Marina Brunetti

Properties and function of the calcium-dependent incorporation of choline, ethanolamine and serine into the phospholipids of isolated rat brain microsomes.

Abstract— The calcium‐dependent incorporation of choline, ethanolamine and L‐serine into the phospholipids of isolated rat brain microsomes has been studied in vitro, and various properties of the incorporation have have been examined. The optimum pH for the incorporation of each base was found to vary inversely with the Ca2‐ concentration. Conversely, the optimal Ca2 + concentration for the exchange of the bases increased with decreasing pH values. The enzymic system for the incorporation of ethanolamine appeared to be saturated by two substrate concentrations, i.e. 0‐2 and 1‐7‐2‐0 mM. At low ethanolamine concentration (0‐2 mM] much less incorporation of the base occurred into the alkenylacyl‐ and alkylacyl‐derivatives of ethanolamine phosphoglycerides compared to that into the diacyl species, whereas the difference becomes smaller at a high substrate concentration (1‐7 mM). At pH 81 and 2 mM‐Ca2+ the apparent Km of ethanolamine at low substrate concentration was 80 × 10‐5 M, and this value increased to 16‐2 × 10‐4.viat 10mM‐Ca2+ concentration. At similar pH the Km values for choline and L‐serine were 5.88 × 10‐4M and 40 × 10‐4 M at 2 mM‐ and 10mM‐Ca2 + concentrations, respectively. The properties of the enzyme system show differences for the three substrates when various factors are changed during incubation. These and other results indicate that more than one enzyme is probably involved in the Ca2+‐medialed exchange of nitrogenous bases.

The synthesis in vivo of choline and ethanolamine phosphoglycerides in different brain areas during aging.

The biosynthesis of choline and ethanolamine phosphoglycerides was tested in vivo in different brain areas of the rat during aging. Mixtures of [2−3H] glycerol and [Me-14C] choline or [2-3H] glycerol and [2-14H] ethanolamine were injected into lateral ventricle of the brain as lipid precursors and their incorporation into corresponding phospholipid was examined. A significant decrease of synthesis of both phosphoglycerides takes place in cerebral cortex and in the striatum, and is already apparent at 9 months of age with no further decrease or change therafter. No significant change takes place in the cerebellum. The unchanged absorption of injected water-soluble precursors, together with the lack of any significant change of phospholipid/protein ratio in all examined brain areas, suggests that the incorporation of both glycerol and nitrogen bases are affected by aging.

Platelet MAO-B activity as a marker of behavioural characteristics in dementia disorders

Both low and high platelet MAO-B (pMAO-B) activity is considered an indicator of increased vulnerability in psychopathology. How the activity of this peripheral enzyme can be linked with the sophisticated functions of the central nervous system (CNS) is not clear; in man, evidence exists that the genetic mechanisms determining the size or capacity of the central serotonin system are common to platelet and brain MAO. In the present study pMAO- B activity was evaluated in demented patients suffering from early- onset Alzheimer’s disease (AD), late- onset Alzheimer’s disease (SDAT), vascular dementia (VD), and controls. In these dementia categories, the relationship between pMAO- B activity and clinical features, and between pMAO- B activity and cerebrospinal fluid (CSF) monoamine metabolites (3-methoxy-4-hydroxyphenyl-glycol, MHPG; 5-hydroxy-in-doleacetic acid, 5-HIAA; homovanillic acid, HVA) was also investigated. pMAO-B activity was significantly higher in SDAT patients, compared to controls and AD. Age, as covariate, failed to show any significant effect, and no association was found between pMAO-B activity and CSF monoamine metabolites. The correlation analysis between pMAO-B and neuropsychological scores showed a highly significant positive relationship with GBS- emotional impairment (N=40, r=0.72, p<0.01 in the SDAT group. This result suggests the importance of platelet MAO- B activity as biological marker also in old- age dementias, namely senile dementia of Alzheimer type, where the increased activity of this enzyme might constitute a marker for vulnerability toward behavioural disturbance, i.e., emotional deterioration. (Aging Clin. Exp. Res. 6: 201–207, 1994)

Synthesis of phosphatidylcholine and phosphatidylethanolamine at different ages in the rat brain in vitro.

The de novo synthesis of choline and ethanolamine phosphoglycerides in brain microsomes from 18 month-old male rats was investigated in vitro by using labeled cytidine-5′-diphosphate choline and cytidine-5′-diphosphate ethanolamine as lipid precursors. The rate of synthesis of the two phospholipid classes was found to be noticeably decreased, as compared to that of adult animals. The addition of exogenous diacyl glycerols to microsomes from ageing rat brain brings the rate of synthesis nearly to the adult levels. The synthesis of choline and ethanolamine phosphoglycerides is not affected in the liver microsomes of ageing rats. The molar distribution of fatty acids in brain microsomal diacyl glycerols of ageing rats is noticeably different from that of adult animals. The content of monoenoic and dienoic species is increased, whereas that of the tetraenoic species is decreased. Base exchange reaction for choline and ethanolamine incorporation into respective phospholipids is not affected in the brain microsomes of the aged rats.

The relationships between the phospholipid pool and the base-exchange reaction in the Ca2+-stimulated incorporation of ethanolamine into brain microsomal phospholipids

It has been shown in recent years that a Ca”-dependent base-exchange system occurs in purified brain microsomes which converts in vitro, at the expenses of membrane-bound phospholipids, labelled free ethanolamine, serine and choline into the corresponding phosphoglycerides [ l-41. Little information has been given, however, about the type of phospholipid which participates in the reaction at the nerve membrane level [2]. The experiments described here are aimed therefore at an examination of the type and degree of exchange that takes place in vitro at the brain microsomal level between endogenous phospholipid and choline, ethanolamine or L-serine, when the microsomal membranes are prelabelled in vitro in their PE either with 1,2[ r4 C] ethanolamine by the base-exchange reaction [ 1,2] or with radioactive CDPE by de novo synthesis of PE (see [ 51). It is shown that different degrees of displacement of the lipid-bound labelled ethanolamine are obtained by the exchange in vitro dependent on the mechanism of prelabelling the PE-containing rnicrosomes. The result points to the existence in brain of at least two biochemically different pools of microsomal PE.

Platelet MAO-B activity and vitamin B12 in old age dementias

Platelet MAO-B activity, serum vitamin B12 levels, and plasma folate were measured in patients suffering from presenile (AD) and senile (SDAT) dementia of Alzheimer-type, and vascular dementia (VD). MAO-B was higher in the SDAT group than in AD and controls. An inverse relationship between MAO-B activity and vit. B12 levels was documented in the whole group and in each category studied; furthermore, MAO-B was positively related to age. All the patients were then divided into two groups, according to vit. B12 levels (Group I: less than 200 pg/mL; Group II: greater than or equal to 200 pg/mL); Group I showed a significantly higher MAO-B activity with respect to Group II. The results indicate the existence of a negative association between platelet MAO-B activity and serum levels of vitamin B12 and confirm the existence of biological differences between presenile and senile dementia of Alzheimer type.

Arachidonic and palmitic acid utilization in aged rat brain areas

We have previously demonstrated that the arachidonic acid (20: 4) incorporation into brain lipids differs according to the age of the animals used and the experimental conditions adopted. These differences led to a further investigation of arachidonic acid uptake in both aged and adult rat brains, its transformation into CoA derivatives, its incorporation into diacyl-glycerols and polar lipids, and finally its oxidation to CO2. These metabolic parameters were then compared with those obtained after using the saturated fatty acid palmitate (16: 0). In both cases slices or mitochondria from different brain areas of 24-month-old and 4-month-old rats were examined.The results obtained indicate that the uptake of the fatty acids into cells is not modified by age. However, the successive metabolic transformations of the acids are altered to a considerable extent. In particular, in 24-month-old animals (compared with 4-month-old rats) there is a significant decrease of 20: 4 in its incorporation into lipids as well as its oxidation to CO2, while arachidonoyl-CoA content increases by about 50%. This increased amount of CoA derivative, which has a potent detergent effect, may interfere with membrane structure and affect membrane physiological functions. Furthermore, because the free arachidonate pool is manteined in a dynamic equilibrium with its esterified forms, the final result may be a perturbation of this equilibrium.

The synthesis of choline phosphoglycerides in the giant fibre system of the squid.

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion‐nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP‐choline in the intact ganglion‐nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base‐exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations.

Relationships between base-exchange reaction and the microsomal phospholipid pool in the rat brain in vitro

The calcium-stimulated incorporation of ethanolamine, choline and L-serine into rat brain microsomal phospholipids has been investigated. The membranes were prelabeled in vitro in their choline or serine phosphoglycerides by base-exchange and then chasing experiments were done by displacing the lipid-bound base by ethanolamine, choline, or L-serine labeled with a different isotope. The results indicate that membrane phosphatidylcholine is presumably a substrate for the exchange with all the three bases, whereas phosphatidylserine exchanges only with ethanolamine and L-serine but not with choline. A small phospholipid pool (3–7% of the total available pool) is active in the calcium-dependent exchange with choline, ethanolamine, and L-serine. When the microsomal membranes are prelabeled in vitro in their phosphatidylcholine moiety through the cytidine-dependent pathway and then chasing experiments are performed with the three nitrogenous bases, as above, the small phospholipid pool is hardly detectable. In view of these and other results (Gaiti et al., FEBS Letters 49:361 1975), it is suggested that at least two different pools of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine might exist in rat brain microsomes.

CSF monoamine metabolites in old age dementias

Cerebrospinal Fluid (CSF) levels of the main metabolites of monoamines (MHPG, 5-HIAA, and HVA) were measured in patients with early onset (AD) and late-onset (SDAT) Alzheimers disease, vascular dementia (VD), and elderly controls. Psychobehavioral assessment was carried out by means of MMSE and GBS. Mean MHPG levels did not differ from controls; 5-HIAA was lower in VD when compared to both controls and SDAT. HVA was decreased in AD, SDAT, and VD with respect to controls. Significant correlations between HVA and psycho-behavioral parameters were observed in SDAT and VD groups, whereas no relationship was documented in AD. The SDAT group was divided in SDAT-A (age at onset: greater than 65 less than or equal to 80 yr) and SDAT-B (age at onset: greater than 80 yr). SDAT-A had significantly lower CSF HVA values than SDAT-B (165 +/- 64 vs 235.7 +/- 85). SDAT-B HVA levels were similar to those observed in controls. Correlation analysis between HVA and neuropsychological variables was significant in SDAT-A, but not in SDAT-B. These results might support the evidence of SDAT heterogeneity.