 For the moment, a biochemist show a weak poorly nourished shot of good health as chemical balances within individual cells are brought to normal. How do we know of these chemical balances? How is it that the daily lack of a microscopic amount of vitamins or minerals can bring about such a departure from the normal? In a laboratory demonstration of intracellular balance and imbalance, the vesicant wargas is used. The arsenic of the lewisite throws out the chemical balance inside the cells of the skin. British anti-lewisite compound was fashioned specifically to inhibit the action of the lewisite and maintain the balance in spite of exposure to the wargas. This compound frees the enzymes of the skin cells from inhibition by the arsenic of the lewisite. Within 24 hours, the severe vesicant effect of a minute application is clearly visible on the unprotected arm. The BAL has effectively prevented vesication on the other arm. BAL is a drug made with a molecular structure designed to perform a specific action inside the cell. The search for new knowledge of the chemistry of life goes on with and beyond the microscope. The individual cell, once considered the ultimate unit of the living body, is now the continent into which the explorers of science are penetrating on their expeditions to map the pathways of molecular change. Many of the secrets of human health and well-being are located along these chains of chemical changes. This scene, the totality of glucose metabolism, is too complex to be grasped at once. We shall soon dissect it and describe the details of each part. Fluency in the language of the chemist is essential to travel inside the cell. Research at the intracellular level is complex. Compare glucose metabolism to the simple burning of glucose outside the cell. Outside, the direct union of oxygen and glucose produces simultaneously carbon dioxide and water and energy in a single burst. Inside the cell, glucose is broken down and energy is released during a complex series of chemical events. These changes provide the energy of life in useful small amounts. Our story starts with glucose. 6 carbon atoms, 6 oxygen, 12 hydrogen. When joined in a particular pattern, these spheres represent glucose. Glucose is first built up for the breakdown through combination with energy-rich phosphate, forming glucose-6-phosphate. A shift of position, more phosphate here. And we have fructose-1-6-diphosphate. And the pathway, which is in truth a chain of chemical changes, begins to be revealed. What organizes and directs these changes? At each step, what catalyzes the change? Enzymes, a special one at each point of change. Louis Pasteur, in saving the wine industry of France, related the Ventner's art to the science of chemistry and contributed to our knowledge of enzymes. Pasteur believed that life itself was a constituent part of enzyme action. His work drew the first faint lines of the modern concept of enzyme action inside the cell. Edward Buchner added to the growing knowledge. By grinding yeast with sand and Kieselgauer and squeezing out the juice with a hydraulic press, he produced a cell-free extract. Chemical compounds, not life, are within these droplets. Dr. Buchner demonstrated that the cell-free liquid retains the enzymes of alcoholic fermentation. Many years later, Dr. J. B. Sumner, working at Cornell University, isolated and purified the first enzyme in crystalline form. Dr. Sumner used a 32% acetone solution as the solvent to extract urease from Jackbean meal. The mixture of Jackbean meal and acetone is filtered. The extract contains the enzyme which crystallizes during refrigeration. The next day, these crystals are centrifuged out of the extract. In the next step, water dissolves the crystals but leaves the impurities undissolved, ready to be separated from the liquid. Centrifuging a second time throws out impurities and leaves the dissolved urease undisturbed in aqueous solution. This time, the supernatant fluid is the important part and the precipitate is discarded. Citrate buffer and acetone are added. During a second night in the refrigerator, the crystals form again. It was in 1926 that Dr. Sumner prepared to view the first isolated enzyme crystals through the microscope. Twenty years later, in 1946, the Nobel Prize in Medicine was awarded for this achievement. Dr. Sumner had fashioned a keystone of the enzyme theory, purified urease crystals. In 1926, there was only one. By 1940, 17 different enzymes had been separated and crystallized. Today, the total stands at well over 30. Infrocellular enzyme molecules usually consist of a large protein part, here represented as a cloud-shaped structure which activates the substrate and a second part called the coenzyme. The current hypothesis is that the coenzyme is the working part. In this step, carbon dioxide is split from pyruvic acid. This coenzyme is composed of the familiar vitamin B1 and two phosphate molecules. The complex protein portion of the enzyme is specific, as a structure into which the pyruvic acid just fits. This protein and the coenzyme together are an oxidative decarboxylase. This enzyme is essential in glucose metabolism. Located at each point of change are other enzymes, many of which have vitamins or minerals as essential parts. For metabolism to proceed normally, complete enzymes are necessary. Specifically, lack of vitamin B1 affects these decarboxylated steps. When the diet is deficient in vitamins or minerals, incomplete enzymes slow metabolism. Less of the energy of life is produced. The resulting symptoms are familiar. Avitaminosis, the doctor calls it. Avitaminosis, deficient enzymes and slow production of life's energy. Small amounts of essential chemicals restore enzymes and speed energy production. The diet must continue to provide vitamins and minerals for continued health because like all molecules, enzymes and vitamins are themselves broken up in other steps of metabolism. Though all steps of metabolism go on together, we study our chart in areas. The cytochrome system, glycolysis, the Krebs cycle and the energy cycle. The energy cycle shows a few units of energy put into metabolism and a great many of these units emerging. The excess is energy freed from the glucose molecule. About half of the energy in glucose takes the form of heat. The other half goes into special high energy phosphate bonds. This phosphate energy keeps the cells and the body alive and active. It is the cells way of capturing some of the glucose energy and making it available for life activity. Glycolysis and the Krebs cycle can be thought of as cellular devices for generating these phosphate bonds from the energy of glucose and other foodstuffs. The cytochrome system is a sort of right hand man, aiding the energy producing sections. In glycolysis, twice as many phosphate bonds are generated as are invested. The very first step requires phosphate energy. When phosphate attaches to glucose, glucose-6 phosphate is formed. Rearrangement of the molecules yields fructose-6 phosphate. Now a second energy-rich phosphate is used to give fructose-1-6 diphosphate. So far it has been all investment and no return. This six-carbon molecule now splits in two, yielding two triose-phosphate molecules in equilibrium, dihydroxyacetone phosphate and glyceraldehyde-3 phosphate. The glyceraldehyde-3 phosphate is changed by the addition of inorganic phosphate to some unstable diphosphate, which is simultaneously oxidized and the investment begins to pay off. Oxidation. The name suggests oxygen addition, but oxidation is actually the loss of electrons. Inside the cell, the loss of electrons most often involves the removal of hydrogen, or dehydrogenation, not the addition of oxygen. Dehydrogenation can be demonstrated not only with little imaginary spheres, but with actual chemical substances. The necessary enzymes are provided by rat brain homogeneate, which is placed in the side arm of a Thunberg tube. Gysceraldehyde-3 phosphate, buffer inorganic phosphate and methylene blue are in the main compartment of the tube. The biochemist knows that methylene blue will decolorize when hydrogen combines with it. Oxygen is removed from the tube with a high vacuum pump. During the removal, the enzyme and substrate remain apart. When the enzyme and glyceraldehyde phosphate are mixed, oxidation begins. The hydrogen atoms join with the methylene blue. The methylene blue is no longer blue. The glyceraldehyde phosphate minus its hydrogen atoms is now 1-3 diphosphoglyceric acid. The enzyme which does this is called triose phosphate dehydrogenase. Its coenzyme is the molecule DPN, which contains a derivative of the B-vitamin niacin. It is the DPN which removes the hydrogen, becoming DPNH2. Then it attaches to a second enzyme and transfers the hydrogen to the methylene blue. The coenzyme is free to repeat. What does the DPN find inside the cell ready to accept the hydrogen atoms? Methylene blue is merely an artificial dye used in laboratory experiments. Pyruvic acid accepts the hydrogen inside the cell. In the absence of oxygen, the hydrogen is transferred to the pyruvic acid. This is the way it works in such cells. The DPN, after taking off the two hydrogens and becoming DPNH2, attaches to another enzyme. Then it switches the hydrogen onto pyruvic acid, changing it to lactic acid. Lactic acid production continues as long as there is pyruvic acid in the presence of the two dehydrogenases and DPN. The DPN continues to transport the hydrogens as long as there is pyruvic acid to accept them. The system is self-sustaining. Pyro's phosphate is oxidized here as DPN transports the hydrogens. Through successive steps, pyruvic acid is regenerated. Here are these steps in detail. Oxidation results in one-three diphosphoglyceric acid. Now a high-energy phosphate bond is split from the acid. The remaining phosphate moves to another part of the molecule. It now loses water and becomes phosphopyruvic acid. At this point, the second high-energy phosphate leaves, and we have again pyruvic acid. In this way, glucose is changed to lactic acid. This is glycolysis. We owe our knowledge of glycolysis to men like Dr. Otto Meyerhoff In his present laboratory at the University of Pennsylvania School of Medicine, he demonstrates that DPN and the high-energy phosphate of ATP are essential in glycolysis. Three Warburg blasts have been prepared with Ringer's solution and sodium bicarbonate before brain extract containing the enzyme is added. Blasts 2 and 3 also contain glucose and a bit of fructose 1,6 diphosphate. The side arms of blasts 1 and 2 contain a mixture of DPN and ATP. Each flask is attached to a manometer, and air is replaced by a mixture of 95% nitrogen and 5% carbon dioxide gas. At Dr. Meyerhoff's left is control, which reveals fluctuations of temperature and barometric pressure. During 10 minutes of preliminary agitation in the water bath, temperature equilibrium is established. Manometer levels are set at a uniform mark. Then the ATP and DPN mixture is tipped in from the side arms of blasts 1 and 2. This starts the reaction. Any acid formed in the blasts reacts with the bicarbonate to release carbon dioxide gas. After 20 minutes, only flask 2 with ATP, DPN, and glucose shows the result of glycolysis, a large carbon dioxide pressure. There is no glycolysis in flask 3, which contains glucose, but no ATP and DPN. Flask 1 shows some positive CO2 pressure. The acid which built up the slight pressure has come not from glycolysis, but from the breakdown of ATP, forming inorganic phosphate and adenilic acid. Without glucose or ATP, the first step is not taken. Without DPN, the oxidative step of glycolysis cannot occur. Dr. Meyerhoff precipitates the enzymes with trichloroacetic acid. This stops the reaction. The precipitate is separated out. Then the supernatant fluids derived from flasks of corresponding numbers are tested for inorganic phosphate. In the supernatant from flask 1, where ATP breakdown occurred, much inorganic phosphate is shown. In tube 2, where glycolysis occurred, the phosphate of ATP was transferred to glucose. Thus no inorganic phosphate is shown. Tube 3 shows a little inorganic phosphate, probably from the tissue extract. During the first few steps of glycolysis, two high-energy phosphate bonds are invested. At this step, two triosphosphate molecules are formed. These molecules take turns going down the ladder. Two high-energy phosphate bonds are generated from a single molecule. Now the second triosphosphate changes and starts down the ladder. Two more energy units are released from this second part of the original glucose molecule. So in glycolysis, the net gain is 4 for 2. This seems like a good return, but actually it is less than 4% of the energy inside the glucose molecule, hardly enough for the continued existence of animal cells. However, in the presence of oxygen, the cytochrome system transports the hydrogen atoms and electrons. This is a much more efficient system than glycolysis. In the first step, the hydrogen is removed by DPN and carried to the enzyme cytochrome C reductase. Here, hydrogen separates into electrons and positive ions. The ions combine temporarily with cell buffers and the electrons with cytochrome C. The electrons reduce the iron of the cytochrome C. Then an enzyme, cytochrome oxidase, combines electrons, ions, and oxygen producing water. The cytochrome system transports hydrogen not only from the early oxidative step, but from the five which follow pyruvic acid breakdown. In the Krebs tricarboxylic acid cycle, about 50% of the energy of glucose is made available for use by the cell. It is in the Krebs cycle that the carbon dioxide of cell respiration is formed. 3 times CO2 is chipped from a molecule. The first time is at pyruvic acid, a 3-carbon acid which is simultaneously oxidized. Now we have a 2-carbon acid. This probably combines with the 4-carbon oxaloacetic acid and the result is the 6-carbon cis-aconitic acid. When this is hydrated, isocentric acid is formed and this is oxidized, forming oxalo-succinic acid. From this, another CO2 is chipped, resulting in alpha-ketoglutaric acid, and then the third CO2 is chipped off. Oxidation has occurred at the same time and succinic acid results. The succinic acid is oxidized to fumaric acid and the fumaric acid hydrated to malic acid. Malic acid is oxidized and a molecule of oxaloacetic acid is regenerated. Yet again combines with the 2-carbon acid to form cis-aconitic acid and the cycle is started again. Carbon dioxide production is demonstrated with the Warburg Manometric Apparatus by Dr. Severo Ochoa of New York University College of Medicine. Into the main compartment of the flask go the tissue preparation containing the enzyme oxalo-succinic carboxylase and other components. The oxalo-succinic acid is placed in the side bulb. This flask contains all components except the enzyme. The one already on the apparatus is a barometric and temperature control. The next flask contains a small amount of the tissue preparation containing the enzyme. The flask on the right, number 4, contains twice as much enzyme as flask 3. The reactants are brought to a constant temperature 15 degrees centigrade in this demonstration. Chipping in the substrate starts the reaction. The enzyme greatly accelerates the chipping off of carbon dioxide from the oxalo-succinic acid molecule. The control has remained stable. In flask 2, without the enzyme, there is little or no reaction. In flask 3, CO2 production is apparent. The manometer of flask 4, which contains the greatest amount of enzyme, indicates the greatest amount of carbon dioxide production. In the Krebs cycle, decarboxylation occurs three times. There are five oxidative steps. Dr. Ochoa shows us one of these. He demonstrates the enzymatic oxidation of isocentric acid with the Beckman spectrophotometer. Quartz cells are used. One is prepared with all components except the enzyme. The other contains all reactants, including the enzyme. They are placed in the spectrophotometer in a movable holder. The ultraviolet light source is set at a wavelength of 340 millimicrons. The substrate, isocentric acid, is added to the mixture in both cells. Only when the beam of light is directed toward the quartz cell containing all components, including the enzyme, is ultraviolet absorbed. The needle shows that DPN has accepted hydrogens. Before oxidation takes place, the DPN of the isocentric dehydrogenase does not absorb ultraviolet. It acquires this ability when it combines with the hydrogen of the substrate. The exact number of high energy phosphate bonds liberated in the oxidative steps of the Krebs cycle is not yet known. There are probably about 30. This is equivalent to perhaps 50% of the total energy in the glucose molecule. It is sufficient to meet the high energy demands of animal cell activity. Most of this energy generated by glucose metabolism goes to the adenillic acid system. Beginning with adenosine monophosphate, it is converted first to diphosphate and then triphosphate ATP. In the presence of the proper enzymes, the terminal phosphate is split from the ATP and begins performing its role in life processes. It phosphorylates glucose and primes metabolism. It phosphorylates vitamins to form coenzymes. It phosphorylates smaller molecules, which synthesize larger ones. The energy of ATP makes possible muscle activity. It is probably the source of energy for nerve conduction, kidney tubule activity, intestinal absorption, and other functions. The supply of ATP in any cell is limited. But there is another molecule, creatin phosphate, which acts as an emergency store of energy-rich phosphate. When more ATP is being built than used, creatin is converted to creatin phosphate. Then when ATP is being used more rapidly than it is being built, the creatin phosphate breaks down, making high-energy phosphate available to the cell. The cell has also a mechanism of storing small glucose molecules by combining them into large glycogen molecules. When there is an abundance of glucose, some of the glucose-6 phosphate is converted to glucose-1 phosphate. Now the enzyme phosphorylates adds the glucose-1 phosphate to a glycogen molecule already present. A second is added, and then a third. One after another, molecules of glucose-1 phosphate are added. A straight chain is formed until a branch occurs. Again, straight chains and branches. And branches and straight chains. The resulting polysaccharide is glycogen. Doctors Carl Corey and Gertie Corey, 1947 Nobel Prize winners in medicine, demonstrate glycogen synthesis. Glucose-1 phosphate, buffer, and a trace of glycogen are in all tubes. A solution of crystalline phosphorylate is added to two of the tubes. One tube also receives branching factors. Fired in addition reveals polysaccharide synthesis. Tube one, no enzyme, no polysaccharide. Tube two, with phosphorylate, the blue color of the straight chain starts. Tube three, with phosphorylate and branching factor, the brown color of glycogen. Glycogen breakdown later requires less high-energy phosphate than does glucose breakdown. The glycogen is changed to glucose-1 phosphate. This is changed to glucose-6 phosphate, which can enter the metabolic steps leading to the release of energy. In the liver, glucose-6 phosphate can be changed to glucose, so the steps from glucose to glycogen can proceed in either direction. The same has been shown to be true of almost all the other steps. Under the proper conditions, glycolysis may reverse. Almost all of the Krebs cycle has been shown to be reversible. Such reversals will not produce energy. On the contrary, energy is consumed. This is the path of glucose metabolism. The pathways of protein and fat metabolism are being drawn. All three paths are interconnected inside the cell. Amino acids of food proteins can produce pyruvic acid, alpha-ketoglutaric, and oxaloacetic acids. The fatty acids of fats can produce the two-carbon acid, which combines with the oxaloacetic acid. The Krebs cycle is common to the paths of metabolism of all three foodstuffs. Through it, fat and protein, as well as carbohydrate, can be oxidized, and some of their energy made available to the cell as energy-rich phosphate bonds. This chart helps us to understand how one foodstuff can be converted to another inside the cell. Carbohydrate to fat, protein to fat, or protein to carbohydrate. The integrated activity of enzymes catalyzes these molecular changes, which release and make use of energy inside the cell. Now that these pathways have been drawn, now that the changes going on within the cell are known, the enzyme content of tissues can be determined without troubling to isolate the enzymes. We can find the specific points of action of hormones, vitamins, genes inside the cell. Enzyme changes in disease can be plotted. The diabetes story is beginning to be understood. Insulin and pituitary and adrenal hormones affect hexokinase, the enzyme functioning in the very first step of glucose metabolism. Work with the isolated enzyme system in Dr. Corey's laboratory showed hexokinase to be inhibited by anterior pituitary extract and in diabetic animals by adrenal cortex extract. These inhibitors are overcome by insulin. This can be demonstrated with intact diaphragm muscle. Dr. Corey and Dr. Creil prepare to test the uptake of glucose from solution by diaphragm. They use this as the measure of hexokinase activity. Glucose is pipetted into three of the flasks. The other receives glucose plus insulin. Sections of rat diaphragm are weighed. A normal animal furnished the muscle placed in one flask. Another receives diaphragm from an adrenal-actamized animal. Alloxand diabetic muscle is in the other two flasks. This is the type placed in the flask containing the insulin. After incubation, samples of solutions are removed and tested for the amount of sugar remaining. The intense blue of the control on the right shows the amount of sugar added. The muscle of the normal animal in the next tube used up the sugar rapidly. That of the diabetic animal, the middle tube, used almost none at all. The addition of insulin in the next tube lifts sugar utilization almost to normal. The adrenal-less diabetic muscle in the tube at the left uses sugar at practically the normal rate. Through knowledge of enzyme action, we are learning how insulin relieves diabetes. Knowledge of enzymes of tumors may help in the continuing fight on cancer. Dr. Van R. Potter at the University of Wisconsin Medical School assays normal and cancer tissues. The Warburg flasks are prepared. Cytochrome C is pipetted. Neutralized succinic acid is the substrate. Oxygen consumption, not CO2 production, is to be measured. So the carbon dioxide produced in metabolism must be absorbed. To do this, alkali is placed in each center cup. Filter paper placed in the center cups with the alkali provides a larger surface for carbon dioxide absorption. The tissues to be assayed were very recently removed from a normal and two cancerous rats. Tissues are individually weighed and placed in the glass homogenizer tube. A 5% homogenate in water is made. The homogenates made from kidney, liver, and heart tissue of the normal animal and from two cancers are the subject of the test. Three flasks are prepared with normal tissue. Two with cancer tissue. The Flexner-Jobling tumor and Walker 256 tumor are used. The manometers will reveal oxygen consumption. The limiting factor is succinic dehydrogenase, a vital enzyme of the Krebs cycle. The thermobarometer is at the right. After 30 minutes, the manometers show that the three normal tissues on the left consume a relatively large amount of oxygen while the two tumors use very little. In cancer, then, the Krebs cycle seems slow, producing less energy. An important lead that must be followed up. The fight against disease continues, with weapons directed more and more rationally at the biochemical events inside the cell.