Isolation and Identification of Cellular Phospholipids

In this chapter, some of the types of methodologies currently in use for isolation and analysis of cellular phospholipids will be outlined. Such techniques can be readily applied to experiments designed to explore the involvement of phospholipids in cellular events, such as stimulus-induced activation. Primary attention will be paid to the human platelet. If you need to justify the choice of human platelets as the cell of choice, a number of highly creditable reasons can be cited. Only three need to be considered at this point. First, the circulating platelet is of paramount importance in hemostasis, and there is convincing evidence that its membrane phospholipids are intimately involved in this process. Second, these cells can serve as excellent targets or model systems for stimulus-induced activation in which the membrane phospholipids play an important role. Third, human platelets can be isolated from whole blood by a simple, convenient centrifugal approach. Human donors are available at a very reasonable cost, and the platelets obtained from a varied spectrum of donors show remarkable consistency. Thus, one can undertake their isolation using the following method and have them available for immediate experimentation. Nonfasting venous blood is drawn (with informed consent) from male or female subjects between the ages of 20 and 40 years, who are considered to be normal and healthy and had not ingested platelet-active medication for at least 10 days prior. Blood is obtained by insertion of a butterfly infusion set (12-in. tubing from Abbott Hospitals, Inc., North Chicago, IL) with a 1-in. x 19-gauge needle into the antecubital vein. A few milliliters of blood is allowed to flow before a 60-ml plastic syringe containing 7.5 ml of an ACD [10.8% citric acid, 2.2% trisodium citrate, and 2% dextrose (w/v)] solution was attached. Four syringes were filled to the 50-ml mark and inverted gently, and the contents were transferred carefully into 50-ml plastic tubes that are then capped. The blood-to-ACD volume ratio is 6:1 (v/v). The tubes are centrifuged at 2000 rpm (830g) in a Sorvall RT 6000 centrifuge at 24°C for 15 min.

Non-Choline-Containing Phospholipids: Diacyl-, Alkylacyl-, Alkenylacyl-Ethanolaminephosphoglycerides, Phosphatidylinositols, and Phosphatidylserine

The diversity of phospholipids present in the mammalian cell membranes continues to titillate one’s scientific senses. In addition to the presence of at least 12 different structural types of phospholipids in a cell that serve to complicate the picture, there are species within species. If one considers the diacyl, alkylacyl, and alkenylacyl variants, plus the number of different fatty acyl and fatty ether combinations, there can be several hundred different species present. Certainly progress is being made in relating certain species with a particular cellular process, and this is no doubt an exciting and important area of study. However, this is only the tip of the “cellular iceberg,” since there is little or no information on the biological role of the majority (certainly over 75%) of the phospholipids. Questions to be asked center on the need for such a spectrum of phospholipids. Are some structural components only, are some vestigial remnants, or do they play a crucial role in biological reactions yet to be discovered? There is no simple answer as yet, but this trend of thought should be kept in mind in any investigation on membrane lipid behavior. An important route to interpreting the role of various phospholipids in a biological milieu is to be certain of the chemical structure and identification of the molecules under study. So in continuation of the general format used in Chapter 4, the chemistry of the ethanolamine-, inositol-, and serine-containing phosphoglycerides will be explored at this point. A limited excursion will be made as to their participation in biological reactions. Though the above three classes of compounds share certain common structural features, there are sufficient differences to warrant separate treatment of each group of compounds. For example, the ethanolamine-containing phosphoglycerides can contain, in addition to the diacyl form, an alkylacyl and/or alkenylacyl form. Inositol-containing phosphoglycerides other than the diacyl type have not been reported, but several other phosphorylated species have been detected. The serine-containing phosphoglycerides have been found only as diacyl derivatives.

Choline-Containing Phospholipids: Diacyl-, Alkylacyl-, and Alkenylacylcholine Phosphoglycerides and Sphingomyelin

In choosing the order for discussion of phospholipids, it is not the intention to single out one particular group as the most important; rather, an initial premise would be that all phospholipids are critical to a cell’s structure and metabolism. Certainly, as has been emphasized before, phospholipids have been shown to have key roles in the process of cellular signal transduction, and it is debatable which of several types of phospholipids is the most important. There is no doubt that the mechanism of involvement of membrane phospholipids in these complex reactions has presented a major experimental challenge, and as such this has titillated the acute scientific senses of many researchers. It is equally true also that an important field of study is emerging in cell signaling, in which unusual cellular disorders have been noted. Certainly the latter will implicate alterations or aberrations in membrane phospholipid chemistry and metabolism in one way or another. This digression was made to show quite simply that it behooves one to understand the chemical/ biochemical characteristics of the phospholipids in order to best meet the challenges of this field (and, of course, other related ones as well). On the basis of undoubted faulty logic on the choice of the order of topics, one simply can retreat to the argument of personal preference. Thus, the first group of compounds will be the choline-containing phospholipids—that is, the choline phosphoglycerides and the choline sphingolipids. As it so happens, these are among the most ubiquitous phospholipids in nature and, at least in the early “chemical” years of investigations on the phospholipids, the best-studied group. It is assumed at this junction that a highly purified phospholipid has been obtained, usually through the use of chromatographic procedures. A frequently asked question is, How do I tell whether the sample is pure? It is a logical question, especially with compounds isolated from naturally occurring sources. In actual fact, there is no simple answer.

Minor Phospholipids: Platelet activating factor (PAF) and PAF analogs, Lysophosphatidic acid and phosphatidic acid, Phosphatidylglycerol, Cardiolipin, Sphingosine-1-P

In the previous chapters, the emphasis was placed on establishing the chemical nature of phospholipids normally found in relatively high concentrations in mammalian cells. The methodology described for identification of these compounds can be applied, perhaps with some minor modifications to the structural characterization of phospholipids found in plants, fish, and bacteria. Interwoven into the fabric of these chapters was the subtle reference to their potential biological role in cellular behavior. As lipid biochemistry has developed over the past several years, there is no doubt that the field of signal transduction has had an enormous impact on phospholipid awareness. As might be expected, the potential for phospholipids or their metabolites to exhibit biological activity loomed large. As will be evident in this chapter, phospholipids can certainly have biological activity and this theme shall be explored in some depth. The title “Minor Phospholipids” is not meant to be belittling, but is only intended to reflect the fact that these phospholipids are present in very low concentrations. In fact, some are considered to be formed only after a cell has been stimulated by an extracellular agonist. As shall be seen, these compounds have considerable biological activity and hence are really of major importance in the cellular metabolic scene. The term platelet activating factor (PAF) was originally applied to a phosphoglyceride capable of activating platelets, leading to their aggregation. In addition, in certain species, there was also discharge of their dense granules (as indicated by serotonin release from the platelets). This nomenclature was unfortunate because it is now well established that many cells can produce this compound and that, likewise, PAF can stimulate many other cells. There are many other compounds, such as thrombin and arachidonic acid, which also can activate platelets. Notwithstanding this problem of nomenclature, there is a widespread (deeply entrenched) usage of this term to indicate a specific type of phospholipid with a particular biological activity. Given the status of this field at the current time, the use of the term PAF will be continued here.

Phospholipid(s) Associations in Cellular Structures

In the preceding chapter, the intent was to provide the reader with a broad perspective on the chemical characteristics of cellular phospholipids. At the same time, emphasis was placed on the potential usefulness of this information in dissecting the importance of phospholipids in cellular events, such as signal transduction. There is no doubt that the large number of observations reported on the close relationship of phospholipids to the transduction process has stimulated a widespread (and gratifying) interest in these compounds. Certainly it is very clear now that stimulus-induced activation of cells leads to the turnover of specific membrane phospholipids. The following diagram reemphasizes several, but not all, possible reaction pathways that can be invoked during an agonist (stimulus)–induced activation of a cell and gives the possible sequelae: In each of the above reactions, the substrates phosphatidylcholine and phosphatidylinositol bisphosphate normally are considered biologically inactive in membranes. Then, subsequent to activation of cellular phospholipases by a stimulus, biologically active products are formed from these compounds. Thus, inositol bisphosphate triggers the release of calcium ions from intracellular stores, diacylglycerol is implicated in the translocation and activation of protein kinase C, arachidonic acid can be converted to biologically active prostaglandins, and phosphatidic acid can be an agonist in its own right. The major point to be stressed here is that phospholipid turnover is intimately associated with the signal transduction pathway in cells. Hence an understanding of the chemistry of these phospholipids is of major relevance to delineating the complicated process of signal transduction. While investigation of the behavior of phospholipids in this pathway in platelets has been a consuming interest of this author, the main thrust in this book will be simply to acquaint the reader with the chemistry of phospholipids of major importance in signal transduction and also to discuss other phospholipids found in mammalian membranes. Inasmuch as most investigations on stimulus response in cells utilize quite small numbers of cells—for example, a typical experiment on human platelets might use 1 x 109 cells, which would yield ∼50 μg of total lipid—this poses a challenge to an investigator to be able to isolate and identify these lipids.

Introduction to Lipids

Concomitant with the explosive development and progress made in the field of molecular biology, initiated by development and proof of the double helix structure of DNA some 40 years ago, there has been a much more subdued, but equally exciting and important, development in the area of signal transduction (Nishizuka, 1992; Berridge, 1984; Exton, 1990). What makes the latter subject so enchanting to biochemists is the finding that membrane phospholipids are intimately involved in the transduction process. Interestingly, the potential role of phospholipids in the signal transduction pathway was formulated some 40 years ago also. (Perhaps all great discoveries occur in 40 year cycles.) In any event the first hint of any possible involvement of phospholipids in cellular stimulus responses was gained from the work of Hokin and Hokin, which first appeared in 1953. In this classic paper, these investigators reported that treatment of pigeon pancreas slices with acetylcholine or carbamylcholine (cholinergic drugs) resulted not only in the secretion of amylases but also in the turnover of two specific membrane phospholipids, namely, phosphatidylinositol and phosphatidic acid. While the entire process of stimulus response in a mammalian cell is now much more complicated, nevertheless the findings by Hokin and Hokin were of major importance in the maturation of this field. Unfortunately, the impact of the Hokins’ observations was not immmediately felt. At that point in time, phospholipids were viewed mainly as semipermeable membrane structures whose main function was to regulate the ion content of the cell. In addition, another deterrent was the limited information on the chemical structure of the mammalian cell phospholipids. Hence there was a hiatus of many years in which low-profile lipid chemists and biochemists labored to solve the chemical nature of membrane lipids and to deduce their physical arrangement in the cell. Then in 1975, Michell published a key paper (Michell, 1975) in which he noted the importance of the inositol-containing phospholipids in the membrane process known as calcium gating. This paper initiated what can be called the “PI” era, which is still very much alive and well today.