Part 8.1: A Basic Introduction to Lipid Chemistry and History the Recognition of Lipids in Nutrition
By Eben van Tonder
2 Junie 2019
For part 1, 2, 3, 4, 5, 6 and 7, click on:
In 2018, I started on a journey to understand the determination of total meat content and the historical roots of the determination. Tonight I begin the last installment in this short overview. Through experimentation, the following rations were determined.
|% N||% Protein||% Lean Meat|
%N x (6.25 x 4.8) = % Lean Meat. This means that,
%N x 30 = % Lean Meat
How was the 4.8 determined?
We know that Lean Meat (fat-free) contains 20.8% protein.
So, % Protein x 100/20.8 = % Lean Meat which is 4.8
Meat Protein contains 16% Nitrogen. So, %N x 100/16 = % Protein
In other words, %N x 6.25 = % Prot.
When we talk about Lean Meat we exclude fat and fat is the final component in determining total meat content to consider. Here we briefly overview biochemistry of lipids as a macromolecule. In the second part of this article, I quote Arthur A. Spector and Hee-Yong Kim’s excellent article, Discovery of essential fatty acids
The macromolecules that form the ingredients of life are responsible for energy generation, storage and the storing and transmission of information. The ingredients of life are carbohydrates, lipids, proteins, and nucleic acid. Let’s focus on the first three.
The Formation of Macro Molecules
Lets first consider how macromolecules are formed. Macromolecules are often polymers (not always). A polymer is the repeat of a monomer and we can write it as (monomer)n or Mn. In the formation of macromolecules, there are two kinds of reactions that are important namely condensation and hydrolysis reactions. Condensation reactions form bonds and hydrolysis reactions break bonds.
Condensation Reactions: A monomer with a hydroxyl group interacts with another monomer with a hydroxyl group and the outcome is a bond between the two monomers with the release of water represented as follows: M – OH + M – OH -> M – O – M + H2O A hydrolysis reaction is exactly the opposite.
A hydrolysis reaction is exactly the opposite. The di-monomer with an ether bond between them adds water and the bond between them is broken. Both of these reactions often require energy to proceed.
The particular class of macromolecules important to us is the lipids.
A lipid is a name given to a host of different biomolecules, all of which can be dissolved in nonpolar solvents. Typically this includes hydrocarbons used to dissolve other naturally occurring hydrocarbon lipid molecules that do not (or do not easily) dissolve in water. Scientists sometimes classify lipids and small hydrophobic molecules which include fatty acids, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, phospholipids, and triglycerides. Note that there are some lipids that have hydrophilic parts. Some lipids are therefore all out hydrophobic (non-polar molecules which do not like water); some lipids have hydrophobic and hydrophilic parts called amphipathic molecules. These substances fare well when we use them as emulsifiers. For a more detailed discussion about emulsifiers, have a look at Emulsifiers in Sausages. About 5% of the dry mass of a cell consists of lipids. They are an important energy store for cells with energy-rich bonds and key to the formation of cell membranes. They are involved in signaling. They are important in insulation in terms of keeping the organism warm and also in insulating nerve cells as the nerve cells transmit their signals.
The condensation reactions during lipid formation often involve the synthesis of triglycerides.
Triglycerides are formed from glycerol to which a fatty acid is bound. A fatty acid is a hydroxyl group (hydroxyl group is denoted by –OH), bound to a carbonyl group (a functional group composed of a carbon atom double-bonded to an oxygen atom: C=O) attached to long-chain hydrocarbons. Out of the glycerol, attached to a fatty acid comes then a triglyceride. The reaction that forms the triglyceride is a condensation reaction as well as a transesterification reaction with the formation of an ester bond. Transesterification is the process of exchanging the organic group R″ of an ester with the organic group R′ of an alcohol.
Let’s look a bit closer at esters. “Esters are an important functional group in organic chemistry, and they are generally written RCOOR’ or RCO2R’. An ester is characterized by the orientation and bonding of the atoms shown, where R and R’ are both carbon-initiated chains of varying length, also known as alkyl groups.
As usual, R and R’ are both alkyl groups or groups initiating with carbon. Esters are derivative of carboxylic acids where the hydroxyl (OH) group has been replaced by an alkoxy (O-R) group. They are commonly synthesized from the condensation of a carboxylic acid with an alcohol.”(courses.lumenlearning.com)
Lipids are not strictly speaking polymers.
Another representation of triglyceride.
Glycerides are esters formed from glycerol and fatty acids that are, as we pointed out before, are very hydrophobic. The fatty acids are nothing more than a more or less long chain of carbon atoms (from 12 to 20 C atoms) with a COOH group at the bottom and have this typical zigzag structure. In organic chemistry, they are also called carboxylic acids.
Why is fat not very soluble in water? If we look at the structure of fat, can we predict if it will be hydrophobic or are there parts that will be hydrophilic? The fat molecule is a hydrocarbon chain with a COOH group at the end and has this typical zigzag structure. In organic chemistry, they are also called carboxylic acids. In the triglyceride I have shown above the three fatty acids are the same, but it is much more common that different fatty acids are present.
There are no obvious charges that will bind to water. Oxygen is a bit more electronegative and we will have a partial positive at the carbon. Then again, carbon is more electronegative than hydrogen. It will therefore not form the kind of hydrogen bonds that one will see if we were dealing with hydroxyl groups as would have been the case if this was an alcohol. The carbon chains are very hydrophobic which is what makes fat not soluble in water. They clump up when you add them to water.
There are a number of important triglyceride chains that are important for the food processor namely saturated fats and unsaturated fats.
– Saturated and Unsaturated Fats
In saturated fats, the triglyceride chains have all single carbon atoms with all of them completely saturated (as in, there are no double bonds). Triglycerides with these kinds of chains all pack tightly. This gives them the property of chemical stability and gives them a high melting point. Saturated fats are often solid. These are bad for humans. A saturated fat is “saturated” with hydrogen atoms. We will see in a minute that unsaturated fats have a double carbon bond somewhere in its structure and wherever a double bond occurs, a hydrogen atom is eliminated which means it is “unsaturated” in terms of hydrogen atoms.
In contrast, unsaturated fats are sometimes good for humans and sometimes not. As we said, unsaturated fats have a double carbon bond. “A fat molecule is monounsaturated if it contains one double bond, and polyunsaturated if it contains more than one double bond.” (sciencedaily) Where double bonds are formed, hydrogen atoms are eliminated which makes them unsaturated in terms of hydrogen atoms.
“The greater the degree of unsaturation in a fatty acid (ie, the more double bonds in the fatty acid), the more vulnerable it is to lipid peroxidation (rancidity). Antioxidants can protect unsaturated fat from lipid peroxidation. Foods containing unsaturated fats include avocado, nuts, and soybean, canola, and olive oils. Meat products contain both saturated and unsaturated fats. Unsaturated fats are liquid at room temperature.” (sciencedaily)
This link with rancidity of great interest to the food scientist. “Rancidity is the oxidation of fats that is caused by hydration (water), oxidation (oxygen), metallic atoms or microbes. Rancidity often produces unusual odor and/or taste.” (Marcus, 2013) “Unsaturated fatty acids are a component of the phospholipids, which we discuss next, in cell membranes and help maintain membrane fluidity.” (Pelley, 2012)
There are two kinds of unsaturated fats. CIS unsaturated fats where the other bonds that are available to the carbons are on the same side of the molecule. Remember that there is no free rotation around a double bond. This means that the molecule is stuck in its configuration. CIS fats paks poorly because they are kinked and have a low melting point and these fats are good for us.
The other kind is TRANS unsaturated fats where the additional valances of carbon are on opposite sides of the molecule. These fats are similar to saturated fats as they too pack tightly with a high melting point. These are particularly bad for us. Trans fats are seldom found in nature. They are, however, found in confectionery products.
One of the important characteristics of lipids is that they can be modified. This happens when one of the fatty acid chains are replaced with something that is polar. The triglyceride is very non-polar, consisting mainly of hydrogen and carbons. Replacing one of the fatty acid chains with something that is polar, dramatically alter the properties of the molecule. A very good example of this is the formation of a phospholipid.
A phospholipid is a great example of an amphipathic molecule (with both hydrophilic and hydrophobic parts). They are similar to triglycerides in structure. One of the fatty acid groups is replaced with a phosphate which is highly charged. On the one end, the molecule is then polar and on the other end, it is non-polar. This causes them to self-associate where the polar groups face water and the non-polar groups face one another. these will self-associate and spontaneously form a lipid bilayer. A bilayered membrane will thus be formed.
One of the fatty acids is replaced with a phosphate group. The chains from the fatty acids (hydrocarbon chains) are hydrophobic. The phosphate end has charge and charged molecules dissolve in water very well. The head is, therefore, hydrophilic with two hydrophobic tails. To qualify as a phospholipid, the phosphate group should be modified by an alcohol. This structure makes them ideal for cell membranes.
Other examples of lipids are waxes and steroids.
Another example of lipids is waxes which also exist as esters.
The reason we call it an ester is because we have an ester functional group where a carbon double bonded to oxygen and single bonded to another oxygen which in turn is bonded to a long hydrocarbon chain. The carbon is also bound to a long hydrocarbon chain. The molecule is obviously very hydrophobic. Such a structure is characteristic of one of the major constituents in Beeswax.
“Most naturally occurring fats and oils are the fatty acid esters of glycerol. Esters are typically fragrant, and those with low enough molecular weights to be volatile are commonly used as perfumes and are found in essential oils and pheromones.” (courses.lumenlearning.com)
Another very common example of lipids is steroids.
Steroids share a common ring structure. They are lipids with a common ring structure. Their precursor is cholesterol which is an essential lipid and is essential for the formation of the membrane and is crucial for signaling. The issue with cholesterol is that too much is bad.
An ester has the characteristic rings. Three are 6 carbon rings and one is 5. If it has an OH group attached to it, it actually is an alcohol and a steroid which is called a sterol.
An example of a familiar sterol is cholesterol. Cholesterol is essential for life. It is a precursor molecule for steroid hormones, for example, testosterone.
Let us briefly return to our discussion on trans fats. Cholesterol is used for membranes and in signalling but is carried through the body by a component called low-density lipoprotein is deposited in the arteries where it clogs up and caused heart attacks. If the cholesterol binds to high-density lipoproteins (a different kind of a transport molecule) then excess cholesterol is secreted by the liver with no adverse effect. Transfats and saturated fats increase the levels of low-density lipoprotein and therefore increases the risk of a heart attack. 80% of cholesterol is produced by our bodies and 20% comes from our food which is why eating a low cholesterol diet does not usually help if you have high cholesterol. One must interfere with its synthesis which is what drugs like statins do.
Another example of lipids is Vitamin D which is important in the prevention of inflammation.
The History of Lipids
As is customary in our blog, we now place some of the concepts we have learned about in the first part of this article in a historical context. A hundred years ago, a key question under consideration was if fat is important in our diet. “In 1929, a young, comparatively unknown assistant professor of plant physiology at the University of Minnesota, George Oswald Burr, reported that the deficiency disease observed in rats fed a fat-free diet was caused by the absence of dietary fatty acids, not by the lack of a lipoid contained in the fat, and he concluded that fat was an essential dietary component. Burr then demonstrated that the addition of a small amount of linoleic acid, the 18-carbon ω-6 polyunsaturated fatty acid containing two double bonds (18:2ω-6), cured this deficiency disease and, therefore, was an essential fatty acid. These two seminal papers are now regarded as classics in biochemistry, but they initially met with considerable skepticism. To understand why one must appreciate the paradigm-changing nature of the discovery and the stature of the experts whose views concerning dietary fat were being challenged by Burr’s findings.” (Spector, 2015)
Views on the Role of Dietary Fat in the Early 20th Century
Proteins and carbohydrates were known to be indispensable dietary components by the first decade of the 20th century. However, dietary fat was not considered to be essential because fatty acids were known to be synthesized from carbohydrates. The evidence concerning fat was not definitive due to the inability to completely extract fat from the other dietary components using the methods available in the early 1900s, and the experimental fat-free diets of that era contained traces of residual fat.
Two of the most prominent physiological chemists of the early 20th century, Thomas B. Osborne of the Connecticut Agricultural Experiment Station and Lafayette B. Mendel of the Sheffield Scientific School at Yale University, began their studies on the role of dietary fat in 1912. Osborne and Mendel were working collaboratively in New Haven and were already recognized world-wide for their pioneering studies on dietary proteins. Their initial findings indicated that rats gained weight normally when fed a fat-free food mixture, and they concluded that “true fats” are not required for growth. However, Osborne and Mendel were aware of the work of Wilhelm Stepp in Strasbourg, who found that a lipoid present in egg yolk was an essential nutrient for mice. MacArthur and Luckett at the University of Illinois also reported that a lipoid extracted from egg yolk was necessary for optimum growth of mice.
Osborne and Mendel realized that the fat-free diet used in their studies may have contained an essential lipoid because it had not been extracted with hot alcohol. They explored this issue and in 1913 found that the growth of rats actually was reduced by a fat-free diet but was restored when an ether-extract of protein-free milk was added to the food mixture. The necessary factor was shown to be present in milk fat, butter fat, egg yolk, and cod liver oil, and extremely small quantities of this “accessory substance” supported growth. Although Osborne and Mendel determined that the substance was not an amine, they suggested that it was similar to the vital amines, then called “vitamines”, that were known to be essential dietary components. Elmer McCollum, who had done a year of postdoctoral study with Osborne and Mendel, but by this time was working independently at the University of Wisconsin, also reported that an ether-soluble substance contained in egg or butter fat restored the growth of rats consuming a fat-extracted diet. He concluded that the growth-promoting effect was due to an indispensible organic complex “in the nature of lipins”, or some substance accompanying lipins, which is an “essential accessory article in foodstuffs”. The substance discovered by Osborne and Mendel, and independently by McCollum, was initially called the “growth-promoting fat-soluble vitamin” and was subsequently designated as vitamin A. Both groups reported that the failure of the rats to grow was not due to the absence of dietary fat, lecithin, or cholesterol, findings that diverted attention away from the possibility that fatty acids might be essential nutrients.
The question of the essentiality of dietary fat was rekindled between 1918 and 1920 by Hans Aron in Breslau, who reported that fats had a specific nutrient value that could not be replaced by other foodstuffs and was not accounted for by caloric value alone. Osborne and Mendel argued that these findings were not convincing because they were obtained with butter, which contained other vital nutrients besides fat. Because of the uncertainty raised by Aron’s findings and the confusion between lack of fats and deficiency of fat-soluble vitamins, Osborne and Mendel decided to reexamine the question of whether “true fat” was an essential dietary component.
Dietary Fat Studies in the Early 1920s
Osborne and Mendel fed young rats diets exceedingly low in true fats, which they defined as compounds soluble in ether. The diets contained adequate amounts of fat-soluble vitamins from dried alfalfa and water soluble vitamins from dried yeast. To reduce the fat content as much as possible, the dried meat present in the food mixture was extracted five times with ether containing alcohol. The rats fed this lipid-extracted diet grew as well as those fed diets with liberal portions of butter fat or lard, and Osborne and Mendel concluded: “If true fats are essential for nutrition during growth the minimum necessary must be exceedingly small.”
While this statement equivocates to some degree, the research community of the 1920s interpreted it as a definitive statement that dietary fat was not essential. Negative results also were reported in 1921 by Jack C. Drummond in London. Drummond fed young rats a diet lacking neutral fat from weaning to maturity and found that they developed normally and exhibited normal behavior. He concluded that neutral fats are not required in the diet provided that the vitamins associated with fat are supplied adequately, and he stated that the real value of fat is that it is a convenient source of energy. Based on the findings of these leading experts, there was general agreement that true fats, that is, glycerides and their fatty acid moieties, were not essential nutrients.
These results and conclusions of Osborne and Mendel, and of Drummond, had a powerful influence on nutritional science in the 1920s. George Burr explained why at the Golden Jubilee International Congress on Essential Fatty Acids and Prostaglandins in 1980. The Congress, organized by Ralph Holman, was held to honor Burr for the discovery of essential fatty acids, and also Ulf von Euler for his part in the discovery of prostaglandins (PGs). In remarks delivered at the Congress banquet, Burr said that: “We had been told on high authority that fats per se were not required in the diet, and our minds were closed.”
Considering the stature of the individuals who concluded that fat was not an essential nutrient, it is easy to understand why Burr considered this as coming from high authority.
Thomas B. Osborne was internationally renowned for his work on dietary proteins and was one of the most prominent American biochemists of the early 20th century. He was a member of the National Academy of Sciences and an Honorary Fellow of the Chemical Society (London). Osborne served as the fourth President of the American Society of Biological Chemists, was awarded a gold medal by the Paris Exposition of 1900, and received an honorary degree from Yale University. Osborne’s collaborator, Lafayette B. Mendel, was an equally prominent leader of American biochemistry and a founder of the science of nutrition
Mendel was head of the Department of Physiological Chemistry at the Sheffield Scientific School. This renowned department was founded by Russell H. Chittenden, considered the dean of American biochemistry, and it was the first scientific department devoted specifically to biochemical studies in the United States. Mendel also was the Sterling Professor of Physiological Chemistry at Yale University, was elected to the National Academy of Sciences, served as the fifth President of the American Society of Biological Chemists and the first President of the American Institute of Nutrition, and received honorary degrees from Michigan, Rutgers, and Western Reserve Universities. Jack C. Drummond was a well-recognized nutritional biochemist who had a large laboratory in London in the 1920s. Drummond was appointed the first Professor of Biochemistry at University College London in 1922, became Dean of the Faculty of Medical Science, and was subsequently elected a Fellow of the Royal Society. A young, relatively unknown investigator had to be mature, self-confident, and willing to take chances to challenge such high authority, and George Burr was such an individual.
GEORGE OSWALD BURR
Burr was born in 1896 in Conway, Arkansas, played cornet in the Conway Juvenile Band, and harvested wheat in Kansas during summer vacations. He received a BA degree from Hendrix College in 1916, where he was a member of the football team. Burr had a variety of experiences between 1916 and the end of 1918. He was Principal of a high school in Crossett, Arkansas, Professor of Science at Kentucky Wesleyan College, attended summer school at the University of Chicago, worked for General Electric in Erie, Pennsylvania, and served in the United States Army Signal Corps.
In 1919, Burr was appointed Chief Chemist of the Arkansas Feed and Fertilizer Inspectors. He resigned shortly thereafter and formed the Little Rock Oil Company to drill for oil, but the company disbanded after hitting a dry well. Burr then obtained a MS degree in chemistry and mathematics at the University of Arkansas and began working for the Missouri Pacific Railroad. He resigned in 1920 after winning a scholarship to the University of Illinois to work on the synthesis of organic arsenicals. In 1921, Burr accepted a job as a science teacher at the Wichita, Kansas high school, but resigned when he was awarded a Fellowship from the Department of Biochemistry at the University of Minnesota to join the laboratory of Professor Ross Gortner. While a graduate student, Burr again showed his entrepreneurial spirit by opening a mill in Wells, Minnesota to produce sugar from corn. Burr also worked for two summers on plant distribution in the Utah and Arizona deserts with Professor J. Arthur Harris, head of the Department of Botany at the University of Minnesota. Although this summer job was unrelated to his thesis project, Burr’s association with Professor Harris had a pivotal influence on his future career. In 1924 at the age of 28, Burr received a PhD in Biochemistry and Chemistry from the University of Minnesota. His thesis characterized condensation products formed during protein hydrolysis called humins.
Burr’s experiences were much more extensive and varied than the average newly minted PhD. He had taught in public schools, studied at four universities, worked in industry and State government, did field work on plants, and had military service. His moves to new locations and ventures in drilling for oil and milling corn indicate a degree of self-confidence and willingness to take chances. These traits would serve him well in his subsequent research studies.
Postdoctoral studies at the University of California, Berkeley
Burr was awarded a National Research Council Fellowship to work with Herbert M. Evans at the University of California, and he headed for Berkeley after receiving his PhD degree. Evans was an anatomist and physiologist who, with Katherine Scott Bishop, had recently discovered a dietary factor essential for reproduction, subsequently called vitamin E. Evans, who directed a large well-funded laboratory, needed a biochemist to isolate and characterize the anti-sterility factor that he and Bishop had discovered, and Burr had the necessary expertise. Burr progressively purified vitamin E from wheat germ, first isolating it to the oil extract and then to the nonsteroid fraction of the nonsaponifiable lipids. Burr stated that by chance the Evans group was having trouble with reproducibility of their vitamin E experiments which they attributed to the presence of variable amounts of lipid containing vitamin E in the basal diet used to produce the sterile female rats for testing.
To investigate this possibility, Burr set up a separate colony of rats that were fed a fat-free diet that he prepared, consisting of sucrose recrystallized from alcohol, purified and reprecipitated casein, salts, and vitamin supplements. The rats fed this diet developed a disease that was different from vitamin E deficiency. Evans and Burr reported this new dietary deficiency, initially only emphasizing the potential usefulness of the experimental diet without speculating on the cause of the deficiency. Burr’s further work demonstrated that, unlike the known fat-soluble vitamins that were present in the nonsaponifiable lipid fraction, the substance which prevented the disease was present in the fatty acid fraction of the lipid extract. Based on this finding, Evans and Burr hypothesized that the active factor was a new vitamin-like substance present in the fatty acid fraction of fat and tentatively designated it as vitamin F.
Burr, in his written comments in 1980, stated: “Over a period of 4 years of work and 3 published papers, it never occurred to us that the deficiency was the lack of a well-known fatty acid.”
A personal event occurred during Burr’s tenure in Berkeley that turned out to be an important factor in his subsequent discovery of essential fatty acids. Burr married Mildred Lawson, an assistant in the Evans laboratory who was in charge of the rat colony. Mildred’s expertise with laboratory rats was vital for Burr’s subsequent studies on fatty acid deficiency, and she was the coauthor of the two classic papers on the discovery of essential fatty acids. Mildred Burr was also a coauthor of the 1932 paper reporting the essentiality of α-linolenic acid (18:3ω-3), the ω-3 analog of linoleic acid that is the parent of the ω-3 family of polyunsaturated fatty acids
Faculty appointment at the University of Minnesota
The University of Minnesota completed a new Botany Building with adequate space in 1926, and Professor Harris, with whom Burr had worked during summers on plant distribution in the desert, was given new positions to expand the Botany faculty. Harris recognized Burr’s talent and succeeded in recruiting him as an Assistant Professor of Plant Physiology. Burr left for Minneapolis in September, 1928, stating: “With deep sorrow and high hopes, the Burr’s left Berkeley in their Model T Ford roadster with two cages of Long-Evans rats…. On cold fall nights, our pets were smuggled into hotel rooms under long overcoats.”
Although Professor Harris hired Burr as a plant physiologist, he told Burr that he didn’t care what type of research he did as long as it was good work. Burr decided to continue his fat nutrition studies, so Harris arranged space for a rat colony in the attic of the Anatomy Building. The attic room was equipped with air conditioning and the finest individual metabolic cages, and Burr set up a small rat colony with the cooperation of C. M. Jackson, Professor of Anatomy. Burr received support from the University of Minnesota Research Fund and a grant from the Graduate College, but funding still was very limited. He states that because of the shortage of research funds, Mildred Burr pitched in and made some of the special observations, including the effects of the fat-free diet on the estrus cycle and fertility. Thus, the paradigm-changing studies on essential fatty acids had their beginning, and the resulting papers were published with Mildred Burr as coauthor
The classic papers of 1929 and 1930
Burr realized that to make further progress, he had to rigidly exclude fat from the diet and describe the new deficiency symptoms in quantitative terms so that the relative curative value of additives could be measured. The paper published in the May 1929 issue of the Journal of Biological Chemistry describes the purification of the fat-free diet in great detail and contains a much more complete description of the deficiency disease than the prior Evans and Burr publications. The results proved that dietary fat was required to stimulate growth and prevent disease in rats fed the fat-free diet. The key finding, shown in Fig. 2 which is reprinted from Burr’s 1929 paper, was that the component of the fat that stimulated growth and prevented disease was the fatty acid fraction, not the nonsaponifiable lipids or the glycerol backbone of the glycerides. Burr concluded that, “The data presented here definitely settle the uncertainty as to the necessity for fats in the diet (of the rat) and prove not only that ingested fats have a beneficial effect upon the animal but that under certain experimental conditions outlined in this paper they are essential constituents of the diet.”