Archive for the ‘glycolysis’ Category

Ketogenic Diets for Cancer III. More background and the Warburg Effect.

Posted: December 4, 2016 in Cancer, Cell Signaling, energy metabolism, gluconeogenesis, glycolysis, ketogenic diet, low-carbohydrate diet, Warburg effect
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This series of posts is a followup to the project that Dr. Eugene Fine and I described in our campaign at Experiment.com. as follow-up to Dr. Fine’s pilot study of ten advanced cancer patients on ketogenic diets and the in vitro projects that we are carrying out in parallel.

The last post described the two major processes in energy metabolism, (anaerobic) glycolysis and respiration. Pyruvate is the product of glycolysis and has many fates. (Remember pyruvate and pyruvic acid refer to the same chemical). For cells that rely largely on glycolysis, pyruvate is converted to several final products like ethanol, lactic acid and a bunch of other stuff that microorganisms make in the fermentation of glucose. (The unique smell of butter, e.g., is due to acetoin and other condensation products of pyruvate). Rapidly exercising muscles also produce lactic acid.

The sudden interest in the metabolic approach to cancer stems from the work of Otto Warburg whose lab in the 1930’s was a center for the study of metabolism. (Hans Krebs was an Assistant Professor in the lab). Warburg’s landmark observation was that cells from cancer tissue showed a higher ratio of lactate to CO2 than normal cells, that is, the cancerous tissue was metabolizing glucose via glycolysis to a greater degree than normal even though oxygen was present. The Coris (Carl and Gerty of the Cori cycle) demonstrated what is now called the Warburg effect in a whole animal. Ultimately, Warburg refined the result by comparing the ratio of lactate:CO2 in a cannulated artery to that in the vein for a normal forearm muscle. He compared that to the ratio in the forearm of the same patient  that contained a tumor. Warburg claimed that this greater dependence on glycolysis was a general feature of all cancers and for a long time it was assumed that there was a defect in the mitochondrion in cancer cells. These are both exaggerations but aerobic glycolysis appears as a feature of many cancers and defects in mitochondria, where they exist, are more subtle than gross structural damage. The figure shows current understanding of the Warburg Effect.

kdforca_blog_iii_warburg_figure

What about this mechanism makes us think that  ketone bodies are going to be effective against cancer? We need one more step in biochemical background to explain what we think is going on. In normal aerobic cells, pyruvate is converted to the compound acetyl-CoA.  Acetyl-CoA represents another big player in metabolism and functions as the real substrate for aerobic metabolism. If you have taken general chemistry, you will recognize acetyl-CoA as a a derivative of acetic acid.

The reaction acetyl-CoA ➛ 2CO2 is the main transformation from which we get energy. Acetyl-CoA provides the building block for fatty acids and for ketone bodies. Conversely, fatty acids are a fuel for cells because they can be broken down to acetyl-CoA. Under conditions of starvation, or a low-carbohydrate diet, the liver assembles 2 acetyl-CoA’s to ketone bodies (β-hydroxy butyrate and acetoacetyl-CoA). The ketone bodies are transported to other cells where they are disassembled back to acetyl-CoA and are processed in the cell for energy. The liver is a kind of metabolic command center and ketone bodies are a way for the liver to deliver acetyl-CoA to other cells.kdforca_blog-iii_dec_4

Now we are at the point of asking how a cell knows what to do when presented with a choice of fuels? In particular, how does the input from fat dial down glycolysis so that pyruvate, which could be used for something else (in starvation or low-carb, it will be substrate for gluconeogenesis), is not used to make acetyl-CoA.  It turns out that acetylCoA (that is, fat or ketone bodies) regulate their own use by feeding back and directly or indirectly turning off glycolysis (in other words: don’t process pyruvate to acetyl-CoA because we already have a lot). The feedback system is known as the Randle cycle and appears (roughly) as the dotted line in our expanded metabolic scheme.

robin_map_2012-2Where we are going. In our earlier work Dr. Fine and I and our assistant, Anna Miller, found that if we grow cancer cells in culture, acetoacetate (one of the ketone bodies) will inhibit their growth and will reduce the amount of ATP that they can generate. Normal cells, however, are not inhibited by ketone bodies and the cells may even be using them. Our working explanation is that the ketone bodies are inhibiting the cancer cell through the Randle cycle. Now, normal cells can maintain energy, that is compensate for the Randle cycle, by running the TCA cycle (in fact, that is the purpose of the Randle cycle: to switch fuel sources). The cancer cells, however, have some kind of  defect in aerobic metabolism and can’t compensate.  How does this happen? That’s what we’re trying to find out but we have a good guess. (A good guess in science means that when we find out it’s wrong we’ll probably see a better idea). We find that our cancer cells in culture over-express a protein called uncoupling protein-2 (UCP2). We think that’s a player. To be discussed in Part IV.

Organic-Biochem-Nutrition – 3. Aldehydes, Ketones, Sugars.

Posted: February 14, 2012 in alcohol, Fructose, glycolysis, Learn organic chemistry
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Last post, we were running with the name game, which emphasizes one of the two features of organic chemistry, its precision and logic.  The other distinguishing feature, as in all chemistry, is the sense of cooking and transformation that  we only hinted at: we had only one group of compounds, the alcohols but we did predict that the more the structure looks like water (the greater the percentage contributed by the OH group) the more water-soluble the compound was.

The last post finished with a quiz, for which I will now provide answers.  If you already know the answers and want to see new stuff, you can jump ahead.

Answers. 

Q1. The rule: find the longest continuous chain of carbon atoms, five in this case.  Consider as if it were derived from the five-carbon hydrocarbon, pentane. (“As if” because the real compound may not have been derived from pentane). Look for substituents (attached to the main chain.  There is a one carbon substituent, that is, a methyl group. Which carbon is it attached to? Count the carbons, trying each end. Use the one with the lowest number : 2-methyl pentane (not 4-methyl pentane). The compound is an isomer of hexane but the name is unambiguous which is the idea.

Q2. Same rule.  Find the longest carbon chain. Four carbons = butane. Find the functional group, alcohol. Always use the lowest number so it is a 1-butanol (not 4-butanol).  One methyl groups, so this compound would be called: 2-methyl 1-butanol.

Q3. Find the longest carbon chain. Don’t be misled by how the structure is laid out on the page.  (The way it is written will be determined from the chemical context).  This is an 8-carbon compound, an octane backbone.  Find the functional group, an alcohol. Always use the lowest number so it is a 2-octanol.  The methyl groups on carbons 4 and 5 means this compound would be called: 4, 5-dimethyl 2-octanol.

Q4. The functional group (if there is only one) is always on carbon 1, so you do not have to specify that.  Note: carbon 3 is indicated twice.

Q5. The variation: propyl is the substituent on carbon 3 of the main structure, but this side chain itself has a substituent on carbon 2 (counting from the point of attachment to the main structure) so 2-methyl is the “adjective” that modifies “propyl.”

Q6, 7. Pretty much obvious variations on the standard rules.

 

 

 

Where we’re going.  The new functional group is the carbonyl, C=O, a group that has two chemical bonds between carbon and oxygen.

When the carbonyl group is at the 1-position, that is, at the end of the chain of carbon atoms, the compound is called an aldehyde.  When, someplace else in the chain, the compound is called a ketone.

The principle: compounds that have more than one functional group have a different classification — sometimes the properties of the compound are the sum of the properties of the functional groups and sometimes they interact to give totally new properties.

Compounds that have both a carbonyl group and an -OH group are called sugars, that is, the sugars are polyhydroxy aldehydes and ketones and are sometimes referred to as aldoses and ketoses.

Sugars and their polymers and derivatives are called carbohydrates. (Alcohol, that is, ethanol is not a carbohydrate).

Start with aldehydes: Once again, the name game is a good idea. If you can give a correct name for the compound, then you have identified where the functional groups are and that’s where the chemistry lives.  A compound with a C=O group at one end is called an aldehyde.

Formaldehyde.The simplest aldehyde could formally be named as if were a derivative of methane. Drop the final -e and add the suffix –al.  Methanal, however, is a very common substance and is always called formaldehyde.

Acetaldehyde. Drop the final -e from the 2-carbon hydrocarbon, ethane. Add the suffix -al.  Like formaldehyde, ethanal is a common compound, especially in biochemistry and it is always called by the familiar name acetaldehyde (accent on third syllable).  The conversion of ethanol to acetaldehyde is the first step in the liver’s processing of ingested alcohol.  Conversely, microorganisms that carry out alcoholic fermentation, convert sugar (in many steps) to acetaldehyde and then to ethanol (faites attention: you are making the transition from organic chemistry to biochemistry).  Aldehydes are chemically reactive; acetaldehyde can react with proteins of the body and is fairly toxic accounting for some of the side effects of excessive drinking.  In normal people it is cleared by the next step which incorporates the compound into metabolism; people with genetic abnormalities in metabolizing acetaldehyde (common in the Asian population) are pretty much incapable of drinking at all because of the severe physiologic responses.

The system of naming aldehydes is perfectly regular and it should be obvious how to name aldehydes of 3-carbon (propanal), 4-carbon (butanal), 5-carbon (pentanal) aldehydes, etc.  The rules for substituents are the same as before and you should be able to write the  structure of, for example, 3,3- dimethyl hexanal.

Aldehydes can be complicated and tend to have fruity or complex aromas and are, in fact, found in many natural products.  Cinnamaldehyde and citronellal smell just the way you think.  Veratraldehyde was probably first isolated from a plant called veratrum but from one of its common names, methyl-vanillin you can guess where it is used in the food industry.

If the carbonyl group appears in the middle of a chain or ring, the compound is called a ketone.  The simplest is the three carbon compound acetone; formal name would be derived by dropping the -e from propane and adding the suffix -one although this is never used.

Ketones should not be confused with the colloquial “ketones” meaning ketone bodies, the compounds produced during starvation or low-carbohydrate diets which include acetone, acetoacetic acid (a keto-acid) and β-hydroxybutyrate which does not have a keto group at all.

Sugars.

Sugars are polyhydroxy aldehydes and ketones. Organic compounds, in general, can have more than one functional group.  For names, there is a hierarchy: carbonyl compounds have precedence over alcohols.  In other words, in a compound containing both an -OH group and a carbonyl, the compound is named as an aldehyde or ketone and the hydroxyl group is treated as if it were a substituent along the lines of a methyl group as in previous exercises. The alcohol groups are called hydroxy– when a substituent in another compound.

The simplest sugars have three carbons.  The suffix -ose is common for sugars and these compounds are called trioses.  The one shown below is an aldose, or combining the classifications, it would be called an aldotriose.  The name of the common aldotriose is glyceraldehyde, a name indicating its relation to sugar but probably discovered before the “ose” terminology became common; the compound is called in German, glycerose (pr. glitzerose)

There is one ketotriose, dihydroxyacetone.

The hexoses.  Looking ahead, the major sugars of interest in biochemistry are glucose and fructose.  They are isomers (the same chemical formula) but glucose is an aldose and fructose is a ketose.  Structures are shown below but there is another level of complication, stereochemistry, that has to wait for the next organic post.

With some of the major players, however, we can start to put together some information on biochemistry.  Glycolysis: the lysis (breaking) part of glycolysis involves the cleavage of a hexose into two triodes.  Both glucose and fructose are connected throughout the triodes. Ingested fructose can be converted to derivatives of the trioses and these, in turn, can be turned into glucose.

Introduction to Metabolism. The Black Box of Life.

Posted: February 2, 2012 in energy metabolism, glycolysis, Krebs cycle, low-carbohydrate diet
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I am currently teaching nutrition and metabolism to first year medical students.  The problem in this subject is the large number of individual reactions which leads students to think of the subject the way somebody described the study of history: just one damned thing after another.  I try to present the big picture and the approach is the systems or “black box”  strategy.  The method is to ask whether we can get some information just by looking at the inputs and outputs to a system even if we don’t know any of the details of what’s going on inside.  In other words, it is a way of organizing limited information.  The method is favored by engineers who are the people most unhappy with the idea that they don’t know anything at all.  First, the big principles.

Metabolism: two goals, two fuels.  

There are two major goals in human energy metabolism: First, to provide energy for life processes in the form of the molecule ATP and second, to provide glucose for those cells that require glucose (particularly brain and central nervous system) and to maintain blood glucose at a relatively constant level: too little is obviously not good but too much is also a problem in that glucose is chemically reactive and can interact with body material, particularly proteins when at high concentrations. Of course, metabolism does many things but these are the two major goals in providing energy.

A second big generalization is that in this process there two kinds of fuels: glucose and acetyl-Coenzyme A (abbreviated acetyl-CoA or sometimes written as acetyl-SCoA; the S, which is meant to show that the compound contains sulfur, is not pronounced).

The black box of life. 

You knew what we do in metabolism even before you started reading this. Putting it into black box terms, you knew: we take in food and we take in oxygen. We excrete CO2 and water.  Somehow this gives us the energy for life as well as the material to build up components of the body.  You don’t have to know too much chemistry to figure out the important conclusion that, inside the black box, living systems use oxidation, just like combustion in a furnace. Lavoisier’s whole animal calorimeter that I described in a previous post was a beautiful real demonstration of this black box.  More technically, this is an oxidation-reduction reaction.  Oxidation, in a biochemical context, means combination with oxygen or loss of hydrogen and reduction means loss of oxygen or gain of hydrogen; we say that the (carbons in the) food gets oxidized and the oxygen gets reduced (to water).  Like the common oxidation reactions you know (combustion in a furnace or an automobile engine), this produces energy which can be used to do work. Some work is mechanical work — moving muscles — but most of the energy is used for chemical: work making body material and keeping biological structures intact and generally keeping things running.  The medium of energy in metabolism is the chemical reaction of synthesis and breakdown of the molecule ATP.  Textbooks frequently refer to ATP as a “high energy molecule” but it is not the compound itself but rather the reaction (synthesis and breakdown (hydrolysis)) that is high energy.  For the moment, we can think of ATP as the “coin of energy exchange in metabolism.”  A heavy-duty thought concept: the challenge for biochemistry historically was to explain how the energy from an oxidation-reduction reaction could be used to carry out the synthesis of ATP which has a different mechanism (phosphate transfer).  The process is called oxidative phosphorylation and was only figured out about fifty years ago.

So again, our two goals in human metabolism: Make energy in the form of ATP and maintain a pretty much constant level of blood glucose for those cells, brain and central nervous system, that require glucose (the brain can’t use fatty acids as a fuel).

Let’s look at energy production first because it is a little easier to understand.  As we look inside the black box, each of the processes uncovered will have its own degree of complexity.  In reading this you have to do what scientists do: hang in there.  Skip over the parts that seem complex and see if you can come back to them later.

The role of redox coenzymes

So, breaking into the black box, the first thing to notice is that the oxidation of food is done in steps, and that there is another player that mediates the process by coupling separate pieces: the food never sees the oxygen.  The intermediaries are called coenzymes or cofactors.  The most important oxidative coenzyme is known as NAD.  It’s always referred to by the acronym, but if you’ve had some organic chemistry and you’re curious, NAD stands for nicotinamide-adenine-dinucleotide; the structure is shown in the figure and the action end of the molecule is indicated. NAD coenzymes are derived from the vitamin niacin.  So   what happens in metabolism is that food is oxidized by NAD+ (the oxidized form of NAD) and the product, NADH (the reduced form) is re-oxidized by molecular oxygen. Although it is still just as we thought (food+oxygen-in, CO2+water-out), the oxygen never sees the food.   Why do we do it this way?  If we did it all in one big blast like an automobile engine, we would have little control over it and we would not be able to capture the energy in a usable chemical form.

It’s easiest to start with glucose, a six-carbon compound. The early steps in metabolism involve a process known as glycolysis (sugar splitting) that ultimately gives you two molecules of a three-carbon compounds known as pyruvic acid. Pyruvic acid is oxidized to a derivative of acetic acid, known as acetyl-CoA. The CoA is short for Coenzyme A, a complicated molecule but, like many coenzymes is always referred to in this way so it is not important to know the detailed structure.  The compound is frequently written acetyl-SCoA to emphasize that it is a thioester (sulfur ester); again, the “S” is not pronounced.

Acetyl-SCoA is the fuel for the major NADH-producing process, known as the Krebs cycle after the major player in its discovery. Without looking into that black box too much the key compound is citric acid, which is, chemically a try-carboxylic acid (TCA) so the Krebs cycle is also called the citric acid cycle or TCA cycle; Krebs called it the TCA cycle so I will generally use that term.  The process whereby NADH is finally re-oxidized by oxygen is known as electron transport.  So, The big black boxes of metabolism:

Where do we get Glucose and Acetyl-CoA?

So far we know: most energy comes from the oxidation of acetyl-CoA and most of the glucose that provides energy does so by first being converted to acetyl-CoA. Where else can we get acetyl-CoA? We’ve taken glucose as synonymous with food but where else can we get glucose from besides the diet?

Looking ahead, the big results that will come out of opening up the black box of metabolism: 1) Acetyl-CoA also comes from fat and to a smaller extent from protein.  2) Glucose can also be formed from protein. 3) Under conditions where there is no dietary glucose (starvation, low carbohydrate diet), glucose will be made from protein or released from stored glycogen, and an alternative fuel ketone bodies will provide acetyl-CoA; ketone bodies are essentially a dimer of acetyl-CoAs and the liver makes and exports ketone bodies to other cells.  Acetyl-CoA and, therefore, glucose can be converted to fat but a major asymmetry that will have profound significance is that 4) glucose cannot be formed from acetyl-CoA.  The significance of the last statement is that: we know all too well that fat can be formed from glucose but, with minor exceptions, 5) glucose cannot be formed from fat. (Chris Masterjohn’s post “We Really Can Make Glucose From Fatty Acids After All!”
indicates the extent to which the exceptions become important but the overriding principle that has the most impact on metabolism is that you cannot make glucose from fat).
So that’s it.  You now have a blackbox view of metabolism.  I will try to open some of the boxes in future posts.

Summary of fuel sources and synthesis and looking ahead.

  1. There are, roughly speaking, two kinds of fuels: glucose and acetyl-CoA.
  2. Carbohydrates and other nutrients, fat (that is, fatty acids) and protein (amino acids) can supply acetyl-CoA.  Glucose is not required for acetyl-CoA and under conditions of low carbohydrate or low total food, fatty acids become the major source of acetyl-CoA.
  3. Not all tissues can use all fuel sources. Brain, CNS and red blood cells, for example cannot use fatty acids. Brain and CNS can use acetyl-CoA but cannot get it from fatty acids.  Red blood cells only use glucose and, to a first approximation, brain and CNS are also dependent on glucose for metabolism.
  4. Under conditions of starvation or carbohydrate restriction, acetyl-CoA can be effectively transported from the liver in the form of  ketone bodies. Ketone bodies, then, are a source of acetyl-CoA that can be used by brain and CNS.  Red blood cells are still dependent on glucose but the brain’s demand for glucose is reduced by the availability of ketone bodies.
  5. There is no dietary requirement for carbohydrate and amino acids can also supply glucose through the process of gluconeogenesis.
  6. Fat as a source of acetyl-CoA also works the other way: acetyl-CoA can be converted to fat.
  7. Whereas glucose can be converted to fat, with a few exceptions, fat cannot be converted to glucose. This will be a key idea behind carbohydrate restriction.
  8. Glucose can also be stored as the polymer glycogen.
  9. Bottom line is the limitation of “you are what you eat.” Metabolism means the interconversion of food and metabolites. Conversely, it will be critical that not everything is interconvertible. In particular, we will emphasize that you can make fat from carbohydrate but, to a large extent,  you cannot make glucose from fat.

Looking ahead on sources of blood glucose:

  1. Glucose from dietary input (referred to as the fed state; in nutrition, as the postprandial period), is depleted after about 8 hours.
  2. Glycogen is a storage/supply source of glucose.  Liver glycogen can supply export glucose to the blood, thereby supplying other tissues.  Muscle glycogen supplies glucose only for the muscle itself.  Glycogen may become largely depleted after 24 hours, depending on the conditions (exercise, for example).
  3. The third source of blood glucose is gluconeogenesis (GNG) which, as the name implies, makes glucose anew from existing metabolites. Depending on the conditions, the source of carbon may be amino acids, lactic acid or glycerol from fat metabolism.  Whereas it is sometimes indicated to be a “last ditch” source of glucose in the textbooks, it goes on all the time. The glucose it synthesizes in GNG may be used to replenish glycogen and only appear in the blood at a later time.