Simple procedures for the rapid cleavage of ester lipids and for the large-scale isolation from brain of cerebroside sulfate

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Multi-dimensional mass spectrometry-based shotgun lipidomics and the altered lipids at the mild cognitive impairment stage of Alzheimer's disease

Multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) is a well-developed technology for global lipid analysis, which identifies and quantifies individual lipid molecular species directly from lipid extracts of biological samples. By using this technology, we have revealed three marked changes of lipids in brain samples of subjects with mild cognitive impairment of Alzheimer's disease including sulfatides, ceramides, and plasmalogens. Further studies using MDMS-SL lead us to the identification of the potential biochemical mechanisms responsible for the altered lipids at the disease state, which are thoroughly discussed in this minireview. Specifically, in studies to identify the causes responsible for sulfatide depletion at the mild cognitive impairment stage of Alzheimer's disease, we have found that apolipoprotein E is associated with sulfatide transport and mediates sulfatide homeostasis in the nervous system through lipoprotein metabolism pathways and that alterations in apolipoprotein E-mediated sulfatide trafficking can lead to sulfatide depletion in the brain. Collectively, the results obtained from lipidomic analyses of brain samples provide important insights into the biochemical mechanisms underlying the pathogenesis of Alzheimer's disease.     

Brain Galactolipid Content in a Patient with Pseudo-Arylsulfatase A Deficiency and Coincidental Diffuse Disseminated Sclerosis, and in Patients with Metachromatic, Adreno-, and Other Leukodystrophies

A 4-year old boy died of diffuse disseminated sclerosis (DDS) of the brain and was found to have also pseudoarylsulfatase A deficiency (PASAD) with about 20% residual arylsulfatase A (ASA) and cerebroside sulfatase (CS) activity. The reexamination of lipids did not show any sulfatide accumulation in the patient's organ extracts. Although the residual CS activity in the patient's extracts was clearly demonstrable only after partial purification, it was concluded that this activity protects organ tissues from sulfatide accumulation in PASAD, since in sulfatide lipidosis (metachromatic leukodystrophy, MLD) no residual CS activity was detectable. The study of residual ASA activity in the patient's fibroblasts by gel electrofocusing resulted in an almost normal enzyme microheterogeneity. However, the detailed study of the brain galactolipids in the patient revealed an elevated ratio of sulfatide/galactocerebroside content, despite the decrease of both lipids. In tissues of other patients with severe demyelinating diseases different from DDS and MLD, this galactolipid ratio was also found to be increased, especially in three patients with adrenoleukodystrophy. A general mechanism of this anomaly in severe demyelination is considered.     

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.     

CHEMICAL SYNTHESIS OF [3H]CEREBROSIDE [35S]SULFATE AND ITS IN VIVO METABOLISM IN RAT BRAIN

—Double-labeled sulfatide containing [3-³H]lignoceric acid and [³⁵S]sulfate was synthesized and injected intracerebrally into 28-day-old rats. The ³H-labeled sulfatide was synthesized by condensing (RS)-[3-³H]lignoceroyl chloride with lysosulfatide which had been obtained by saponification of sulfatide. The ³⁵S-labeled sulfatide was synthesized by using [³⁵S]sulfuric acid for sulfating 2′, 4′, 6′-tri-benzoyl-galactosyl N-fatty acyl, N-benzoyl-3-0-benzoyl-sphingosine, which had been obtained by per-benzoylation followed by solvolysis of calf brain nonhydroxycerebrosides. The perbenzoylated [³⁵S]sul-fatide was then subjected to mild alkaline saponification. Eight hours following the injection, the brain lipids contained various radioactive sphingolipids in addition to sulfatides. Fourteen per cent of the injected ³H was recovered in total lipids, and 26% of this was found in sulfatide. Nonhydroxy- and hydroxyceramides, nonhydroxy- and hydroxycerebrosides, and polar lipids contained 7, 1, 8, 3, and 22 per cent of the ³H found in total lipids, respectively. On the other hand, only 6% of the ³⁵S injected was recovered in total lipids; 63% of this was found in sulfatide, 5% in a mixture of seminolipid and cholesterol sulfate and 10% in a water-soluble material.     

Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

Retention of gangliosides in serum delipidated by diisopropyl ether-1-butanol extraction

Extraction with diisopropyl ether-1-butanol is a rapid method for the delipidation of serum without protein denaturation. We sought to confirm that this solvent system would also extract the highly polar acidic glycosphingolipids, gangliosides. In fact, however, both endogenous serum gangliosides and radiolabeled rat brain gangliosides added to serum were nearly quantitatively retained (87.5% and 98.7%, respectively) in the aqueous phase after two extractions. Therefore, while useful for the extraction of most lipids, this delipidation procedure cannot be used to remove gangliosides from serum or plasma.     

Cerebroside sulfuric ester (sulfatide) induces oxygen radical generation in guinea-pig leukocytes

Previous studies on experimental allergic encephalomyelitis have shown that a number of leukocytes appear in demyelinating lesions of guinea-pig brain. The present studies showed that cerebroside sulfuric ester (sulfatide), a typical component of myelin membranes, stimulated the oxidative metabolism of guinea-pig neutrophils and macrophages, leading to marked generation of oxygen radicals and light emission. Formation of a spin adduct of 5,5-dimethyl-1-pyrroline N-oxide by leukocytes was dependent on the concentration of sulfatide, and correlated well with the generation of superoxide anion and the intensity of chemiluminescence measured in the absence of luminol. The addition of myelin membranes to the sulfatide-stimulated neutrophils amplified the light emission, suggesting an interaction between myelin membranes and those of leukocytes. Assay of the thiobarbituric acid reaction in the mixture of membranes and cells showed that sulfatide-stimulated cells induced lipid peroxidation in myelin membranes. These results suggest that sulfatide released from demyelinating lesions stimulates leukocytes to release toxic oxygen radicals, which attack myelin membranes, leading to a chain reaction of demyelination.     

A CHOLESTEROL-ESTERIFYING ENZYME IN RAT CENTRAL NERVOUS SYSTEM MYELIN1

Aontrary to our earlier finding (Eto & Suzuki , 1971), the myelin fraction purified from young adult rat brain consistently showed cholesterol-esterifying activity. The specific activity in myelin was the highest among subcellular fractions. Extensive washing wiih various aqueous salt solutions failed to remove the activity from myelin. The enzyme was evenly distributed among the arbitrarily defined light, medium and heavy myelin subfractions. The myelin-localized activity showed the pH optimum and heat stability identical to the microsome-bound activity. Although there were minor differences in the effect of detergents or exogenous lipids added to the reaction mixture, no firm evidence was obtained to indicate that the myelin-bound cholesterol-esterifying enzyme is different from that in other subcellular fractions. On the other hand, the distribution among the myelin subfractions and heat stability of the myelin-bound cholesterol-esterifying activity were different from those of the myelin-specific cholesterol ester hydrolase. Therefore, the esterification does not appear to be a mere reverse reaction catalyzed by the previously known myelin-specific hydrolase. The rat brain myelin, therefore, is capable of both synthesizing and hydrolyzing cholesterol esters.     

Lipid microarrays identify key mediators of autoimmune brain inflammation

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.     

ESR studies of the dynamics of dipalmitoylglycerophosphocholine-sulfatide model membranes

The effects of sulfatide on the fluidity and surface dynamics of bilayered and micellar model membranes of dipalmitoylglycerophosphocholine containing sulfatide were studied as a function of gel-to-liquid-crystalline state of the lipids by electron spin resonance. 5- and 15-nitroxystearic acid were employed as spinlabel probes for the region close to the surface and that close to the nonpolar core of lipid structures. The sulfatide effect is completely different above and below the gel-to-liquid-crystalline phase transition point, the glycolipid promoting a more disordered state below it and having a condensing effect above the phase transition temperature.     

Isolation of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine from the bull heart

A novel efficient procedure based on silica gel column chromatography has been developed for isolating chromatographically pure diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. For this purpose the lipid extract is treated with aqueous MgCl2, whereas aqueous ammonia is added to the eluent systems. To raise the yield of lipids, a fractional loading of columns with portions of lipid extracts is employed, each loading being followed by partial elution of neutral lipids with chloroform. Optimal phospholipid--silica gel ratio is 1:20 for such a procedure. The presence of cation exchange between the lipid extract and silica gel is confirmed and its influence on the efficacy of phospholipid separation is discussed.     

Quantitative analysis of trifluoroacetic acid in body fluids of patients treated with halothane

A simple procedure for the quantitative analysis of trifluoroscetic acid (TFA) in urine and serum from patients narcotized with halothane is described. This involves addition of sodium hydroxide to the body fluid, evaporation of the aqueous phase and esterification of TFA in concentrated sulphuric acid with 2,2,2-trichloroethanol. The gaseous phases above the reaction mixture were then analyzed by gas chromatography with a nickel-63 electron-capture detector. The detection limit was 1 μg of TFA per mililitre of body fluid (200 μg of body fluid are analysed) and the relative standard deviation was ±6%. Patients treated with ethrane, another commercial ansesthetic, did not produce any detectable TFA.     

Preparation of spin-labelled sulfatides for EPR studies on model membranes

Using the N-hydroxysuccinimide ester of the fatty acids, galactosylceramide I3-sulfate containing a 5-or 16-doxyl-stearoyl residue was prepared in good yield by acylation of the galactosylsphingosine I3-sulfate (lysosulfatide) obtained from the saponification of the bovine brain sulfatide. The EPR behavior of the two semisynthetic sulfatides was analyzed in natural sulfatide micelles and in multilamellar vesicles of egg phosphatidylcholine. The evaluated parameters demonstrate that these spin-labelled sulfatides can be used for the study of sulfatide behavior in lipid structures.     

Hemolysis and membrane lipid changes induced by xanthine oxidase in vitamin E deficient red cells

A procedure to induce homolysis by the hypoxanthine-xanthine oxidase reaction was developed and applied to vitamin E deficient red blood cells (RBCs) in rats. The reaction system was as follows: 0. 16 mM hypoxanthine, 0.05 U/ml xanthine oxidase in 2.5% RBC suspensions with an isotonic buffer (pH 7.4) containing 10 mM phosphate buffer and 125 mM saline (227 mOsm). Hemolysis was observed to depend on the vitamin E concentrations in the RBCs. Hemolysis was inhibited by catalase but not by SOD. After the reaction with vitamin E deficient RBCs, an increase in TBARS in the aqueous phase of the reaction mixture was observed. This accompanied the increase in fluorescent substances in the lipid extracts, in associatioon with a significant decrease in the PE and PS of the RBCs, and a decrease in arachidonic acid in membrane lipids. The above changes were almost completely inhibited by tocopherol incorporated into vitamin E deficient RBCs.     

Evaluation of the binding of serotonin by isolated CNS acidic lipids

Binding of serotonin by rat lipids was examined in an organic solvent-aqueous partition system. Only phospholipids and sulfatide were found to have appreciable activity: this technique was unsuitable for gangliosides due to their poor extractibility. Binding by phospholipid was abolished and that by sulfatide was greatly inhibited by increasing ionic strength in the aqueous phase. At an ionic strength of 0.3 M the apparent affinity of sulfatide for serotonin was about 3×10³ M. Both tryptamine and 5-methoxytryptamine were much more effective than serotonin in inhibiting the binding of radioactive serotonin, suggesting that the observed binding is simply a charge neutralization with little specificity. Binding of serotonin by mixed brain gangliosides was examined in an equilibrium dialysis system. Without adequate precautions, the chemical lability of serotonin was found to produce spurious data when binding was assessed by the distribution of radiolabel. Binding of serotonin by ganglioside was also greatly inhibited by increasing ionic strength: at 0.3 M an apparent affinity of about 10³ M was found. While dopamine did not inhibit the binding of radioactive serotonin, tryptamine, 5-methoxytryptamine, and serotonin were equally effective inhibitors.     

Membrane lipids of human peripheral nerve and spinal cord.

Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20-70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17-91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 +/- 11 and 96 +/- 9 nmol/g fresh weight; of cholesterol 70 +/- 15 and 101 +/- 16; of cerebroside 19 +/- 3 and 41 +/- 7; of sulfatide 10 +/- 1 and 11 +/- l; and of gangliosides 0.80 +/- 0.08 and 0.40 +/- 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.     

THE CHARACTERIZATION OF SPHINGOLIPIDS OF OLIGODENDROGLIA FROM CALF BRAIN

Abstract— Purified oligodendroglia isolated from bovine brain white matter were found to contain, in addition to galactosylceramide, sulfatide and sphingomyelin, significant quantities of glucosylcerai-mide, dihexosylceramide and esterified galactosylceramide. These sphingolipids were isolated and quan-titated and their fatty acid and long chain base patterns compared with those from sphingolipids isolated from bovine myelin, white matter and gray matter.
The minor glycosphingolipids, glucosylceramide, dihexosylceramide and esterified galactosylceramide, constituted a higher percentage of glial lipids than of myelin lipids. Glucosylceramide accounted for 12% of the total glial monohexosylceramide fraction and 0.8% of total lipids; dihexosylceramide was 0.9% of total glial lipids. Both of these lipids had small quantities of α-hydroxy fatty acids. The unsubstituted fatty acids of glucosylceramide were mostly short chain (16 and 18 carbons) and were different from those of the dihexosylceramides which were a mixture of short and long chain. The hydroxy acids of each of these lipids were, however, similar and resembled those of galactosylceramide.
The fatty acid patterns of galactosylceramide, sulfatide and sphingomyelin from glial cells resembled those of the corresponding lipids from myelin and white matter. The amide-linked acids of esterified galactosylceramide contained both unsubstituted and α-hydroxy chains. Their patterns were not identical to those of galactosylceramide, but were similar in all brain fractions.
With the exception of sphingomyelin and dihexosylceramide, which contained small amounts of C₂₀-sphingosine, all sphingolipids analyzed contained mostly sphingosine and dihydrosphingosine.
We conclude that the distribution of sphingolipids in the oligodendroglia is characteristic, but the lipophilic residues of these lipids are not cell-specific.     

Irrigation of Solanum lycopersicum L. with magnetically treated water increases antioxidant properties of its tomato fruits

Antioxidant effects of tomatoes (Solanum lycopersicum L.) have been studied and an association between dietary intake of tomatoes and lowered risk of cancer, neurodegenerative, and cardiovascular diseases has been suggested. Here we used magnetically treated water (MTW; 0.03–0.15 T), which promotes better germination and productivity in tomatoes, and we investigated the effects of aqueous and ethanolic (10–400 μg/ml) extracts of S. lycopersicum as potential antioxidant against 10 μM Fe(II)-induced thiobarbituric acid reactive species (TBARS) in liver and brain homogenates from rats. The ethanolic extracts from magnetically treated plants were more effective than aqueous extracts in preventing TBARS formation in brain and liver. The protective effects of ethanolic extract can be associated with antioxidants (polyphenols and flavonoids), lycopene and other lipophilic components found in the extract. In effect, magnetically treated plants had higher content of polyphenolic and flavonoid compounds than nontreated plants and they can be a better source of antioxidants than nontreated plants. Consequently, MTW can be used to produce functional foods with high contents of antioxidant components and may have better beneficial health effects than traditionally produced foods.