http://science.sciencemag.org/content/sci/168/3934/939.full.pdf

Cryobiology: The Freezing of Biological Systems

Lipid metabolism in the rumen

Summary. 

Malonyldialdehyde (MDA) formation, a measure of polyunsaturated fat autoxidation, was estimated in normal human red cells incubated in vitro. Exposure to oxygen under a variety of conditions did not induce autoxidation. Exposure to hydrogen peroxide was either by the addition of a hydrogen peroxide solution or by incubation in an atmosphere saturated with hydrogen peroxide vapour. A pattern of MDA formation was established with both methods. Lack of reproducibility, a feature of previous methods of measuring the susceptibility of red cells to exogenous peroxide, could be overcome by (i) catalase inhibition, (ii) attention to the non-linear relation between packed-cell volume and MDA formation, and (iii) elimination of potentially misleading coloured complexes on spectroscopy. A number of recognized antioxidants inhibited peroxide-induced MDA formation. Inhibition was proportional to the logarithm of the antioxidant concentration. Under certain conditions pre-incubation with nucleosides and with lecithin protected the cells against autoxidation on subsequent exposure to peroxide. Peroxide-induced cell autoxidation was also influenced by plasma, bovine albumin, and ascorbic acid. Haemolysis was an invariable consequence of autoxidation. Similarities and dissimilarities between autoxidation and antioxidation in free lipid emulsions and in organized biological structures were examined and discussed in the light of the experimental findings.

The autoxidation of human red cell lipids induced by hydrogen peroxide.

Polge, Smith and Parkes1 first reported the protective action of glycerol against the adverse effects on spermatozoa of freezing and thawing. Lovelock2 found that the protective action of glycerol was shared with a number of other neutral solutes of low molecular weight, including methanol, acetamide and glyceryl monoacetate. Using the extent of haemolysis of red blood-cells as a measure of damage by freezing, it was concluded that the principal protective action of the neutral solutes was a simple consequence of their ability to prevent the excessive concentration of electrolytes and other substances that otherwise occurs on freezing. The protective property is limited to substances that are in themselves not toxic, have a low molecular weight, a high solubility in aqueous electrolyte solutions and an ability to permeate living cells. The number of solutes capable of fulfilling these conditions is small, and so far glycerol most closely approaches the ideal protective agent. Some cells, however, are impermeable to glycerol, for example, bovine red blood cells, and with these glycerol is of little use in their preservation by cold storage. An alternative solute with a greater permeability to living cells is dimethyl sulphoxide. This communication reports the protective action of dimethyl sulphoxide against freezing damage to human and bovine red blood cells and to bull spermatozoa.

Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide

Protectants used in the cryopreservation of microorganisms

The protective action of neutral solutes against haemolysis by freezing and thawing.

It was discovered by Polge, Smith & Parkes (1949) that spermatozoa could be frozen and thawed without loss of motility if glycerol was included in their suspending medium. Following this discovery the spermatozoa of the domestic fowl (Polge, 1951), ofthe bull (Polge & Rowson, 1952) and ofthe herring (Blaxter, 1953) have been preserved without loss of function for long periods in the frozen state. The importance of the practical applications of this method has been discussed (Polge & Parkes, 1952). The investigation of the protective action of glycerol was facilitated by the observation that red blood cells also were protected against the adverse effects of freezing and thawing by means of glycerol (Smith, 1950), and the nature of the damage occurring to red blood cells when they are frozen and thawed, and of the protection given by glycerol was then described (Lovelock, 1953a, b). Briefly, haemolysis by freezing and thawing is due to the destructive action of the concentrated salt solution to which the cells are exposedwhen water is removed as ice. In the presence of glycerol the electrolyte concentration at temperatures below the freezing point is sufficiently reduced to explain in full the protection afforded by glycerol. The apparent simplicity of the processes causing the haemolysis of red blood cells by freezing and thawing, and of the protective action of glycerol, made it ofsome interest to observe the behaviour of other living cells on freezing and thawing. It seemed that this provisional explanation of the effects of freezing could best be tested using living cells with a wide range of sensitivity to hypertonic salt solutions. It also seemed desirable to use a criterion of damage more delicate than haemolysis. Both of these requirements are met by the spermatozoa of different animals. Those of the rabbit are particularly sensitive to hypertonic salt solutions (Emmens, 1948), while those of the herring clearly will tolerate salt solutions at least as strong as sea water. In addition, the observation of the motility of spermatozoa, even if somewhat lacking in precision, provides a different and sensitive criterion for the assessment of freezing damage. This paper records the effects of freezing and thawing upon the motility ofthe spermatozoa of the rabbit, the bull, the fowl and the herring. The modification of these effects by glycerol is also described.

The immobilization of spermatozoa by freezing and thawing and the protective action of glycerol

The lipids of whole blood. 1. Lipid biosynthesis in human blood in vitro

In Part 1 (James, Lovelock & Webb, 1959) and in the earlier work of Altman, Watman & Solomon (1952), it was reported that the cellular constituents of the blood are able to incorporate activity from sodium [Me-14C]acetate into their lipid components. It was also shown that the radioactive lipids synthesized by the cells are transferred during the course of incubation in vitro to the plasma lipoproteins. From this earlier work little or no information is available on the contribution of the celLs to the lipid metabolism of the blood, or indeed to that of the whole animal. This paper reports experiments designed to determine quantitatively the turnover of the major lipid components, namely the phospholipids, the neutral fat and the unsaponifiable lipids, between the cells and the lipoproteins of the plasma.

/pdf/the-lipids-of-whole-blood-2-the-exchange-of-lipids-between-2xmxvyfrh7.pdf

The lipids of whole blood. 2. The exchange of lipids between the cellular constituents and the lipoproteins of human blood.

/pdf/the-physical-instability-of-human-red-blood-cells-o4gvmxgojx.pdf

J. E. Lovelock

Papers

The protective action of neutral solutes against haemolysis by freezing and thawing.

The immobilization of spermatozoa by freezing and thawing and the protective action of glycerol

The lipids of whole blood. 1. Lipid biosynthesis in human blood in vitro

The lipids of whole blood. 2. The exchange of lipids between the cellular constituents and the lipoproteins of human blood.

The physical instability of human red blood cells.