Biochemistry

From FETAL PRECURSOR CELL TRANSPLANTATION (FPCT)
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An important benefit of fetal precursor cell transplantation is the restoration of normal digestion and metabolism, i.e. improved function of the entire digestive system, and harmonization of the regulation of metabolic function of all cells of the body, as one of the key requirements for regeneration of damaged organs and tissues.


Digestion and metabolism are enzymatic events. Enzymes increase the biochemical reaction rates by at least a factor of one million. An enzyme is a protein that takes a molecule of a substrate and converts it into a product molecule by catalytic action. Enzymes usually work in series, in pathways, or cycles, so that the product of one enzyme is used as a substrate by the next enzyme to produce a new product. In turn, this new compound is used by the third enzyme to yield a third product. This occurs because each enzyme catalyzes a specific reaction, and usually a string of different reactions is required to generate the desired end product.


When a product of chemical reaction is more complex than the initial substrate that entered the chain of reactions, such sequence of enzymatic reactions is called anabolic, i.e. conversion of glucose molecules into a long-branched polymer called glycogen, assembly of polypeptides and proteins from amino acids, synthesis of lipids from fatty acids and glycerol. All such reactions require energy provided by the molecule of adenosine triphosphate, ATP.


Similarly, larger molecules can be taken apart by a sequence of specific enzymatic actions to produce smaller, simple molecules. Such pathways are usually associated with the production of energy via generation of energy storing molecule of ATP. Such sequences of enzymeatic actions involved in the breakdown of substrates are called catabolic, i.e. breakdown of glucose molecule into a smaller pyruvate, that is then converted into acetyl-CoA, breakdown of amino acids, or nucleotides, from disassembly of nucleic acids, or breakdown of fatty acids.


All catabolic pathways contribute to the production of acetyl-CoA, that can be used in further catabolic sequence of Krebs cycle, in which CO2 is released, and energy is conserved with the formation of reduced co-enzymes nicotinamide-adenine dinucleotide (NADH) and nicotinamide-adenine dinucleotide phosphate (NADPH). This cycle is associated with respiratory electron transport cascade that produces water, and oxidative phosphorylation that drives the formation of ATP using the power supplied by electron transport cascade.


Metabolism is the sum total of all enzyme reactions in the body, both anabolic and catabolic.


Digestion:


Enzymes secreted in gastrointestinal tract facilitate the breakdown of ingested food components. Proteolytic enzymes in the stomach and intestine, mostly coming from exocrine pancreas and stomach, break down peptide bonds in proteins, down to amino acids and a few di- and tri-peptides, that are taken up by the cells of intestinal mucosa.


High carbohydrates are degraded by amylase from saliva and exocrine pancreas to simple sugars, and other glycosidases break bonds between the simple sugars in the intestine to yield glucose, that is then absorbed by the intestinal mucosal cells.


Ingested fat, as triacylglycerides, is broken down by lipases, mostly from exocrine pancreas, down to fatty acids and monoacylglycerides (one fatty acid attached by ester linkage to glycerol) and all these are absorbed by the mucosal cells of the intestine.


Simple molecules that are absorbed following digestion are used by the body to construct its own complex molecules. Glucose is used by various tissues for synthesis of glycogen, while amino acids for synthesis of proteins. Glucose is the first fuel substrate used for generation of ATP.


Fatty acids and monoacylglycerides are re-synthesized back into triacylglycerides within the same intestinal mucosal cells that absorbed them, and then released to blood as parts of lipoprotein particles to be distributed to various body tissues. At their target locations triacylglycerides are broken down in the blood into simpler molecules yet again, that are then taken in by the nearby cells to be used in triacylglyceride synthesis, and as fuel substrates for generation of ATP.


Cell Metabolism:


Complex molecules are broken down in various catabolic pathways. Glycogen is degraded to glucose-1-phosphate, that is subsequently broken down in a pathway called glycolysis. In glycolysis, glucose is converted to pyruvate, that is further metabolized to acetyl-CoA.


Acetyl-CoA enters Krebs cycle, or citric acid pathway, or circular enzyme pathway, that eventually releases CO2, to be exhaled by the lungs, and produces also the reduced co-enzymes NADH and flavin adenine dinucleotide FADH2. These co-enzymes have energy of reduction, as the passage of their electrons down the electron transport chain releases energy. Oxygen is the final recipient of these electrons, and its reduction yields water. The respiratory chain is linked to oxidative phosphorylation that uses energy released during the passage of electrons to drive the synthesis of ATP from ADP and inorganic phosphate.


Proteins can be degraded by proteolytic enzymes, that hydrolyze peptide bonds and yield amino acids. In turn, amino acids can be stripped of their amino groups, and the carbon skeletons, as pyruvate, acetyl-CoA, oxaloacetate, etc., are further broken down in Krebs cycle. So proteins can be used to provide energy as well, but because of their diverse functional roles and importance in muscle, they are used as major fuel sources only under extreme circumstances, i.e. starvation.


Quantitatively, the most important stored molecules that act as fuel substrates, are fats, i.e. triacylglycerides. They are broken down to fatty acids, that can be degraded through a pathway of beta-oxidation. Acetyl-CoA is produced in beta-oxidation that enters Krebs cycle.


All normal and usual catabolic paths converge to a small number of simple molecules, of which acetyl-CoA is central to all three: proteins, fats, and carbohydrates.


Under normal circumstances, there is a balance between catabolic and anabolic processes. While a protein molecule, for example that of albumin, is broken down within the circulation after 20 days, there is a balancing synthesis of albumin by the liver so that albumin concentration in blood remains the same.


Triacylgyceroles and glycogen build up in the body after food ingestion by synthesizing these complex storage molecules from glucose and fatty acids. Between meals the body can draw on the reserves to provide fuel for the ongoing daily activities. If food ingestion is higher than necessary for daily activities, a fat storage ensues. During starvation the fat stores are diminishing.


Metabolic pathways:


Glycolysis is splitting of six carbon glucose-6-phosphate into two three carbon molecules of pyruvate. Two molecules of ATP are created per one molecule of glucose entering glycolysis.


Within the mitochondria, pyruvate loses CO2, and acetyl-CoA is formed by re-cycling of Co-enzyme A. Acetyl-CoA enters the Krebs cycle, with the production of reduced co-enzymes NADH and FADH2, that serve as a source of electrons for the respiratory chain cascade, that in turn provides energy for the ATP synthesis. In a complete breakdown of one molecule of glucose through glycolysis, Krebs cycle, and respiratory chain cascade altogether 36 to 38 molecules of ATP are made (38 molecules of ATP in the liver, while only 36 molecules of ATP in the brain and muscles). So eventually the molecule of glucose is broken down to CO2 and water, and about 40% of energy is conserved in ATP.


Gluconeogenesis occurs when the level of glucose is falling, and glucose is needed for the brain function. It starts by conversion of pyruvate into oxaloacetate and then steps of glycolysis are reversed.


When there is too much glucose in the blood, as after food ingestion, glycogen synthesis pathways opens up to turn excessive glucose into glycogen.


Glucose can also go through hexose monophosphate pathway, also known as pentose phosphate shunt, in which five carbon sugars are produced, that are important for the synthesis of nucleic acids. .


With excessive food ingestion fatty acyl-CoA, along with a glycerol derivative, synthesizes triacylglyceride. During starvation triacylglycerides degrade down to acyl-CoA, that is further degraded by beta-oxidation pathway in the mitochondrial matrix to acetyl-CoA to be used in Krebs cycle. The complete breakdown of fatty acids through beta-oxidation and Krebs cycle generates considerably more ATP than that of glucose.


A complete breakdown of 16 carbon palmitic acid requires 7 repetitions of beta-oxidation sequences, that generates 7 molecules of each FADH2 and NADH, and those are then used in respiratory electron transport chain cascade and oxidative phosphorylation to generate 14 and 21 molecules of ATP, respectively. Then 8 acetyl-CoA molecules enter Krebs cycle, and each turn generates 12 ATP’s on the basis of generation of FADH2 and NADH, and their passage through electron transport chain and oxidative phosphorylation. Thus one molecule of palmitoyl-CoA yields 131 molecules of ATP via beta-oxidation, Krebs cycle and electron transfer with oxidative phosphorylation.


With 131 molecules of ATP 146 molecules of water is released in mitochondria.


Acetyl-CoA can also turn into ketone bodies. When there is more acetyl-CoA from beta-oxidation than Krebs cycle can handle, then excessive acetyl-CoA enters a secondary pathway that generates acetoacetate, beta-hydroxybutyrate and acetone, i.e. ketone bodies. This occurs primarily in the mitochondria of hepatocytes. They are water-soluble fuel substrates, than can reverse to acetyl-CoA in any tissue, even in brain, and enter Krebs cycle.


Acetyl-CoA can also be formed during the breakdown of carbon residues of various amino acids. Carbon skeletons of amino acids can enter Krebs cycle as pyruvate, fumarate, oxaloacetate, or succinyl-CoA.


Amino groups of amino acids are converted via urea pathway in the liver into urea, eliminated in urine. Without this pathways amino acids would turn into toxic ammonia.


Anabolic and catabolic reactions make use of different pathways as the former require energy, while the later generate energy. Anabolic and catabolic pathways are usually located in different compartments of cell: anabolic reactions take place in cytosol, while catabolic in mitochondria. This physical separation optimizes the conditions for each type of reactions, as well as regulatory controls.


Catabolic pathways lead to the formation of ATP, NADH, and NADPH, and these molecules are then used as sources of electrons or energy in various steps of anabolic pathways. Concentration of these co-factors, and ATP, help regulate the speed of anabolic and catabolic pathways within the cell. The ADP/ATP ratio, AMP/ATP ratio, NAD+/NADH ratio, are critical in control of glycolysis and Krebs cycle, and rising values of these ratios trigger increased rates of glycolysis and Krebs cycle.


Glycolysis is present in all cells, while Krebs cycle is absent in erythrocytes that lack mitochondria, and is not too active in leukocytes. There is no activity of beta-oxidation pathway of fatty acids in the brain, except in starvation, as brain prefers glucose as fuel source. Gluconeogenesis takes place predominantly in liver and kidneys.


There is often a cooperation of various cell types of different organs in processing substrates. Lactate, the end product of glucose catabolism in erythrocytes and muscles during strenuous physical activity, enters blood, and is picked up by the liver and converted by gluconeogenesis to glucose.


Various catabolic pathways do not function simultaneously. Following food ingestion there is usually sufficient glucose in the body for energy needs, so that there is no need to degrade triacylglycerides. On the contrary fat stores are built-up after food ingestion. Likewise there is no need to use protein for energy after food ingestion. In contrast, fasting leads to a breakdown of triacylglycerides, and breakdown of fatty acids provides ATP for most tissues, sparing glucose to be used as fuel for the brain. During fasting brain can use as a source of fuel also ketone bodies.


Different organs behave differently when it comes to fuel metabolism. Brain prefers only one fuel, glucose, and the metabolic regulation assures that there is sufficient supply of glucose for the brain. Liver is the main provider of fuel for all organs. If blood sugar drops down, liver turns on glycogenolysis, and gluconeogenesis for glucose synthesis. Muscle likewise consumes fuel, except it will take in fatty acids from triacylglycerides as well, along with glucose. Muscles do not provide glucose for blood, i.e. for other organs and tissues. Adipose tissue collects all fuel substrates and stores them as energy reserves.


Since all cells of the body require a continuous supply of fuel substrates to maintain adequate levels of ATP, and there is no continuous ingestion of food to supply these fuels, fuels derived from food have to be stored in a convenient form. The granules of glycogen are in abundance in the liver and muscle. But triacylglycerides are the most abundant form of stored fuel, and fat makes up the bulk of fuel reserves.


In adult male there are on average 135,000 kcal stored in fat, 900 kcal in glycogen, and 24,000 kcal in protein. One kilogram of fat can sustain a starving 70-kg male for 3 – 4 days, and his total fat reserves last for 2 months.


Carbohydrate yields 4 kcal/g, while fat 9 kcal/g.


The advantage of fat is that it is stored in anhydrous condition, whereas the strongly polar glycogen must be stored in hydrated form, which takes much more space. On the other side, simple sugars can be produced much quicker from glycogen while fatty acids are not mobilized as fast from stored fats.


The caloric requirements depend upon the level of physical activity, and environmental temperature. More calories are needed for survival in Arctic than on tropical island.


Fat metabolism and carbohydrate metabolism are directly connected, and under hormonal control. Many tissues use glucose as fuel source when food ingestion is adequate, and fat in times of fasting and starvation.


Under normal conditions there is a dynamic balance between protein breakdown and synthesis in protein metabolism. There is a constant turnover of proteins, old molecules are destroyed and new ones made to take their place.


Protein synthesis relies on availability of the 20 amino acids, derived from food ingestion or from the breakdown of existing in the body proteins. Non-essential amino acids can be made from other amino acid, or intermediates formed in glycolysis, Krebs cycle or hexose monophosphate pathway. Essential amino acids have to be supplied by food.


Breakdown of proteins in the body is carried out by proteases, which utilize water to break peptide bonds between amino acids. Inside the cells proteolysis takes place within two compartments: lysosomes, and cytosol.


Only in prolonged fasting/starvation, and in diabetes mellitus, is there a net loss of proteins, when they are used to provide fuel or carbon sources for gluconeogenesis.