up to its passage. The key ethical components of SOX will be identified and explained. The social responsibility implications of the mandatory publication of corporate ethics will be assessed. One of the main criticisms of SOX has been its implementation costs, and this specific criticism will be addressed in regards to smaller organizations. Finally, potential improvements to the SOX legislation will be explored, basedRead MoreThe Enron and Worldcom Scandals875 Words   |  4 PagesE. Boos – Week 2 – Assignment February 17, 2013 The Enron and WoldCom Scandals ENRON 1. The segment of Enron’s operations that got them into difficulties had several parts. They published misleading financial reports. They could not meet their bridge financing commitment with Barclay Bank because outside investors were not found. Because of this, they restated activities of JEDI and Chewco SPEs so they could be retroactively consolidated into Enron’s accounts. The SPEsRead MoreThe, Greed, And Hubris Of Action1236 Words   |  5 Pagescorrupts absolutely†. There were three specific corporate scandals that led to failed confidence in the financial sector and the subsequent legislation known as Sarbanes-Oxley Act of 2002 which attempted to address this malfeasance: Enron, WorldCom, and Arthur Andersen. Enron Notably, the most widely recognized scandal of all time because it led to a systemic lack of trust in corporations and the financial markets in general. Enron’s fraud was twofold; it included complex financial maneuvering through

Titration Journal Free Essays

E r J. Biochem. 40,177-185 (1973) u. We will write a custom essay sample on Titration Journal or any similar topic only for you Order Now Intracellular Titration of Cyclic AMP Bound to Receptor Proteins and Correlation with Cyclic-AMP Levels in the Surviving Rat Diaphragm Lien DO KHAC,Simone HARBON Hubert J. CLAUSER and lnstitut de Biochimie, Universit6 de Paris-Sud, Orsay (Received April 9/July 17, 1973) Extracts prepared from rat diaphragms incubated with or without theophylline and/or epinephrine have been tested for their total cyclic AMP content and for their ability to bind exogenously added cyclic [â€Å"]AMP. Less cyclic [3H]AMP can be bound inthe extracts after theophylline and/or epinephrine treatment indicating that the rise in cyclic AMP level was accompanied by a n increase in the quantity of cyclic AMP bound intracellularly to the cyclic AMP-dependent protein kinases. Maximum cyclic AMP binding capacities, as measured by total cyclic AMP exchanges, were however identical in all cases. Accurate estimations of intracellular binding of cyclic AMP have been correlated with the level of cyclic AMP in the tissue : the reaction seems to obey simple saturation kinetics, a n apparent intracellular K d for cyclic AMP has been evaluated as 330 nM. The findings are consistent either with a real difference in the intracellular binding constant as compared to that measured in vitro (28 nM) or with the fact that the cyclic nucleotide in the cell may not all be available for the kinase protein receptors. They also suggest that the method described may prove useful for studying any possible intracellular control beyond the step of cyclic AMP synthesis. Regulation of cellular metabolism by adenosine 3†² :5†²-monophosphate (cyclic AMP) [I], its mediation through complex protein kinases [2,3] and the mechanism of the activation of these enzymes [4–61 have been well documented within the past years in the eukaryotic cell. Activation has been demonstrated to occur according to Equation (1) through a n interaction of cyclic AMP with the regulatory subunit (R) of the enzyme, leading to a dissociation of this subunit from the catalytic subunit (C) which is thus activated. RC cyclic AMP + R cyclic AMP C . (1) + + However completely satisfactory correlations between the levels of intracellular cyclic AMP and its ultimate metabolic effects have been in many cases difficult to obtain. Striking examples for this situation are to be found in the results of Craig et al. [7] in rat diaphragm, of Stull and Mayer [8] in rabbit skeletal muscle concerning the regulation of phosphorylase activation, of Schaeffer et al. [9] and Miller et al. [lo] concerning regulation of glycogen metabolism in adrenalectomized rats, and of Harbon and Clauser [Ill This work is dedicated to Professor E. Lederer for his 65 th anniversary. Abbreviations. Cyclic AMP; adenosine 3†²: 5†²-monophosphate. in the rat uterus stimulated by prostaglandin El or E,. I n all these cases, cyclic AMP levels may be elevated without eliciting the expected metabolic responses. Two hypotheses have been formulated to explain these obvious discrepancies, either a decrease in the activation of the enzymes mediating cyclic AMP action within the cell, or a compartmentalization of the intracellular nucleotide. Hence it seems necessary to measure directly the degree to which the first step of the activation sequence (Equation 1)reflects the apparent intracellular cyclic AMP concentrations. This might be achieved by establishing in intact cells or tissues, correlations between the levels of intracellular cyclic AMP under welldefined physiological conditions, the extent to which it is bound to the specific receptor protein and the extent to which the complex protein kinases are in the active state. Satisfactory correlations between cyclic AMP levels and protein kinase activation have been recently established in various tissues by Corbin et al. [I21 and Soderling et al. [13]. The present work was to investigate if correlations could also be obtained between intracellular cyclic AMP levels and the amounts of intracellular cyclic AMP bound to receptor protein (R cyclic AMP) in the surviving rat diaphragm incubated with or without theophylline and epinephrine. The results reported demonstrate that – E r J. Biochem. 40 (1973) u. 178 Intracellular Titration of Cyclic AMP-Receptor Protein Binding precise titrations of endogenous cyclic AMP bound versus cyclic AMP present in the intact tissue may be obtained. An apparent Kd value for the intracehlar cyclic AMP binding is observed which differs widely from the K d of the same binding established in vitro [14-161. This method may prove to be useful for studying the modification of cyclic AMP binding under conditions where the formation and breakdown of cyclic AMP does not seem to be affected. A preliminary report of these results has been presented [17]. MATERIALS AND METHODS Cylic AMP was obtained from P L Biochemicals Inc. , theophylline and Tris from Merck (Darmstadt), Na,ATP 4 H,O, L-epinephrine bitartrate from Calbiochem. Cellulose ester membrane filters (HA 0. 45 pm, 24 mm) were purchased from Millipore Corp. All reagents used were products of Prolabo (reagent grade). Cyclic [3H]AMP was a product of New England Nuclear Inc. , specific activity 24 Ci/ mmol. Animals were Wistar rats weighing about 200 to 300 g and fasted 24 h before the experiments. Tissue homogenizations were performed with an Ultra Turrax homogenizer. – The reaction mixture for the binding assay contained in a final volume of 250 p1, 20 mM TrisHC1 buffer pH 7. 5, 10 mM MgCI,, 6. 7 mM theophylline and cyclic [3H]AMP a t various concentrations as indicated. The reaction was initiated by the addition of a n aliquot of diaphragm extracts equivalent to 70- 150 pg protein. Method B. I n this case, cyclic [3H]AMPwas added to the homogenizing medium a t saturating concentrations up to 0. 2 p M a t 0 â€Å"C, centrifugation was carried out immediately and cyclic [3H]AMP bound measured directly on the extract. Cyclic [3H]AMP bound to the proteins, under either condition, was determined after different incubation times at 0 â€Å"C: the reaction mixtures were then diluted to 3 m l with cold buffer (20mM TrisHC1, 10mM MgCl,, pH 7. 5) and passed through cellulose acetate Millipore filters (0. 45 pm). The filters were washed with 25ml of the same buffer, dried and counted in i 0 ml scintillation fluid, in a Packard Tri-Carb liquid scintillation spectrometer. Results were expressed as pmol cyclic AMP bound/mg protein ; the concentration of endogenous unlabelled cyclic AMP has been always taken into account for the estimation of the specific activity of cyclic [3H]AMP present in the incubation medium. Incubation Procedures The animals were killed by decapitation. The diaphragms were rapidly removed, freed from connective tissue, cut to small pieces, pooled and divided into equal parts. 200-250 mg tissue were preincubated in 2. ml Krebs-Ringer-bicarbonate buffer pH 7. 4, gas phase (95O/, O,, 5O//, CO,) for 30 min a t 37 â€Å"C, in the absence or presence of 10 mM theophylline. Incubations were then performed in the absence or presence of epinephrine (5 pM) for varying periods of time. Extraction of the Tissue Standard Binding Assays for Cyclic A M P Two methods have been deviced to extract the tissue and estimate the binding of exogenous cyclic [3H]AMP to the extracted proteins, both slightly modified from the method defined by Walton and Garren [15]. Method A . The tissue was homogenized a t 0 â€Å"C in 3 ml of one of the following solutions: 20 mM TrisHCl buffer pH 7. or 20 mM sodium acetate pH 7. 5 or 4 mM EDTA pH 6. 0. Theophylline (10 mM) was always present in the various homogenizing media in order to minimize any degradation of cyclio AMP by phosphodiesterase present in diaphragm extracts. A first centrifugation was carried out for 5 min a t 3000 x g , followed by a second one a t 50000 x g for 30min. The supernatants will be referred to as Tris extract, acetate extract and EDTA extract. Assay for Cyclic-AMP Levels For cyclic AMP assay, the tissue was homogenized in 3 ml cold 7 trichloroacetic acid and centrifuged for 30 min a t 50000 xg. After addition of 0. 1 ml N HC1, the supernatants were extracted 7-8 times with twice their volume of cold ether and evaporated to dryness. Total levels of cyclic AMP in the tissue trichloroacetic acid extract were determined according to Gilman using a protein b a s e and the heatstable inhibitor prepared from rabbit skeletal muscle [161. I n some instances, cyclic AMP content was also evaluated in the Tris and acetate extracts. Proteins were precipitated by trichloroacetic acid and extracts processed as described above. Proteins in the extracts were determined according to Lowry et al. 18] using bovine serum albumin as a standard. RESULTS AXD DISCUSSION Total Cyclic-AMP Levels in Rat Diaphragm. Effects of Epinephrine and Theophylline In order to study the cyclic AMP binding capacity of rat diaphragm proteins and its possible rnodification under the influence of epinephrine, it seemed necessary to test the first effect of the catecholamine, viz. the rise in the tissue cyclic AMP lev el under our experimental conditions. Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 1. Total cyclic A H P levels in trichloroacetic acid extracts of rat diaphragm. Effect of epinephrine and theophylline R a t diaphragms (200-250 mg) were preincubated for 30 rnin a t 37 â€Å"C in 2. 5 ml Krebs-Ringer-bicarbonate buffer (0, 95 °/0-C0, 50/0) in the absence or presence of 10mM theophylline, Incubation was then performed for 5 rnin with or without 5 pM epinephrine. The tissue was then homogenized in 7O/, trichloroacetic acid for cyclic AMP assay as described under Methods. Levels of cyclic AMP were expressed as pmol cyclic AMP/100mg wet tissue and as pmol cyclic AMP/mg soluble protein (as estimated by the Lowry procedure in the Tris extract. Values are means f S. E. M. of 5 different experiments Incubation condit,ions Total cyclic AMP TheoDhvlline EDineDhrine pmo1/100 mg pmol/mg wet tissue soluble protein 41 f 8. 0 20. 5 f 4. 7 104 1. 1 52 0. 47 93 f 4. 5 46 2 350 f 21 170 f 10. 7 179 Table 3. Distribution of cyclic [3H]AMP-bindingfractions i n different hom. ogenutes from rat diaplwagms incubated with or without epinephrine Preincubation and incubation conditions as described in Table 2. Tissues were homogenized in 3 ml 20mM TrisHCI, p H 7. 5, 4 mM EDTA or 20 mM sodium acetate pH 7. and centrifuged for 5 rnin at 3000 x g, the supernatants were centrifuged once more at 500OOxg for 30 min yielding extract 1 and pellet 1. The sediment of the first centrifugation was resuspended in 1. 5 ml of the corresponding buffer and centrifuged at 500OOxg for 30 min giving extract 2 and pellet 2. Binding activity for cyclic rSH]AMP was measured in each fraction as described in the text under method A and was expressed as pmol cyclic AMP bound/l00 mg wet tissue Fraction Cyclic AMP bound in EDTA Acetate Tris extract extract, extract, 5 yM noepinoepino epinephrine nephrine nephrine – + + + + – :Lhrine pmo1/100 mg wet tissue Extract 1 Extract 2 Pellet 1 Pellet 2 15. 70 1. 47 0. 76 1. 49 14. 90 1. 54 0. 83 1. 50 15. 30 1. 35 0. 80 1. 10 9. 40 0. 80 0. 44 0. 39 Table 2. Cyclic A M P levels in different extracts obtained from epinephrine-treated and untreated rat diaphragms Preincubation with 10 mM theophylline and incubation conditions in the absence or presence of 5 pM epinephrine as in Table 1. Diaphragms were homogenized in three different solutions: cold 7O/, trichloroacetic acid, Tris-HC1 pH 7. 5 or acetate p H 7. 5 as described under methods. Centrifugation was carried out for 30 rnin at 50000 x g. Soluble Tris extract, acetate extract and their corresponding sediments were deproteinized by 7 o/o trichloroacetic acid before cyclic AMP assay Incubation with epinephrine None 5wM Total cyclic AMP in Trichloroacetic 20 mM acetate acid extract pellet 57 280 – 20 mM Tris extract pellet 48 218 9. 5 26 extract pellet 45 242 pmo1/100 mg wet tissue – 8. 5 8. 3 As shown in Table 1, epinephrine (5 pM) in the absence of theophylline increases (by a factor of 2. 5) the total cyclic AMP content of rat diaphragm extracted by trichloroacetic acid. Theophylline alone (10 mM) had a stimulating effect, double; when both compounds were used together, the rise in cyclic AMP levels was 8- t o 9-fold, reaching 350pmol cyclic AMP/100 mg wet tissue. When cyclic AMP was assayed in either acetate or Tris extracts after deproteinization with trichloroacetic acid the values obtained were identical t o those found when the diaphragms were directly extracted with trichloroacetic acid ; hence almost none of the cyclic nucleotide in these extracts was associatcd with membrane-bound fractions (Table 2). Eur. J. Biochem. 0 (1973) Location of Cyclic AMP-Binding Fractions Table 3 shows the distribution of cyclic AMP binding activity in various fractions of three rat diaphragm homogenates measured by method A : in all cases more than goo/, of this activity was recovered in the 50000 x g supernatant, almost no cyclic AMP binding occurred in the pellets. Preincubation of the diaphragm with epinephrine did not modify the percentage distribution of the radioactive nucleotide between the supernatants and the pellets, hence subsequent experiments have been performed on the soluble extracts. On the other hand, in the case of epinephrine-treated diaphragms, less exogenous labelled cyclic AMP (about 50-60 °/0) was bound to the various fractions, indicating a decrease in the binding capacity of the extract as compared to the untreated diaphragm. Dilution by endogenous cyclic AMP cannot explain the effect of epinephrine, since allowance was made for this parameter (see Methods) ; the phenomenon was consistently reproducible and will be further substantiated and discussed below. The binding capacities of the various extracts for cyclic E3H]AMP have also been verified in the absence of any free endogenous cyclic AMP after removal of the latter by filtration through Sephadex G 50 (1x 37 cm) columns, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7. 5 a t 4 â€Å"C. I n these experiments, the detail of which w l not be reported in i l the present manuscript, the effect of epinephrine was still observed, when binding was measured on the main protein peak emerging with the void volume of the columns. When the corrections outlined in the 180 Intracellular Titration of Cyclic AMP-Receptor Protein Binding Z A 0. 51 / 0 20 40 60 Time ( m i n ) l / f r e e cyclic AMP (nM-‘) l / f r e e cyclic A M P (nM-‘) Fig. 1. The time wurse and cyclic-AMP-concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method A ) . (A) Diaphragms were incubated for 30 min in the presence of 10 mM theophylline and extracted with Tris HCI buffer (meth od A). Cyclic AMP binding was estimated in the presence of various concentrations of cyclic E3H]AMP: 20nM ( 0 – 0 ) ; 60nM ( – ) 0 0 ; SO (A-A); 100 nM ( –) #-. , a t 0 â€Å"C. The react,ion mixtures contained in a final volume of 2. 5 ml, 20 mM Tris-HC1 buffer, pH 7. , 10 mM MgCI,, 6. 5 mM theophylline. The reaction was initiated by the addition of 930 pg protein. At the indicated times, aliquots were pipetted, immediately diluted with cold 30 mM Tris-HC1buffer pH 7. 5,lO mM MgCl, and passed on the Millipore filters. Filters were washed with the same buffer, dried and counted. Binding activity is expressed as pmol cyclic AMP bound/mg protein. (B) Data obtained from similar experiments where binding for cyclic AMP was performed a t 0 â€Å"C, for 1 h, in the presence of cyclic [aHIAMP ranging from 12 nM to 110 I. Double-reciprocal plot, according to Klotz [25] Fig. 2. Cyclic-AMP-Concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method B ) . Binding assays were carried out as described under method B. Various concentrations of cyclic [3H]AMP ranging from 12nM to 200 nM were added directly to the homogenizing medium for preparing extracts from epinephrine treated (A-A) and untreated (0-0) rat diaphragms. Aliquot,s of the extracts were filtered through Millipore filters, dried and counted. Double-reciprocal plot, according to Klotz [25] present paper were applied to these figures, the results were essentially identical to those obtained with the unfiltered extracts. Specificity. Kinetics and Concentration Dependence of Exogenous Cyclic-AMP Binding in the Extracts Specificity of cyclic AMP binding has been assessed by dilution experiments of cyclic [3H]AMP (100 nM) with unlabelled nucleotides (adenine, AMP, ATP, cyclic AMP) a t molar concentrations equalling up t o 100 times cyclic [3H]AMP concentrations. I n no case, except with unlabelled cyclic AMP, the amount of radioactive material bound to proteins by either method A or B was significantly reduced (the details of these experiments are not reported). When various concentrations of cyclic [3H]AMP were added to diaphragm extracts (after homogenization and centrifugation) and the binding reaction (method A) carried out for different incubation times at 0 â€Å"C (Fig. I), it appears that saturation was obtained at a concentration of 80 nM for the cyclic nucleotide which essentially coincides with previously published data [14-161 and that binding equilibrium was reached a t p H 7. 5 and 0 â€Å"C after less than 60 min incubation. It has also been verified that with the protein concentration used (70-150 pg in 250 pl) binding of cyclic AMP was directly proportional to the amount of added proteins. From a reciprocal plot of cyclic AMP binding versus cyclic AMP concentration (inset of Fig. I), an apparent Kd of 33 nM can be calculated. When similar experiments were performed by adding various concentrations of cyclic [3H]AMP into the homogenizing medium (method B) and using diaphragms which have been incubated in the presence and absence of epinephrine, the double-reciprocal plots of Fig. 2 were obtained. The apparent Kd values calculated with this method (45 nM) are in the same range as with method A. I n addition this figure shows that epinephrine treatment of the diaphragms does not modify this Kd but decreases the amount of exogenous cyclic AMP which can be bound to the extract proteins. By comparing exogenous cyclic AMP binding values obtained with methods A and B, it appears (Table 4) that when cyclic [3H]AMPwas added to the Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 4. Comparison of exogenous binding of cyclic [SII]AMP to diaphragm extracts by method A or method B. Rat diaphragms were incubated with theophylline in the absence or presancc of 5 p M epinephrine. Extracts in Tris-HC1 were prepared as described under method A for subsequent binding of cyclic [3H]AMP (100 nM), 1 h, a t 0 â€Å"C. A second series of extracts were prepared in the same way but in the prescnce of 100 nM cyclic [3H]ABIP in the homogenizing medium (method R); binding of cyclic [3H]AMP was measured in a n aliquot immediately after centrifugation at 0 â€Å"C (about 1 h after the end of incubation). Values are expressed as pmol bound cyclic AMP/mg protein. Numerals within brackets indicate number of experiments Method Cvclic A P bound with M 5 pM epinephrine no epinephrine pmol/mg protein 4 f 0. 22 (9) 4. 80 5 0. 2 (5) 181 6 t e . ;? 4 Q Q E A B 2 f 0. 13 (9) 3 f 0. 19 (5) 0 I I I 30 60 90 * Time (rnin) homogenization medium (extract B) higher binding values were obtained both with epinephrine-treated and untreated diaphragms, than with method A. This demonstrates that some additional binding of endogenous cyclic AMP occurred during the homogenization and fractionati on procedures, which tends to decrease the amount of unoccupied binding sites available for exogenous cyclic [3H]AMP. Hence method B has been currently used to measure exogenous cyclic AMP binding, since the values obtained with this method seem to reflect intracellular conditions more accurately. Fig. 3. Time course of cyclic [3H]AMP binding in extracts from rat diaphragms incubated in the absence or presence of theophylline orland epinephrine. Half rat diaphragms were preincubated in the absence (m, A ) or in the presence ( 0 , 0 ) of 10 m31 theophylline for 30 min at 37 â€Å"C. Epinephrine (5 pM) was added ( A , 0 )and incubation continued for 5min. Tissue was homogenized in 1. 5 ml Tris-HC1 buffer containing 200 nnf cyclic [3H]AMP and centrifuged at 5000xg for 10 min at 0 â€Å"C. Binding of cyclic [3H]AMP was measured in aliquots of the supernatant at the times indicated, through Millipore filtration, t = 0 corresponds to the onset of the extraction. Results are expressed as pmol cyclic AMP bound/ mg protein (without correction for cyclic AMP exchange) Effect of Theophylline and Epinephrine Treatment on the Binding of Exogenous Cyclic [3H]AMP by Diaphragm Extracts Fig. 3 shows the results of a typical experiment in which diaphragms have been incubated in the absence or presence of theophylline and epinephrine. Homogenization has been performed according to method B, the centrifugation time of the homogenate kept to a inimum (10 min), and the binding capacity for cyclic [3H]AMP determined a t different times. As may have been expected, this cyclic [3H]AMP binding (which measures the residual binding capacities of the extracts) was, in the course of the whole titration period, inversely related t o the amount of endogenous cyclic AMP present in the relevant ext racts (see Table 1). Hence the agents which increase the intracellular cyclic AMP level appear to decrease the amount of binding sites available for exogenous cyclic [3H]AMP, probably through an increase of endogenous cyclic AMP binding to the receptors. I n order to titrate endogenous binding of cyclic AMP accurately, experiments were designed to estiEm. J. Biochem. 40 (1973) mate the total binding capacities of the extracts through complete exchange of endogenously bound cyclic AMP with cyclic [3H]AMP, and also to estimate the actual amount of exchange occurring in the extracts between endogenous bound unlabelled cyclic AMP and exogenous cyclic [3H]AMP during the titration period. A precise knowledge of these two parameters is required for the determination of the binding sites occupied by endogenous cyclic AMP at the moment where the tissues are homogenized. Cyclic-AM P Exchange and Determination of Maximal Binding Capacities Total cyclic AMP exchange has been measured under the conditions defined by Wilchek et al. [19] for parotid gland and skeletal muscle : extracts from both treated and untreated diaphragms were f i s t incubated at 0 â€Å"C with cyclic [3H]AMP (100 nM) under binding conditions of method A and then allowed t o exchange with 1 pM unlabelled cyclic AMP at 20 â€Å"C in the presence of 100p. M ATP and 10mM MgCl,. Fig. 4 shows that almost complete exchange of the bound labelled nucleotide occurred within 30 min, 182 Intracellular Titration of Cyclic AMP-Receptor Protein Binding 0 10 20 30 40 50 60 Time (min) 70 80 90 Fig. 4. Exchange of bound cyclic [SHIAMP. Extracts were prepared from epinephrine-treated ( + o ) and untreated (0-0) rat diaphragms. Binding of cyclic [3H]AMP was carried out a t 0 â€Å"C in a volume of 2. 5 ml with 500 pg proteins, and 100 nM cyclic r3H]AMP in Tris-HC1 buffer, MgCl, and theophylline a t the concentrations described for the standard binding assay. After 1-h incubation, 1 pM unlabelled cyclic AMP and 100 pM ATP were added and the mixture allowed to stand at 20 °C. At the different times indicated in the figure, aliquots corresponding t o 50 pg protein were pipetted, rapidly diluted with 20 mM Tris-HC1 buffer, 2. 5 mM MgC1, p H 7 5 and filtered through Millipore filters. The filters . were washed with the same buffer, dried and counted. Results are expressed as pmol/mg protein 0 30 60 90 120 Time (rnin) 180 240 – Total binding capacities of the proteins could thus be measured by incubating the extracts first with 100 nM unlabelled cyclic AMP a t 0 â€Å"C and carrying on the exchange reaction in the presence of 1 pM cyclic I13H]AMP at 20 â€Å"C for 1-2 h ; the values obtained averaged 8. -9. 5 pmol cyclic [3H]AMP/mg soluble protein, both with epinephrine-treated and untreated diaphragms. These results were confirmed by direct assay of bound cyclic AMP: the extracts have been fully saturated with unlabelled 1pM cyclic AMP and filtered as described. After washing the Millipore filters, bound cyclic AMP was extracted by cold 7 O/, trichl oroacetic acid and the cyclic nucleotide was directly assayed according to Gilman [16]. The average value was 9. 8 f 0. 4 pmol cyclic AMP bound per mg protein, which is of the same order of magnitude as the amount of bound cyclic [3H]AMP calculated above. Previously published data are in close agreement with these values. Walton and GarFen [15] reported maximal binding capacities of 9. 8 pmol/mg protein for adrenal extracts, whereas Gilman [l6] found a total binding of 12pmol/mg protein in muscle extracts. The values for maximal cyclic AMP binding are very low as compared t o the total endogenous cyclic AMP present in the extract (46 pmol/mg protein with the theophylline-treated diaphragm and 170 pmol/mg protein with the epinephrine theophylline-treated diaphragm). It must be added that the binding proteins, saturated with cyclic AMP or not, were almost completely retained on the Millipore filters, and that endogenous cyclic AMP, not Fig. 5. T i m e course of cyclic A M P exchange under binding (0 â€Å"C) and exchange (20 â€Å"C} conditions. Extracts were prepared from epinephrine treated (0,A ) and untreated ( 0 , A) r a t diaphragms. Binding of cyclic AMP was performed as described in Fig. 2 in the presence of 100 nM cyclic AMP for 60 min at 0 â€Å"C. A t the end of the binding reaction 1 pM cyclic [3H]AMP was added t. the different extracts, in the absence (A, A ) or presence ( 0 , 0 ) of l00p. M ATP. The reaction mixtures were maintained a t 0 â€Å"C for 2 h and then at 20 â€Å"C (arrow) for 2 more hours. At the different times indicated on the figure, aliquots corresponding t o 70 pg protein were pipetted and treated as in Fig. 4. Results are expressed as cyclic rH]AMP bound in pmol/mg protein. bound to these fractions, was quant itatively recovered in the Millipore filtrates after trichloroacetic acid extraction. The extent t o which this â€Å"free† cyclic AMP may or not be bound to other proteins is presently not known. Cyclic-AMP Exchange under Binding Conditions The extent of cyclic AMP exchange under binding conditions (0 â€Å"C, 1 h, 100 nM cyclic AMP) must be controlled if corrections for simultaneous exchange have to be applied t o binding data: extracts of rat diaphragms treated with theophylline and theophylline epinephrine were first saturated with 1 O O n M unlabelled cyclic AMP (binding conditions) and then exchanged with 1 pM cyclic [3H]AMP but a t 0 â€Å"C. After 2 h, the temperature was raised to 20 â€Å"C and completion ofthe exchange measured after 1-2 h further incubation. Fig. 5 shows that a t 0 â€Å"C, within 1h incubation time, which are the conditions described above for the binding assay, about 200/, of total sites were exchangeable. Under these conditions, ATP and Mg ions slightly increase the exchange velocity. I n addition, this figure confirms that a t 20 â€Å"C total exchange capacities were identical for epinephrine-treated and untreated diaphragms ; hence initial + + Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser 183 Table 5. Relationship between intracellular cyclic A M P levels and cyclic A M P binding in extracts from diaphragm incubated under various conditions Diaphragms were incubated with or without 10 mM theophylline for 30 min at 37 â€Å"C, 5 pM epinephrine was added where indicated and incubation continued for varying times. From each incubation, half a diaphragm was extracted by trichloroacetic acid for cyclic AMP estimation. The other half was homogenized with Tris-HC1buffer lOOnM cyclic [3H]AMP(method B) for exogenous cyclic AMP binding after 1 h a t 0 â€Å"C; maximal binding capacities were determined in the same extracts a t 20 â€Å"C in the presence of 1 pM cyclic [3H]AMP under conditions described for cyclic A P exchange. R. esults are expressed as pmol cyclic AMP/mg M protein. Endogenous binding values were calculated as the difference between maximal binding capacities ( A )and exogenousbinding ( B ) and corrected for the 200/, exchange + Incubation conditions Theophylline 10 mM Epinephrine 5t*M Time Cyclic AMP Total level Maximal binding Exogenous capacity binding (a) (b) Endogenous binding (a-b) corrected min pmol/mg protein – – – + + + + + + 0 2 10 30 5 5 20. 5 52 43 38 46 170 f 4. 7 0. 47 f2 f 10. 7 9. 6 f 0. 9 9. 4 f 0. 1 9. 20 9. 40 8. 9 5 0. 73 8. 9 0. 85 5. 35 f0. 40 4. 50 f 0. 133 4. 40 4. 70 4. 46 f 0. 20 2. 7 f0. 224 5. 31 6. 13 6 5. 5 5. 53 7. 77 differences in residual binding capacities reflect variations in the degree of saturation of the receptor proteins by endogenous cyclic AMP, rather than modifications of their maximal binding capacity. 1 Titration o Endogenous Cyclic-AMP Binding in Rat f Diaphragm. Effects of Theophylline and Epinephrine Since total bindin g capacities of the receptor proteins in the extracts and the amount of exogenous cyclic [3H]AMP bound by these extracts after homogenization may be estimated, it appears possible to calculate endogenous cyclic AMP bound in the intact organs, correcting for a 2001, exchange during the titration period. Table 5 summarizes the results of a series of experiments where diaphragms have been incubated under conditions which modify endogenous levels of cyclic AMP :in every case, half of the diaphragm was extracted with cold trichloroacetic acid (see Methods) for the assay of intracellular cyclic AMP levels: the second half was extracted according to method B for the estimation of exogenous cyclic [3H]AMP binding and of total cyclic AMP binding capacities. The endogenous cyclic AMP bound was calculated from the latter experimental data. This table definitely establishes that the average values obtained for the intracellular binding of endogenous cyclic AMP in the intact organ seem to correlate with its cyclic AMP levels. A reciprocal plot of intracellular binding versus intracellular cyclic AMP concentrations (Fig. 6) shows that this correlation fits simple saturation kinetics very accurately. I n the unstimulated diaphragm (no theophylline nor epinephrine added to the incubation medium) about 50 °/, of the available binding sites are occupied by endogenous cyclic AMP; this Eur. J. Biochem. 40 (1973) -0. 002 I 0. 002 l/Free cyclic AMP (nM-‘) 0 0. 004 . Fig. 6. Reciprocal plot of intracellular cyclic A M P levels and cyclic A M P binding in rat-diaphragm extracts. Data arc obtained from experiments performed as described in Table 5 and replotted according t o the Klotz equation. The intercept on the y axis yields a n estimate of the number of binding sites and the x intercept provides a n estimation of the in tracellular apparent dissociation constant. Statistical analysis of the data were performed according to Cleland [26] using a Wang electronic calculator alue increases to almost goo/,, when the diaphragms have been fully stimulated with both theophylline and epinephrine. Various treatments with one of the agonists alone cause endogenous bindings ranging between these two extreme values. The apparent Kd value for intracellular binding according to this plot was estimated to 330 nM f 50, as compared to the apparent Kd (33-45 nM) when binding was assayed in the extracts (Fig. l and 2). Hence a difference of about one order of magnitude appears to obtain between the Kd values calculated within the cell and the 84 Intracellular Titration of Cyclic AMP-Receptor Protein Binding same constant measured with diaphragm homogenates. The double-reciprocal plot may also be used to calculate the intracellular maximal binding capacities, from its intercept with the ordinate axis. A value of 8. 9 pm ol/mg protein was found which coincides with the values measured in the extracts by total cyclic [3H]AMP exchange. This discrepancy between the intracellular Kd and the Kd measured in vitro in a variety of tissue extracts including diaphragm may a t first sight seem surprising. It has however repeatedly been pointed out that cyclic AMP concentration even in the unstimulated cell was far in excess of the concentration which should result in almost maximal stimulation of protein kinases and compartmentalization of the nucleotide within the cell has usually been postulated to explain this contradiction [8,9,20]. The present work shows that despite these high intracellular concentrations of cyclic AMP, protein kinases could indeed not be fully activated, since under the same conditions, the receptor proteins appear not to be fully saturated with cyclic AMP. Concluding Remarks As might have been expected from Equation (1) (if this reaction truly reflects intracellular conditions) a rise in cyclic AMP should be paralleled by an increase in the amount of cyclic AMP bound to receptor protein in the cell. The results reported show this indeed to be the case in the isolated rat diaphragm: when this tissue is stimulated by various agents which increase the level of cyclic AMP the amount of protein receptors endogenously saturated by cyclic AMP (R cyclic AMP) rises, as indicated in our experiments by a decrease in their ability to bind exogenously added cyclic [3H]AMP after tissue extraction. Maximal binding capacities for cyclic AMP do not seem to be affected under any circumstance. A parallel approach t o the study of this problem has been undertaken by Corbin et al. [12] and Soderling et al. [13] who investigated in adipose tissue under various stimulatory conditions, the state of activation of the catalytic subunit (C) by assaying the cyclic AMP dependence of the protein kinase in tissues extracts. These authors demonstrated that under well-defined xperimental conditions, there was a quantitative relationship between the intracellular level of cyclic AMP and the amount of the active C unit which could be separated from the complex protein kinase RC. However in their experiments high concentrations of NaCl had to be added to the extracts, since in its absence R and C tended to reassociate almost immediately, indicating that cyclic AMP is no longer bound to its receptor protein (R). The situation in various other tissue xtracts has been found to be analogous, except wit h skeletal muscle, where preliminary results obtained by the authors led them to suggest that the protein kinase subunits do not readily reassociate. This seems also to be the case for the diaphragm, since under the conditions of the present work, it has been possible to titrate for R * cyclic AMP in the crude extracts even in the absence of high salt concentrations : acccurate estimations of intracelM a r binding of cyclic AMP have been obtained and correlated with the absolute amounts of the nucleotide present in the stimulated and unstimulated cell. The binding seems t o obey simple saturation kinetics but the apparent Kd of this binding is about10 times higher as compared with the crude extracts. These results may be explained by cyclic AMP compartmentalization within the cell ; in this case, however, the simple saturation kinetics would indicate that the various pools of the cyclic nucleotide attain equilibrium very rapidly. Or else, if cyclic AMP within the cell is not compartmentalized, and if the reaction described by Equation (1) may be applied, without any modification, to intracellular equilibria, a decrease in the apparent Kd could be merely a consequence of the dilution (about 10-fold) of the protein components during extraction of the tissue, while cyclic AMP concentrations are maintained by the addition of exogenous cyclic [3H]AMP. However these two hypotheses are certainly oversimplified, since they do not take into account factors like the intracellular concentration of the heat-stable kinase inhibitor [21,22], ATP or Mg2+ [19,23], which are known to affect cyclic AMP binding either in crude extracts or with purified protein kinase preparations. It seems impossible to decide at present which of these interpretations is most likely to reflect true intracellular conditions. It is noteworthy that the apparent Kd estimated is close to the intracehlar cyclic AMP concentration of the nstimulated tissue, a fact which should account for maximal sensitivity of the regulatory mechanisms under physiological conditions. Hormonal controls at the level of cyclic AMP-receptor protein interaction have hitherto never been described; the data reported above provide a suitable means for investigating such problems. The authors are very much indebted to Mrs Ginette Delarbre for her excellent technical assistance and to Mrs Marie -ThBrBse Crosnier for preparing the manuscript. The present work has been performed thanks to two official grants of the C. N. R. S. Paris, France: ERA No 33 and ATP No 429. 914), to a grant obtained from the D. G. R. S. T. (No 72. 7. 0135), to a generous contribution of the Fondation pour la Recherche Mf? dicale Franpise and to a participation of the CEA (Saclay, France) in the purchase of radioactive compounds. The work has been performed as a partial fulfillment of a thesis (Doctorat Bs-Sciences) submitted by L. D. -K. Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser REFERENCES 1. Robison, G. A. , Butcher, R. W. Sutherland, E. W. (1968) Ann. Rev. Biochem. 37, 149-174. 2. Walsh, D. A. , Perkins, J. P. Krebs, E. G. (1968) J. Biol. Chem. 243, 3763-3765. 3. Kuo, J. F. Greengard, P. (1969) Proc. Nut. Acad. Xci. U . S. A. 64, 1349-1355. 4. Reimann, E. M. , Brostrom, C. O. , Corbin, J. D. , King, C. A. Krebs, E. G. (1971) Biochem. Biophys. Res. Commun. 42, 187-194. 5. Tao, M, Salas, M. L. Lipmann, F. (1970) Proc. Nut. Acad. Sci. U . S. A. 67, 408-414. 6. Gill, G. N. Garren, L. D. (1970)Biochem. Biophys. Res. Commun. 39, 335-343. 7. Craig, J. W. , Rall, T. W. Larner, J. (1969) Biochim. Biophys. Acta, 177, 213-219. 8. Stuil, J. Mayer, S. E. (1971) J. Biol. Chem. 246, 5716-5723. 9. Schaeffer, L. D. , Chenoweth, M. Dunn, A. (1969) Biochim. Biophys. Acta, 192, 292-303. 10. Miller, T. B. , Exton, J. H. Park, C. R. (1971) J. Biol. Chem. 246, 3672-3678. 11. Harbon, S. Clauser, H. (1971) Biochem. Biophys. Res. Commun. 44, 1496-1503. 12. Corbin, J. D. , Soderling, T. R. Park, C. R. (1973) J. Biol. Chem. 248. 1813-1821. 185 13. Soderling, T. R. , Corbin, J. D. Park, C. R. (1973) J. Biol. Chem. 248, 1822-1829. 14. Gill, G. N. Garren, L. D. (1969) Proc. Nut. A d . Sci. U. 8. A. 63, 512-519. 5. Walton, G. M. Garren, L. D. (1970) Biochemistry, 9, 4223-4229. 16. Gilman, A. G. (1970) Proc. Nut. Acad. Sci. U. 8. A. 67, 305-3 12. 17. Do Khac, L. , Harbon, S. Clauser, H. (1973) Ninth Int. Congr. Biochem. p. 354. 18. Lowry, 0. H. , Rosebrough, N. J. , Farr, A. L. Randall, R. J. (1954) J. Biol. Chem. 193, 265-275. 19. Wilchek, M. , Salomon, Y. , Lowe, M. Selinzer, Z. (1971)Biochem. Biophys. Res. Commun. 45,1177-1184. 20. Chambaut. A. M. , Lerav, F. Hanoune, J. (1971)PEBS . . â€Å". Lett. 15,’328-334. Walsh, D. A. , Ashby, C. D. , Gonzalez, C. , Calkines, D. 21. Fisher. E. H. Krebs. E. G. (1971)J. Biol. Chem. 246, i977-1985. 22. Ashby, C. D. Walsh, D. A. (1973) J. Biol. Chem. 248, 1255-1261. 23. Haddox. M. K. , Newton, N. E. , Hartler, D. K. Goldberg, N. D. (1972) Biochem. Biophys. Res. Commun. 47,-653-661. 24. Klotz, I. M. (195 3)in The Proteins (Neurath, H. Bailey, K. , eds) p. 772, Academic Press, New York. 25. Cleland, W. W. (1967) Advan. Enzymol. 29, 1. , L. Do Khac, S. Harbon and H. J. Clauser, Institut de Biochimie, Universit6 de Paris-Sud, BLtiment 432, F-91405 Orsay, France Eur. J. Biochem. 40 (1973) How to cite Titration Journal, Papers

Critical Appraisal of Epidemiological Study †MyAssignmenthelp.com

Question: Discuss about the Critical Appraisal of Epidemiological Study. Answer: Introduction Schizophrenia is a serious mental disorder that interferes with the ability of a person to think, make decisions, manage emotions and relate to others. It manifests commonly in the form of hallucinations or delusions. Cognitive issues such as, disorganized thinking, struggling to remember things and lack of insight (anosognosia) are often observed. Clozapine is one of the most commonly used atypical antipsychotics to treat schizophrenia (Leutwyler et al., 2014). It leads to a decrease in suicidal ideation. However, there are some serious side effects associated with its administration. One such effect is weight gain (Gressier et al., 2016). People under this medication report significant weight gain. This drug-induced weight gain is identified as a major risk factor for disorders that can increase morbidity and mortality rates of schizophrenic patients (Sagy, Weizman Katz, 2014). This report aims to critically appraise a study that was conducted to evaluate the effects of physical a ctivity and diet control on obese schizophrenic patients, under clozapine treatment (Wu et al., 2007). The paper focuses on a randomized, controlled study that was conducted to evaluate the effects of regular physical activity and continuous dietary control, for six months, on obese patients who were suffering from schizophrenia. These patients were being administered clozapine to reduce their mental disorder. The study tried to establish an association between clozapine use and weight gain among schizophrenia patients. It assessed biochemical and anthropometric parameters such as, triglyceride, serum glucose, insulin, cholesterol, prolactin, cortisol, and growth hormones for three months and six months. The study was a novel research as no other study had been conducted prior to this research that investigated the effects of dietary control among the target population, who were under clozapine medication (Wu et al., 2007). The study recruited 753 hospitalized patients who had been diagnosed with DSM-IV schizophrenia (McLean et al., 2014). The age of the participants ranged between 18-65 years. Respondents who were under administration of 300mg oral clozapine per day, for at least one year and had a BMI higher than 27 kg/m2 were included in the study. A registered dietitian implemented dietary control among the respondents and restricted the caloric intake to 1,600-1,800 kcal per day for men and 1,300-1,500 kcal per day for women. Minimum dietary requirements for men and women were 1,500 kcal and 1,200 kcal per day respectively (Dipasquale et al., 2013). The types of foods consumed by the participants were assessed, which included an evaluation of the vegetable, fruits, sugar free drinks and, artificial sweeteners (Kim et al., 2017). The calorie intake was measured. A minimum of 30 minutes of moderate intensity physical activity like brisk walking is recommended for people belonging to all age groups for most days of the week. The intervention involved performing physical activities for six months, three days a week. The patients were made to take part in activities that involved 1.62 km level walking for 40 minutes and walking up and down the stairs for 20 minutes, under supervision (231 steps upstairs and 330 steps downstairs). The speed and distance of these activities were maintained at a constant level, throughout the intervention period. The participants were encouraged to complete them in an hour. The guidelines proposed by the American College of Sports Medicine were used to estimate the rate of energy expenditure per week (Thompson et al., 2013). The effects of the interventions were assessed by anthropogenic measurements, which included measuring the body fat percentage, weight, height, hip and waist circumference and BMI (Lau et al., 2016). Serum glucose, insulin, cholesterol, cortisol, triglyceride and prolactin levels were measured by ELISA tests. On comparing the result values of the sample and control group using ANCOVA and SPSS software version 10.0, no significant difference was observed among the two groups at baseline. No significant changes in body fat percentages were observed between men and women or during the 3 month and 6 month intervention period. However, significant reduction (p0.05) was observed in body weight, waist circumference in the study group, after 3 months. Waist circumference showed significant reduction after 6 months. Metabolic analysis and ELISA failed to show any reduction at baseline or during the 3 month intervention period. However, triglyceride levels were significantly lower in the control group after 6 months (p0.05). A high IGF-1 to IGFBP-3 molar ratio was observed in the study group than the control group, after 6 months. The study found out that physical activity and dietary are responsible for normalizing metabolic abnormalities, attenuating neuroleptic side effects and minimizing hormonal changes. However, the researchers found presence of low motivation among psychiatric patients for weight reduction, in absence of supervision (Vancampfort et al., 2015). They also proposed that it is difficult to suppress appetite for a long period of time. Therefore, they suggested that long-term adherence to such lifestyle modification programs are necessary for putting these interventions to practice. Bias and Confounding variables Randomised control studies are generally less susceptible to sample bias, when compared to other study designs that assess the effect of several therapeutic interventions. The study avoided bias on the basis of baseline prognostic variables. Randomisation ensured that the treatment groups were balanced and as similar as possible. This was supported by the fact that all 53 participants selected from 753 hospitalised patients had a DSM-IV schilzophrenia diagnosis, were 18-65 years of age, had BMI higher than 27 kg/m2 and was under the medication of 300 mg oral clozapine intake for more than a year. No patients were included in the study if they were found to suffer from organ failure, abnormal ambulatory functions, and severe mental retardation or presented vented walking. Neither of the sample or control group included patients who were under medication of antipsychotics apart from clozapine. Moreover, bias due to presence of confounding variables was also removed by performing the two way mixed designs ANCOVA. This eliminated the influence of any external factors on the measured outcomes. The results obtained were therefore least likely to get affected. However, one major bias associated with the study was the recruitment of participants from inpatient settings. The rates of compliance to the 6 month intervention and the success rate of the study would have been different if the sample was selected from outpatient settings. Thus, it can be stated that the randomized controlled study did not remove bias with respect to selection of participants from a larger population. Chance variations are inherent errors in predictive statistical models. They are defined as the difference between actual and predicted values of the variable being investigated. Similar to other epidemiological studies, this research also included participants from a larger population. There was a chance of the small sample differing from the wider patient population (Nuzzo, 2014). To show that the difference in results between the sample and the control group reflected a real difference in the parent population, a statistical test was performed. The p Causal association between exposure and outcome The primary goal of most epidemiological studies is assessment of a particular disease cause. However, owing to the concept that most epidemiological studies are based on observation, rather than experiment, several possible explanations are considered before drawing inference for any cause and effect relationship. Causal relationships are more likely to demonstrate a stronger association between the cause and outcome in a particular study. Plausibiity establishes the cause-effect relationship between a biological factor and an adverse health outcome or effect. In this research study, a relationship was established between use of clozapine and weight gain among patients with schizophrenia. The study was built on the basis of several scientific researches that proposed that clozapine and olanzapine induced mean weight gain among patients who were under administration of these drugs for more than 6 months (Samara, Leucht, 2016). The research was based on other findings that such incre ase in mean weight, induced by the action of antipsychotics like clozapine often leads to noncompliance (Olfson et al., 2016). This results in treatment discontinuation and relapse of psychotic symptoms. Furthermore, scientific evidences suggest that weight control is effective in reducing health risks among schizophrenia patients who are overweight. The presence of existing biological research on the use of clozapine among such patients and their subsequent weight gain explains the association of interest of this research. Demonstrating the plausibility of causal relationships is complex since a particular health outcome is the result of balance and interplay between different factors. The study showed consistency with other findings, which indicated that schizophrenia patients who were treated with clozapine, reported a gain in weight (McNamee et al., 2013). These patients also demonstrated an increase in body fat deposits and BMI. A marked increase in the waist-to-hip ratio and central adiposity was reported by other studies. These outcomes were consistent with the findings of the current epidemiological research (Rosenbaum et al., 2014). Moreover, the results are also consistent with other studies that indicate a loss in weight among inpatients who took clozapine. The study also showed agreement with several other findings in the low levels of IGFBP-3 and high IGF-1 to IGFBP-3 molar ratio after six months of intervention. These results were consistent with research that displayed a reduction in IGFBP-3 levels with exercise. The special mechanism illustrated in the study focused on the effect of IGF-1 on the cardiovascular system. IGF-1 is a peptide hormone predominantly produced by the liver, in response to pituitary growth hormone. Several studies have elaborated on the role of low levels of IGF-1 in increasing the likelihood for cardiovascular diseases (Arcopinto et al., 2014). Low levels have been shown to promote atherosclerosis and stroke. This in turn increases the mortality rates. An increase in the level of IGF-1 in macrophages works to removed the plaques from clogged arteries and prevents the incidence of cardiovascular diseases (Troncoso et al., 2014). This study therefore elaborated on the fact that IGFBP-3 (insulin-like growth factorbinding protein-3) is the most abundant protein that carries the maximum amount of IGFs that circulate in the bloodstream. The study performed an ELISA test to investigate the molar ratio of IGF-1 to IGFBP-3, to determine the risk of cardiovascular diseases among the participants taken from inpatient settings, who were under clozapine treatment for schizophrenia. Although, previous studies did not investigate the role of IGF-1, IGFBP-3 and growth hormones among schizophrenic patients, this research illustrated the effects of long term clozapine therapy on the factors. Appraising external validity External validity measures the validity of the inferences obtained from the particular study to wider population. A research study is considered to be externally valid if the relevant results can be extrapolated to a larger population with similar characteristic features. The population, setting, interventions and treatment outcomes of this study can be applied to the source population of schizophrenia patients who adhered to clozapine medication. The interventions followed in this study could therefore be followed in the source population. The effects of dietary control and physical exercise could be applied to monitor the health effects of obese inpatients with schizophrenia, who were subjected to clozapine drugs (Mizuno et al., 2014). However, the external validity cannot be completely established before the interventions are applied to outpatient settings. Participants belonging to outpatient settings have a less likelihood of showing adherence to the interventions and following a strict dietary and exercise regime. The results of the intervention on such participants are therefore most likely to get altered. Such patients would show less success rate of the proposed intervention. Furthermore, other metabolic effects of the interventions were not diagnosed before the 6 month period. Therefore, before being applied to a larger population, the study parameters should be tested on outpatients and the secondary metabolic effects of the lifestyle modifications should be measured. Quality of the study The discussions prove that the study was successful in measuring the effects of physical activity and dietary control on the 53 participants, randomly distributed in the sample and control group. It effectively demonstrated the benefits of a 3 month and 6 month intervention that consisted of regular physical activity and integrated dietary control on obese patients, who suffered from schizophrenia and were subjected to clozapine treatment. The interventions were successful in showing significant reduction in body fat percentage, BMI and waist and hip circumference (Amiaz et al., 2016). In addition, the study effectively measured the insulin, triglyceride, cortisol, serum glucose, prolactin, IGF-1 and IGF-3 levels and their molar ratio among the participating patients. The results showed significant improvement in their metabolic profiles. In contrast, negligible or no improvement in the control group results for anthropometric measurements established the fact that the interventions were effective in managing weight gain in the sample. It can also be suggested that the study was of a good quality owing to the fact that the dietary control intervention was applied on the participants by following dietary guidelines. Approximately 200-300 fewer kilocalories were present in the diet of the participants during the 6 month intervention period and they were made to spend 600-750 kcal more energy per week by regular physical activity. The study chose these levels for calorie consumption or energy expenditure to minimize occurrence of adverse effects due to diet changes. These adverse events could be manifested in the form of emotional and mental instability among inpatients that were given fewer calories. Moreover, the study was of a good quality in selecting physical activities that were suitable for the patients. The activities chosen were uncomplicated and did not pose any danger. Furthermore, it utilized the role of efficient health professionals to evaluate and ensure the proper application of the weight management t echniques among the patients. A minimum body weight reduction by 5% to 10% produces significant health benefits among the patients (Manu et al., 2015). The results showed that there was a significant reduction in for obese patients with schizophrenia who are taking clozapine, the intervention resulted in body weight (5.4%), BMI (5.4%), hip circumference (3.3 cm) and waist circumference (3.3 cm) after the 6 month intervention period. The research was effective in lowering BMI and improving other outcome measures by the end of the intervention period. A reduction was observed in some of the parameters just after 3 months of intervention, while others showed significant changes in results after 6 months. These discussion state that the research considered appropriate intervention strategies to evaluate the intended outcomes. Moreover, the study focused on regular monitoring the anthropometric measurements, dietary behavior and physical activity of the sample group, by the ward staff and the investigators. At the end of the 6 month period, none of the participants displayed worsened conditions. Female patients with schizophrenia under clozapine medications are likely to get affected with cardiovascular diseases. The findings provided evidence for low IGFB-3 levels after the 6 month intervention but, failed to show any alteration of the levels of IGF-1. Therefore, it can be stated that the exercise intensity used in the intervention was inadequate to create changes in the levels of the growth hormone. Conclusion Thus, it can be concluded that the study helped to establish the benefits of regular physical activity and dietary control among the target population. The intervention showed significant improvements among obese, schizophrenia inpatients that were treated with clozapine. However, a strong health monitoring and lifestyle modifications are required to observe the long term effect of the interventions. References Amiaz, R., Rubinstein, K., Czerniak, E., Karni, Y., Weiser, M. (2016). A diet and fitness program similarly affects weight reduction in schizophrenia patients treated with typical or atypical medications.Pharmacopsychiatry,26(03), 112-116. Arcopinto, M., Isgaard, J., Marra, A. M., Formisano, P., Bossone, E., Vriz, O., ... Cittadini, A. (2014). IGF-1 predicts survival in chronic heart failure. Insights from the TOS CA.(Trattamento Ormonale Nello Scompenso CArdiaco) registry.International journal of cardiology,176(3), 1006-1008. Dipasquale, S., Pariante, C. 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W., Uchida, H. (2014). Pharmacological strategies to counteract antipsychotic-induced weight gain and metabolic adverse effects in schizophrenia: a systematic review and meta-analysis.Schizophrenia bulletin,40(6), 1385-1403. Nuzzo, R. (2014). Statistical errors.Nature,506(7487), 150. Olfson, M., Gerhard, T., Crystal, S., Stroup, T. S. (2016). Clozapine for schizophrenia: state variation in evidence-based practice.Psychiatric Services,67(2), 152-152. Rosenbaum, S., Tiedemann, A., Sherrington, C., Curtis, J., Ward, P. B. (2014). Physical activity interventions for people with mental illness: a systematic review and meta-analysis. Sagy, R., Weizman, A., Katz, N. (2014). Pharmacological and behavioral management of some often-overlooked clozapine-induced side effects.International clinical psychopharmacology,29(6), 313-317. Samara, M. T., Leucht, S. (2016). Use of Clozapine in SchizophreniaReply.JAMA psychiatry,73(10), 1098-1099. Thompson, P. D., Arena, R., Riebe, D., Pescatello, L. S. (2013). ACSMs new preparticipation health screening recommendations from ACSMs guidelines for exercise testing and prescription.Current sports medicine reports,12(4), 215-217. Troncoso, R., Ibarra, C., Vicencio, J. M., Jaimovich, E., Lavandero, S. (2014). New insights into IGF-1 signaling in the heart.Trends in Endocrinology Metabolism,25(3), 128-137. Vancampfort, D., De Hert, M., Stubbs, B., Ward, P. B., Rosenbaum, S., Soundy, A., Probst, M. (2015). Negative symptoms are associated with lower autonomous motivation towards physical activity in people with schizophrenia.Comprehensive psychiatry,56, 128-132. Wu, M. K., Wang, C. K., Bai, Y. M., Huang, C. Y., Lee, S. D. (2007). Outcomes of obese, clozapine-treated inpatients with schizophrenia placed on a six-month diet and physical activity program.Psychiatric services,58(4), 544-550.