Lars Svennerholm

Quantitive estimation of sialic acids: II. A colorimetric resorcinol-hydrochloric acid method☆☆☆

Abstract A new method for the quantitive determination of sialic acids is described. Optimal conditions for the colour formation were investigated. The molar absorbancy indices of N-acetylsialic acid, N-glycolysialic and N, O-diacetylsialic acid and several naturally-occuringg carbohydrates were determined. The method is about 50% more sensitive than the orcinol-hydrochloric acid method generally used. The influence of other carbohydrates is also considerably lower with the resorcin reagent. The recovery of N-acetylsialic acid from known mixtures with galactose and fructose was determined. The detemination of sialic acid in body fluid proteins is briefly discussed.

CHROMATOGRAPHlC SEPARATION OF HUMAN BRAIN GANGLIOSIDES

GANGLIOSIDES are defined as acylsphingosyl oligosaccharides containing sialic acid (KLENK, 1942). For several years brain gangliosides were considered to contain a common carbohydrate moiety (KLENK and LAUENSTEIN, 1953 ; BOGOCH, 1958). The occurrence of brain gangliosides with different carbohydrate moieties was suggested (SVENNERHOLM, 1956, 1957a), since different ganglioside preparations varied in their composition and behaviour on cellulose columns. KUHN and EGGE (1959) confirmed the chromatographic separation of brain gangliosides and showed that the slowmoving gangliosides had a considerably more complicated structure than the fastmoving ones. During the last two years reports have appeared from many laboratories on the complexity of brain gangliosides. SVENNEMOLM and RAAL (1961) isolated monoand disialogangliosides from human brains, KUHN, WIEGANDT and EGGE (1961) found one inonosialoganglioside, two disialogangliosides and one trisialoganglioside in human and calf brains, and DAIN, et al. (1962) isolated four components from ox brain with similar composition to those of KUHN et al. (1961). KLENK and GIELEN (19614 isolated the hexosamine-containing and the hexosaniinefree monosialogangliosides originally postulated by SVENNERHOLM (19576). In a recent communication (SVENNERHOLM, 1962) it was shown that predominantly the gangliosides have a common basic structure : N-acyl-sphingosine-glucose-galactoseN-acetylgalactosamine-galactose to which one or more molecules of N-acetylneuraininic acid are bound. In the present report chromatographic methods for the quantitative isolation and separation of human brain gangliosides are described. These methods have been applied to studies of the ganglioside fraction of normal foetal, infant and adult brains while several different gangliosides have been isolated and further characterized. A knowledge of the normal ganglioside pattern is important in the study of the metabolism of the gangliosides but it is also necessary for the detection of disturbances in ganglioside metabolism. In infantile amaurotic idiocy (SVENNERHOLM, 1962) for example, there are no abnormal gangliosides but excessive amounts of a ganglioside are present which normally constitute only a few per cent of the total ganglioside fraction. It is likely that in several other mental or neurological disorders there is a disturbance of the normal ganglioside pattern. As this can now be determined by simple chromatographic methods it will be quite easy to detect minor deviations from the normal ganglioside metabolism.

A procedure for the quantitative isolation of brain gangliosides

In a systematic study of the optimal conditions for the quantitative isolation of gangliosides from brain tissue and their further purification the yield of gangliosides obtained by extraction of the tissue twice with twenty volumes of chloroform/methanol/water (4 : 8 : 3, v/v) was larger than that obtained with all other solvents tested, including tetrahydrofuran/phosphate buffer. The gangliosides were separated from other lipids by phase partition, water was added to the total lipid extract to give a final chloroform/methanol/water volume ratio of 4 : 8 : 5.6. Isolation of gangliosides from the total lipid extract with the aid of anion-exchange resins was not practical as a routine procedure on a large scale. The crude gangliosides extract was freed from low molecular weight contaminants by dialysis against water. This method was superior to the purification on gel filtration media or on anion-exchange resins, which required large columns with selective losses of gangliosides as a result. The present method has been applied to human brain, and the concentration and distribution of gangliosides in the human forebrain in infancy and old age are given.

Biochemical changes in Dementia disorders of Alzheimer type (AD/SDAT)

In postmortem investigations of patients with dementia of Alzheimer type (AD/SDAT) (n = 14) the brain weight was significantly reduced when compared to controls (n = 16). In four AD/SDAT-brain parts investigated the concentrations of 5-hydroxy-tryptamine and noradrenaline were significantly reduced while 3-methoxy-4-hydroxyphenylglycol was significantly increased. In the caudate nucleus of the AD/SDAT-brains the concentrations of dopamine and homovanillic acid were significantly reduced. The activity of monoamine oxidase B was increased suggesting a proliferation of extra neuronal tissue in the AD/SDAT-brains. The activity of choline acetyl transferase was reduced in the four brain parts investigated, showing a general reduction in the acetylcholine system in the AD/SDAT-brains. The ganglioside concentration was significantly reduced suggesting a reduced density of nerve endings in the demented brains. The AD/SDAT-group was according to rating scales severely demented. Patients with an early onset of the dementia disease were more severely intellectually reduced and had more pronounced biochemical disturbances than those with a late onset of the dementia.

DEVELOPMENTAL PROFILES OF GANGLIOSIDES IN HUMAN AND RAT BRAIN

Abstract— The developmental profiles of individual gangliosides of human brain were compared with those of rat brain. Interest was focused mainly on the pre‐ and early postnatal development. Human frontal lobe cortex covering the period from 10 foetal weeks to adult age and the cerebrum of rat from birth to 21 days were analysed. Lipid‐NANA and lipid‐P were followed; in the rat, also protein and brain weight. A limited number of samples of human cerebral white matter and cerebellar cortex were also studied. The following major results were obtained:

Membrane Lipids, Selectively Diminished in Alzheimer Brains, Suggest Synapse Loss as a Primary Event in Early‐Onset Form (Type I) and Demyelination in Late‐Onset Form (Type II)

Abstract: Major membrane lipids were quantified in frontal (Brodmann area 9) and temporal (Brodmann areas 21 and 22) cortices, caudate nucleus, hippocampus, and frontal white matter of 12 cases with Alzheimers disease (AD) type I (early onset), 21 cases with AD type II (late onset), and 20 age‐matched controls. The concentration of gangliosides—a marker for axodendritic arborization—was reduced to 58–70% of the control concentration in all four gray areas (p < 0.0001) and to 81 % in frontal white matter (p < 0.01) of AD type I cases, whereas it was only significantly reduced in temporal cortex (p < 0.01), hippocampus (p < 0.05), and frontal white matter (p < 0.05) in AD type II cases. The concentration of phospholipids was also significantly reduced (p < 0.01–0.0001) in all four gray areas of AD type I cases but in no area of AD type II cases. The loss of cholesterol was only 50% of the corresponding phospholipid diminution in AD type I. These results suggested a pronounced loss of nerve endings in AD type 1. The characteristic membrane lipid disturbance in AD type II was a loss of myelin lipids. This is the first time a fundamental biochemical difference has been shown between the two major forms of AD.

Accumulation of Glucosylceramide and Glucosylsphingosine (Psychosine) in Cerebrum and Cerebellum in Infantile and Juvenile Gaucher Disease

Abstract: Three major clinical variants of Gaucher disease have been defined: Type I, chronic nonneuronopathic; Type II, acute neuronopathic; and Type III, subacute neuronopathic. In a search for the underlying molecular basis of the neurological manifestations, the concentration and composition of cholesterol, phospholipids, neutral glycosphingolipids, and gangliosides were examined in cerebral and cerebellar cortices of five cases of Type II, eight cases of Type III, and one case of presumed Type I/III. In Type II the concentration of glucosylceramide was 140‐530 μmol/kg in cerebral cortex and 51‐450 μmol/kg in cerebellar cortex, the highest values found in the most fulminant cases. These concentrations were 20‐80 times greater than normal in cerebral cortex and 5‐40 times normal in cerebellar cortex. In Type III the concentration of glucosylceramide was 37‐65 and 59‐1750 μmol/kg in cerebral and cerebellar cortex, respectively. The highest concentrations were found in the cerebellum of patients who had survived splenectomy for several years. The ceramide composition of the accumulated glucosylceramide suggested that brain gangliosides were the major precursors of the glucosylceramide in brains of Type II but in cerebellar cortex in Type III was partly of extracerebral origin. The levels of lactosylceramide and oligohexaosylceramides were slightly raised in all brain specimens from the Gaucher cases. The ganglioside concentration was normal, whereas there was a certain increase in the proportion of GM2 and GM3 gangliosides. The brain glycosphingolipid changes in the Type I/III case were similar but slightly less than those in Type III cases of corresponding age. Glucosylsphingosine (psychosine), never detected in normal human brain, was demonstrated in brains from all the Gaucher cases. The psychosine concentration was highest in Type II cases, 3.8‐8.8 and 3.9‐12.3 μmol/kg in cerebral and cerebellar cortex, respectively, with the highest values found in the most fulminant cases. In Type III the psychosine concentration varied more widely, 0.8‐4.6 and 1.4‐6.3 μmol/kg in cerebral and cerebellar cortex, respectively. The lowest value, 0.7 μmol/kg, was found in the Type I/III case. Our method detected psychosine down to 0.01 μmol/kg, which means that the concentration of psychosine was increased at least 100‐ to 1000‐fold in Gaucher grey matter. We suggest that the accumulation of the cell‐toxic substance psychosine is the basis for the extensive neuronal cell loss in Gaucher disease, which is most striking in Type II disease.

THE DISTRIBUTION OF LIPIDS IN THE HUMAN NERVOUS SYSTEM. I. ANALYTICAL PROCEDURE. LIPIDS OF FOETAL AND NEWBORN BRAIN.

MOST studies of inherited mental diseases have been concerned with the clinical signs, genetics or gross pathology. In recent years several investigators have applied modern analytical biochemical techniques to the study of degenerative disease, and several symposia have been devoted to this subject (see CUMINGS, 1957; VOLK, 1959; FOLCHPI, 1961; ARONSSON and VOLK, 1962; FOLCH-PI and BAUER, 1963). The lack of representative figures for the normal lipid composition of human nervous tissue has been a limitation in the studies. JOHNSON, MCNABB and ROSSITER (1948) and BRANTE (1949) have made important contributions to the knowledge of lipid distribution within the normal central nervous system. CUMINGS (1953) and CUMINGS, GOODWIN, WOODWARD and CURZON (1958) and TINGEY (1959) determined the lipids in normal brains as well as in brains from patients with various diseases. Nevertheless, their results have not been in complete agreement, the number of patients have been rather small, and more accurate and sensitive methods have been developed during the last decade. The aim of the present investigations has been to determine the lipid pattern-with special emphasis on the sphingolipids-in nervous tissue and some paranchymatous organs, in healthy humans and in patients with inherited mental diseases. The different sphingolipids have been quantitatively isolated and their components characterized on representative samples of the material. The main interest has been the study of their fatty acid composition, as it may be of considerable importance for membrane function (SVENNERHOLM, 1963~7, STENHAGEN and SVENNERHOLM, 1965). The present procedure for the quantitative lipid determination has been developed for use with fresh, deep-frozen and formalin-fixed tissues. Rather large tissue samples have been used, 0.5-1-0 g of fresh tissue, as the lipids were analysed chromatographically, and otherwise characterized at each step. The procedure to be described has, however, already been slightly modified for analyses of amounts less than onethousandth of those used here. This modified technique is still much less sensitive than the ultra microprocedure developed for the analyses of lipids in samples weighing less than 1Opg by LOWRY and collaborators (LOWRY, ROBERTS, LEINER, Wu and FARR, 1954; ROBINS, LOWRY, EYDT and MCCAMAN, 1956). Although their methods can also be applied to formalin-fixed tissues they have so far still been of limited application for the study of lipid disturbances in the nervous system. Recently, KOREY, GOMEZ, STEIN, GONATAS and SUZUKI (1963) described a procedure for the biochemical study of small brain biopsy samples. Their efforts have been directed

The chemical structure of normal human brain and Tay-Sachs gangliosides☆

Abstract Normal human brains contain mono- and disialogangliosides. The latter can be converted to the major monosialoganglioside by enzymic hydrolysis with neuraminidase. Partial acid degradation of the monosialoganglioside has given four neutral ceramide — saccharides which have been isolated and characterised. The ganglioside accumulated into the brain in Tay-Sachs disease has been shown to lack the galactose in end position.

Membrane Lipids of Adult Human Brain: Lipid Composition of Frontal and Temporal Lobe in Subjects of Age 20 to 100 Years

Abstract: The membrane lipid composition of human frontal and temporal cortices and white matter has been studied in 118 subjects, age 20–100 years. The brain specimens were selected from subjects who lived a normal social life and died suddenly and unexpectedly with no history of neurologic or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The sudden death eliminated all risk of changes over a long agonal stage. The data for total solids and major lipids are summarized in graphic form. Total solids, phospholipids, and cholesterol diminished linearly from 20 years of age in frontal and temporal cortices, whereas total solids phospholipids, cholesterol, cerebroside, and sulfatide showed a curvilinear diminution in frontal and temporal white matter. Gangliosides differed from the other lipids, showing an almost constant concentration between 20 and 70 years of age with a slight peak around 50 years of age. The ganglioside pattern showed continuous change with aging, with decreasing proportions of GM1 and GD1a and increasing proportions of GD1b, GM3, and GD3. Equations are given that can be used to calculate the lipid composition of normal human frontal and temporal cortices and white matter at any age between 20 and 100 years of age. These data can be used where data by direct analysis are not available for comparison with values for various pathological states.