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Waxes



To my knowledge, there is no satisfactory definition of the word "wax" in chemical terms. It is derived from the Anglo-Saxon word "weax" for beeswax, so a practical definition of a wax may therefore be "a substance similar in composition and physical properties to beeswax". Technologists use the term for a variety of commercial products of mineral, marine, plant and insect origin that contain fatty materials of various kinds, but only those from living organisms are described here. Waxes of mineral origin, such as montan wax, are not discussed.


1.  Introduction

Biochemists often link waxes with the thin layer of fatty constituents that cover the leaves of plants or provide a surface coating for insects or the skin of animals for which primary requirements are spreadability and chemical and metabolic stability. Such surface waxes are produced by specialized cells or glands, and all tend to contain wax esters as major and perhaps defining components, i.e. esters of long-chain fatty alcohols with long-chain fatty acids.

Formula of a wax ester

The nature of the other lipid constituents can vary greatly with the source of the waxy material, but they include hydrocarbons (including squalene), sterol esters, aliphatic aldehydes, primary and secondary alcohols, 1,2-, 2,3- and α,ω-diols, ketones, β-diketones, triacylglycerols, and many more.

Formulae of common wax constituents

Also, the chain-length and degree of unsaturation and branching of the fatty acids and the other aliphatic constituents, varies with the origin of the wax, but other than in some waxes of marine origin or from some higher animals, the aliphatic moieties tend to be saturated or monoenoic.


2.  Plant Waxes

Plant leaf surfaces are coated with a thin layer of waxy material that is microcrystalline in structure and forms the outermost boundary of the cuticular membrane, i.e. it is the interface between the plant and the atmosphere. It serves many purposes, for example to limit the diffusion of water and solutes and control gas exchange, while permitting a controlled release of volatiles that may deter pests or attract pollinating insects. The wax provides protection from ultraviolet light, disease and insects, and helps the plants resist drought and other environmental stresses. As plants cover much of the earth's surface, it seems likely that plant waxes are among the most abundant of all natural lipids.

The range of lipid types in plant waxes is highly variable, both in nature and in composition, and Table 1 illustrates some of this diversity in the main components.

Table 1. The major constituents of plant leaf waxes.
  n-Alkanes CH3(CH2)xCH3 21 to 35C - odd numbered
  Alkyl esters CH3(CH2)xCOO(CH2)yCH3 34 to 62C - even numbered
  Fatty acids CH3(CH2)xCOOH 16 to 32C - even numbered
  Fatty alcohols (primary) CH3(CH2)yCH2OH 22 to 32C - even numbered
  Fatty aldehydes CH3(CH2)yCHO 22 to 32C - even numbered
  Ketones CH3(CH2)xCO(CH2)yCH3 23 to 33C - odd numbered
  Fatty alcohols (secondary) CH3(CH2)xCHOH (CH2)yCH3 23 to 33C - odd numbered
  β-Diketones CH3(CH2)xCOCH2CO(CH2)yCH3 27 to 33C - odd numbered
  Triterpenols Sterols, α-amyrin, β-amyrin, uvaol, lupeol, erythrodiol
  Triterpenoid acids Ursolic acid, oleanolic acid, etc
 

In addition, there may be hydroxy-β-diketones, oxo-β-diketones, alkenes, branched alkanes, acids, esters, acetates and benzoates of aliphatic alcohols, methyl, phenylethyl and triterpenoid esters, and many more. Polar complex lipids are rarely encountered.

The amount of each lipid class and the nature and proportions of the various molecular species within each class vary greatly according to the plant species and the site of wax deposition (leaf, flower, fruit, etc.), and some data for some well-studied species are listed in Table 2.

Table 2. Relative proportions (wt %) of the common wax constituents in some plant species.
Arabidopsis
leaf
Rape leaf Apple fruit Rose flower Pea leaf Sugar cane
stem
 
  Hydrocarbons 73 33 20 58 40-50 2-8
  Wax esters - 16 18 11 5-10 6
  Aldehydes 14 3 2 - 5 50
  Ketones 4 20 3 - -
  Secondary alcohols 1 8 20 9 7 -
  Primary alcohols 8 12 6 4 20 5-25
  Acids 1 8 20 5 6 3-8
Other components present include various diol types and triterpenoid acids

Carnauba - The leaves of the carnauba palm, Copernicia cerifera that grows in Brazil, have a thick coating of wax, which can be harvested from the dried leaves. It contains mainly wax esters (85%), which constitute C16 to C20 fatty acids linked to C30 to C34 alcohols to give C46 to C54 molecular species, and these are accompanied by small amounts of free acids and alcohols, hydrocarbons and resins. This is the only leaf wax available in sufficient quantities to be of commercial value.

Jojoba - The jojoba plant (Simmondsia chinensis), which grows in the semi-arid regions of Mexico and the U.S.A., is unique in producing wax esters rather than triacylglycerols in its seeds, and it has become a significant crop. These consist mainly of 18:1 (6%), 20:1 (35%) and 22:1 (7%) fatty acids linked to 20:1 (22%), 22:1 (21%) and 24:1 (4%) fatty alcohols. Therefore, it contains C38 to C44 esters with one double bond in each alkyl moiety. As methylene-interrupted double bonds are absent, the wax is relatively resistant to oxidation.

Bayberry (Myrica pensylvanica) fruits are covered with a thick layer of crystalline wax (30% of the dry weight) that consists of triacylglycerols and diacylglycerols esterified exclusively with saturated fatty acids. It is believed that this may attract birds as an aid to seed dispersal. Biosynthesis and secretion differ from conventional plant waxes and are more closely related to cutin production. The Japanese sumac tree (Rhus verniciflua) produces a similar wax ('Japan wax').


Biosynthesis of Plant Waxes

Because of their biochemical importance and relative ease of study, the waxes of the plant cuticle have received most study. All the aliphatic components of plant waxes are synthesised in the epidermal cells from saturated very-long-chain fatty acids (commonly C20 to C34). 16:0 and 18:0 Fatty acids are first synthesised in the stroma of plastids by the soluble enzymes forming the fatty acid synthase complex. In the second stage, multiple elongation steps are catalysed by membrane-associated multi-enzyme complexes, known as fatty acid elongases, outwith the plastids (see our web page on the biosynthesis of saturated fatty acids). As in fatty acid synthesis de novo, each two-carbon extension of the chain involves four reactions: condensation between a CoA-esterified fatty acyl substrate and malonyl-CoA, followed by a β-keto reduction, dehydration and an enoyl reduction to produce saturated very-long-chain fatty acids with 24 to 36 carbon atoms.

There are then two main pathways for biosynthesis of wax components in the endoplasmic reticulum of plastids of the epidermal cells: an acyl reduction pathway, which yields primary alcohols and wax esters, and a decarbonylation pathway that results in synthesis of aldehydes, alkanes, secondary alcohols and ketones. In the reductive pathway, acyl-CoA esters produced by chain elongation are reduced in a two-step process via a transient aldehyde intermediate, catalysed by a single enzyme, an acyl-CoA reductase (though it was once thought that two distinct enzymes were involved).

Biosynthesis of fatty alcohols and wax esters

The fatty alcohol produced can then be esterified via an acyl-CoA alcohol transacylase to form a wax ester. An enzyme of this type has been isolated from microsomal fractions of seeds of the jojoba plant that is responsible for production of the storage wax, but also appears to be structurally related to the wax synthases involved in the assembly of the epicuticular waxes. Similar mechanisms have been observed in studies with insects, algae and birds (uropygial glands).

In the decarbonylation pathway for the synthesis of wax constituents, the first step is again believed to be the reduction of acyl-CoA ester to an aldehyde by means of an acyl-CoA reductase. Removal of the carbonyl group by an aldehyde decarbonylase yields an alkane, with one fewer carbon atom than the fatty acid precursor. However, the enzymes involved have not been characterized.

Biosynthesis of hydrocarbons, secondary alcohols and ketones

Further metabolism of the hydrocarbon is then possible, for example by insertion of a hydroxyl group into the chain via a hydroxylase or mixed-function oxidase to form a secondary alcohol, with the position of the substitution depending on the species and the specificities of the enzymes involved. Secondary alkanols can in turn be esterified to form wax esters. Alternatively, the hydroxyl group can be oxidized with formation of a long-chain ketone, while an associated pathway leads to the formation of β-diketones and 2-alkanols. Again, these processes have been studied most in plants, but similar biochemical reactions appear to occur in insects and birds.

Once synthesised, the wax components must be exported from the sites of lipid synthesis in the plastid and the endoplasmic reticulum to the plasma membrane and through the cell wall. They must then pass into the cutin layer that provides a matrix, consisting of a polymer of hydroxy and dibasic fatty acids, within and upon which the waxes are deposited. Less is known of how wax is exported, but two groups of transport molecules have been identified that are known to facilitate this process, i.e. ATP binding cassette (ABC) transporters and members of the lipid transfer protein (LTP) family. There is also evidence for vesicular transport.


3.  Waxes from Animal Tissues

Waxes associated with skin

In mammals, the main wax production is associated with the sebaceous glands of the skin, most of which are associated with hair follicles, although there are also related structures on the eyelids termed Meibomian glands. Sebaceous glands secrete mainly non-polar lipids in the form of sebum onto the skin surface, where they are easily recovered for analysis. In humans, sebaceous glands are distributed throughout the body with the exception of the palm and sole of the foot. They consist of three type of cells, peripheral undifferentiated cells, cells that produce lipid bodies, and lysing cells loaded lipid that empty their contents into the lumen. Although relatively few species have been studied in real detail, it is evident that a wide range of lipid classes are present and that these vary greatly in amount and nature between species (there may also be variation with age). The composition of human sebum differs appreciably from that of other species, especially in the high content of triacylglycerols and in the nature of the fatty acids. Some typical data are listed in Table 3.

Table 3. Relative composition (wt % of the total) of the non-polar lipids of from the skin surface of various species.
Squalene Sterols Sterol esters Wax esters Diesters Glyceryl ethers Triacyl-
glycerols
Free acids Free alcohols
 
  Human 12 1 3 25 41 16
  Sheep 12 46 10 21 11
  Rat 1 6 27 17 21 8 1
  Mouse 13 10 5 65 6
Adapted from Downing, D.T. Mammalian waxes. In: Chemistry and Biochemistry of Natural Waxes. (Ed. P.E. Kolattukudy, Elsevier, Amsterdam) (1976).

Sebaceous glands are the only appreciable source of wax esters in mammalian tissues and the only tissue where squalene accumulates in significant amounts. The alcohol components of the wax esters are C24 to C27 iso- and anteiso-methyl-branched, while the fatty acids are mainly C12 to C29 saturated and monounsaturated with a relatively high proportion being branched chain, although the last tend to be trace components only in other organs. Very-long-chain ω-hydroxy acids are also present, and in some the hydroxyl group is esterified with a conventional fatty acid. The biosynthesis of long-chain and very-long-chain fatty acids is discussed in our web page dealing with saturated fatty acids. Fatty alcohols are synthesised from the corresponding acids by the action of fatty acyl-CoA reductases with NADPH as the reductant, apparently without formation of an aldehyde as an intermediate. Finally, fatty acids and alcohols are coupled by multifunctional wax synthases, designated AWAT1 and AWAT2, although an enzyme involved in triacylglycerol biosynthesis, i.e. an acyl CoA:diacylglycerol acyltransferase (DGAT1), can also be used to synthesise wax and retinol esters.

Human sebum is unique in containing cis-6-hexadecenoic acid (6-16:1 or sapienic acid), which is the single most abundant component indeed, and is accompanied by an elongation and desaturation product 5,8-octadecadienoic acid (‘sebaleic’ acid), also unique to human skin. Sapienic acid is formed in the sebaceous glands by a Δ6 desaturase FADS2 involved in the formation of polyunsaturated fatty acids; it has powerful antibacterial properties, for example against the organisms responsible for acne and the opportunist pathogen Staphylococcus aureus.

Skin also contains a wide range of distinctive but more polar lipids based on the ceramide backbone. They have been most studied in the skin of the pig and human, where a range of unusual ceramides have been identified, some of which contain linoleic acid (a) esterified to a hydroxy acid (b) that is in turn linked to a long-chain base (c). In addition, several molecular forms of glucosylceramide, based on similar structures, have been characterized.

Formula of a skin ceramide

The composition depends on the particular layer of the skin (epidermis, stratum corneum, etc.). Whether they should truly be called waxes is doubtful (see our web pages on ceramides, where there is much more information).

Vernix caseosa is a waxy material that coats the skin of the human fetus and newborn and is produced during the third trimester of gestation. In utero, it acts as waterproofing, to control the flux of water across the skin and as a protective agent, facilitating the final stages of development of the skin. Following birth, it protects the skin against bacterial attack and aids the neonate to adapt to exposure to air. The lipid component includes a high proportion of triacylglycerols and wax esters. The latter have a rather unusual pattern of fatty acids in a range of chain-lengths (C10 to C16) with methyl substituents in different even-numbered positions in the alkyl chain, presumably formed during biosynthesis by the replacement of malonyl CoA with a molecule of methyl malonyl CoA at irregular intervals on the growing alkyl chain.

Wool wax (lanolin) - The grease obtained from the wool of sheep during the cleaning or refining process, and presumably derived from the sebaceous glands and/or stratum corneum of the skin, is rich in wax esters (of 1- and 2-alkanols and of 1,2-diols), sterol esters, triterpene alcohols, and free acids and sterols. The nature of the product varies with the degree and type of processing involved, but can contain up to 50% wax esters and 33% sterol esters. A high proportion of the sterol component is lanosterol, while the fatty acid components are mainly saturated with straight and iso- and anteiso-methyl-branched-chains. This is the only animal wax of commercial value.


Other animal waxes

Meibomian glands are holocrine glands that are located in the upper and the lower eyelids of humans and most animals and produce an oily, lipid-enriched secretion, often termed meibum. This is excreted onto the eye, mixing with tears to form the outermost surface layer as a protection from desiccation and bacterial infection. The lipids constitute a mixture of many different classes, including wax esters, cholesterol esters, acylglycerols, diacylated diols, free fatty acids and cholesterol. A multitude of different fatty acid components are present, including very-long chain (C16 to C36) branch-chain, mono-unsaturated (often with a double bond in position 6), and ω-hydroxy and (O-acyl)-ω-hydroxy fatty acids. While many aspects of the biosynthesis remain to be elucidated, it is evident that mice and humans have similar gene products and there must be active fatty acid elongases (ELOVL) and reductases. Cholesterol synthesis de novo occurs and there are wax ester and cholesterol ester synthases.

Harderian glandsScottish thistle are exocrine glands located deep in the orbit of the eyeball in most terrestrial vertebrates that possess a nictitating membrane, i.e. a protective translucent third eyelid. The gland secretes lipids onto this membrane, and in rodents these consist mainly of alkyldiacylglycerols and wax esters with many different fatty acid constituents from isovaleric up to C30 in chain length and often with hydroxyl groups in position 2. In the rat, the main molecular species of wax ester consists of a 24:1(n-7) alcohol attached to a 20:1(n-7) fatty acid.

Marine waxes. Many marine animals from invertebrates to whales contain appreciable amounts of waxes in the form mainly of hydrocarbons and wax esters. In addition, glycerol ethers and sterols could be classified as wax components in some species. They are found in a variety of tissues from fish roe, to liver and muscle tissues. The wax esters consist of the normal range of saturated, monoenoic and polyunsaturated fatty acids typical of fish, esterified to mainly saturated and monoenoic alcohols often with the 18:1 fatty alcohol as the main component. The orange roughy, a fish found predominantly in the seas around New Zealand, has more than 90% of its total lipid in the form of wax esters. In the hydrocarbon fraction, squalene and other terpenoid hydrocarbons are frequently major constituents, and they can be accompanied by saturated (straight-chain and methyl-branched), monoenoic and polyenoic components. Waxes appear to have a variety of functions in fish, from serving as an energy source to insulation, buoyancy and even echo location. Spermaceti or sperm whale oil (wax esters, 76%; triacylglycerols, 23%) was once in great demand as a lubricant but now is proscribed. Zooplankton can produce large amounts of wax esters at some stages of their life cycle, and they make a major contribution to the marine food chain.

Bird waxes. The uropygial or preen glands of birds are similar structurally to sebaceous glands, and they secrete waxes that consist largely of wax esters. With monoesters, the fatty alcohol components are usually relatively simple in nature, consisting largely of normal C16 and C18 saturated compounds, although those with branched-chains can make up an appreciable proportion in some species. The chicken and related species contain fatty acid esters of 2,3-alkanediols. Depending on species, the fatty acids can be highly complex and are often shorter chain than usual with up to four methyl branches (see our web page on branched-chain fatty acids). Mono- and bifunctional wax ester synthases have been characterized, the latter catalysing both wax ester and triacylglycerol synthesis, and both forms differ in their substrate specificities with regard to branched-chain alcohols and acyl-CoA thioesters. The main purpose of the waxes is presumed to be to give a water-proof layer to the feathers, but other functions have been suggested.


4.  Other Waxes

Insect waxes. The surface of insects is covered by a layer of wax that serves to restrict movement of water across the cuticle and prevent desiccation and provides a physical barrier to protect against abiotic and biotic stresses. The nature of this lipid is dependent on species, but in general a high proportion tends to be saturated alkanes (C23 to C31) often with one or two methyl branches. In addition, wax esters, sterol esters, and free fatty alcohols and acids may be present. Some species of insect secrete triacylglycerols in their waxes together with free sterols and other terpenoid components; aphids are distinctive in that they secrete triacylglycerols containing sorbic (2,4-hexadienoic) acid.

Beeswax: the archetypal wax. Glands under the abdomen of bees secrete a wax, which they use to construct the honeycomb, and this is recovered as a by-product when the honey is harvested and refined. It contains a high proportion of wax esters (35 to 80%), which consist of C40 to C46 molecular species, based on 16:0 and 18:0 fatty acids some with hydroxyl groups in the ω-2 and ω-3 positions. In addition, some diesters with up to 64 carbons may be present, together with triesters, hydroxy-polyesters and free acids (which are different in composition and nature from the esterified acids). The hydrocarbon content is highly variable, and much may be "unnatural" as beekeepers may feed some to bees to improve the yield of honey.

Microbial waxes. Waxes, often accompanied by triacylglycerols, are known to be produced by many species of prokaryotes, including both Gram-negative and Gram-positive bacteria. The mycobacteria especially produce waxes, termed 'mycoserosates', based on branched-chain alcohols or 'phthiocerols'. These are C34 or C36 branched-chain compounds with hydroxyl groups in positions 9 and 11, or 11 and 13, which are esterified with long-chain fatty acids varying in chain length from C18 to C26 and with two to four methyl branches that may occur at the 2, 4, 6, and 8 positions. In pathogenic mycobacteria such as Mycobacterium tuberculosis and M. leprae, the dimycocerosate esters are major virulence factors. Related structures have a phenol group at the terminus of the alkyl chain, and the hydroxyl of this can be attached to carbohydrate moieties of varying complexity. Mycobacteria are unusual in a number of other ways in that their lipids can comprise triacylglycerols, complex trehalose-containing lipids, and the distinctive mycolic acids, for example.

Formula of a phthiocerol

Biosynthesis of wax esters in the Gram-negative Acinetobacter baylyi consists of three enzymatic steps; a fatty acid CoA ester is reduced by an NADPH-dependent fatty acyl-CoA reductase to a long-chain aldehyde, which is further reduced by an as yet uncharacterized aldehyde reductase to a fatty alcohol for esterification with a fatty acyl-CoA by the well characterized bifunctional enzyme wax ester synthase/diacylglycerol acyltransferase to produce a wax ester.


5.  Digestion and Absorption of Wax Esters

Wax esters in mammalian food are poorly hydrolysed in the digestive system by pancreatic lipase, so they have little nutritional value. However, fish are obviously well adapted to their diet of wax-rich zooplankton, and have little difficulty in hydrolysing wax esters and assimilating their components into their tissues, presumably with the aid of so-far uncharacterized lipases or esterases.


6.  Analysis

Thin-layer and high-performance liquid chromatography have been used widely to isolate individual classes of waxes for more detailed analysis. On the other hand, much of the more recent published work has made use of high-temperature gas chromatography following trimethylsilylation, often in combination with mass spectrometry, so that simultaneous identification and quantification of the various molecular species can be achieved.


Further Reading

The best sources of information on waxes, from which much of the data in this web document have been culled, are the following books (now out of print, unfortunately) -

In addition -



Lipid listings Credits/disclaimer Updated: July 20th, 2017 Author: William W. Christie LipidWeb icon