The effect of moderate hypoxia on free sterols: content and pattern in white matter.

Wistar rats underwent moderate hypoxia by exposing them to a respiratory gas mixture containing 7% oxygen, for 30 min. The myelin was analyzed at 4 h 24 h, day 14, and day 60 after hypoxia. The group of neutral lipids isolated from myelin was analyzed by GC-MS (gas chromatography-mass spectrometry) to separate and measure free sterols. In the myelin of hypoxia treated animals the pattern of free sterols was variable. Cholesterol concentration decreased with the changes in the content and composition of sterols present in smaller amounts. These changes may indicate the disturbances in some steps of cholesterol biosynthesis and metabolism in pathologic conditions. The appearance of sterols characteristic for plants (beta-sitosterol) was noticed in myelin of experimental animals. The possible reason for the presence of this sterol in animals may be the result of its intake during feeding, however, biosynthesis can not be excluded.

[Lipid image in glioblastoma multiforme]. / Obraz lipidów w glejaku wielopostaciowym.

Changes in the content and composition of lipids in brain tumours of different degree of malignancy are still the subject of numerous scientific studies. It is known that in developing brain tumours structural and functional changes of its cells, take place, in which lipids play a crucial role. The examination of the lipid spectrum was conducted in a material extracted from tumour adjacent areas (by means of fenestration) as well as from tumours themselves in 11 patients (mean age 56.8 yrs). Based on histological studies all the tumours were qualified as glioblastoma 4th degree of malignancy. Lipids were isolated from the tumours as well as from their adjacent areas. Later on they were separated, using column chromatography and thin-layer chromatography, into three classes: phospholipids, galactolipids and neutral lipids. Separated lipids were subjected to quantitative analysis by spectrophotocolorimetric method. In comparison to adjacent areas, in tumours distinct changes of lipid levels were noticed in all analysed lipid classes. Statistical analysis showed significant decrease of phospholipids (elements stabilizing cell membranes) in tumours in comparison to their adjacent areas (Wilcoxon's test: p < 0.05). At the same time in tumours an increase of level of plasmalogens took place: phosphatidylcholine (PC) (PPC), phosphatidylethanolamine (PE) (PPE) typical elements of malignant tumours, responsible for cross cell membrane transportation processes. This phenomenon was accompanied by changes of levels of PC/PE and PC/SM (sphingomyelin) ratios connected with adhesiveness and other membrane features. Changes in the level of lipids, and phospholipids in particular, in glioblastoma 4th degree of malignancy in comparison to adjacent areas can indicate the pathological processes in cells of these tumours.

Patterns of free and esterified sterol fractions of the cerebral white matter in severe and moderate experimental hypoxia.

Cerebral sterols were examined in Wistar strain rats, subjected 4 h, 24 h, 14 days or 2 months earlier to severe (2% oxygen) and moderate (7% oxygen) experimental hypoxia. From brains of the experimental animals myelin was isolated and examined by gas chromatography coupled with mass spectrometry (GC-MS) to separate and to identify free and esterified (following hydrolysis) cerebral sterols. In both types of hypoxia, the same compounds were identified in fractions of free and esterified sterols. Slight differences in sterol content were noted between the two experimental models, involving cholesterol, the main sterol of the myelin sheath, its smaller sterol precursors (desmosterol, lanosterol) and the less abundant sterols (beta-sitosterol, 24-ethyl-4-cholesten-3-on, cholesta-3,5-dien, cholest-4-en-3-on, 22-propyl-3 beta-hydroxy-5,24-cholestadien). Both types (severe and moderate) of hypoxia were found to induce similar alterations in patterns of cerebral sterols 24 h, 14 days and 2 months following the exposure. Quantitative differences in the sterol pattern were detected only at the very early stage (4 h) of the experiment.

Free sterols in the rat white matter following experimental global ischemia.

The pattern of free sterols occurring in the cerebral white matter was studied in an experimental model of global ischemia that was induced in rats by means of the method described by KORPACHEV et al. (1982). Sterol isolation, separation and identification was accomplished by suitable extraction procedures followed by gas-chromatography-mass spectrometry of the purified sterol extracts. It has been shown that the percentage of lanosterol happened to decrease sharply following the ischemic state and other sterols, typically occurring in maturating brains and absent in the control brain specimens from adult rats, such as 4,4-dimethylcholesterol, 4,4-dimethylcholest-5,8,24-triene-3 beta-ol and 4,4,14-trimethylcholest-5,8-diene-3 beta-ol happened to appear. The appearance of these forms of free sterols in the postischemic brain is interpreted as a biochemical exponent of regeneration processes occurring in the white matter membranes following the injury suffered during the experimental heart arrest.

Free sterols of the cerebral white matter in experimental severe hypoxia.

Wistar rats, aged 3.5 months, were subjected to severe hypoxia by placing them for 3 minutes in an atmosphere containing 2 kPa of oxygen. The myelin fraction was isolated from brains of the experimental animals at the following periods after the hypoxia event: 4 and 24 hours, 14 days, 2 months. The myelin neutral lipid extracts were fed into GC-MS system (gas chromatography-mass spectrometry) for identification of the separated free sterols. The free sterol spectrum of the myelin fraction of rats following the experimental hypoxia was variable unstable. The content of free cholesterol decreased and the contribution of minor sterols (24-ethylcholesterol, 24-ethyl-4-cholesten-3-on, 22-propyl-3 beta-hydroxy-5,24-cholestadien) changed. Lanosterol, one of the natural precursors in cholesterol biosynthesis happened to appear, and the content of the other precursor, desmosterol-increased. These changes may be indicative of some disturbances in the process of cholesterol biosynthesis induced by the experimental hypoxia. The occurrence of ethyl- and propylderivatives of cholesterol (compound found mainly in plant sterol spectra) in both the control and experimental myelin samples in either of the examined post-hypoxia periods is most likely the result of incorporation into myelin lipids of ingested plant sterols, nevertheless, endogenous synthesis cannot be ruled out.

Composition of the cerebral white matter sterol ester fraction in severe experimental hypoxia.

Severe hypoxia was induced in adult Wistar rats by means of exposure to an atmosphere containing 2.0 kPa of oxygen for a period of 3 minutes. The animals were sacrificed at 4 different periods following the hypoxia incident: 4 and 24 hours, 14 days and 2 months, respectively, and sterol esters (ES) were isolated from the myelin fraction of the cerebral white matter. After hydrolysis, the isolated sterols were fed into a GC-MS system (gas chromatography-mass spectrometry). The following sterols were identified in the hydrolysates: cholesterol, cholesta-3,5-dien, cholesta-4-en-3-on, 22-propyl-3 beta-hydroxy-5,24-cholestadien. The induced severe hypoxia lead to a twofold increase of the SE content in the myelin fraction of experimental rats (24 hrs, 14 days and 2 months samples). Cholesterol was the dominating sterol species in this fraction, however other minor sterols were found as well. The dynamic equilibrium between free and esterified sterols in the brain is controlled by the activity of respective sterol ester hydrolases. Esterification of free sterols renders them inaccessible for other metabolic changes, such as demethylation, reduction, isomerisation of double bonds, hydroxylation, and thus may lead to compositional changes of the myelin sterol spectrum under conditions of severe hypoxia.

Transformation of sterols pattern in course of late development of rat brain.

Free sterol content was determined in the late period of development of the rat brain using the method of gas chromatography-mass spectrometry. The results obtained have shown that the free sterol pattern is very unstable in course of late period of ontogenic development. In contrast to young adult rats, in which apart from cholesterol only trimethylcholestenon and trace amounts of desmosterol were visible, in the brain of 1 and 2 years old rats some new sterols are appearing. There are cholestadien, cholest-5-en-3-on, 4 methyl-cholesten, cholest-5-en-3-ethoxy and 4,4,14-trimethyl-5-alpha-cholest-24-en-3-en. There are also qualitative differences in the structure of some sterols (various isomers) between both groups of late development. These changes, indicating some transformation of cellular membranes seem to be the result of biologically programmed late ontogenic development.

Myelin lipids and proteins in experimental global ischemia.

The experiments were performed on white rats, in which clinical death was induced according to the method described by Korpatchev et al. (1982). After cardiac arrest and cessation of respiratory function lasting 5 minutes resuscitatory action was performed. The lipids and proteins of the cerebral myelin fraction were studied in animals sacrificed 1, 9 and 14 days after global ischemia. The obtained results lead to the following conclusions: 1. Myelin lipids in postreanimation syndrome are characterized by a marked increase in cholesteryl esters and a mild one of lysophosphatidylcholine content. 2. Myelin lipids in the predemyelinating period demonstrate a general pattern of reaction, notwithstanding to the character of the primary noxious agent. 3. After global ischemia a great fall of small basic protein in the myelin fraction and subsequently of SBP to LBP ratio occurred. 4. Various noxious agents affect different proteins of the myelin membranes and the resulting changes seem to be characteristic for various pathological processes.

Free sterols in senile human brain.

Autopsy material of 18 patients, who died between 66-86 years of age and of 4 ones, who died between 20 and 38 years of age was investigated. The white matter of frontal lobe, corpus callosum and cerebellum was studied using histological and biochemical methods. According to the results of neuropathological studies, the material of aged patients was divided into two subgroups: patients with vascular changes only and brains with senile atrophy of Alzheimer type. Chemical changes in all aging brain regions studied included higher proportion of desmosterol and differences in the pattern of cholesterol isomers. These findings are interpreted as suggesting some chemical transformations of cellular membranes in the cerebral white matter of the aging. The above mentioned changes were almost identical despite presence of the signs of Alzheimer's type senile atrophy, therefore it is not possible to explain their significance in developing of dementia process.

Myelin proteins in aging human brain.

Autopsy material of 13 persons who died between 70 and 89 yrs old and of patients who died between 27 and 44 yrs old was studied. White matter of temporal, parietal, and occipital lobes was investigated using histological and biochemical methods. According to results of neuropathological studies, the material of aged patients was divided into two subgroups: (a) brains with vascular changes only and (b) patients with senile atrophy of Alzheimer type. Chemical changes found in all studied brain lobes included a mild decrease in Wolfgram protein content with reciprocal increase in large basic protein content, together with a marked decrease in myelin yield. The abovementioned chemical changes were almost identical whether they were only vascular changes or whether senile atrophy of Alzheimer type was also present. It seems, therefore, that the degeneration of vessels is the decisive factor in the pathogenetic mechanism of myelin lesions in the aged brain.

Myelin lipids in ischemic stroke.

Slices of ischemic focus (infarct area) and of the contralateral frontal lobe were submitted to histological and biochemical studies. The obtained results indicate that in stroke cases the necrotic focus as well as contralateral brain hemisphere are characterized by a marked decrease of cholesterol and cerebrosides content and an increase of lysophosphatidylcholine and cholesterol esters in the myelin fraction. We conclude that ischemia as well as the degenerative aging process are both responsible for the abnormal lipid pattern in the myelin of the white matter in stroke cases. The long lasting hypoxia resulting from cerebral vessel atheromatosis contributes to biochemical changes in the myelin of the apparently healthy white matter of the contralateral hemisphere in brain infarction.

Pattern of myelin proteins in moderate hypoxia.

Myelin proteins isolated from the brain of Wistar rats subjected to moderate hypoxia (7% of oxygen in a respiration gaseous mixture) for 30 min were investigated. Among the various protein fractions Wolfgram protein happened to be most markedly affected, demonstrating considerably lowered percentages in all experimental groups, starting from 24 h after hypoxia. Our findings compared with those obtained in other experimental conditions lead to the conclusion that changes in the myelin protein pattern are characteristic for particular morbid processes evoked by various pathogenic factors. The demyelination is concomitant with severe loss of basic proteins, whereas myelin lesions of other types are connected with changes in protein of higher molecular mass.

Influence of experimental hypoxia on content and composition of free fatty acids in cerebral white matter.

The content and spectrum of free fatty acids were studied in the cerebral white matter, on Wistar rats exposed to acute hypoxia (2% of oxygen in a respiratory mixture) for 3 min. The total content of fatty acids, especially of tetraenoic ones, rose sharply already 4 min after hypoxia and persisted elevated even 2 months after the hypoxia. The results showed that the degradation of phospholipids-containing biological membranes in the nervous tissue of the white matter starts immediately after hypoxia, and is continued for months.

Myelin lipids in aging human brain.

The studies were performed on autopsy material of 18 patients who died between the ages of 70-89 y, and of 5 patients who died between 23-44 ys of age. White matter of the frontal lobe and of oerebellum was submitted for histological and biochemical analysis. The neuropathological data provided a rationale for dividing the material into two subgroups: one including patients mainly with vascular changes in the brain, and the other consisting of patients with senile atrophy of the Alzheimer type. Chemical alterations noted both in frontal lobe and in cerebellum were an increase in lysophosphatidylcholine content and a marked decrease in myelin yield. Additionally, in cerebellum a decrease in sulphatide content was observed. The chemical results were almost identical in the two subgroups of patients, although they differ in neuropathological patterns of lesions. It is emphasized that the general decrease in myelin yield as well as some minor changes in myelin lipid pattern seem to be a sign of aging and are not connected with atrophy of the Alzheimer type.