Lipids

Lipids R. Kennedy Keller University of South Florida, Tampa, USA ã 2007 Elsevier Inc. All rights reserved.

Chemistry: Lipids are a major class of biomolecules that often bring to mind negative connotations, such as the correlation of excess dietary fat intake to heart disease. Nevertheless, lipids play essential roles in the body. They serve as insulation for retention of heat and nerve conduction, they are the major form of energy storage, and they are the major components of cell membranes. Most recently, lipids have received considerable attention because of their role in signal transduction. Definition: The most common definition of a lipid is that it is a material that extracts from biological samples with organic solvents. Such solvents usually include mixtures of either chloroform and methanol or hexane and isopropanol. This definition covers a wide range of substances, including some compounds that are present in trace amounts. This report is confined primarily to those lipids generally considered as ‘‘bulk lipids’’ as opposed to those occurring in trace amounts that belong to other molecular classes. Accordingly, steroid hormones, eicosanoids, coenzymes Q , and the fat-soluble vitamins are not dealt with here. Classification: Although there are several ways to categorize lipids, a useful starting point is into two major groups, neutral and polar. The neutral lipids include waxes, fatty esters, mono-, di-, and triglycerides, squalene, sterols and sterol esters, carotenes, dolichols, long chain alcohols, and alpha-glyceryl ethers. Polar lipids include glycerophospholipids, glyceryl ether phospholipids, plasmalogens, sphingomyelin, glycolipids (glycosylceramides and gangliosides), cardiolipin, bile acids, and lysophospholipids. Structures of the most commonly occurring lipids are shown in Fig. 1. For an exhaustive list of lipid structures, see the Cyberlipid Center website at: http://www.cyberlipid.org/. Extraction: The extraction of lipids from a variety of sources has been reviewed St. John and Bell (1989). The most common procedures use chloroform and methanol mixtures Folch et al (1957), Bligh and Dyer (1959). Despite the broad use of chloroform for extraction, it is a suspected carcinogen. Alternative methods of extraction, such as using hexane-isopropanol mixtures Hara and Radin (1978), have been described, but they are rarely used. Those working with chloroform should be aware of its deleterious effects and perform extractions in a well-ventilated hood. Following extraction, nonlipid material is usually removed by washing the organic phase with aqueous solvent. Highly polar lipids, such as gangliosides (which contain ionizable sialic acid), dolichol-oligosaccharides (intermediates in glycoprotein synthesis), bile acids, and phosphorylated phosphatidyl inositols are incompletely extracted with the usual methods and require a more polar solvent mixture, often containing water. Cabrini et al. (1992) compared different lipid extraction procedures for different tissues and found that recovery varies considerably. They concluded that the procedure chosen depends on the tissue being extracted and which lipids are being analyzed Cabrini et al (1992). Once the extract is obtained, total lipid can be determined gravitimetrically by drying the organic extract to constant weight or using colorimetric assays. Isolation: There are a variety of published procedures to separate the different classes of lipids and to purify a particular lipid for analysis. Separation using thin layer chromatography is a common procedure that has the advantage of allowing multiple samples to be run on a single plate Skipski and Barclay (1969). Classic column chromatography on Florisil, silicic acid, or DEAE-cellulose has also been used Lowenstein (1969). Over the 1

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Lipids

Fig. 1. Structures of the most commonly occurring lipids.

last two decades, the use of prepoured sorbent extraction cartridges (e.g., Sep Pak, Bond Elute) has gained wide acceptance Kaluzny et al (1985). High-pressure liquid chromatography (HPLC) is also commonly used. For example, Markello and others Markello et al (1991) described the use of HPLC to separate all major lipid classes in a single run. Special techniques are described in the literature for the isolation of particular species within a lipid class. For example, HPLC in the presence of silver ion is used for the separation of sterol intermediates that differ in double bond number and position Ruan et al (1997). Analysis: For lipids isolated on TLC or column chromatography, the isolated species is often directly quantified (e.g., individual phospholipids can be assayed using a colorimetric assay for phosphate). For other species, HPLC, GC, or GC-mass spectroscopy is used. If the aim is to recover the lipid species in its native (underivatized) form, and if the lipid can be quantified using a nondestructive property (e.g., UV absorption), HPLC with online detection is often the method of choice (e.g., sterol and phospholipid analysis).

Biochemistry Dietary considerations—Because of increasing concerns about obesity, health officials have recommended a substantial reduction in the amount of fat in the diet to less than 30% of total calories. In fact, most lipids used in vivo are synthesized from nonlipid sources. The exceptions include the fat-soluble vitamins, A, D, E, and K, and fatty acids possessing double bonds 6 carbons or less from the o end (the o end is opposite to the carboxyl end; e.g., linoleate, linolenate). The latter compounds are required for synthesis of the eicosanoids, a family of lipids that includes leukotrienes, prostaglandins, prostacyclins, and thromboxanes.

Lipids

Digestion and Transport—Since lipids are not water soluble, they must be solubilized for digestion. This is the role of bile acids, secreted from the liver via the gall bladder. Bile acids emulsify dietary triglycerides and cholesterol esters for hydrolysis by intestinal lipases. Following uptake into the enterocyte, these fats are re-esterified to trigylcerides and cholesterol esters and packaged into chylomicrons, large lipoproteins with a density less than water. Chylomicrons are transported by the lymph and enter the bloodstream through the thoracic duct. They are rapidly cleared, due to the action of lipoprotein lipase which hydrolyzes the triglycerides to free fatty acids. The free fatty acids are used for energy production by various tissues with the excess stored in adipose tissue as triglyceride. The liver clears the remaining ‘‘chylomicron remnant’’. This part of lipoprotein metabolism is often referred to as the exogenous pathway. In the endogenous pathway of lipoprotein metabolism, the liver synthesizes and secretes very low-density lipoproteins, which are also degraded by lipoprotein lipase. The immediate product of this action is intermediate density lipoprotein (IDL) and then low-density lipoprotein (LDL), which is accumulated by liver (predominantly) and peripheral tissues by way of the LDL receptor. In the absence of an active receptor, LDL becomes oxidized and binds to a scavenger receptor on macrophages, a process that increases the risk for atherosclerosis. Metabolism—The bulk of the lipids synthesized in vivo are derived from the two carbon precursor acetyl-Co A. The first and regulated step of fatty acid synthesis is catalyzed by acetyl-CoA carboxylase, which produces malonyl-Co A. Malonyl-CoA and acetyl-CoA are the starting substrates for fatty acid synthase, which generates palmitoyl-Co A. The latter is a starting point for further elongation and desaturation reactions. The CoA derivatives so generated are used for the formation of triglycerides and the various phospholipids, the latter of which are requisite components of membranes and lipoproteins. Acetyl-CoA is also the precursor to sterols. In the formation of cholesterol, 15 acetylCoA units are required to generate the 27-carbon product. The regulated step of this pathway is HMG-CoA reductase, a primary site for drug targeting to lower cholesterol levels (HmG-CoA reductase inhibitors). Cholesterol serves as the precursor to bile acids in the liver, the regulated step being cholesterol 7-a hydroxylase. In the adrenals and gonads, cholesterol serves as a precursor to steroid hormones, via the action of cytochrome P450 enzymes. The rate-limiting enzyme of steroidogenesis is mitochondrial cholesterol 22,23 desmolase (side chain cleavage enzyme). Regulation of this pathway appears to be controlled by the transport of cholesterol into the mitochondria by a shortlived mitochondrial import factor referred to as the steroidogenic acute regulatory protein (StAR). The degradation of fat begins with the action of hormone-sensitive lipase, which catalyzes the breakdown of triglycerides in adipose tissue. The released fatty acids are transported to the liver via albumin and then taken into mitochondria using carnitine as a carrier. Very long chain fatty acids are shortened in the peroxisomes and are released as octanoyl-CoA, which enters the mitochondria. Once in the mitochondria, fatty acyl CoAs are degraded via b-oxidation to acetyl-CoA, which can be further oxidized via the Krebs cycle. Complete degradation of fat yields about 9 Kcal/g, more than twice that derived from glycogen or protein degradation. When the ratio of glucagon to insulin is elevated and the intracellular concentration of oxaloacetate is low, a portion of liver acetyl-CoA derived from fatty acids is shunted to ketone body synthesis. In the diabetic, the rate of this flux can become severe, resulting in ketoacidosis. Endocrinology and signal transduction: Until a few years ago, the only lipids considered to be involved in cell signaling were steroid hormones, eicosanoids, and

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platelet activating factor (a water-soluble ether phospholipid). However, it is now recognized that cholesterol, fatty acids, and other dietary lipids serve as precursors for ligands that bind nuclear receptors and participate in signal transduction (for a review see Chawla et al (2001)). The nuclear receptors that bind lipid derivatives are part of a superfamily that includes the steroid hormone receptors, with the major difference that they bind their respective ligands with much lower affinity (~10-6 M). The lipid receptors dimerize with retinoic acid receptors to regulate genes involved in lipid metabolism and transport. For example, cholesterol is metabolized by cholesterol 7-alpha hydroxylase in the liver to generate bile acids, which in turn bind to the FXR receptor. The activated FXR receptor then mediates a series of events that results in the down-regulation of CYP genes involved in bile acid synthesis (Table 1, taken from Chawla et al (2001) provides a list of lipid receptors, their ligands, and the action mediated).

Table 1. The nuclear receptor ligand metabolic cascade. The RXR heterodimers, their ligands, and regulated target genes are shown. Question-marks (?) indicate that a member of this family has not yet been identified as a target for this ligand/receptor. Arrows denote whether the gene is up- or down-regulated by its cognate ligand. CYP, cytochrome P450; ABC, ATP-binding cassette. Nuclear receptor

Retinoid X receptors*

Peroxisome proliferatoractivated receptors

Ligand

CYP enzyme

Cytosolic binding protein

ABC transporter

RXRa,b,g

9-cis Retinoic acid

-

-

-

PPARa

Fatty acids

"CYP4A1

"L-FABP

PPARd

Fibrates Fatty acids

"CYP4A3 (?)

(?)

"ABCD2, ABCD3 "ABCB4 (?)

Liver X receptors

LXRa,b

Carboprostacyclin Fatty acids Eicosanoids Thiazolidinediones Oxysterols

Famesoid X receptor

FXR

Bile salts

#CYP7A1

"IBABP

SXR/ PXR

Xenobiotics

#CYP8B1 "CYP3A

(?)

"ABCB1, ABCC2

Steroids

"CYP2C "ABCC3

Hexamerins

"E23

RARa,b,g

Retinoic acids

"CYP2B "CYP2C "26-(OH) ase "CYP26A1

(?)

EcR

Xenobiotics Phenobarbital 20(OH)-ecdysone

"CRABPII

(?)

VDR

1,25(OH)2-vitamin D3

"CYP24 #CYP27B1

"CRBPI (?)

(?)

PPARg

Xenobiotic receptors CAR Ecdysone receptor Retinoic acid receptors Vitamin D Receptor

"CYP4B1

"ALBP/aP2 "H-FABP

(?)

"CYP7A1

OSBPs?

"ABCA1, "ABCG1, ABCG4 "ABCG5, ABCG8 "ABCB11

*RXRs serve as common heterodimer partners with other receptors

Lipids

Table 1. Lipid receptors, their ligands, and the action mediated. (Reproduced with permission from the American Association for the Advancement of Science http://www. sciencemag.org/). Lipid diseases and pharmacology: By far the most common clinical presentation related to altered lipids is hyperlipidemia (actually lipemia), which is an important risk factor in developing atherosclerosis and heart disease. There are six types of hyperlipidemias (I, IIa, IIb, III, IV, and V), which are differentiated by the type(s) of lipids elevated in blood. Some types may be caused by a primary disorder such as a familial lipoprotein lipase deficiency. However, it must be appreciated that monogenic causes of hyperlipidemia are rare. Secondary causes of hyperlipidemia are related to disease risk factors, dietary risk factors, and drugs associated with hyperlipidemia. Disease risk factors include Type I and Type II diabetes mellitus, hypothyroidism, Cushing’s syndrome, and certain types of renal failure. Dietary risk factors include dietary fat intake greater than 40% of total calories, saturated fat intake greater than 10% of total calories, cholesterol intake greater than 300 milligrams per day, habitual excessive alcohol use, and obesity. Drug risk factors include birth control pills, hormones such as estrogen and corticosteroids, certain diuretics, and beta adrenoceptor antagonists. Cigarette smoking with hyperlipidemia increases the risk for heart disease. For more information, see The Medline Information Web site at http://www.nlm.nih.gov/medlineplus/ency/article/000403.htm The first line treatment in the management of hyperlipidemia usually involves changing the risk factors associated with diet, disease, and drugs. However, such changes often do not reduce serum lipid levels into the normal range. Pharmacological approaches include the use of statins (which competitively inhibit HMG-CoA reductase), fibric acid derivatives (which bind to the peroxisomal proliferation activator receptor PPAR and enhance catabolism of triglyceride-rich particles and reduce secretion of VLDL particles) (see Table I), bile acid resins (which block the interhepatic circulation of bile acids, thus increasing the conversion of hepatic cholesterol to bile acids), and nicotinic acid (which blocks VLDL synthesis). Recent studies indicate that statins alone can achieve the desired change in serum lipids with few side effects Henley et al (2002). There are several rare inherited diseases of lipid metabolism (e.g., Refsum’s disease, adrenoleukodystrophy, various lysosomal or peroxisomal protein deficiencies), which are described in detail by Scriver and others Scriver et al (2001).

Other Information – Web Sites http://www.cyberlipid.org/ http://www.nlm.nih.gov/medlineplus/ency/article/000403.htm

Journal Citations Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem., 37, 911–917. Cabrini, L., Landi, L., Stefanelli, C., Barzanti, V., Sechi, A.M., 1992. Extraction of lipids and lipophilic antioxidants from fish tissues: a comparison among different methods. Comp. Biochem. Physiol. B, 101, 383–386. Chawla, A., Repa, J.J., Evans, R.M., Mangelsdorf, D.J., 2001. Nuclear receptors and lipid physiology: opening the X-files. Science, 294, 1866–1870. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem., 226, 497–509. Hara, A., Radin, N.S., 1978. Extraction of tissue lipids with a solvent of low toxicity. Anal. Biochem., 90, 420–426.

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Lipids Henley, E., Chang, L., Hollander, S., 2002. Treatment of hyperlipidemia. The Journal of Family Practice, 51, 370–376. Kaluzny, M.A., Duncan, L.A., Merritt, M.V., Epps, D.E., 1985. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J. Lipid Res., 26, 135–140. Lowenstein, J.M., 1969. Various articles. Methods Enzymol, 14, 244–598. Markello, T.C., Guo, J., Gahl, W.A., 1991. High-performance liquid chromatography of lipids for the identification of human metabolic disease. Anal. Biochem., 198(2), 368–374. Ruan, B., Gerst, N., Emmons, G.T., Shey, J., Schroepfer, G.J. Jr, 1997. Sterol synthesis. A timely look at the capabilities of conventional and silver ion high performance liquid chromatography for the separation of C27 sterols related to cholesterol biosynthesis. J. Lipid Res., 38, 2615–2626. Skipski, V.P., Barclay, M., 1969. Thin layer chromatography of lipids. Methods Enzymol., 14, 530–598. St. John, L.C., Bell, F.P., 1989. Extraction and fractionation of lipids from biological tissues, cells, organelles, and fluids. Biotechniques, 7, 476–481.

Book Citations Scriver, C.R., et al. 2001. Various chapters. Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D., Kinzler, K., Vogelstein, B. (Ed.), The metabolic and molecular bases of inherited disease, Edition 8. , McGraw-Hill, New York.