Estimation of tissue phospholipids by natural abundance 13C-NMR

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance ¹³C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after ¹³C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: (1) normal and nonspecific muscle diseases, (2) myotonia and myopathy, (3) Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

Characterization of the broad resonance in 31P NMR spectra of excised rat brain

31P NMR spectra of excised rat brain showed a broad resonance between-12 and -13 ppm. Subcellular fractions of brain, rich in membranes, exhibited the broad resonance and it was also present in isolated myelin, the major membrane component of brain. However, it was absent in brain cytosol (161,100 X g supernatant). Raising the temperature of the brain above 50 degrees C caused a gradual downfield chemical shift of the broad resonance, to about -1 ppm at 90 degrees C. An even larger downfield shift was produced by halothane or deoxycholate with concomitant narrowing of the line width of this resonance. Vesicles prepared from the phospholipids of excised brain or isolated myelin showed the broad resonance, and halothane produced the same downfield shift and peak sharpening in brain phospholipid vesicles as that in the intact brain. The chemical shift anisotropy was estimated to be 45 ppm for both myelin and the brain, as characteristic for biological membranes. The T1 and T2 relaxation times of the perpendicular 31P chemical shift tensor component of the broad resonance were 0.66 sec and 1.6 msec, respectively, in the same range as those for other biological membranes. Halothane-treatment of the brain increased both the T1 and T2 times considerably, as expected from the disruption of the phospholipid bilayer in a membrane. These data indicate that the broad resonance in the 31P NMR spectrum of excised rat brain originates exclusively from the phosphate head group of membrane bound phospholipids. Similar broad resonances were found in autopsied human brain and porcine spinal cord and to a lesser extent in excised rat liver and kidney.     

Characterization of lipids from human brain tissues by multinuclear magnetic resonance spectroscopy

Multinuclear ((1)H, (13)C, and (31)P) magnetic resonance spectroscopy are applied to the biochemical characterization of the total lipid fraction of healthy and neoplastic human brain tissues. Lipid extracts from normal brains, glioblastomas, anaplastic oligodendrogliomas, oligodendrogliomas, and meningiomas are examined. Moreover, the unknown liquid content of a cyst adjacent to a meningioma is analyzed. Two biopsies from glioblastomas are directly studied by (1)H-NMR without any treatment (ex vivo NMR). The (1)H- and (13)C-NMR analysis allows full characterization of the lipid component of the cerebral tissues. In particular, the presence of cholesteryl esters and triglycerides in the extracts of high grade tumors is correlated to the vascular proliferation degree, which is different from normal brain tissue and low grade neoplasms. The (31)P spectra show that phosphatidylcholine is the prominent phospholipid and its relative amount, which is higher in gliomas, is correlated to the low grade of differentiation of tumor cells and an altered membrane turnover. The ex vivo (1)H-NMR data on the glioblastoma samples show the presence of mobile lipids that are correlated to cell necrotic phenomena. Our data allow a direct correlation between biochemical results obtained by NMR and the histopathological factors (vascular and cell proliferations, differentiation, and necrosis) that are prominent in determining brain tumor grading.     

Changes in the natural abundance 13C NMR spectra of intact frog muscle upon storage and caffeine contracture

The carbons of phospholipids have limited mobility in fresh, resting, gastrocnemius frog muscle, but a population of phospholipids gains considerable mobility upon storage in the muscle or in contracture induced by caffeine. In parallel with the appearance of sharp phospholipid resonances, lactic acid also appears in the 13C NMR spectra. There is a correlation between the mobility of phospholipids and the depletion of phosphocreatine and ATP in muscle.     

Effect of denervation on the phospholipid content in subcellular fractions of tonic and tetanic muscles.

Four weeks after denervation, various changes were observed in the phospholipid composition of the sarcolemmal and sarcoplasmic fractions of skeletal muscles with different functions. Neurotomy also affected the innervated contralateral muscles and produced opposite changes in the phospholipid content of subcellular fractions. The increase in the amount of phospholipids in the sarcolemmal fractions of the denervated muscles was only apparent. The difference between the denervated and contralateral muscles was also due to the decrease of phospholipids in the contralateral muscles. These changes were more pronounced in the tetanic (fast-twitch) than in the tonic (slow-twitch) muscles. In the sarcoplasmic fraction of the denervated tetanic muscle an increase, while in that of the tonic one a slight decrease of phospholipids appeared. In contrast, the phospholipid content in the sarcoplasmic fractions of contralateral muscles did not decrease, while it increased slightly in the tonic muscle. The amount of plasmalogens (fatty aldehyde: lipid phosphorus ratio) decreased only in the subcellular fractions of the denervated muscles while there was no change in those of the contralateral muscles.     

Effect of halothane on the natural-abundance 13C NMR spectra of excised rat brain

Halothane increases the intensity of the 30.5- and 129-ppm resonances in 13C nuclear magnetic resonance spectra of excised rat brain, of phospholipid vesicles prepared from chloroform-methanol extract of rat brain, and of brain excised from rats anesthetized with halothane. The 13C spin-lattice relaxation times of the 30.5- and 129-ppm resonances are increased in excised brain, or phospholipid vesicles, upon addition of halothane, and they are also increased in brain excised from rats anesthetized with halothane. Excised brain and its membrane-rich subcellular fractions interact with [14C]halothane reversibly. The interaction is virtually abolished when the phospholipids are extracted from the brain. The [14C]halothane content of the brain membranes is correlated with the halothane-induced increase in the integral of the 129-ppm resonance. From this correlation and from the phospholipid content of the membranes, a halothane concentration of 3.34 mM and a partition of 0.057 mol of halothane/mol of phospholipid may be calculated in the brain of anesthetized rats.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.     

Lithium increases turnover of phospholipids in flight muscles of Periplaneta americana L

The effect of prolonged lithium administration on the phospholipid metabolism of flight muscles of the cockroach Periplaneta americana has been studied. Following daily injections of LiCl in a dose of 19.25 mumol LiCl per gram of wet weight [32P]- orthophosphate were injected and its incorporation into the phospholipids was measured 2, 12 and 24 h later. Lithium administration did not change the content of phospholipids but increased the 32P incorporation into phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and sphingomyeline 1.87, 2.13, 2.02 and 1.87 times, respectively, as compared with the control values. These increases were neither due to an increased permeability of the tissue for inorganic phosphate nor to an increased turnover of gamma-P-ATP. It is concluded that prolonged lithium treatment increases the turnover of all phospholipids in insect flight muscle tissue.     

Phosphorus NMR analysis of human white matter in mixed non-ionic detergent micelles

In order to study the lipid composition of human white matter, we have developed a 31P NMR spectroscopy method, which allows the determination and quantitation of the main phospholipids found in biological membranes. The technique is based upon the use of a non-ionic detergent (Triton X-100) which induces, in aqueous media, the formation of mixed micelles that are magnetically isotropic. The linewidths and chemical shifts depend on both the molar ratio detergent/phospholipid and the pH of the suspension. After determination of the optimum values for these two parameters, 31P NMR spectra were recorded, in which all phospholipid resonances were resolved. After determining precise chemical shifts for each phospholipid, concentrations were measured by comparing the peak areas with that of an internal standard. Analysis of the complex phospholipid composition of human white matter using this method gave values very close to that found in the literature for such tissue. Moreover this nondestructive method proved to be very sensitive since less than 1 mg of a mixture of phospholipids was needed.     

Phospholipid metabolism of monkey smooth muscle cells grown in hyperlipemic serum.

The phospholipid content and synthesis of monkey smooth muscle cells grown in tissue culture with normal or hyperlipemic monkey serum were examined. The pattern of incorporation of radioactively labeled inorganic phosphate into the phospholipids of these cells was measured using a 4 h pulse of 32P. Phosphatidylcholine and phosphatidylinositol were the predominant phospholipids labeled. Although phosphatidylcholine constituted 45% of the cellular phospholipid, phosphatidylinositol had the highest specific activity. Exposure of the smooth muscle cells to hyperlipemic monkey serum did not alter the phospholipid content, composition or synthesis of these cells. The total phospholipid content of the smooth muscle cells was independent of the concentration of lipid in the media. The distribution of 32P into the phospholipids of monkey alveolar macrophages, L-cell mouse fibroblasts, and segments of the intima-media from monkey aortas is reported.     

13C isotopomer analysis of glutamate by J-resolved heteronuclear single quantum coherence spectroscopy

13C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of 13C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. (1)H NMR is intrinsically more sensitive than 13C NMR and can potentially supply the same information via indirect detection of 13C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for 13C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct 13C detection but with improved sensitivity.     

Incorporation of [32P]orthophosphate into brain-slice phospholipids and their precursors. Effects of electrical stimulation

1. The incorporation of [(32)P]phosphate into phospholipids was measured in slices cut from the pial surface of guinea-pig cerebral cortex; incorporation into the phosphorus of some water-soluble precursors of phospholipid was measured under similar conditions. 2. Slices subjected to overall electrical stimulation at a frequency of 5pulses/sec. differed from control slices in their pattern of phospholipid labelling. After 1hr. of stimulation, incorporation of [(32)P]phosphate into phosphatidylcholine, ethanolamine phospholipid and cardiolipin was respectively 54, 55 and 58% of the control value, and that into phosphatidylinositol was 186% of control. Phosphatidic acid labelling tended to increase with electrical stimulation, but the statistical significance of this change was marginal. Labelling of phosphatidylglycerol and di- and tri-phosphoinositides was not affected significantly by electrical stimulation. 3. Electrical stimulation of the tissue altered the specific radioactivities of water-soluble precursors of phospholipid. 4. The turnover rates of the phosphate groups of phospholipids were estimated approximately from the specific radioactivities of phospholipids and their precursors. Phosphatidylinositol (and its lipid-soluble precursors) showed the largest change in turnover rate in response to electrical stimulation of the tissue; the turnover rates of other lipids were also affected. Changes in the specific radioactivity of phospholipids did not correspond to changes in turnover in these experiments.     

Effect of triiodothyronine on phospholipid metabolism in skeletal muscles of the rat.

The aim of the present study was to examine the effect of treatment with triiodothyronine (T3) on certain aspects of phospholipid metabolism in skeletal muscles. Rats were injected with triiodothyronine (T3) daily (10 microg x 100 g(-1) b.w., s.c.) for six days. Saline-treated rats served as controls. 24 h after the last dose of T3, 14C palmitic acid suspended in the serum of a donor rat, was administered intravenously. Thirty min later, samples of the soleus, white and red section of the gastrocnemius and blood from the abdominal aorta were taken. The muscle phospholipids were extracted and separated into different fractions by means of thin layer chromatography. The following fractions were obtained: shingomeylin, phosphatidylcholine phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and cardiolipin. The phospholipids were quantified and their radioactivity was measured. The plasma free fatty acid concentration and radioactivity was also determined. Treatment with T3 reduced the content of phosphatidylinositol and phosphatidylserine in each muscle type, whereas the concentration of other phospholipids remained stable. T3 increased markedly incorporation of the blood-borne fatty acids into each phospholipid fraction in the muscles. It is concluded that an excess of T3 influences the metabolism of phospholipids in skeletal muscles.     

Restoration by phospholipids of dolichol pyrophosphate N-acetylglucosamine synthesis in delipidated rat lung microsomes

The effects of phospholipids on the reaction catalyzed by UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase have been studied with delipidated rat lung microsomes. Deoxycholate-solubilized enzyme was depleted of measurable phospholipid by either gel filtration on Sephadex G-100 or affinity chromatography on pentyl-agarose. The latter procedure also removed nucleotide and sugar nucleotide hydrolases. Delipidated protein fractions were devoid of GlcNAc-1-phosphate transferase activity unless supplemented with phospholipids. Maximal recovery of enzyme activity was obtained with an approximate 1:1 weight ratio of phosphatidylglycerol:phosphatidylcholine, with the observed rate being synergistic as compared to rates observed for each individual phospholipid. Variable recoveries of enzyme activity were obtained with mixtures containing other acidic phospholipids and phosphatidylcholine. Enzyme activity in the fraction eluted from pentyl-agarose could be recovered, after removal of Triton X-100, with sedimented phospholipid vesicles. Significant stabilization of enzyme activity associated with the phospholipid vesicles was obtained by the inclusion of dolichol phosphate.     

31P-NMR study of brain phospholipid structures in vivo.

31P-NMR has been used extensively for the study of cytosolic small molecule phosphates in vivo and phospholipid structures in vitro. We present in this paper a series of studies of the brain by 31P-NMR, both in vivo and in extracts, showing the information that can be derived about phospholipids. 31P-NMR spectra of mouse brain at 73 mHz are characterised by almost a complete absence of the large phosphodiester peak in comparison to equivalent spectra at 32 mHz. Proton decoupled spectra in vivo, and spectra of extracts, show that the phosphodiester peak observed in 32 mHz spectra in vivo is mainly due to phospholipid bilayers. Homogenates of quaking and control mouse brains, and of bovine grey matter, show another narrower phosphodiester peak possibly from small phospholipid vesicles. This peak is increased in intensity in the affected mice. These experiments demonstrate the presence of three major components contributing to the phosphodiester resonance: bilayer phospholipids, more mobile phospholipids, and the freely soluble cytosolic molecules glycerophosphocholine and glycerophosphoethanolamine. These NMR methods for non-invasive investigation of phospholipid structures in the brain might be extended to studies of patients with membrane involved diseases such as multiple sclerosis.     

31P NMR and freeze fracture studies of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pigs: effect of halothane and dantrolene.

The effects of halothane and dantrolene on sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia pig muscle have been investigated using 31P NMR and freeze fracture electron microscopy. The dynamical and structural changes are estimated from the second moment, as calculated from 31P NMR spectra. For both membranes, addition of halothane induces a similar decrease in the spectral second moment. At high concentration of halothane, freeze fracture replicas show small unilamellar vesicles or mixed micelles, uniformly sprayed in the case of malignant hyperthermia membranes but mainly aggregated for the normal ones. The effect of halothane on both membranes is partially inhibited by adding dantrolene. These results suggest that (i) the malignant hyperthermia syndrome is not directly related to the polar heads of phospholipids and (ii) dantrolene counteracts unspecifically the disturbing effect of halothane at the lipid level.     

Effect of acute streptozotocin diabetes on fatty acid content and composition in different lipid fractions of rat skeletal muscle.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on long chain fatty acid content and composition in different lipid classes of particular muscle types in the rat. Two days after streptozotocin administration, rats were anesthetised, and the white and red sections of the gastrocnemius, the soleus and the blood were taken. Lipids were extracted with chloroform/methanol and separated into different fractions (phospholipids, free fatty acids, di- and triacylglycerols) by means of thin layer chromatography. Fatty acids of each fraction were identified and quantified by means of gas-liquid chromatography. The diabetes resulted in elevation of the concentration of blood glucose (over four-fold) and the plasma free fatty acid (over two-fold). Total free fatty acid content in the muscles of diabetic rats increased by 26% in the white, 24% in the red gastrocnemius and 21% in the soleus. There were also changes in the composition of that fraction in each muscle. Diacylglycerol fatty acid content was elevated in both parts of the gastrocnemius (the white part by 15%, the red part by 44%) and remained stable in the soleus of the diabetic rats. The content of triacylglycerol fatty acids was elevated only in the red gastrocnemius in the diabetic group (by 112%), but changes in fatty acid composition in this fraction occurred in each muscle. The content of phospholipid fatty acids was elevated in the white gastrocnemius (by 13%) and remained stable in other muscles. There were only minor changes in phospholipid fatty acid composition in the diabetic rats. We concluded that acute insulin deficiency changes fatty acid content and composition in skeletal muscle lipids. The changes depend both on lipid fraction and muscle type.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Changes in fatty acid composition of phospholipids and triglycerides of Musca domestica resulting from choline deficiency

The fatty acid composition of the phospholipids and triglycerides extracted from housefly larvae reared on diets containing no added fatty acids but containing differing concentrations of choline has been determined. Reducing the choline content of the diet resulted in a graded reduction of the percentage of phosphatidylcholine present in the phospholipids of the larvae. This was accompanied by changes in the fatty acid composition, choline deficiency causing an increased utilization of 16-C rather than 18-C acids by the phospholipids. Changes in the fatty acid composition of the triglyceride fraction were also observed but these were associated with insects containing very low levels of phosphatidylcholine. Examination of the fatty acids in the different classes of phospholipids showed that the major change resulting from choline deficiency was in the fatty acids of the phosphatidylethanolamine fraction—the phospholipid which increased as the phosphatidylcholine decreased.Although the fatty acid composition of the different classes of phospholipids was not completely fixed, some preferential utilization of certain fatty acids by certain classes was observed, in both larval and adult insects. The fatty acid composition of the phospholipids extracted from larval gut, muscle, fat body, cuticle, trachea, nervous tissue, and haemolymph was determined. Changes resulting from choline deficiency similar to those seen in the whole larva were observed in all tissues except the nervous tissue. The effect of rearing larvae at temperatures between 24 and 35°C resulted in only minor changes in the fatty acid composition of both phospholipid and triglyceride fractions but the difference due to choline deficiency was observed at all temperatures. The possibility that the observed changes in the fatty acids of the phospholipids are compensatory to the changes in the proportion of the choline to the ethanolamine phospholipids is discussed.     

Muscle fiber number in biceps brachii in bodybuilders and control subjects

Muscle fiber numbers were estimated in vivo in biceps brachii in 5 elite male bodybuilders, 7 intermediate caliber bodybuilders, and 13 age-matched controls. Mean fiber area and collagen volume density were calculated from needle biopsies and muscle cross-sectional area by computerized tomographic scanning. Contralateral measurements in a subsample of seven subjects indicated the method for estimation of fiber numbers to have adequate reliability. There was a wide interindividual range for fiber numbers in biceps (172,085-418,884), but despite large differences in muscle size both bodybuilder groups possessed the same number of muscle fibers as the group of untrained controls. Although there was a high correlation between average cross-sectional fiber area and total muscle cross-sectional area within each group, many of the subjects with the largest muscles also tended to have a large number of fibers. Since there were equally well-trained subjects with fewer than normal fiber numbers, we interpret this finding to be due to genetic endowment rather than to training-induced hyperplasia. The proportion of muscle comprised of connective and other noncontractile tissue was the same for all subjects (approximately 13%), thus indicating greater absolute amounts of connective tissue in the trained subjects. We conclude that in humans, heavy resistance training directed toward achieving maximum size in skeletal muscle does not result in an increase in fiber numbers.