Gianfranco Trovarelli

Effect of cytidine diphosphate choline (CDP-choline) on ischemia-induced alterations of brain lipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries. Definite changes in the energy status of brain demonstrated that carotid occlusion was effective. Five minutes before ligation, an intraventricular injection of either saline or cytidine diphosphate choline (CDP-choline, 0.6 μmol/brain, 3μl) was given to groups of animals. Control animals, with and without CDP-choline, together with the ischemic groups, were decapitated directly into liquid nitrogen; 10 min after arterial ligation. Brain free fatty acids, neutral lipids and phospholipids, which were labeled in vivo by the intraventricular injection of [1-14C] arachidonic acid (0.4–0.6 μCi, 6–9 nmol) 2 hr prior to ligation, were extracted, purified, and separated by thin-layer chromatographic procedures. The CDP-choline treatment noticeably corrected the increase of total and individual fatty acids due to ischemia and the increase of their radioactivity content. The changes in neutral lipids, particularly in the diacyl glycerol fraction, were also corrected by the injection of the nucleotide. CDP-choline partially reversed the decrease of brain phosphatidylcholine and of its labeling, which was due to ischemia. All the data indicate that the prior injection of CDP-choline stimulates the choline phosphotransferase reaction of brain towards synthesis of phosphatidylcholine and prevents the release of free fatty acids, particularly of arachidonic acid, associated with ischemia.

Breath alkanes determination in ulcerative colitis and Crohn's disease

PURPOSE: By considering the pathophysiologic basis of inflammatory bowel diseases, a role for excessive lipid peroxidation caused by oxygen free radical compounds has been proposed repeatedly. However, to date only a few studies are available on this topic in human beings. This study was designed to assess breath alkanes in a group of patients with active inflammatory bowel disease by a technique that clearly distinguishes pentane from isoprene, to prevent overestimation of values as in previous studies. PATIENTS: Twenty patients with a diagnosis of active inflammatory bowel disease (10 with Crohns disease and 10 with ulcerative colitis) were studied. Extension of the disease was similar between patient groups, and all were treated with equivalent doses of steroids and salicylates. METHODS: Breath alkanes determination was performed by a standard procedure involving a gas cromatography column able to separate pentane from isoprene. RESULTS: Overall, significant differences between patients with inflammatory bowel diseases and controls were found for ethane, propane, and pentane, but not for butane and isoprene. Isoprene was clearly distinguished from pentane, demonstrating that the significant elevation of pentane levels in patients with inflammatory bowel diseases is a real phenomenon and not an artifact caused by coelution with isoprene. CONCLUSIONS: An excess of lipid peroxidation is probably an important pathogenetic factor in inflammatory bowel diseases, and this may be assessed through a nonivasive method. Because this method previously also has been shown to be able to evaluate disease activity, it could be a useful tool for studying patients with inflammatory bowel diseases.

Volatile Alkanes and Increased Concentrations of Isoprene in Exhaled Air during Hemodialysis

In this study we examined breath volatile hydrocarbon concentrations in exhaled air of hemodialysis patients. We assessed both C2–C5 alkanes – among them ethane and pentane the production of which in man is essentially due to the action free radicals exert on polyunsaturated fatty acids – and isoprene, an unsaturated hydrocarbon the biosynthesis and biological effects of which are the subject of controversy and mounting interest. Twenty patients were studied. Evaluation was performed intrapatient in the breath of patients with chronic renal failure, before and after dialysis (20 patients) and, in the same cases, during hemodialytic treatment (10 patients). Breath concentrations of these volatile hydrocarbons, determined before dialysis, were not different from those of normal subjects. Dialysis did not modify the levels of the C2–C5 saturated hydrocarbons ethane, propane, butane and pentane. Instead, there was a marked increase in isoprene in all patients (basal values rose by a mean of 270%). Since isoprene was not present in the fluids or filters used for dialysis and there were only traces in the ambient air, the isoprene must have been produced endogenously during hemodialysis. As no situation has previously been reported to increase endogenous production of isoprene in humans, patients in hemodialysis offer a unique opportunity to investigate in depth the medical, biological and toxicological aspects of isoprene.

The effect of S-adenosyl-l-methionine on ischemia-induced disturbances of brain phospholipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries with reported procedures. Changes in the energy status of brain demonstrated that carotid ligation was effective. At different time intervals from ligation, groups of ferbils were given either saline or S-Adenosyl-l-methionine (SAMe) by the intraventricular (i.v.) route (1.6 mg/Kg body wt. twice, at each 10 min interval), or by the intraperitoneal (i.p.) administration (200 mg/Kg body wt.) or subcutaneously (s.c.) with 40 mg/Kg body wt, daily, for two weeks. Control animals, with and without SAMe, together with the ischemic groups, were decapitated directly into liquid nitrogen, 10 min after ligation. Brain neutral and polar lipid, together with free fatty acids, which were all labeled in vivo by the intraventricular injection of [1-14C]arachidonic acid 2 hr prior to ligation, were extracted, purified and separated by conventional procedures. SAMe when injected i.v. or i.p. noticeably corrected the changes in polar lipid by reversing the decrease of brain phosphatidylcholine and choline plasmalogen, as well as of their labeling, which was due to ischemia. Concurrently with this action, SAMe treatment (i.v. and i.p.) also provided to some extent to re-establish the normal level of labeling of ethanolamine lipids. When SAMe was given s.c., no effect was present. SAMe had no effect on the increase of free fatty acid and diglyceride due to ischemia. The prevention by SAMe of the changes of choline lipids suggests that a stimulation of the methyltransferase reaction may occur in the ischemic brain, due to increased substrate (SAMe) availability. This effect may be important for cell survival, since membrane phospholipid derangements alter the properties of the membrane.

Fatty acid dietary intake and the risk of ischaemic stroke: a multicentre case-control study

Abstract A low dietary intake of unsaturated fatty acids has been found in male patients with stroke as compared with controls in Italy, and a high consumption of meat has been associated with an increased risk of stroke in Australia. We present a case-control study, comparing the unsaturated and saturated fatty acids content of red cell membranes (which reflects the dietary intake of saturated and unsaturated fats) in 89 patients with ischaemic stroke and 89 controls matched for age and sex. In univariate analysis, besides hypertension, atrial fibrillation, ischaemic changes in ECG and hypercholesterolaemia, stroke patients showed a lower level of oleic acid (P = 0.000), but a higher level of eicosatrienoic acid (P = 0.009). Conditional logistic regression (dependent variable: being a case) showed that the best model included atrial fibrillation, hypertension, oleic acid and eicosatrienoic acids. These results confirm a possible protective role of unsaturated fatty acids against vascular diseases; however, we did not find any difference in the content of omega3 acids, which have been considered in the past to protect against coronary heart disease. We conclude that the preceding diet of patients with ischaemic stroke may be poor in unsaturated fatty acids (namely, oleic acid), and this defect is independent of other vascular risk factors. Only further studies will show whether changes in diet and/or supplement of unsaturated fatty acids might reduce the incidence of ischaemic stroke.

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.

Onset, time course, and persistence of increased haemodialysis-induced breath isoprene emission.

Recent findings of increased isoprene emission in the exhaled breath of patients undergoing haemodialysis and experimental evidence of the potential toxic and cancerogenic effects of isoprene hydrocarbon led us to assess how long haemodialysis patients are exposed to how much isoprene after a single haemodialysis session. Patients with end-stage renal failure on regular 4-hour (from 08.00 to 12.00 h) maintenance haemodialysis three times weekly were monitored. The breath isoprene content was analyzed by gas chromatography. Intrapatient evaluations were performed by collecting samples before, during, and immediately after the haemodialysis session, during the following hours, and on the following nondialysis day. The breath isoprene content increased in all patients. Isoprene overproduction showing a biphasic pattern was first detected soon after the dialysis session ended. These data show that haemodialyzed patients seem to be consistently exposed to high endogenous isoprene concentrations. The mechanisms and implications of this endogenous isoprene overproduction need to be elucidated with regard to the mevalonic pathway and in the physiopathological setting of the uraemia-dialysis syndrome.

Synaptosomal phospholipid pool in rabbit brain and its effect on GABA uptake

Rabbit synaptosomes have been used to study the effect of the base-exchange reaction in membrane phospholipids on γ-aminobutyric acid (GABA) transport in vitro. The uptake of GABA was measured after a base-exchange reaction with ethanolamine, choline, orl-serine and after subsequent displacement of these exchanged moieties from lipid by bases of similar or different structures which were added to the synaptosomal medium. Serine incorporation stimulated GABA transport, but its displacement from membrane lipid by choline or ethanolamine induced an inhibition of GABA transport. Ethanolamine incorporation inhibited GABA transport, but its displacement by serine or choline resulted in stimulation of GABA uptake. Choline incorporation also inhibited GABA transport, although less than ethanolamine. The pool size of synaptosomal phospholipids, presumably involved in GABA uptake, accounted for 0.2 to 10% of the total content of membrane phospholipid. Thus, alteration of phospholipid compositior by exchange of the lipid hydrophilic head-groups influences the extent GABA uptake into rabbit synaptosomes.

Biochemical studies on the nootropic drug, oxiracetam, in brain.

Summary: The aim of this work was to study the effects of the nootropic drug oxiracetam, on lipid metabolism in rat brain. Twenty-month-old rats and spontaneous hypertensive (SHR) rats with cerebrovascular lesions were used, which showed an impaired learning and memory rate if challenged with behavioral tests. Oxiracetam improves the in vitro and in vivo synthesis of phosphatidylcholine (PhC) and phosphatidylethanolamine (PhE) impaired by aging, when respectively added to the incubation medium or administered subacutely to animals. SHR rats drinking saline, and with cerebrovascular lesions, have a reduced choline incorporation into cerebral phospholipids and an increase of arachidonic acid release from the same lipids if compared to SHR rats (without cerebrovascular lesions) drinking water. They also show a decreased incorporation rate of arachidonic acid into PhC, PhE, and PhC plasmalogen and PhE plasmalogen. If oxiracetam is chronically administered (200 mg/kg/day for 14 weeks) a significant variation in the incorporation of both precursors takes place. In the first 2 h after the intracerebroventricular (i.c.v.) injection of choline and arachidonic acid the values are comparable to those observed in SHR rats with lesions; at longer time intervals, however, the rates of incorporation are similar and even better than those of SHR rats without lesions. Since the drug does not seem to influence the incorporation of the precursors in the first 2 h after their administration, we may assume that oxiracetam acts on the turnover of the phospholipids more than on their rate of synthesis from injected precursors.

MEMBRANE-BOUND BASE-EXCHANGE REACTIONS IN ANIMAL TISSUES

The calcium-stimulated incorporation of ethanolamine, L-serine and choline into rabbit synaptosomal phospholipids in vitro has been investigated. The synaptosomal membranes were prelabelled in vitro in their choline-, ethanolamine- or serine-phosphoglycerides by base-exchange and then chasing experiments were performed by displacing the lipid-bound base by ethanolamine, choline or L-serine labelled with a different isotope. The results indicate that membrane phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine are substrates for the exchange with all the three mentioned bases. A very small phospholipid pool (0.5-2% of the total available pool) is active in the calcium-dependent exchange between membrane phosphatidylcholine or phosphatidylethanolamine and free bases, whereas the pool of exchanging phosphatidylserine is sensibly larger (2-9%). In another series of experiments the effect of the base-exchange reaction upon the production of cyclic-AMP at the level of rat brain synaptic membranes has been examined. An exchange with ethanolamine produces a significant decrease of the NaF-stimulated production of the cyclic nucleotide, whereas it increases the noradrenaline-induced production. With some exceptions, the exchange with L-serine produces opposite effects. The possible physiological importance of phospholipid pool at the synaptosomal level is discussed.