Glycolysis is the first phase of a series of reactions for the catabolism of carbohydrates. Catabolism is the breakdown of larger molecules into its respective smaller constituents. Glycolysis is the first part of cellular respiration that generates pyruvate to be used in either anaerobic respiration in the absence of oxygen or in the TCA cycle in aerobic respiration which yields useable energy for cells. This will be a general outline of the steps in glycolysis.

The whole process can be broken down into an energy investment phase where ATP is being used and an energy payoff phase where ATP is being generated. Fructose-1,6-biphosphate is where the energy investment phase ends. That is where the last ATP has to be used for energy to drive glycolysis.

A simple equation can be remembered as a summary of glycolysis.

Glucose + 2 ADP + 2 phosphate ions + 2 NAD+ —-> 2 Pyruvate + 2 ATP + 2 NADH + 2 H20 + 2 H+.


In the first step of glycolysis an addition of a high energy phosphate from ATP yields glucose-6-phosphate and ADP. This step is initialized by the enzyme hexokinase. G6P is more reactive than glucose.

In the next step, glucose-6-phosphate is converted to its isomer, fructose-6-phosphate by phosphoglucose isomerase.

In the third step of glycolysis, fructose-6-phosphate is converted to fructose-1,6-biphosphate by phosphofructokinase (PFK) and the addition of ATP. This is the committed step, meaning that fructose-1,6-biphosphate MUST be converted to pyruvate. This is also the end of the energy investment phase of glycolysis.

Fructose-1,6-biphosphate is converted to glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate catalyzed by aldolase. Glyceraldehyde-3 phosphate maintains a reversible reaction with dihydroxyacetone phosphate through triode phosphate isomerase. The resulting reaction generates two molecules of glyceraldehyde-3-phosphate. A key point going forward is that two molecules of each substrate are produced.

Each G3P molecule gains an inorganic phosphate and with the addition of NAD+ to form the energized carrier molecules NADH. The resulting reaction catalyzed by glyeraldehyde-3-phosphate dehydrogenase generates two molecules of 1,3-bisphophoglycerate which yield two high energy phosphates.

Through the addition of two low energy ADP molecules and the enzyme phosphoglycerate kinase, the two molecules of 1,3-bisphophoglycerate are converted to 3-phosphoglycerate and yields two molecules of ATP. This reaction is called the break even reaction because at this point the energy input is equal to the energy output. Two molecules of ATP were expended and at this step there was a generation of two ATP molecules.

In the next step the two molecules of 3-phosphoglyercate are converted to 2-phosphoglycerate through the enzymatic properties of phosphoglycerate mutase.

The molecules of 2-phosphoglycerate are converted to phosphoenolpyruvate catalyzed by enolase. This step yields H20 molecules.

In the final step of glycolysis, the molecules of phosphoenolpyruvate are converted to pyruvate catalyzed by pyruvate kinase. ATP is generated from the addition of ADP and the two high energy phosphates from the molecules of phosphoenolpyruvate.

Upon the completion of glycolysis, the pyruvate molecules can be oxidized to carbon dioxide in cellular respiration to generate 28 molecules of ATP.

The NADH that is produced is turned back into NAD+ to drive further glycolysis. There are two ways to accomplish this. In the presence of oxygen NADH passes it electrons into the electron transport chain, which regenerates NAD+ for use in glycolysis. In the absence of oxygen, cells regenerate NAD+ by undergoing fermentation.



Lactate Dehydrogenase

Lactate dehydrogenase (LD, LDH) is an enzyme that is found in all cells in all tissues of the body. It catalyzes the reversible conversion of lactate to pyruvic acid in glycolysis and gluconeogenesis. It is released from various anatomical sites of the body in response to cellular injury and damage. It is used as a common marker for tissue damage and disease such as heart failure. Its down-fall is that it isn’t very specific to which tissue is damaged, but there are subtle hints that can clue a physician in particular directions.

Lactate dehydrogenase is structurally composed of four subunits, but the two common subunits are LDHA known as LDH-M, and LDHB, known as LDH-H. The only difference between the two subunits is that their is an amino acid substitution of alanine with glutamine within the H subunit. This amino acid change slightly changes the biochemical properties of the two subunits slightly in that the H subunit can bind faster, but the catalytic activity of the M subunit does not deteriorate at the same rate as the H subunit, it holds well.


The two subunit of LDH can form five isomers which are found in various sites within the body;

LDH-1 (4H)- Found in the heart, RBCs, and the brain.

LDH-2 (3H1M)- Found in the RES.

LDH-3 (2H2M)- Found in the lungs.

LDH-4 (1H3M)- Found in the kidneys, placenta, and the pancreas.

LDH-5 (4M)- Found in the liver and striated muscle.

LDH is a protein that is found in small amounts normally in the body and there are various conditions that can cause an elevation. Cancer can raise the LDH levels within the body. Cancer cells rely on increased glycolysis due to their high energy demand. LDH elevation in cancer is often times referred to the Warburg effect which allows malignant cells to convert glucose stores into lactate even in the presence of aerobic respiration. This shifts glucose metabolism from simple energy production to accelerate cell growth and proliferation.

Hemolysis can be measured as LDH is abundant in RBCs and can be measured. Although measures should be taken to correctly receive the sample as incorrect procedures an cause hemolysis and a false-positive elevation in LDH levels among other substrates and electrolytes.

It can also be used as a marker for myocardial infarction. Normally LDH-2 is at a higher level than LDH-1. When someone experiences a myocardial infarction, levels of LDH-1 will be significantly elevated to a level higher than LDH-2. This is known as the LDH flip and is diagnostic in patients who have experienced myocardial infarction. Elevation of LDH peaks 3-4 days after MI, and can remain elevated for up to 10 days. LDH has since been replaced by the troponin test, which is a much more specific and sensitive test in diagnosing MI.

High levels of LDH in the cerebrospinal fluid (CSF) can indicate bacterial meningitis. Elevated LDH levels in viral meningitis is indicative of a poor prognosis.

LDH is an important tool that physicians don’t always utilize to its lack of specificity, but it can still be helpful in a diagnosis. Its important not to ignore any test and any result as it still contributes to the whole picture.



Hemoglobin, What is it?


Hemoglobins main function is to carry oxygen to the tissues and cells of the body. Hemoglobin can bind and transport four molecules of oxygen. Hemoglobin is made up of 4 polypeptide globin chains, two alpha and two beta that forms a tetramer heme group with iron located within the tetramer. Globin is the protein part of hemoglobin. The different globin chains are typically 141-146 amino acids in length and each chain is designated by a greek letter. Each chain is subdivided into eight helices designated an alphabetical letter with each helice divided by seven non-helical segments. The globin chains loop to form a cleft pocket for heme which is suspended between the E and the F helices of the polypeptide chain. The ferrous iron in each heme molecule reversibly binds to one oxygen molecule.

Heme is a four ring consisting of carbon, hydrogen and nitrogen atoms called the protoporphyrin IX. The single carbon atoms act as connecting bridges. There are alternating double bonds where the electron resonation absorbs light which is the reason why heme is colored. There are two propionic acid side chains on end rendering it polar, with the rest of the heme molecule being non-polar and hydrophobic.

Globin Synthesis

Globin is a protein so naturally translation and synthesis occurs in the ribosomes while transcription occurs in the nucleus. Transcription of the alpha globin gene which occurs on chromosome 16 produces more mRNA than the beta globin genes which is transcribed on chromosome 11. There are four alpha genes, and only two beta genes. To make up for that discrepancy, translation of the alpha globin is less efficient than that of the beta globin so there are equal amounts of both produced in Hgb A.

Heme Synthesis

Heme is synthesized in the mitochondria and cytoplasm of the bone marrow erythrocyte precursor cells. Biosynthesis begins in the mitochondria with the condensation of glycine and succinyl CoA catalyzed by aminolevulinate synthase (ALA synthase) to form ALA in the cytoplasm. Porphobilinogen synthase converts ALA to porphobilinogen (PBG). Porphobilinogen synthase is the enzyme that is inhibited by lead. PBG is then converted to hydroxymethylbilane which is further converted to uroporphyrinogen III. Uroporphyrinogen III is converted to coproporphyrinogen III. Synthesis then continues back in the mitochondria by the conversion of coproporphyrinogen III to protoporphyrinogen IX. Protoporphyrinogen IX is then converted to protoporphyrin IX by protoporphyrinogen oxidase. From there protoporphyrin IX is converted to heme in the presence of ferrous iron and ferrochelatase. Heme has a negative feedback mechanism on ALA by inhibiting the transcription of the ALA synthase enzyme.

Hemoglobin Synthesis

With the synthesis of globin and heme covered the next step is the assembly of the hemoglobin molecule as one. After the globin is released by the ribosomes, each polypeptide chain binds to a single heme molecule. An alpha globin:heme complex and a beta globin:heme polypeptide then combine to form a heterodimer. Now remember that step is repeated as there are four polypeptide chains in a hemoglobin molecule. Two heterodimers then combine to form a tetramer to complete the assembly of the hemoglobin molecule.


Oxygen Binding

When in the deoxygenated state, the iron within the heme molecule is pulled out of plane of the heme ring. When oxygen binds it pulls the iron back into the plane of the heme ring and also causes a shape change in the polypeptide chains which causes a ripple effect among all four polypeptide chains. This phenomenon is allosteric regulation. The allosteric effect is the conformational change in the entire hemoglobin molecule caused by the binding of one oxygen molecule to one ferrous iron molecule within the heme cleft. In the deoxygenated state ionic bridges form creating a stable and rigid configuration. A single molecule of 2,3-DPG binds adjacent polypeptides to further stabilize the hemoglobin molecule. In the oxygenated state there is that allosteric effect which alters the shape of the hemoglobin molecule enough so that ionic bridges are broken which causes the globin molecules to relax and heme cleft enlarges. This allows the remaining three oxygen molecules to bind readily to the ferrous iron.

Oxygen/Hemoglobin Dissociation Curve Effects

The oxyhemoglobin is formed when oxygen binds during physiological respiration within the pulmonary capillaries. Various factors such as pH, CO2 concentration, and 2,3-DPG concentration affect the way that oxygen binds. When talking in terms of the oxygen/hemoglobin dissociation curve it is generally said that curve is sigmoidal; meaning that there is low hemoglobin affinity for oxygen at low oxygen tension and high affinity for oxygen at high oxygen tension. There are values such as the P50 which is defined in terms of the amount of oxygen needed to saturate 50% of the hemoglobin molecule. The PO2 of the lungs are ~ 100 mm/Hg so that means that hemoglobin is 100% saturated when the RBC is in the lungs. Normally a P02 of 27 mm/Hg results in 50% hemoglobin saturation. You can also have shifts to the right or left of the curve. These shifts come from the various factors that were mentioned above. Hemoglobin exists as two forms, a taut (tense) phase and a (R) form or relaxed form. Low pH, high CO2 (such as in the tissues), and high 2,3-DPG initiate a shift to the right, meaning that there is less affinity for oxygen and the oxygen is released into the tissues resulting in the taut form of hemoglobin. Conversely a high pH, low CO2 (such as in the lung capillaries), and low 2,3-DPG results in the relaxed form and a shift to the left creating a higher affinity to oxygen. The partial pressure of the system also affects the affinity to oxygen. At high PO2 levels, such as those present in the lungs, a high affinity relaxed state is favored. In a low PO2 state, such as in the tissues, the low affinity, taut form is favored. Hemoglobin needs to be able to bind oxygen and release it. There is no point in the molecule binding if there is no chance at releasing it. The sigmoidal curve causes it to be efficient at taking up oxygen in the lungs and efficient at releasing it in the tissues

oxygen-dissociation curve

Hemoglobin is an essential aspect of homeostasis for the cells. It needs to functioning and be assembled correctly to efficiently do its job. This is just a brief overview of what it does and the structure of it. More will come on the different hemoglobinopathies (qualitative) and thalassemias (quanitative).