G. B. Ansell

THE ALKALINE HYDROLYSIS OF THE ETHANOLAMINE PLASMALOGEN OF BRAIN TISSUE.

I N THE course of experiments on the enzymic breakdown of brain plasmslogens in uitro (ANSELL and SPANNER, 1963b) the method of DAWSON, HEMINGTON and DAVENPORT, (1962) was adopted for determining ethanolamine plasmalogen, which is the predominant plasmalogen in brain tissue. In this ingenious method, which stems from earlier work by the same group (DAWSON, 1954; DAWSON, 1960), the diacyl glycerophospholipids are completely deacylated by brief alkaline hydrolysis to yield watersoluble glycerylphosphoryl esters. The alkali-stable phospholipids are then heated with trichloroacetic acid (TCA)* solution containing mercuric chloride which liberates glycerylphosphoryl esters from the lysoplasmalogens. This method gives higher values for plasmalogens than the earlier (DAWSON, 1960) method since the formation of cyclic acetals during the acid hydrolysis of lysoplasmalogens is largely prevented. It was found that, even using this modification, the yield of glycerylphosphorylethanolamine (GPE) from the alkali-stable lipid on acid hydrolysis was much lower than would be expected from the amount of ethanolamine plasmalogen present in the tissue when this was independently determined from the vinyl ether and ethanolamine content of the lipids surviving the initial alkaline hydrolysis. These independent determinations gave values for total plasmalogen and ethanolamine plasmalogen which agreed more closely with those of RAPPORT and LERNER (1959) and WEBSTER (1960). In this paper the discrepancy is explained and the explanation exploited to prepare a native ethanolamine plasmalogen of approximately 90 per cent purity.

THE EFFECT OF INSULIN ON THE FORMATION OF PHOSPHORYLCHOLINE AND PHOSPHORYLETHANOL-AMINE IN THE BRAIN

IT was shown by DAWSON and RICHTER in 1950 that the uptake of m]-orthophosphate into brain phospholipids in vivo was depressed in insulin h y p o g l y d ANSELL and Do(1957) measured the depression in individual phospholipids, and showed that the depression covered phosphatidyl choline, phosphatidyl ethanolamine (and presumably the ethanolamine-containhg plasmalogen), and diphosphoinositide. In an attempt to locate the point of action in the phospholipid synthetic chain the effect of insulin on the uptake of into phosphorylethanold and phosphoryicholine has been measured.

The long‐term metabolism of the ethanolamine moiety of rat brain myelin phospholipids

IN A PREVIOUS paper (ANSELL and SPANNER, 1967), it was shown that when [14C]ethanolamine was injected intracerebrally into mature rats, the label was rapidly incorporated into the phospholipid fraction of the brain. Over 90 per cent of this labelled phospholipid was recovered in the ethanolamine-containing lipid fraction. In the same paper the iticorporation of labelled ethanolamine into the lipids of the subcellular fractions of brain including myelin was described. It was seen that the ethanolamine-containing lipids of myelin, in particular the small myelin, were rapidly labelled. This work has now been extended and the fate of the [14C]ethanolamine studied in brain, and in particular in the two myelin fractions of brain, over a period of 9 weeks. Female rats, 12-14 weeks old, were injected with 0.817 pc (equivalent to 0.1665 pmoles) of [1,2-14C]ethanolamine and the animals killed 1, 7, 14, 21, 35, 49 and 63 days after the injection. No significant change in the brain weights was observed over this period and this was reflected in the ethanolamine lipid content (Table 1). The large and small myelin fractions were prepared and the

The determination of free ethanolamine in brain tissue and its release on incubation

ALTHOUGH there are good spectrophotometric methods for the determination of lipid-bound ethanolamine in animal tissues (AXELROD et al.. 1953; SIAKOTOS, 1967) they can only be used because the only interfering substance present in washed lipid solutions is serine and this can be estimated separately. The determination of ethanolamine as a component of the pool of free amino compounds in ti.+ sues has proved tedious since it has to be separated from numerous other substances. including ammonia, which will be determined as ethanolamine in the methods mentioned above. DE ROPP & SNEDEKER (1960) used l o g of rapidly frozen brain tissue to prepare a brain extract with either picric acid or 80% ethanol. Amino compounds were then separated by sequential unidimensional chromatography, reacted with ninhydrin and the eluted coloured product measured spectrophotometrically. They found 0.2 pmol ethanolamine/g fresh weight. However. OJA (1966). who also used this method, found ethanolamine to be immeasurably small in brains rapidly removed from rats and frozen in liquid nitrogen. KNAUFF & WK (1961), on the other hand, found a value as high as 3 p o l / g fresh weight even for rats whose heads had been frozen in liquid nitrogen after decapitation. They used a chromatographic method similar to that of DE ROPP & SNEDEKER (1960). except that the chromatograms were scanned with a photometer. A value of 3-4 prnol/g fresh weight was also found for cat brain by TALLAN qt a/. (1954) who used ionexchange chromatography. Large amounts of tissue were required in all these methods. These observations suggested to us that (a) a better method for the determination of ethanolamine might be developed and (b) that there is rapid release of ethanolamine from brain tissue post mortem, a possibility also noted by OJA (1966). This communication describes a method for the determination of free ethanolamine in brain tissue and its application to demonstrating the release of the base when brain tissue is incubated in Ringer-bicarbonate solutions.

The Application of Zonal Centrifugation to the Study of Some Brain Subcellular Fractions

Publisher Summary This chapter discusses the application of zonal centrifugation to the study of some brain subcellular fractions. The first attempt to separate subcellular fractions from brain tissue was by Brody and Bain, but with the benefit of hindsight, it is clear that the fractions they obtained were extremely heterogeneous. The major impetus for improving the techniques of subcellular fractionation stemmed from the observation of Hebb and Smallman that a high proportion of the choline acetyltransferase in brain tissue could be located in the mitochondrial fraction when this was prepared by the method of Brody and Bain. Hebb and Whittaker then demonstrated that acetylcholine (ACh) was also associated with the crude mitochondrial fraction and made the important observation that the ACh-containing particles could also be separated from the mitochondria. This led to an intensive investigation by Whittakers group and by De Robertis and his colleagues of which Whittaker gives an excellent account.