Adenosine triphosphate (ATP) is an endogenously occurring nucleoside triphosphate, which is ubiquitous in all cell types and constitutes the natural precursor molecule of adenosine, (AD) a purine nucleoside formed by adenine and ribose. One ATP molecule consists of three phosphate groups, and is synthetized by several enzymes, namely ATP synthase, from adenosine diphosphate (ADP) or adenosine monophosphate (AMP). ATP is generated during cellular respiration by substrate level phosphorylation and oxidative phosphorylation.1 The actions of ATP are different in the intracellular and extracellular compartment. The main role of intracellular ATP is as a coenzyme in many fundamental cellular processes, such as cellular metabolism and energy production. The extracellular ATP however acts a molecular mediator between cells, after being released from endothelial cells, erythrocytes, activated platelets, muscle and nerve fibers, ischemic, inflammatory and apoptotic cells. Experiment data point towards an increased cellular formation of AD when either the local tissue metabolic demand increases or the regional blood flow and oxygen delivery decreases, especially in tissues which rely to a large extent to oxidative phosphorylation for energy production. AD and ATP exert their physiologic signaling effects via binding two purinergic receptor families in the cell membrane, named adenosine receptor or P1 receptor and ATP receptor or P2 receptor. P1 receptors are G protein-coupled receptors and are further classified into A1R, A2AR, A2BR, and A3R. With regard to P2 receptors two types have been identified: P2X, which are ion channels, and P2Y which are G protein coupled receptors. The half-life of extracellular ATP is extremely short as it is catabolized rapidly by ecto-nucleotide enzymes which rapidly dephosphorylate extracellular ATP to ADP, AMP and AD, the latter in turn being subsequently transported back to the cytoplasm.2 Another secondary source of AD production within cells is the intracellular degradation of S-adenosyl-homocysteine, which is derived from S-adenosylmethionine via transmethylation reactions.3
Electrophysiologic effects of ATP and adenosine
In the cardiac conduction system, ATP and AD exert distinct negative chronotropic and dromotropic effects, by suppressing the sinus nodal automaticity and prolonging the conduction interval through the atrioventricular node (AVN). Intravenous administration of AD in humans has been demonstrated to cause sinus bradycardia and sinus arrest.4 Adenosine can also cause sino-atrial exit block at high concentrations, as well as a relocation of the earliest site of atrial activation from the sinus nodal region to the crista terminalis area.5 Interestingly, the sinus node (SN) is not the only site of the cardiac conduction system which manifests decreased automaticity after AD administration. The His-bundle and the Purkinje fibers have been shown to be even more responsive to AD, exhibiting a similar degree of decrease in automaticity with considerably lower doses of AD. With regards to the negative dromotropic action of AD it has been shown to increase the A-H interval in a dose-dependent manner, while it has no effect in the H-V interval.6 More specifically, it has been found that a suppression of nodal (N) cells action potentials accounts for 83% of the prolongation of the A-H interval caused by AD.7 Notwithstanding the inhibitory effects of AD in action potential propagation in the sinoatrial and (AV) node, AD has no impact in signal transduction through the atrial cell tissue.7 At the cellular level, AD induces a hyperpolarization of the resting potential across the membrane, a decrease in the slope of phase 4 depolarization, and a reduction in the action potential duration. In clinical settings the above effects are typically transient, with an approximate duration of 30 seconds followed by heart rhythm recovery without any clinically significant side-effects.8 Finally, a negative inotropic effect in atrial myocytes has been described.9
Extending beyond the cardiac conduction system, there is also a well-described effect in the coronary arteries, where ATP and AD induce vasodilation. Additional physiologic effects of AD comprise inhibition of platelet adhesion, anti-catecholaminergic actions, inhibition of renin production and sodium retention in the kidneys.10
Pathophysiogic differences in the effects of ATP versus adenosine
hen comparing the cardiac effects of ATP versus AD, a clear difference relies in the fact that the actions of ATP are evidently associated with the vagal tone. Specifically, maneuvers which enhance the parasympathetic afferent stimuli to the heart, such as physostigmine administration and increased plasma calcium levels, trigger an augmented effect of ATP over AD in the cardiac conduction system in experimental animal models.10 On the other hand, interventions which eliminate the vagal stimulation to the heart, such as administration of atropine or surgical denervation, practically render the ATP effects similar to those of AD.11 Furthermore, when the parasympathetic action in the heart is eliminated, the effects of ATP are counteracted by xanthine derivatives like aminophylline, which is a nonselective competitive antagonist of AD receptors, and upregulated by dipyridamole, which acts as an AD reuptake inhibitor.12 These data suggest that without the effects of the parasympathetic system, the actions of ATP in the heart are identical to those of AD. Of interest, the effects of ATP in the heart vary depending on the anatomical site of administration. In the left coronary artery, the vagal component of ATP action prevails while AD administration has no effects to SN automaticity.13 On the other hand, when administered to the sinus nodal artery, the effects of ATP are purely dependent in its subsequent degradation to AD.14 Detailed experimentation with regards to the potential targets and inhibitors of ATP binding revealed that ATP elicits a vagal depressor reflex response in the heart by means of upregulating specific receptors in the left ventricle.15
Safety and side-effects
During exogenous administration, ATP and/or AD are in general very well tolerated, can cause however transient bradyarrhythmias, as sinus bradycardia, sinus arrest or atrioventricular block. Facial flushing, headache, chest discomfort, sweating, dizziness and hyperventilation with dyspnea are also relatively common symptoms, but typically last for less than one minute and rarely are of clinical concern.16 However, the above effects are often pronounced in elderly patients, and therefore caution should be taken. In a few cases, acute exacerbation of asthma or chronic obstructive pulmonary disease with bronchospasm lasting for more than 30 minutes has occurred after AD administration.17,18 Also, AD has minor proarrhythmic effects and may cause atrial and ventricular ectopy as well as bradycardia-dependent polymorphic ventricular tachycardia, especially in patients with long QT syndrome.19 Rarely, AD may induce atrial fibrillation due to a suppression of the atrial refractoriness.20,21 This is potentially dangerous in the co-existence of ventricular preexcitation due to an accessory pathway which could rapidly conduct the atrial signal to the ventricles leading to ventricular arrhythmias. Hypersensitivity to AD has also been reported. Concomitant use of carbamazepine, digoxin, verapamil or dipyridamole increase the pharmacologic effects of AD.
Pathophysiologic basis of the usefulness of adenosine and ATP in the diagnostic investigation of syncopal attacks
The aforementioned cardiac effects of ATP and AD largely account for their widespread and recognized value in the diagnostic workup of neurally mediated syncope (NMS) and syncope of unknown origin (SUO). Their usefulness is explained by considering the proposed pathophysiology of NMS. In specific, it is postulated that an initial drop of systemic arterial pressure elicits an activation of the sympathetic system, which is in turn ensued by a disproportionate increase of parasympathetic discharges with concomitant sympathetic withdrawal, mediated by specialized cardiopulmonary mechanosensitive and chemosensitive receptors in the left ventricle.22 This paradoxical reaction stimulates a profound vasodilation and bradycardia which manifest clinically as presyncope and/or syncope. Exogenous administered ATP mimics this mechanism by inducing initially a sympathetic activation through a direct triggering of cardiac excitatory afferent fibers, followed by activation of vagal sensory nerve terminals that are localized in the left ventricle, which ultimately trigger a cardiocardiac central vagal depressor reflex.23-25 Noteworthy, AD exerts direct negative chronotropic and domotropic actions, but in contrary to ATP has no vagal activity.12 Instead, causes a continued sympathetic withdrawal that in susceptible individuals results finally in vasovagal syncope.26
S It has been postulated therefore, that ATP and AD endogenous production may be related to the clinical presentation and their exogenous administration would unmask syncopal symptoms in patients with NMS and SUO. In support of with this concept, patients with positive tilt test had higher AD plasma concentration and a positive association between the increase in AD levels and the onset of syncope exists.27 Also, patients with unexplained syncope and positive tilt test exhibit an overexpression of the AD receptor A2AR.28,29 Indeed, in some patients the cardiac effects of exogenous ATP/AD administration are exaggerated and result in paroxysmal AV block with long pauses. Therefore, the induction of clinically evident paroxysmal AV block with long periods of ventricular asystole following the injection of ATP/ AD has been suggested as a surrogate of increased risk in patients with syncope not been attributed elsewhere.
Campbell NA, Williamson B, Heyden RJ. Biology: exploring life. Boston, Massachusetts: Pearson Prentice Hall: ISBN 0-13-250882-62006.
Pelleg A, Belhassen B. The mechanism of the negative chronotropic and dromotropic actions of adenosine 5?-triphosphate in the heart: an update. J Cardiovasc Pharmacol2010;56:106-109.
Schrader J. Metabolism of Adenosine and Sites of Production in the Heart. Springer1983:133-156.
DiMarco JP, Sellers TD, Berne RM, West GA, Belardinelli L. Adenosine: electrophysiologic effects and therapeutic use for terminating paroxysmal supraventricular tachycardia. Circulation1983;68:1254-1263.
West GA, Belardinelli L. Sinus slowing and pacemaker shift caused by adenosine in rabbit SA node. Pflugers Arch1985;403:66-74.
Belardinelli L, West GA, Clemo SHF. Regulation of Atrioventricular Node Function by Adenosine. Springer1987:344-355.
Clemo HF, Belardinelli L. Effect of adenosine on atrioventricular conduction. I: Site and characterization of adenosine action in the guinea pig atrioventricular node. Circ Res1986;59:427-436.
Perennes A, Fatemi M, Borel ML, Lebras Y, L’Her C, Blanc JJ. Epidemiology, clinical features, and follow-up of patients with syncope and a positive adenosine triphosphate test result. J Am Coll Cardiol2006;47:594-597.
Rockoff JB, Dobson JG, Jr. Inhibition by adenosine of catecholamine-induced increase in rat atrial contractility. Am J Physiol1980;239:H365-370.
Shryock JC, Belardinelli L. Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology. Am J Cardiol1997;79:2-10.
Pelleg A, Hurt CM. Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals. J Physiol1996;490 ( Pt 1):265-275.
Pelleg A, Belhassen B, Ilia R, Laniado S. Comparative electrophysiologic effects of adenosine triphosphate and adenosine in the canine heart: influence of atropine, propranolol, vagotomy, dipyridamole and aminophylline. Am J Cardiol1985;55:571-576.
Katchanov G, Xu J, Hurt CM, Pelleg A. Electrophysiological-anatomic correlates of ATP-triggered vagal reflex in the dog. III. Role of cardiac afferents. Am J Physiol1996;270:H1785-1790.
Pelleg A, Mitsuoka T, Michelson EL, Menduke H. Adenosine mediates the negative chronotropic action of adenosine 5?-triphosphate in the canine sinus node. J Pharmacol Exp Ther1987;242:791-795.
Wang Y, Li G, Liang S, Zhang A, Xu C, Gao Y, Zhang C, Wan F. Role of P2X3 receptor in myocardial ischemia injury and nociceptive sensory transmission. Auton Neurosci2008;139:30-37.
DiMarco JP, Miles W, Akhtar M, Milstein S, Sharma AD, Platia E, McGovern B, Scheinman MM, Govier WC. Adenosine for paroxysmal supraventricular tachycardia: dose ranging and comparison with verapamil. Assessment in placebo-controlled, multicenter trials. The Adenosine for PSVT Study Group. Ann Intern Med1990;113:104-110.
DeGroff CG, Silka MJ. Bronchospasm after intravenous administration of adenosine in a patient with asthma. J Pediatr1994;125:822-823.
Drake I, Routledge P, Richards R. Bronchospasm induced by intravenous adenosine. Human
Growth and Lipid Production of L. Starkeyi Mutants
Diesel is one of the components in fossil fuel. However, the over-use of diesel is producing greenhouse gases such as carbon dioxide gases which are the major elements leading to global warming. Hence, due to increase in demand and source limitation, biodiesel is introduced as a substitute for diesel fuel (Wild et al., 2010).
Biodiesel is a diesel fuel substitute that is extracted from renewable biomass. Biodiesel can be produced from plant oils, animal fats and microorganisms. Traditionally, biodiesel is produced from plant oils which were transesterify with methanol (Dai et al., 2007). However, production of biodiesel from plant oils is not suitable due to the quality of tillable land (Li et al., 2008) and competition with food production (Wahlen et al., 2012). Furthermore, the increase in animal fats prices due to the increase in animal feed makes it not suitable as biodiesel feedstock (Li et al., 2008). Hence, oleaginous microorganisms have been introduced as good candidates for biodiesel feedstock.
Oleaginous microorganisms can accumulate lipid up to 20% of its cell dry weight (Ageitos et al., 2011). Oleaginous microorganisms have the ability to utilize different carbon source (Ageitos et al., 2011). In this study, Lipomyces starkeyi will be used. This type of yeast has the ability to produce lipid up to 70 % of its cell dry weight (Wild et al., 2010). L. starkeyi can utilize different types of carbon as its sole carbon and it is flexible in terms of culture conditions (Ageitos et al.,2011). However, L. starkeyi is still not economically practical because of the limitations in the wild-type strains (Ageitos et al., 2011). Therefore, in our research, we will be using L. starkeyi mutants in an attempt to produce more lipid more lipid in the fungal cells.
The L. starkeyi mutants will be cultured in modified media consists of glucose, (NH4) SO4, yeast extract, Na2HPO4.7H20, KH2PO4, MgSO4. 7H20, CaCl2. 2H20, FeSO4, ZnSO4.H20 and CuSO4 supplied with 2.5% (w/v) and 5.0% (w/v) of glucose and sago effluents in separated schott bottles. pH 5 and pH 6 will also be used in order to optimize the production of lipid. The temperature that will be used is room temperature (± 27°C). In this experiment, sago effluent and glucose would serve as carbon source for L. starkeyi. The total carbohydrate that would be consumed by L. starkeyi will be tested using phenol-sulphuric test.
Our objectives in this research are:
To optimize growth and lipid production of L. starkeyi mutants
To measure the amount of lipid produced by L. starkeyi mutants cultured in 2.5 % and 5 % of glucose medium
To measure the amount of lipid produced by L. starkeyi mutants cultured in sago effluent
CHAPTER 2: LITERATURE REVIEW
Biodiesel consists of alkly ester of fatty acids or triglycerides. Conventionally, triglyceride is produced from soybeans oil with the addition of alcohol and acid or base catalyst. This process is known as transesterifications which will produce Fatty Acid Methyl Ester (FAME) (Wahlen et al., 2012). Basically, biodiesel can be derived from 3 sources which are plants oil, animal fat and microorganisms (Meng et al., 2008).
Plant oils that involve in the production of biodiesel are rapeseed, palm oil, soybeans, cottonseed, sunflower and many possible crops (Perritano, 2010). However, the practical used of plant oils raises critical issues on the decreasing in quality of land that is needed to plant the crops could affect the quality of the crops produced (Li et al., 2008). In addition, it also competes with the food production (Wahlen et al., 2012). Animal fat is also not a good biodiesel feedstock due to economical reasons (Meng et al., 2008). Hence, oleaginous microorganisms stand out as a potential feedstock provider.
2.2 Oleaginous microorganisms
Oleginous yeasts (OY) are known producers of single cell oil (SCO). SCO produced from this organism are triacylglycerides (TAG) that have long-chain of fatty acids and have similar properties with plant oils. TAG acts as source of energy and it assist in phospholipid membrane formation. OY also utilizes various its carbon sources from waste substrate thus the cost to culture this microorganism is low (El-Fadaly et al., 2009).
There are four groups of oleaginous microorganisms that capable of producing biodiesel which are bacteria, algae, filamentous fungi and yeast (Kitcha and Cheirsilp, 2011). The genera of oleaginous yeast are Yarrowia, Candida, Rhodotorula, Rhodosporium, Crytococcus, Trichosporon and Lipomyces (Ageitos et al., 2011). The specific name for the most preferable candidates for production of lipid are Cryptococcus albidus, Rhodosporidium toruloides, Rhodotorula glutinis, Lipomyces starkeyi and Yarrowia lipolytica. These microorganisms are capable of producing intracellular lipid more than 20% of its cell dry weight (Tapia et al., 2012).
The duplication rate of yeast is lower than 1 hour and it is easy to culture compared to other microalgae. Other than that, certain oily yeast also has the ability to produce lipid up to 80% of their dry weight, while utilizing different carbon source including the lipid present in media (Ageitos et al., 2011).
2.3 Factors affecting lipid accumulations in Oleginous yeast
Lipid accumulations occur when yeast is cultured under high amount of carbon source but in limited source of nitrogen. This is due to the nutrient imbalance that helps in triggering the accumulation of lipid because the remaining substrate would be assimilated by the yeast’s cells hence convert it into fat for storage (Ageitos et al., 2011). The fat that accumulated could be extracted to produce biodiesel. In addition, the accumulations of lipid also affected by other factors such as the present of microelements and inorganic salts in media. These elements help in ATP (AdenosineTriPhosphate) citrate lyse which important in lipid production (Ageitos et al., 2011).
2.4 Lipomyces starkeyi
L. starkeyi is one of the members of Saccharomycetales and considered as true inhabitant of soil which have a worldwide distribution (Ansschau et al., 2014). L. starkeyi have the ability to accumulate lipid up to 70% of its dry weight (Wild et al., 2010). It also has a high flexibility in utilization of carbon source and culture environment. Other than that, fatty acid produced by L. starkeyi is almost similar to the vegetable oil (Tapia et al., 2012). According to Wild et al. (2010), L. starkeyi need a high ratio of carbon to nitrogen in order to optimize the production of lipid. The lipid bodies (LB) of L. starkeyi will receive the excess carbon source in the form of triglycerides (TAGs) (Ageitos et al., 2011)
2.5 Sago effluent
Sago effluent is a form of sago liquid waste. In normal processes, this effluent would be channeled into the river, thus polluting the river and environment (Awang-Adeni et al., 2010). The releasing of sago effluent into the river can cause decreasing in water pH and increase in biochemical oxygen demand (BOD) and chemical oxygen demand (COD) (Ayyasamy et al., 2008)
Sago effluent contains a high amount of organic materials and non-starch polysaccharide (NSP) (Awang-Adeni et al., 2010). NSP are made of cellulose, hemicellulose and lignin. In cellulose, the sub-components are 89% glucose and small amount of xylose, rhamnose, arabinose, mannose, fructose and galactose. In contrast to cellulose, hemicellulose main components are glucose and xylose accompanied with arabinose, galactose, rhmnose, fucose and uranic acid. Lignin functions in rigidity and stability of the wood. To sum up, sago effluent contains up to 66% of starch, 14 % fiber and 25 % lignin (Awang-Adeni et al., 2010).
Sago effluents which flow from the sago mill usually have the ratio of carbon to nitrogen high which is 105: 0.12 (Awang-Adeni et al., 2010). As stated by Ageitos et al. (2011), L. starkeyi have the ability to utilize starch as its sole carbon. Hence, sago effluent is an excellent choice because it has a high amount of starch which can helps in optimizing the lipid production.
2.6 Phenol-sulphuric test
Phenol-sulphuric test is the quantitative assays which often used in estimation of carbohydrate. This test could detect the presence of neutral sugar in oligosaccharides, proteoglycan, glycoproteins and glycolipids (Albalasmeh et al., 2013). When phenol-sulphuric is added, the glucose that presence in samples would dehydrate thus forms hydroxymethyl furfurax. It would yield a yellow-brown product and the OD could be checked at 490 nm (Albalasmeh et al., 2013).
CHAPTER 3: MATERIALS AND METHOD
Modified media as suggested by Wild et al. (2010).
Lipomyces Starkeyi mutants (LS R1 and LS R2)
2.5 % (w/v) and 5.0 % (w/v) of glucose (Ee Syn, Malaysia)
2.5 % (w/v) and 5.0 % (w/v) of sago effluent (Pusa, Malaysia)
80 % (w/v) of Glycerol stock (HmbG, Germany)
5 % Phenol (Nacalai Tesque, Japan)
Hexane (Reagents, USA)
Isopropanol (Amresco, USA)
Microcentrifuge (Hettich EBA 21, England)
Schott’s bottles (Duran, Germany)
3.2 Glycerol stock
A single colony of L. starkeyi mutants R3 will be inoculated into 100 ml of modified media. 800 μl of L. starkeyi mutants R3 that have grown will be transferred into vial that contained 1200 μl of glycerol stock. The glycerol stock steps of L. starkeyi will be repeated for L. starkeyi mutants R4. The solution will be stored in freezer at -20 °C.
3.3 Propagation of cell
1.5 L of modified media with pH 5 will be prepared into two Liter schott bottles and L. starkeyi mutants R3 and R4 will be inoculated in respective bottles (Wild et al., 2010). This step will be repeated for pH 6.
For day 1 until day 6, three (3) falcon tubes will be autoclave and weight. After that, 50 ml of the cultured from first bottle will be transferred into each three (3) falcon tubes and it will be weighted again. The sample will be sent for centrifuge for 5 minutes at 5000 rpm. The supernatant will be discarded and the pellet with falcon tube will be weight again for its wet weight. The sample will be dry in the oven for 1 or 2 days. After that, the sample will be weight again for its dry weight. All experiments will be performed in duplications.
3.4 Standard curve for L. starkeyi
1 ml of culture which will be incubated for 3 days earlier will be added into 9 ml of modified media in test tube. Serial dilution will take place with the factors of 10-1 until 10-7. For factors of 10-1 until 10-7, their OD will be checked for 600 nm. For factors 10-5 until 10-7, 300 μl from each sample will be taken and poured onto plate count agar. The plate will be incubated overnight before colony counting will be performed.
3.5 Lipid accumulation stage for L. starkeyi mutants
The L. starkeyi mutants culture will be incubated for 3 days (optimum growth) at room temperature. After 3 days, 750 ml of 10.0% (w/v) of glucose will be added into 750 ml modified media to achieve final concentration of 5% (w/v) in the schott bottle and it will be incubated further for 6 days. From day 1 to day 6, 150 ml of cultured will be harvested into each three (3) falcon tubes. This step will be repeated for pH 5 with 5.0% (w/v) of glucose and pH 6 with 10.0% (w/v) and 5.0% (w/v) of sago effluent.
3.6 Sampling biomass
The samples will be weighted in wet condition before dry in the oven. After that, the samples will be dried in the oven for 3 days. The dried mass will be taken and weighted again for dry weight.
3.7 Lipid extraction
Hexane: propanol in the ratio of 3:2 will be added into the falcon tubes consists of the dry mass. The mixture will be homogenized for 2 minutes. The homogenized sample will be incubated for 1 hour before centrifuge for 5 minutes. The supernatant will be taken and placed in an empty beaker and weight. The supernatant will be heated until the hexane and propanol solution have evaporated completely. The remaining oil will be weighted again. This step will be repeated for 5.0% (w/v) of glucose, 2.5% (w/v) of sago effluent and 5.0% (w/v) of sago effluent.
3.8 Phenol-sulphuric carbohydrate test
Phenol test is used to detect the amount of carbohydrate that is not consumed by L. starkeyi. For each sample, phenol-sulphuric carbohydrate test will be performed by adding 0.2 ml of 5% (w/v) of phenol and 1 ml of 96% (w/v) of sulphuric acid. After that, 1 ml from each mixture will be placed into a clean cuvette and read at 490 nm in a spectrophotometer.
By the end of this experiment, we expect to measure the amount of lipid produced by Lipomyces starkeyi mutants in 2.5% (w/v) and 5.0% (w/v) concentration of glucose and sago effluent at different pH.
Proposal writing and presentation
Bench work and sample processing
Data validation: Statistical analysis
Report writing and presentation
â-º: In progress
â- : End of progress
Ageitos, J.M., Vallejo, J.A., Veiga-Crespo, P.,