Sunday, December 30, 2012

Biochemistry Tutorial Essays: The Krebs Cycle

The Tricarboxylic acid (TCA), or Krebs’ Cycle, is the ultimate oxidation pathway for compounds such as sugars, amino acids, fatty acids, in most of the living organisms on Earth. It primarily involves the conversion of these nutrients to a form that can enter the Citric Acid Cycle, in this case Acetyl-CoA. Subsequently Acetyl-CoA is oxidized in a cyclic sequence of steps followed by the oxidative phosphorylation and electron transfer processes.

Acetyl-CoA production

Acetyl Coenzyme A is the major common metabolic product of several pathways, including the glycolytic pathway, the metabolism of fatty acids, and the cellular digestion of amino acids. Acetyl-CoA is basically a modified ADP molecule, having the external hydroxyl group substituted with a pantothenic acid residue, also connected to a β-mercaptoethylamine group. In the acetylated form CoA-SH is converted to a thioester, thus opening its possibility of being further oxidised in the cell.

In order to gain access to the TCA cycle, pyruvate undergoes conversion to Acetyl-CoA, a reaction that occurs when catalysed by Pyruvate dehydrogenase multienzyme complex (PDC). PDC acts as a sequence of enzymes whose action is corroborated altogether. It basically involves the reaction of pyruvate and CoA-SH to form Ac-CoA and CO2. By radioactive labelling of CO2 it has been shown that the reaction is irreversible as the labelled CO2 would not reconvert back to radioactively labelled pyruvate.

The PDC multi-enzyme presents a number of advantages when it comes to catalytic efficiency. First and foremost, as the reaction rates are generally limited by the frequency of enzyme-substrate collisions, the PDC has created the circumstances of reducing the distance between the substrate and the enzyme. In this way, once the substrate enters the multi-enzyme complex, it is very easy for it to be passed from one enzyme to another, thus enhancing the reaction rates.

The mechanism of the reaction involves three enzymes working together as integrant partd of the PD complex and five co-factors, four of which happen to be derived from vitamins. These are thiamine pyrophosphate (TPP), flavin adenin dinucleotide (FAD), CoA-SH, NAD and lipoate. The reaction by itself involvs three major steps and two regeneration steps.

The first step of the PDC reaction is the reduction of pyruvate to Hydroxyethyl-TPP•E1. In this step, pyruvate dehydrogenase(E1), a TPP demanding enzyme, decarboxylates the pyruvate into the formation of hydroxyethyl-TPP•E1. However, TPP•E1 does not release acetaldehyde. Instead, the hydroxyethyl-TPP•E1 is sent to the next enzyme in the complex, dihydrolipoyl tranacetylase (E2). The lipoamide group binded through an amide bridge to the lys residue of E2 reacts with the hydroxyethyl-TPP•E1. The outcome of the reaction is the regeneration of TPP and the conversion of the reactant into acetyl-dihydrolipoamide-E2, through an oxidation process.
The third reaction of the PDC series is the last major step of the reaction. The acetyl-dihydrolipoamide-E2 recently created is transesterificated. In thir reaction, the sulfhydryl group of CoA attacks the acetyl group of acetyl-dihydrolipoamide-E2 to form an intermediate that undergoes decomposition to Acetyl-CoA and dihydrolipoamide-E2.

Image from Wikipedia

The last two steps of the PDC reaction are regeneration steps in order to reconvert dihydrolipoamide-E2 to lipoamide-E2. With this view, dihydrolipoamide-E2 is first oxidised in a reaction at the expense of FAD, regenerated itself by its reaction with NAD+.

Experimental Evidence for the existence of the TCA Cycle

Since the elucidation of glycolysis in the early ‘30s, not much progress had been made in the field of glucose oxidation, especially while considering its conversion to carbon dioxide and oxygen uptake. However, what was already known was that several metabolic intermediates play a certain role in this process but it wasn’t still clear how do these reaction take place and how are they interconnected in the metabolic pathway that deals with glucose digestion.

Then, in 1935, the Hungarian researcher Albert Szent-Gyorgyi proved experimentally that small amounts of succinate, fumarate, L-malate, or oxaloacetate would determine a drastic increase of cellular respiration and oxygen uptake in finely cut muscle cells. He suggested that the compounds were converted from one to another according to the sequence: Succinate → fumarate → L-malate → oxaloacetate. In the same year, C. Mauritius and F. Knoop discovered another part of the tricarboxylic acid cycle, from citrate → cis-aconitate → isocitrate → α-ketoglutarate.

Since it was already known that α-ketoglutarate can in certain catalytic conditions be converted into succinate with the release of a molecule of CO2, what was necessary now, was to close the circle, to bring sufficient proof that oxaloacetate would undergo a certain reaction in order to convert itself into citrate. As Mauritius and Knoop demonstrated that the reaction is theoretically possible in non-enzymatic conditions (H2O2 in basic environment), Krebs formulated a hypothesis that these reactions would form a cycle. He sustained the hypothesis by arguing that the reduction of fumarate to succinate is inhibited by malonate, therefore a cyclic pathway could exist in order for this conversion to become possible, as predicted by his pigeon breast muscle respiration experiments. Moreover he inferred that this would also represent the main pathway for pyruvate oxidation in muscle given the reaction rates observed.

The Cycle and its steps

Once Acetyl-CoA has been produced, the tricarboxylic acid cycle can take place. As we are discussing the steps of a cycle, in the first step of the cycle, Acetyl-CoA will suffer condensation with the oxaloacetate produced in the last step of the cycle. The product of this initial reaction is citrate, also giving the name of this cyclic metabolic pathway.

The reaction is catalysed by citrate synthase, also known as citrate condensing enzyme. It is a dimer enzyme that binds Acetyl-CoA and oxaloacetate respectively in two steps. In the first step, oxaloacetate is binded to this enzyme in the deep cleft to be found at the junction between the two subunits that form the enzyme. Therefore, the enzyme is transformed through a conformational change from the open state in which it stood until right before the binding of oxaloacetate, to a closed state, moment in when Acetyl-CoA can also bind to the synthase. The order of the binding in this enzyme has been experimentally proven by the competitive inhibition of the enzyme by Acetonyl Co-A (CoAS-CH2C(O)CH3) or by carboxymethyl-CoA (CoAS-CH2COOH). By binding these two components, the enzyme catalyses an aldol Claisen condensation. Within the mechanism, the Acetyl- CoA enol formed is stabilised by a low-barrier hydrogen bond formed with the Hydrogen atom the Histidine residue found in the active site of the protein.

The last step of the cycle involves a reversible reaction whose equilibrium is displaced far to the left. Luckily, the reaction rate is sufficient to keep the cycle functioning as the continuously substance consuming step involving the synthesis of citrate keeps the oxaloacetate levels in the cell extremely low, generally less than 1 micromolar, creating the conditions so that this last step of the Krebs cycle to be complete.


The tricarboxylic acid cycle is largely regulated by substrate availability in the intracellular environment as well as by product inhibition. Moreover regulation is done by rate determining enzymes, which are, in this particular case of the TCA cycle, Citrate Synthase, Isocitrate Dehydrogenase and α-Ketoglutarate Dehydrogenase. As identifying a pathway’s rate-determining reactions demands the determination of the free energy of each reaction from the concentrations of its products and reactants, it is very hard to analyse the thermodynamics of the citric acid cycle as the concentrations of the intermediates involved are present in both mitochondria and cytosol in varying proportions. Nonetheless, if equilibrium between compartments is assumed, three reactions can be identified as displaced from their equilibrium points, may mean that these three reactions can be considered the rate-determining reactions of the TCA cycle.

Then, the TCA cycle is also regulated by the availability of NAD+ as there are 3 reactions that consume NAD+ and produce NADH. So, it would be reasonable to say that these reactions are regulated by NAD+ concentration. Moreover, four other reactions of the cycle are regulated through negative feedback loops by the concentration of NADH. Calcium ions also play a vitally important role in regulating the process by means of enhancing the reaction rates of the dehydrogenase reactions as it is an important cofactor in these reactions.

Nonetheless, maybe the most important regulators are oxaloacetate and Acetyl-CoA. Since the equilibrium of the reaction displaced so far to the left, discouraging the reaction to take place, it is essential that the concentrations of oxaloacetate are regulated so that the reaction can take place. In order for the reaction to be favoured, the reaction product concentrations in the cell have to be kept really low so as to promote the forward reaction and not the backwards one as predicted by thermo dynamical calculations. This is why the concentration of citrate is kept really low in cells, less than 1 microb Molar.

One of the most toxic small organic substances there is, fluoroacetate, is highly toxic not due to its native structure but due to the metabolic products in the body upon digestion. Since acetates are digested using an enzyme called acetate thiokinase, fluoroacetyl-CoA will be thus generated, this being further metabolized by citrate synthase intro fluorocitrate. The problem with this is that the C2 position is occupied by the fluoride atom and aconitase cannot catalyse the reactions in the cycle further-on. Since the fluorocitrate fits into the active site, we can thusly conclude that it’s a competitive inhibitor of citrate.

Additional Remarks

  • E1 is also regulated as it comes in two forms: active (dephosphorylated), inactive (triphosphorylated). The enzyme which catalyzes this rather unusual phosphorylation by inactivation is Pyruvate Dehydrogenase Kinase, which generated three ADP and the phosphorylated enzyme at the cost of 3ATP. This enzyme is at its turn catalyzed by the PDC products, or by the acetylated E2 PDC enzyme. In contrast, it is inhibited by the PDC products (Pyruvate, NAD+, CoA). In reverse, the phosphorylation process is reversed by the activity of PD phosphatase, hydrolyzing the enzyme in three loci corresponding to the serine residues undergoing phosphorylation. The process is enhanced by the presense of Mg and Ca ions.
  • The lipoic acid is covalently linked to a Lys residue in E2 and the lipoyllysyl arms transfer the metabolic intermediates between enzyme subunits.
  • Arsenic, in the form of inorganic arsenite (H2AsO3-) or the organic arsenicals (R-As=O) are toxic as they covalently bind to the dilydrolipoamide freeing water and blocking the function of PDC. With this view, organic arsenicals are more toxic to bacteria, reason for which it was used as an antibiotic in the early 20th century.

Monday, December 24, 2012

Biochemistry Tutorial Essays: Glycogen Metabolism (Glycogenolysis and Glycogenogenesis)

Glucose, a major metabolic fuels used by living systems is degraded through glycolysis with a view to yield ATP. Nevertheless, until oxidised, glucose cannot stay dissolved in cytosol. This is due to the fact that, if glucose were to be dissolved in cytosol, the molar concentration of glucose in cytosol would be 0.4M, severely tampering with the cells’ osmotic properties. Instead, living organisms have developed two major ways of storing sugars.

Plants store sugars in the form of starch, a mixture of α-amylose and amylopectin, a polysaccharide with branches in the structure every 24 to 30 residues. Instead, animals have developed a way of keeping sugars in their cells, by storing them as glycogen, a chemical compound similar to amylopectin, with the only difference that the branches are to be found every 8 to 14 residues.

Glycogen is insoluble in cytosolic compounds and is therefore maintained in cytosol under the form of small glycogen β-particles, composed of around 55000 glucose residues each, with about 2000 non-reducing ends. Every 20 to 40 β-particles are clustered into α-rosettes. This means of keeping the glucose in a polymerised form so that is basically insoluble in cytosol, reduces the concentration from the 0.4M calculated in the case when all the glucose were to be dissolved in the cytoplasm, to a mere 0.01 μM.

An interesting fact about glycogen is that it basically exists only in skeletal muscles and liver, the latter containing glycogen up to 10% of its mass, as opposed to skeletal muscles, where the total mass of glycogen does not surpass 1-2% out of the muscular mass. Glycogen is an important resource for the human body, as the body would use its glycogen deposits after the glucose levels in the bloodstream have dropped. In this way, the glycogen deposits in muscle can be depleted in no more than one to two hours of intense physical effort and no more than in 24 hours of fasting if were are to talk about the glycogen reserves stocked in the liver.

Glycogenolysis - Glycogen Breakdown

Upon breakdown, the glycogen stored in muscles and liver undergoes three major steps. Firstly, the glycogen chain is shortened one glucose molecule at a time by the use of an enzyme called glycogen phosphorylase, which essentially cleaves the inter-glucose bonds. As a result glucose-1-phosphate is released. The reaction will only take place if the glucose unit is at least five units away from the branching point.

The mechanism for the action of glycogen phosphorylase involves the maintaining of configuration for the glucose units. Therefore, it has been suggested that this occurs via a double nucleophilic attack mechanism, each occurring with inversion of configuration. Here, pyridoxal-5-phosphate plays an essential role as a cofactor. It binds to the Lys 679 residue of the enzyme and it is part of the active site of the enzyme.

The enzyme catalysing this reaction is a dimer that catalyses the controlling step in glycogen breakdown. It comes in two forms, phosphorylase a and phosphorylase b, with the only difference that phosphorylase a has phosphoryl groups esterified to Ser 14 in both of the subunits. The enzyme might assume the inactive form T or the active form R. The reason for the difference in activity between the R and the T state is that the inactive T state has an active site buried into the molecule, inaccessible for the substrate, whereas the R state has an accessible catalytic site and a high affinity for substrate.

In the R form, the enzyme may only be subject to allosteric regulation; however, it may not undergo phosphorylation. For this, the enzyme has to be converted into the inactive T form that can be phosphorylated through the use of phosphorylase kinase, which will take 2 molecules of ATP and convert them to ADP, simultaneous to the phosphorylation of the inactive form of the enzyme. The reverse reaction is essentially a hydrolysis reaction catalysed by phosphoprotein phosphatase. This reaction will, however, not restore ATP as is only releases inorganic Pi. The b form of the active enzyme will suffer allosteric effects from ATP and G6P in what concerns the inhibition of it function, whereas the same form will be allostericaly activated by AMP.

In this specific case, when the action of the phosphorylase enzyme is controlled by phosphorylase kinase, its actions are also highly regulated by the so called glycogen phosphorylase bicyclic cascade. Phosphorylate kinase is controlled at its turn by the activity of two enzymes: an activator, cAMP-dependent protein kinase, which phosphorylates and thus activates phosphorylate kinase, and an inhibitor, phosphoprotein phosphatase-1, which dephosphorylates and thereby deactivates both phosphorylate kinase and glycogen phosphorylase.

Similarly, phosphorylase kinase is also controlled by the concentration of calcium ions in the cytosol. As calcium is an essential metal present in muscle contraction, its action on the glycogen breakdown enzymes would be expected to be one of activation, and in reality, this is also the case. Virtually, a calcium concentration as low as 10-7 M can activate the phosphorylase kinase enzyme, which is subject to covalent modification and significant conformational change. The phosphorylase kinase enzyme is essentially a tetramer with each unit made up of four different sub-units, one of which has full catalytic abilities (γ) and three of which play regulatory roles (α,β and δ). Nonetheless, the δ sub-unit, also known as calmodulin, is sensitive to calcium ions. When calcium ions bind to any of the four calmodulin sub-units of the protein, these undergo extensive conformational changes that will have as a major outcome, the activation of the phosphorylase kinase enzyme.

The next step of glycogen breakdown is the conversion of G1P to G6P by phosphoglucomutase, a reaction that involves glucose-1,6-bisphosphate as an intermediate. The mechanism involves the phosphorylation of the C6 on the G1P by the enzyme, followed by rephosphorylation of the enzyme by the substrate, thus removing the C1 phosphoryl group. It is known that the dissociation of G1,6P from the enzyme deactivates the enzyme. Therefore, we can draw the conclusion that catalytic quantities of G1,6P are necessary for keeping the enzyme in the fully active state.

Glycogen debranching enzyme, the next enzyme in this reaction chain acts as a glucosyl transferase, by transferring an α(1→4) trisaccharide unit from a branch of glycogen to the non-reducing end of another branch, in order for the phosphorylase to pursue the cleavage of sugar-sugar bonds. The remaining residue in the branch to the main chain is hydrolysed by the action of the same debranching enzyme and is set free into the cytosol as glucose.

Glycogenogenesis - Glycogen Synthesis

Glycogen breakdown and synthesis are both important metabolic processes. But when the question whether or not they are the same process just in reverse is raised, two approaches are available to answer the question. From a thermodynamic point of view it is better to have different pathways for glycogen synthesis and degradation for two reasons. First, reactions catalysed by different enzymes can be independently regulated which allows for a very accurate control of glycogen flux. Secondly, both the pathways may be required under similar concentration of cytosolic metabolites, an impossible situation in the case of product/reactant concentration dependent reversible reactions.

From a medical perspective, it has been shown that in the case of McArdle’s disease, the muscle tissue presents no glycogen phosphorylase activity and is therefore unable to perform glycogen breakdown, although glycogen is present in the tissue. This shows that in the case of glycogen, breakdown of glycogen is not associated with synthesis, since in the case of this specific disease, synthesis occurs without breakdown.

The steps of the glycogen synthesis pathway were elucidated after the discovery of an important compound, relevant in the process, uridine diphosphate glucose (UDPG), a high-energy compound capable of spontaneous donation go glucosyl units to the growing glycogen chain. The reaction is catalysed by UDP-glucose phosphorylase. What happens, is that the phosphoryl oxygen on G1P attacks the alpha phosphoryl group of UTP to form UDPG and release PPi, which is further converted to 2Pi by inorganic pyrophosphatase.

The subsequent reaction is the actual synthesis of glycogen, by glycogen synthase. This is achieved by the transferring of the glucosyl unit of UDPG to the C4 hydroxyl group on a non-reducing end to form a glycosidic bond. The mechanism is thought to involve a glucosyl oxonium ion since the reaction is inhibited by 1,5-gluconolactone, a similar compound that acts as a competitive inhibitor for this enzyme.

Interesting enough is the fact that glycogen synthase cannot initiate the reaction between the first two glucose units. For this, the first step towards the synthesis of glycogen is catalysed by glycogenin, an enzyme that binds to a glucose residue. Glycogenin extends the chain to up to seven units, thus allowing glycogen synthase to take the process from here.

Glycogen branching, the last step of glycogen synthesis, is an important step as it confers the 3D branched structure of glycogen as we know it in all living animals and some of the bacteria. This reaction occurs as a result of the action of the amylo-transglucosylase, better known as branching enzyme, which is distinct from the glycogen debranching enzyme. Branches are formed by the transfer of terminal chain glucose residues, consisting of about 7 glucosyl residues to the hydroxyl groups attached to the C6 of glucose residues. Each transferred segment must come from a chain at least 11 residues long and it must be at least 4 residues away from other branch points.

Hormone Regulation

Hormones also play an important role in the regulation of glycogen breakdown or synthesis. We have seen that several proteins, controlling each other’s actions as enzymes regulate the glycogen phosphorylase reaction, the starting point of the glycogen breakdown process. We have also underlined the importance of calcium as a regulatory agent, in strict connection to its relevance to muscle contraction.

Nonetheless, hormones play an equally important role in the assertion of control on processes such as glycogen breakdown or glycogen synthesis. This has to do with the fact that the two fore-mentioned processes directly affect the concentration of blood sugars, especially that of glucose. Moreover, as the body has to respond very quickly to the fight or flight stress situations, the exertion of an action of adrenergic hormones is beneficial for the organism as it mobilises the glycogen deposits to be rapidly converted into fuels that can undergo the glycolysis pathway, such as glucose or G6P.

First and foremost, when it comes to hormones, the most effect on the liver is exerted by glucagon, while in muscles control is imposed by the action of epinephrine (adrenalin) and noradrenalin (norepinephrine). It has been shown in this specific direction that hormones act at cell surfaces to stimulate adenylate cyclase, thus increasing the cytosolic levels of cyclic AMP. When the cytosolic levels of cAMP increase, the cAMP-dependent protein kinase activity increases, thus increasing the rate of phosphorylation and decreasing the rate of dephosphorylation at the same time.

Because of the amplifying properties of the cyclic cascades, a tiny change in the concentration of cAMP would cause a major change in the levels of phosphorylation of different proteins and enzymes, thus explaining the major impact that hormones have on the body over such a short period of time. The opposite is also true, meaning that when cAMP levels drop, glycogen synthase is activated through the same process, just in reverse.

An important task for the liver to achieve is the maintaining of blood sugar levels around a constant 5mM, so that the brain will have sufficient fuels so as to keep its activity in normal physiological ranges. The process is mediates by glucagon and insulin, the first of which acts according to the following process: low concentrations of blood glucose cause the secretion of glucagon by α cells of the pancreas (the islets of Langerhans). Glucagon receptors on the hepatocytes surfaces respond to the presence of the hormone by activating adenylate cyclase that will increase the levels of cAMP slightly, so that the breakdown of glycogen would be favoured with a view to restore blood sugar levels into normal ranges.

When the opposite situation occurs, meaning that the blood levels are too high and homeostasis is not achieved, insulin comes into action. It is produced by the β cells of the islets of Langerhans. In interesting effect of insulin on glucose is that it greatly increases the rate of glucose transport across cell membranes so that the concentration of sugar in blood will encounter a drop significant enough to see a physiologic effect. It is thought that this mechanism would also inhibit glycogen breakdown, as glucose inhibits phosphorylase and binds to the active site of the inactive T form of the enzyme, but in a manner different from that of the substrate. This would cause a conformational shift so that the Ser-14 residues would be exposed and subject to dephosphorylation. Above a concentration of 7mM this process takes place, almost totally reversing the flux of glycogen metabolism. In this way, excess glucose can be stored as glycogen.

Image from Biochemistry, Voet & Voet, Wiley

Monday, November 19, 2012

Biochemistry Tutorial Essays: Glycolysis

I have been for almost seven weeks here at Oxford, an now that the first term is rapidly approaching its end, I would like to draw some conclusions and share some experiences from here.

As we center our study on biochemical essay writing, this term on carbohydrate and lipid metabolism, I thought I could share the experience, by sharing the essays and the essential aspects outlined in them as well as the key aspects of certain topics.

Essay 1: Glycolysis & Gluconeogenesis

In the first week, we were assigned an essay with the topic "How is glucose metabolized to pyruvate? Describe how the pathway can be reversed to accommodate gluconeogenesis in the liver, indicating the importance of key enzyme steps."

I began my essay as following:

Glycolysis, an essential process for cellular respiration, represents the main metabolic pathway of converting glucose into pyruvate. In this way, glucose, one of the most used biological fuels by living systems, is converted into a compound with lower energy potential, pyruvate. Also, by the means of glycolysis, essential metabolic intermediates of the process are supplied to the cells for bio-synthetic purposes.

Chemically speaking, glycolysis is a set of ten reactions that transform glucose, one step at a time, into pyruvate. The reaction involves ten metabolic intermediates, all of which offer access to entry points in the process for products of other metabolic reactions. This plays a key role in the metabolism of sugars as the intake of sugar in living organisms also consists of saccharides other than glucose.

Glycolysis is comprised by a set of ten chemical steps, seven of which take place near their equilibrium state, the other three being far from equilibrium. This fact is useful in the process of glycolysis as the process thus becomes irreversible in 3 major points of the process. By this means, not only metabolic intermediates resulting from glucose are stopped from being transformed into other intermediates previous in the chain, but also those from other metabolic chains are prevented from being converted to glucose or other intermediates, originating from glucose. In other words, the irreversible reactions ensure the unidirectionality of the process, an important mechanism in the cells’ efforts of producing energy in the form of high-energy phosphate molecules (ATP).

The first five steps of the glycolytic pathway are preparatory steps. They are also energy consuming, in the form of ATP, their aim being the consecutive phosphorylation of the glucose molecule followed by the cleavage of the molecule. In opposition to this, the last five steps, also known as the pay-off phase, return the ATP investment and generate high-energy phosphorylated molecules simultaneous to the degradation of intermediate products, which eventually leads to pyruvate.

The first step of glycolysis is the transformation of glucose to glucose-6-phosphate, by hexokinase, an enzyme responsible of transferring phosphoryl groups from high-energy phosphorylated molecules, such as ATP, to lower-energy acceptor molecules, in this case glucose. Nonetheless, hexokinase would also catalyse the reaction between ATP any other hexoses (D-mannose or D-fructose), as it is a rather nonspecific enzyme. With this born in mind, it is also important to notice the presence of more specific kinases, in the liver, such as the glucokinase, an enzyme only responsible for the conversion of glucose to glucose-6-phosphate with a view to maintaining the blood-sugar levels within normal physiological ranges.

The hexokinase enzyme, catalysing this reaction, will form a complex with glucose and the ATP-Mg2+ complex, both essential for the on-set of the reaction. A very important factor in the outcome of the reaction is the presence of the Mg2+ ion, as it will form a complex with two oxygen atoms placed on the two outermost phosphoryl groups of the ATP molecule. This way, the negative charges of the oxygen atoms will be shielded and nucleophilic attack on the phosphorous atom will be facilitated. Even more, the binding of glucose to the active site of the protein will create a conformational change of the protein, thus placing the hydroxyl on the 6th carbon atom of glucose and the external phosphoryl group of the ATP molecule very close one to another, making the reaction possible. Also, this prevents smaller molecules, such as water, that could cause the hydrolysis of ATP, to undergo this reaction as this would be energetically unfavourable for the cell.

The next step of the reaction-chain is the isomerisation of glucose-6-phosphate in order to be transformed into a metabolisable form. In this case, the reaction will be facilitated by an enzyme called phosphoglucose isomerase and will convert glucose-6-phosphate into fructose-6-phosphate. As the reaction is pH dependent it is believed that the mechanism of the reaction involves electrically charged side-chains, localized in the active site of the protein, that are bound to facilitate ring opening of the glucose derivative so that it can be isomerised. Subsequently, the Hydrogen atom α to the aldehyde group, slightly acid due to its location, will be absorbed by a basic side-chain (such as that of Histidine). The cis-enediolate thusly formed will rapidly suffer a replacement on the C1 carbon atom, simultaneous to the tautomeric shift of the C=O double bond to C2. This step is followed by ring closure and release of the newly from intermediate product: fructose-6-phosphate.

It is essential for the on-going of the glycolytic process that the multiple types of sugar (lactose, galactose, sucrose, starch, etc.) inherent to the nutritive intake of the cells be transformed into fructose-6-phosphate at this point in time, since this compound is the only phosphorylated monosaccharide able to undergo further chemical transformations along the glycolytic pathway.

In the third step, fructose-6-phosphate is converted to fructose-1,6-bisphosphate. This third step of the pathway is also determinant for the on-going of glycolysis, as it is also essentially irreversible under normal, physiological conditions and in the intracellular environment. This is due to the very large negative free energy of the reaction. It also represents a major point of regulation within the process of glycolysis, being influenced by the ATP/ADP& and fatty acid concentrations in the cytosol, a very significant issue to be covered later on in the essay.

Again, the course of this reaction is ATP and Mg2+ dependent, since the enzyme catalysing the reaction is also a kinase, called phosphofructokinase-1 (PFK-1). This is the second ATP consuming reaction, a necessary energetic investment in the glycolytic process.

The following reaction in the process is that involving the cleavage of the fructose-1,6-bisphospate molecule, caused by aldolase. The reaction products of this reaction are glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The reaction, basically the reverse of an aldol condensation, is a reversible reaction not necessarily demanding the presence of an enzyme as it can be catalysed in-vitro also by the OH- anion.

In order for the pathway to yield pyruvate, it is essential that the cleavage reaction take place between C3 and C4. This way, only products with a three carbon atom backbone will be generated in the cleavage process. Furthermore, this also explains why glucose-6-phosphate has to be converted to fructose-6-phosphate, as a carbonyl group on C2 and a hydroxyl on C4 are required for the cleavage to take place exactly between C3 and C4.

There are two classes of aldolases, able to perform this cleavage. Out of the two, Class I Aldolase is found in animals and plants, whereas Class II Aldolase is to be found in less evolved biological systems, such as fungi, algae and prokaryotes. It is thought that the reason for being more than one enzyme able to do this specific task is probably that both enzymes evolved early and separately in the evolution. Proof sustaining this theory exists, as some organisms have assimilated both types of aldolase, a physiological aberrance eliminated in the course of millions of years by natural evolutionary forces in most of the living organisms.

For Class I Aldolase, the mechanism of action includes five major steps, beginning with substrate binding. The active site of the enzyme shows one Lys and one Asp side chain. These play a very important role, as the carbonyl group of fructo-1,6-bisphosphate will condensate will the ε-amino group of lysine. A Schiff base will thus be created. Consequently, cleavage takes place simultaneously to the subtraction of a Hydrogen atom from the C4 hydroxyl group, which will become an aldehyde group for glyceraldehyde-3-phosphate, the triose produced in this cleaving step. The next step involves the tautomerization and protonation of the enzyme-Schiff base product, immediately followed by the releasing of dihydroxyacetone.
When it comes to Class II Aldolase, the enzyme does not form a Schiff base with the substrate. Instead, through stabilization of the enolate oxygen by the means of metal ions, such as Zn2+ or Fe2+.

Since the fourth step yields two products, only one of them can go to be further metabolized into pyruvate. Therefore, this fifth and final step of the preparatory phase of glycolysis is bound to convert dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. The reaction is catalysed by an enzyme called triose phosphate isomerase, which has been described in the literature as a perfect enzyme. This statement relies on the fact that the reaction occurs spontaneously and immediately on collision of the substrate with the enzyme, the reaction rate not being able to rise any further.

The sixth reaction of the ten reactions of the glycolytic pathway is rather special due to the fact that the esterification taking place shadows the real chemical process, the oxidation reaction. In this reaction, glyceraldehyde-3-phosphate is converted into 1,3-bisphosphoglycerate. The reaction happens only in the presence of NAD+ which is transformed into NADH. What happens is an initial nucleophilic attack of the SH group on the aldehyde group, followed by the formation of the thiohemiacetal. The thiohemiacetal undergoes further oxidation by the donation of a hydride ion to NAD+. By doing this, the previous thiohemiacetal becomes a thioester, which will undergo subsequent nucleophilic attack by inorganic phosphorous. In the end, the 1,3-bisphosphoglycerate thusly formed will be released by the enzyme.

Very important from an energetic point of view, step 7 is the first step to produce ATP instead of consuming it. This is achieved through a reaction catalysed by phosphoglycerate kinase. Essentially, the action of the enzyme here is opposite to the one of hexokinase in the first step of glycolysis: it removes a phosphoryl group that gets transferred to a new molecule of ATP, leaving only 3-phosphoglycerate as a product. Since the reactions can go both ways, this reaction is generating ATP. Moreover, since the number of moles of chemical substance has doubled ever since the cleavage of the bi-phosphorylated fructose, it is intuitive that for every glucose molecule that underwent glycolysis, two ATP molecules are generated in this step. It can be easily observed that the initial investment of two ATP molecules was fully returned.

In the following step, phosphoglycerate mutase plays a very important role in moving the phosphoryl group from the oxygen atom attached to C2 to the one on C3. The mechanism of the reaction involves phosphorylation of the C2 hydroxyl group, followed by decomposition with a view to form only the 2-phoshoglycerate (2PG) compound. The next reaction is transforming 2PG intro phopshoenolpyruvate (PEP). The reaction occurs in a magnesium rich environment. It is a basic elimination reaction that dehydrates the compound in order to yield PEP. This reaction is significant as there is a rate-limiting step to it: the OH elimination.

As an energy producing metabolic pathway, the last step also produces ATP, in order not only to return the investment of ATP, but to also gain energy from the molecules of glucose undergoing this pathway. For this to become true, phopshoenolpyruvate is converted into pyruvate with the aid of the enzyme pyruvate kinase. This is also a very important step as it is essentially irreversible and thus a regulatory step in the metabolism of glucose.

By analysing the free energy change of the 10 steps of glycolysis we can see two categories of reaction.

First of all, most reactions have varying ∆G’s, but close to 0, a fact that indicates that most reactions, take place close to equilibrium, a fact that is indeed true for seven of the ten reactions. Even though the standard free energy change ( ∆G°) of the reactions may not be close to 0, most of the reactions also depend highly on the concentrations of products versus reactants in cytosol. Therefore the free energy change can be calculated by means of the Gibbs free energy formula: ∆G=∆G°+RTlnKeq . In these circumstances, even some reactions that don’t have their free energy change close to null, given the concentration ratio of products and reactants (the Keq), they still are close to their equilibrium because their ∆G is null or close to zero.

Secondly, there are the reactions whose ∆G and ∆G° are far from 0 and fairly negative. This means that the products have lower energy than the reactants and the forward reaction is favoured if not compulsory as the reverse reaction is not physically possible in the normal, physiological activity of cells.

Finally, due to the overall gain in high-energy molecules such as ATP, glycolysis is an essentially exergonic process (more than -63 kJ/mol) as the initial reactant, glucose, loses its energy when converted into pyruvate, a molecule with much lower energy.


Gluconeogenesis is the process that takes place with a view to creating new glucose from organic chemicals like pyruvate, the intermediate metabolites of the glycolytic pathway, or even from gluconeogenetic amino acids (alanine). Generally, gluconeogenesis is an energetically expensive process run by the cells, especially when they are in need of glucose.

The gluconeogenetic pathway is opposite and similar to the glycolytic pathway, but still, not identical. This is due to the impossibility of cells to reverse the unidirectionality of glycolysis in key regulatory steps. So, gluconeogenesis has also developed means of literally by-passing the glycolytic pathway in those key points, that will be described further on in this essay.

The first by-passing point with the gluconeogenetic pathway is the conversion of pyruvate to PEP, a process that happens in two separate steps, catalysed by two different enzymes. The first step takes place in the mitochondria of cells. This step uses pyruvate obtained either from glycolysis, or from a transamination process if it comes from alanine.

Pyruvate is transformed intro oxaloacetate, by the reaction of pyruvate and acid carbonate, in the presence of pyruvate carboxylase. Other elements are necessary for the reaction to take place, such as the biotin co-factor, or the Acetyl-CoA positive effector. This step is activated when Acetyl-CoA is present in cytosol in concentrations high-enough, as Acetyl-CoA is the direct result of fatty acid degradation. This further means that fatty acids have been metabolized, making the generation of glucose a priority for the cell, so as not to run of chemical fuel.
After oxaloacetate is synthesized in the mitochondria, as there are no protein carriers for it, it is converted through a fairly reversible reaction into malate, driven out of the cell, and reconverted back to malate. Now, the second step of pyruvate to PEP conversion may proceed. In this step, oxaloacetate reacts with GDP, another high-energy molecule, and it gets phosphorylated while giving of carbon dioxide and GDP.

The second bypass mechanism involves the transformation of fructose-1,6-bisphosphate to fructose-1-phosphate, a hydrolysis reaction that occurs in the presence of fructose bisphosphatase-1. Being a simple hydrolysis reaction, the secondary product is inorganic phosphorous.

The same process happens with the conversion of glucose-6-phosphate into glucose, a process catalysed by the glucose-6-phosphatase enzyme, again, a hydrolytic enzyme. Still, an interesting issue about this last by-pass route is the fact that this reaction only takes place in liver and kidney cells. The glucose produced this way is only afterwards transported towards target cells such as muscle or brain tissue, unable of sustaining this process by themselves.

Gluconeogenesis is thus a very expensive process, costing the cells 6 high-energy molecules (4ATP and 2GTP molecules) for every glucose molecule produced. Due to the different reactions catalysed by different enzymes in the major nodes of these two pathways, it is clear that both glycolysis and gluconeogenesis are irreversible processes by themselves, made thermodynamically possible by enzymatic catalysis.


Homeostasis is an essential aspect of cellular function, and therefore, metabolism. Because of the fact that thousands of substances are being repeatedly anabolised and catabolised in the cells of all living beings, a strict control in the concentration of those substances is needed. This is very important, as any fast drop or increase in a particular substance could have great impact on the cells (damage or even cellular death).

This is also applied to glycolysis, the one of the processes responsible directly of maintaining the concentration of ATP within biological ranges. This is a difficult task to achieve, as ATP is the main fuel of the cells, hundreds of processes being linked to the usage of ATP: a major drop in ATP would cause the ceasing of most metabolic pathways, case in which the cell would probably die of starvation. This is why the glycolytic pathway is very important, and regulation of the glycolytic pathway is an essential task for maintaining the homeostasis of the sugar levels in the cell.

There are several enzymes involved in the control of allosteric feedback, such as hexokinase, phosphofructo-1-kinase (PFK) or pyruvate kinase. Any defect in function of any of these enzymes can limit the recycling of ADP to ATP, resulting in a progressively bigger loss of catalytic capacity.

The mechanism of allosteric regulation involves a binding site, different from the active site, where so called allosteric effectors bind into place, thus modifying the conformation of the enzyme. By this means, enzyme activity is increased of inhibited (non-competitive enzyme inhibition).

In the case of hexokinase, the allosteric inhibitor is actually the reaction product, phosphate-6-glucose. An elevation in the concentration of phosphate-6-glucose causes a drop the activity of hexokinase and vice versa, creating a direct negative feedback loop.

Also, a peak in ATP productions inhibits the reaction rates of hexokinase, PFK and pyruvate kinase. In contrast, rises in AMP give a signal that energy is needed into the cell; therefore the glycolytic processes are stimulated. The lack of high-energy molecules is suggested by the high concentration of AMP due to the fact that when ATP reserves run low, two ADP molecules can react and form an ATP molecule and an AMP molecule. In other words, the presence of AMP generally accompanies the depletion of ATP.

Some allosteric effectors are used for the sole purpose of regulation, not playing otherwise any role in glycolysis. For example, 2,3-bishphosphoglycerate (BPG) controls by a negative feedback loop the activities of hexokinase and PFK. Others are also part of the glycolytic pathway but are part of a positive feedback loop control system. Such an example is 1,6-bisphosphate that acts as a positive effector for pyruvate kinase.

Afterwards, in the tutorial itself, great importance and attention was paid to allosteric inhibitors and effectors. As the glycolytic pathway is very important for producing both energy and prime sources for other metabolic pathways, such as the TCA cycle, it it very important that it is very strictly regulated so that normal levels of blood sugar will be maintained (around 5 mM). It has been remarked therefore that certain enzymes in this glycolytic pathways, such as PFK, function in a similar way to hemoglobin. The substrate binding to one of the active sites of the PFK tetramer is causing a conformational change that would further cause the activation of the other sub-units. Therefore, the binding of substrate to the enzyme would represent a sigmoidal curve, rather than a normal Michaelis-Menten hyperbola. This is a very important observation as it influences enzyme kinetics, and thus the fate of glycolysis.

Much attention was also paid to the reason why the regulatory steps have to be unidirectional. According to Le Chatelier's Principle, in the case of a hypothetical regulatory enhancement of the reaction, the concentration of products would increase as opposed to the concentrations of reactants, causing a shift in the equilibrium of the reaction towards the left, thus canceling of diminishing the effect of allosteric regulation. For this reason, we concluded that only an irreversible reaction would be suitable for the regulatory steps.

Sunday, September 2, 2012

DNA Damage Repair

As a fresher in University starting this year, I have received a preparatory reading list useful for me to go through before arriving in University. All the books are due to cover the basic principles of Biochemistry, representing the course that I'll be reading at the University of Oxford, but also Organic and Physical Chemistry.

As I am currently reading a chapter concerning the flow of genetic information, I found out how the cell is responsible for the maintenance of the DNA, an essential feature without whom the cells would suffer chaotic mutations and potentially hazardous transformations.

It turns out, that there are two ways through which the DNA gets damaged. First, there is the danger of "misspelling" a nucleotide during replication. For this, the cell has a system of post-replicative mismatch repair that corrects the eventual errors appeared in the replication of DNA by polymerase.
During cellular division, DNA replication takes place at a speed of about 50 nucleotides per second, leaving almost no room for error. In other words, post-replicative mismatch repair is the "proof-reading" technique used by the cell. Incompatible pairs of nucleotides are identified, the parent strand is kept, while the daughter strand is cut and re-synthesised by DNA Polymerase.

With this "proof-reading" technique, errors still slip at a rate of about 1/107. Still, there are other factors that contribute to the damaging of the chromosomal information, such as UV degradation, normal cellular activity, hazardous chemicals, etc.

For instance, UV damage causes the dimerization of adjacent pyrimidinic nucleotides in a process as follows:

The formation of a Thyminodimer out of two T adjacent nucleotides

The dimers are removed and replaced by excision repair using a special protein (Excision Repair Nuclease) that identifies the affected area (by geometric abnormal conformation), excises the malfunctioning DNA, fills the gap using a polymerase and then joins the bits together with a ligase.

Another common form of degradation of DNA is the transformation tautomerization of nucleotides of by their natural degradation in the cell.

The transformation of Cytosine (C) into Uracil (U) as part of DNA Damage

In this case, the DNA strands are repaired by employing a technique called Base Excision Repair (B.E.R.). In the process the damaged base is first removed. Using the aid of an endonuclease, a gap is made into one of the two strands that are comprised within the helix structure. An exonuclease will enlarge the gap, also removing adjacent nucleotides in the process. Then, DNA polymerase fills the gap, and ligases bind the fragment into place.

These two extra mechanisms further reduce the incidence of errors in the genetic code to a rate equal to 1/109, which is 1 error in one billion replicated nucleotides, thus making the damage repairs 100 times more precise. As a matter of fact, the whole human genome is appreciated to be 3.2 billion nucleotides long, according to the study "Parameters of the human genome" by N. E. Morton, published in 1991.

In conclusion, given the 1/109 incidence rate of errors in DNA, every human has a rate of about 3.2 mutated or erroneous nucleotides per the entire genome, which is very little given the fact that all proof-reading is performed through only these three above mentioned mechanisms at incredible speeds.

Sunday, November 27, 2011

Camphor and Rivanol (Ethacridine Lactate) - An interesting reaction

Two days ago, while trying to clean a pimple with an antiseptic camphor solution, I accidentally used ethacridine lactate (Rivanol) instead. After realizing this, instead of first cleaning the rivanol stain and then applying the camphor, I directly cleaned it with camphor using a cotton pad. Suddenly a new white layer formed at the contact surface between the two layers of substance. The occurrence of a chemical reaction was obvious, and after repeating the experiment in lab conditions, I tried to understand the formation of this new product.

Camphor structure

Rivanol structure

As far as my chemistry knowledge could tell, camphor is a tricyclic ketone while ethacridine is a heterocyclic compound with two amino groups. Maybe, what happened was an amino-carbonyl condensation with the elimination of one molecule of water, I thought.

But then another question came to my mind. If so, which one of the two amino groups was the one responsible for the reaction? Which one would be more reactive? And last but not least, how would the nitrogen atom influence the occurence of the reaction?

What happened

Well, let's first consider all the possible occurring reactions. There are three main possibilities. One of them, the amino group on the eccentric ring would suffer an amino-carbonyl intermolecular condensation. The second scenario is the amino group on the heterocyclic ring undergoing the same process. Last but no least, the third possibility includes both groups reacting with camphor.

Scenario I

Scenario II

Scenario III

Obvious to me, was the fact that the heterocyclic group was more reactive than the eccentric one for two reasons. One of them is the fact that the inner ring is less aromathic than the outer ones, therefore more reactive.

The second reason involves the electronic effects applicable for the substance, more precisely the static inductive effects for the ethacridine lactate and the π electron displacements in the double bond of camphor.

Electronic effects in camphor

Firstly, we will take camphor. The massive hydrocarbon radical in C8H14C=O generates a rather strong -I (electron attracting effect) towards the Oxygen atom. This happens due to the fact that Oxygen is more electronegative than Carbon, having a tendency to attract electrons. Due to the same reason, the π bond of the double bond, composed of two electrons is attracted more towards oxygen. Therefore, oxygen develops a  δ- partial charge, while carbon is doing the opposite, accumulating  δ+.

Electronic effects in rivanol

While talking about rivanol, we must take two effects into consideration. One for the left outer ring, and one for the central ring. For the central ring, the positive charge of the Nitrogen atom attracts electrons greatly, therefore, a -I attracting inductive effect is felt by the heterocyclic ring. At the same time, the amino group charges with δ+ making it more electrophilic, thus more reactive.

On the other hand, the outer amino group undergoes the opposite process. The outer phenyl ring, has an aromathic nature, electrophilic by definition. In this case, a very small -I effect is affecting the -NH2 group, thus accumulating a small δ- partial charge.

Out of the two possible reaction loci, after the previous analysis of the effects in both substances, we can easily draw the conclusion that the locus on the inner ring would be far more plausible for the reaction to occur there, than the other one. In other words, probably, all three reactions occur, but the most probable one would be the one in scenario I as shown below:

The real preponderant reaction

The New Compound

The new compund was a precipitate, initially yellow in appearance, but only due to ethacridin lactate coloration in the test tube.

Before and after the reaction

In fact, after subsequent washing of the precipitate, the yellow coloration faded away almost entirely, what remained being only a very pale yellowish tone of white.

The washed and dried precipitate

After washing and drying, I grounded the precipitate into a very fine powder. Looking under a microscope to the crystals of the compound I found out that they are long, thin and tend to stick together in the form of a stack.
The crystals of the compound viewed in blue light under an optic microscope

Another image of the crystals

In the end, I would like to add a few more images of the compound:

Wednesday, May 25, 2011

Strepsils - A FLurbiprofen pathway to synthesis

Recently, I caught a cold which is why I took some pills (Strepsils) for sore throat. Curious by nature, I took a glimpse at the chemical composition of the medication. What I observed, was that the active compound in the pills was a substance called Flurbiprofen. Due to the phonetical resemblance to ibuprofen, a well known pain-killer, I made some research about its nature and its uses.

Not surprisingly, it resembles very much to (RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid (more shorter ibuprofen). The difference lays in the substitution of the isoproyl radical with phenyl and the hydrogen atom in ortho with a fluoride atom.

Firstly, I thought of a method to synthethise the compound from phenyl lithium and fluorobenzene as main building blocks, both of which had to be produced from benzene.


Fluorobenzene, was to be obtained by reducing the nitration product of benzene followed by diazotation. The diazonium salt was treated with tetrafluoroboric acid. To obtain the desired compound the reaction product was put to high temperatures.

Phenyl Lithium

The phenyl lithium, is more easily available. All is needed to do in order to produce it is to bromurate benzene in a FeBr3 environment and after this to react the product with fresh lithium well kept under petrol before the reaction.

The main synthesis

The two building blocks must be first reacted in a moderate to highly acid environment. This way, biphenyl is produced. If directly available, all previous steps may not be taken into consideration, instead the usage of a more pure biphebyl reagent would be preferred.

The biphenyl will then undertake nitration in ortho. This way, when we alkylate with cloroacetic acid, the acetyl radical will be prone to go into the para position. The nitrate is then reduced with an iron and HCl mixture, next following a process similar to that in the innitial synthesis of fluorobenzene.

It is crucially important to reduce the nitro group after the alkylation due to the fact that in the opposite case, the acetyl radical would go into the ortho position yielding a totally different compound. Finally, flurbiprofen is obtained.

Generally, the sore throat medicines are made of an antiseptic and of an antiinflammatory drug. The antiseptic will reduce the microbian flora in the pharynx, while the antiinflammatory will reduce the prostaglandines and thromboxanes production, the root cause of the painful inflammation.

Despite being used for curing sore throat, its uses exceed this limited applicability. Due to the mechanism of action, flurbiprofene makes possible its use for treating the pain involved in different arthritis types of disease, a major relief for the great number of people suffering from a reumatical condition.

Sunday, May 15, 2011

A Comparative Study of the Romanian Milk Brands

About a month ago, I participated in a national chemistry contest, ChimeXpert for which I had to make a scientific poster based on my personal observations regarding a certain area of applied chemistry. As a consequence, I tried to find a subject close to my chemical study at school at that given moment, proteins, which is why I have chosen milk as a subject of study.


The poster aimed to create an accurate comparative analysis of different Romanian milk brands' quality. The analysis was bound to answer to the need of information amongst the ordinary buyers. The analytical methods consisted in checking the compliance to quality standards of each dairy product selected.

Milk Facts

Milk is a complex liquid used as a food source by young mammals. Chemically speaking, the exact composition of milk varies from species to species, but it generally consists of water, fat, proteins and minerals.


Cow milk contains numerous types of proteins (~3.4%), classically divided into two groups:

Globular Proteins: albumins, lyzozime, lactoferin, all playing vital roles in providing immunity for the newly born.
Heteroproteins - Caseins (proteins containing phosphorylated groups)
Caseins are proteins which are made of up to almost 200 amino-acid residues. H2PO3- groups are attached to the serine residues, thus enhancing the reactivity of caseins towards calcium salts.

Caseins from milk come in many different sorts (αS1 - casein, αS2 - casein, k - casein, β - casein or λ - casein), all of which present coagulation properties at a pH = 4.6, except k - casein (due to a small number of phosphorylated serine residues in k - casein).


Raw milk has about 3.5% fats of which butyric acid is the most predominant fatty acid. It has been proved that butyric acid has anticarcinogenic properties, mechanism yet to be understood. Apart from butyric acid, milk also contains linoleic acids, octadecanoic acids and others as well.

Analytical Methods

For doing the study, I used six different brands: Milli, Zuzu, Rarăul, Covalact, Brenac and Tnuva. Two analytical methods have been used. The first one, is a quick but quite unaccurate method for analysing dairy products. It uses a device called LactoScan, based on the interpreting of ultrasonic resonance patterns of the milk being scanned.


The following charasteristics were measured: acidity, fat content, total dry substance (TDS), ash content (SOL), lactose content, proteic content, freezing point (crioscopic point) and density of the samples.

 The coulours indicate the quality of the product:
bad, unsatisfactory, medium, satifactory, optimum

It can be easily noticed that the 6 brands generally comply to the ISO 22000 standard, except Brenac, which has a potential value of 17% added water (so as to reduce acidity of old milk), which is illegal. Moreover, almost all values for Brenac are out of standards.

The second approach to this analysis was the classical method. Through this method, I determined the pH and the acidity (in Torner degrees), the calcium content, and the eventual counterfeiting with NaHCO3.


Acid milk, generally old, is not proper for general consumption due to the excessive lactic acid formed by the Lactobacillus acidophilus in the milk.

Therefore the pH of the samples was tested (3 weeks before the expiry date). Normal milk pH is around 6.5. The six samples had a pH varying between 6.17 and 6.58, both values of which are within standards.


Generally, milk forgery is done in order to trick the acidity tests. Although illegal, the producers sometimes add NaHCO3 or starch.

Testing for forgery with baking soda can be done by adding bromthymol blue. A normal milk would stay yellow-orange while a counterfeited one would turn green. All the samples have proven to be counterfeited with NaHCO3.

When testing for starch, after boiling for two minutes and adding vinegar (to enhance coagulation) and iodine (starch detectiong purpose), the counterfeited tubes should have turned blue (as in the M tube used for demonstration purpose only), all the test-tubes turning out not to have been counterfeited with starch.

Calcium ions detection

Calcium is determined through a rather complex method. First, we have to reduce milk to ashes by heating in an oven at 600oC for three hours.

The ashes are then incorporated into 50 ml solutions further brought to a pH af about 11.

Then EDTA (Ethylenediaminetetraacetic acid) is titrated into the milk ash solution in the presence of a few drops of murexide. The pink solution will turn purple.

The reaction involved in this mechanism is the following:

The calcium levels measured were at normal levels 800-1300 mg/L.


All the brands were more or less within standards, except Brenac, where severe disconcordances have been noticed. The best quality milk has turned out to be Covalact, respecting virtually all issues stipulated in standard with the exception of baking soda counterfeiting.


At the contest, I have won the best poster award and a special award from the Romanian Inventors Forum.

Finally, you can find the poster here, in the version presented by me at the contest.

Friday, November 19, 2010

BizCamp 2010 - Day 1

I am writing to share with you the experience I lived in the last two days, BizCamp 2010, a business camp for students. Here, through a series of workshops and sessions on the topics of success and how to achieve it, how to set goals, how to follow your dreams and many other personal development skills, I have met a whole bunch of extraordinary people and it was an incredible experience, really helpful for me.

Day 1

Day one was focused generally on the idea of reaching success. There were many speakers, each of them with a creative but very different style from the others. Among the ones who presented there was Alina Constantinescu, who talked about the importance of social media in gaining success and Mihail Musat who had some outstanding performances when being in front of the public.

She stated three principles that can lead to a success also determined by the personal network of relationships with the others. These principles are FIND OUT (be aware of the existing opportunities that surround you), CHECK OUT (show interest towards the others, create your network) and WORK OUT (get involved and be proactive).

Beside these steps, there were also some ideas that were outlined throughout the entire presentation:
  • Precaution can be dangerous when it doesn't make room for new opportunities.
  • When you start something new, begin with whatever you have and improve it later on in the process of building it.
  • The worst thing that may happen to you is not the lack of success, but the lack of anything happening to you.

Then, Mihail Musat, the founder of tried to send us the message that success is a relative term. Therefore, my definition of success may be totally different from yours and so on. Interpreted in another way, even failure may be a means of succeeding due to the fact that we all should learn from our mistakes.

Through some simple examples and stories from his own experience, he succeeded (of course from my point of view due to the relativity of the term :D ) in getting across some simple but important ideas:
  • Never be impeded by your own thoughts. Never try finding reasons for a thing I want to do but for a particular reason it is "not recommended".
  • What will I choose? A potential failure or the lack of a potential success? (no pain, no gain)
  • It is much more important to know what I DO want than what I DON'T want.
  • Do things that you are passionate about.
  • "Experience is not what happens to a man but what a man does with what happens to him" (make proper use of your personal experience)
After that, there came a series of workshops that emphasized the importance of a mix between passion, work and creativity in your way to success, three elements that make a difference.

There were also some funny parts of the day, like when we were the first in our country to see the new Coca Cola cold season commercial :)

In the end it turned out to have fulfilled our expectations and it was definitelly a really nice thing to take part in.

Coming next - BizCamp 2010 Day 2 :D

Monday, November 8, 2010

Barbeque Chemistry

Hello there! It's me again with a pretty unusual subject for this rather cold period of the year: barbeque chemistry. Why? Because it reminds us of the hot summer weekends when we attend outdoor barbeques and because I have read an interesting science paper regarding barbeque fuel tablets.

When we go out and make a camp fire we might use the traditional method of matches and wood or if we are more lazy, we could use those camp fire lighters such as liquid petrol or solid fuels for rapidly starting up a fire. Some are based on phosphorus while others rely on an organic compound, hexamethylentetramine, also called hexamine or urotropine and this is the compound I am going to talk in the next few lines.

Urotropine has a spatial structure, having the form of adamantane, the simplest diamondoid with the only difference that the four methine groups are replaced by nitrogen atoms. Therefore, the structure is the following.

Industrially it is synthesized from methane oxidized in air at 400 to 600 oC on nitrogen oxides so as to obtain formaldehyde. Four molecules of formaldehyde react afterward with six molecules of ammonia and urotropine is the result. The conditions under which the reaction occurs are temperatures high enough so as the formaldehyde and the ammonia to be in gaseous form.

The possible mechanism for this reaction might involve three steps. The first one would be an addition of NH3 to the formaldehyde. Therefore, NH2-CH2-OH is obtained. After this, the compound eliminates water and the aldimine CH2=NH is formed. Through trimerisation a cyclic compound is formed and further reacted with 3 molecules of formaldehyde and one molecule of NH3 will finally make urotropine.

The urotropine synthesized this way is the major product in the solid fuel tablets. They have the advantage of burning on any type of weather with a rather hot flame at about 760 oC. Moreover, these tables do not give off fumes due to the fact that the only gases released are CO2, N2 and vapors of water: (CH2)6N4 + 9 O2 → 6 CO2 + 2 N2 + 6 H2O.

Image from:

It is important to mention that urotropine is used also to treat urinary infections due to its activation by the acids in urine but also as a precursor to many explosives such as HMTD, a compound suspected to have been used in the London underground terrorist attacks in 2005.

All in all, it's highly useful but it can turn out to lead to dangerous products so it is important to be used with care.

Thursday, November 4, 2010

Colorant Synthesis

Hello there once again! I have recently started a new chapter in my preparation for the Chemistry Olympiad, the azo compounds, very important for the production of azo colorants, mainly used as food colorants but also as dyes and other kinds of pigments. I was solving some synthesis problems until I got stuck at the synthesis of Naphtol Blue Black (structure shown below) from inorganic and organic substances with no more than two carbon atoms.

From the very beginning it was clear to me that I first had to make the naphthalene ring. Therefore I started from acetylene and made the necessary trimerisation in order to obtain benzene. Next, I used a Friedel- Crafts alkylation and obtained o-diethylbenzene, which through severe dehydrogenation at 400-600 oC yields naphthalene.

It is important now to arrive at the H-Acid structure, also called 1-Amino-8- Naphthol-3,6-Disulfonic Acid. In its synthesis it is vital to leave the formation of the amino group at the end so as not to be involved in any unwanted reaction made possible by the increased sensitivity of amines.

In its formation, firstly the naphthalene is nitrated and then trisulphonated. The third acid sulphite group in the α position is used for creating the -OH group through a process known as alkaline melting. The rest of the groups do not undergo this process because they are in less reactive β positions. Nevertheless, the other -SO3H groups suffer neutralization with NaOH and form ionic bonds between oxygen and sodium, thus replacing the hydrogen atom.

Finally the -NO2 group is reduced on iron and hydrochloric acid using 6 [H], produced by the following reactions:

Fe = Fe 3+ + 3 e-
HCl = H+ + Cl-
H+ + e- = [H]

The H-acid is ready. Now comes the tricky part where I got stuck. You now have to do the coupling, but there is the problem of the lack of symmetry within the compound.

The preparing method for the diazonium salts to be used in the coupling reaction. Firstly a nitration followed by reduction on Fe/HCl and then diazotation with NaNO2 and HCl after the reaction:
Ar-NH2 + NaNO2 + 2HCl = Ar-N+≡N]Cl- + NaCl + 2 H2O

If you couple in normal conditions a diazonium salt to our naphthalenic ring there will be two problems. First, it won't couple into the β position, α being definitely preferred by the reaction itself. Second, even if you succeed in coupling a diazonium salt into the β position, it will immediately occupy both β carbons.

But there is one thing that makes the H-Acid so suitable for this multiple coupling. On one of the rings in the naphtalenic cycle, we have phenolic -OH, while on the other we have an amino group -NH2. We can clearly see the difference, one is moderately alkaline while the other is acid.

This does matter because in the coupling we have to choose between acidic or alkaline environment. Therefore, the coupling near the alkaline -NH2 group will take place in acidic conditions whereas the coupling next to the phenolic -OH in an alkaline environment.

The first coupling in acidic conditions

The second coupling in NaOH environment and the final product

A sample of the colorant. Image from

Finally, the synthesis is done and we have our Naphtol Blue Black colorant. I considered this useful enough to be posted due to this special property of the H-Acid dictated by the presence of the two different groups that influence drastically the course of the reaction and because it is quite complex enough to bring in front an element of challenge. I'll also try to make it in the lab although I'm not sure I will succeed, being a quite difficult synthesis. See you next time!