An enzymatic method for the assay of serum argininosuccinate lyase

A rapid enzymatic method was developed for the assay of serum argininosuccinate lyase (ASAL: EC 4.3.2.1) which is a useful marker enzyme for diagnosis of parenchymal liver diseases. Fumarate, liberated from argininosuccinate in the lyase-mediated reaction, was converted to pyruvate via L-malate by the actions of fumarase and malic enzyme in the presence of NADP+. The NADPH formed was then oxidized with a diaphorase-resazurin system to give a highly fluorescent resorufin. All the enzymatic reactions proceeded continuously in 0.1 M Tris-HCl buffer (pH 7.5) and allowed direct assay of ASAL in serum by monitoring the increase in the fluorescence intensity due to resorufin. The method is rapid and sensitive; only 50 microliter of serum is required. This method was used to detect increases in the activities in sera from patients with liver diseases.     

Production and standardization of Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus antigens to be used in immunodiagnosis. Comparison between immunodiffusion and immunoelectrophoresis

Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.     

Specific and sensitive enzymatic measurement of sphingomyelin in cultured cells

Sphingomyelin (SM) is the most abundant sphingolipid in mammalian cell membranes and plays multiple physiological roles. In this study, we improved the sensitivity of the enzymatic measurement of SM and validated its specificity and accuracy. The enzymatic reaction sequence of the method involves the hydrolysis of SM by sphingomyelinase, dephosphorylation of phosphorylcholine, oxidation of choline, and reaction of hydrogen peroxide with Amplex Red. The calibration curve was shown to be quadratic and linear at low (0-10μM) and high (10-100μM) concentrations, respectively, and the detection limit was 0.5μM (5pmol in the reaction mixture), which was more sensitive than all other SM assays reported previously. This SM measurement using Triton X-100 detected only SM, but not other choline-containing phospholipids, sphingosylphosphocholine, phosphatidylcholine, and lysophosphatidylcholine, and quantified SM regardless of the length and double bonds of the acyl chain. By using this method, we demonstrated that an increase in the density of HEK293 cells was accompanied by an elevation in the cellular content of SM, and that the treatment of HEK293 cells with tumor necrosis factor α significantly decreased the SM content. This specific and sensitive method for measuring SM will be helpful in studying various cellular processes.     

Serum carboxypeptidase B: A spectrophotometric assay using protamine as substrate

A quantitative colorimetric assay for serum carboxypeptidase B (SCPB, anaphylatoxin inactivator, kininase I) is described. SCPB is known to possess an enzymatic specificity for cleaving COOH-terminallysyl and arginyl residues which is similar to the specificity of bovine pancreatic carboxypeptidase B. One function of SCPB involves the inactivation of C3a and C5a, the two complement derived anaphylatoxins. Since cobalt markedly enhances the activity of the enzyme, serum is treated with CoCl₂ before the SCPB assay is performed. Salmine, a protamine from salmon sperm, was selected as the substrate because it contains multiple COOH-terminal arginyl residues and is digested more rapidly by SCPB than other common substrates of carboxypeptidase B, including hippuryl-arginine and benzyl-glycylarginine. The kinetics for arginine release from salmine were first-order throughout the course of the assay and the colorimetric values obtained were related to micromols of arginine released. A unit of SCPB is defined as one nanomol of arginine released per minute per milliliter of serum. The range of SCPB activity in serum from healthy individuals was found to be 318 to 466 units. The medians of SCPB activity in sera obtained from patients with Dengue shock syndrome and with shock following intravenous dextran infusion were both lower than the mean SCPB activity of healthy individuals. SCPB levels in patients homozygous and heterozygous for cystic fibrosis were within the normal range.     

Positive Fluorescent Treponemal Antibody Reactions in Diabetes

In a survey of 129 diabetic patients and 142 normal individuals, a significantly higher percentage of positive reactions in the fluorescent treponemal antibody-200 (FTA-200) test was found among diabetic patients than in the normal population. Absorption of all FTA-200-reactive sera with an extract of Reiter's treponeme eliminated most of the positive reactions in sera from diabetic patients, and three of the five positive reactions detected in sera from apparently normal subjects. On immunoelectrophoresis, precipitin bands developed most frequently between the Reiter sorbent and sera from diabetic patients positive in the FTA-200 test. Serum components responsible for FTA reactivity and precipitin reactions against the sorbent were resistant to treatment with mercaptoethanol, suggesting antibody of the IgG class. Cross-reacting antibodies produced in response to normal treponemal flora, and perhaps acquiring enhanced reactivity by means of nonspecific interacting substances in sera peculiar to the altered physiological state of diabetes, are suggested as possible causes of positive reactions of unabsorbed sera. No correlation could be made between age of the diabetic patient, treatment or duration of the disease, and FTA or precipitin reactivity of the patient's serum.     

Simplification and standardization of dot-ELISA for human schistosomiasis mansoni

Dot-ELISA, a technique that shares the same principles as the enzyme immunoassay, is useful for detection of anti-Schistosoma mansoni antibodies in the sera of patients with Schistosoma mansoni infections. The antigens were fixed to the nitrocellulose strips, blocked with 1% bovine serum albumin in 0.05% Tween 20. Patient sera (40) and normal laboratory personnel sera (9) were applied to the sheet directly, without cutting the strips into small discs. The nitrocellulose sheets are kept in a humid chamber for 30 min and then washed. After incubation with peroxidase-conjugated goat anti-human antibody, washing, and addition of substrate, positive reactions appear as brown dots against the white background. The room temperature assay takes about 2 hr. The optimum antigen concentration is 20-80 ng per dot and the optimum serum dilution is 1:100-1:400. The sensitivity and specificity of the assay are 90-95% and 90%, respectively. The level of positivity of the dot-ELISA by an arbitrary scale compares with standard micro-ELISA. The single positive reaction in a normal serum sample in dot-ELISA is also positive in micro-ELISA. Cross-reactivity between the S. mansoni antigen and human fascioliasis sera was noticed in 2 out of 8 patient sera. Good correlation between the arbitrary level of dot-ELISA and the absorbance of standardized micro-ELISA shows that the dot-ELISA is useful both for laboratory and field studies.     

Immunodiagnosis of human trichinellosis using excretory-secretory (ES) antigen.

Infective first stage larvae of Trichinella spiralis were recovered from muscles of laboratory infected mice by digesting the muscles with 1% HC1-1% pepsin and collecting the larvae by modified Baerman's method. The larvae were cultivated in a serum-free medium for 18 h. The ES antigen obtained from the culture medium was used in an enzyme-linked immunosorbent assay (ELISA) for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 100%. The sensitivity of the test was also 100% when performed on sera of group 1 collected at days 57 and 120 after infection. Sera collected earlier (day 23) and those collected 700 days after infection had negligible reactivity. Thus IgG-ELISA using ES antigen of the L1 was useful not only for diagnosis but also in evaluation of cure. Western blot analysis revealed that specific antigens of T. spiralis were 94, 67, 63, and 39 kilodalton components.     

Application of 31P MRS to the analysis of phospholipid changes in plasma of patients with acute leukemia

The aim of the experiment was to evaluate the changes of phospholipid concentrations in patients (n=30) with acute leukemia compared with reference group of healthy volunteers (n=21). The analysis focused on the following phospholipids (PL) collected from plasma: phosphatidylcholine (PC), plasmalogen of phosphatidylcholine (CPLAS), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Phospholipid extracts were obtained by Folch's method from 4 ml of plasma. 31P MR spectra were obtained on an AMX Bruker 300 MHz (7.05 T) spectrometer. Calculation of concentration based on integral intensity of the phospholipid relative to an internal concentration standard of MDPA. For healthy volunteers, the following values of phospholipid concentrations were obtained: (5.18+/-1.615) mmol/l for PC+CPLAS; (0.364+/-0.178) mmol/l for LPC; (1.211+/-0.411) mmol/l for SM; (0.343+/-0.124) mmol/l for PI+PE. PLs of patients were assayed at least twice: at the time of diagnosis and, when appropriate, at the time of complete remission from the disease (CR). At the time of diagnosis, the mean concentrations of studied compounds were: (1.602+/-0.716) mmol/l for PC+CPLAS; (0.041+/-0.048) mmol/l for LPC; (0.398+/-0.198) mmol/l for SM; (0.045+/-0.071) mmol/l for PI+PE. After attainment of complete remission (CR), the respective values were as follows: (4.094+/-1.886) mmol/l for PC+CPLAS; (0.295+/-0.139) mmol/l for LPC; (1.123+/-0.634) mmol/l for SM; (0.230+/-0.125) mmol/l for PI+PE. All concentrations found in patients at the time of diagnosis were significantly lower than in reference group and in those benefited from complete remission (CR). By contrast the differences in concentrations of phospholipids in plasma between patients with complete remission (CR) and healthy volunteers were no statistically significant.     

Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.     

Use of the dot-immunogold assay for the rapid diagnosis of acute enteric infections

A simple method based on an immunodot assay using colloidal gold labels is proposed for the rapid diagnosis of a range of acute enteric infections. Owing to its rapidity, high sensitivity, and specificity, the method can be recommended for routine use in the laboratory diagnosis of enteric infections.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

Phospholipase C purification and specificity with respect to individual phospholipids and brain microsomal membrane phospholipids

Phospholipase C was purified from a crude preparation derived from Cl. perfringens utilizing a one-step polypreparative electrophoresis procedure. The purified enzyme has a molecular weight of 46,500 ± 500 and is essentially free of proteolytic and phospholipase A enzymatic activities. It exhibited the following substrate specificity: PC ≥ SM > PS > PI, lyso PC. PE was hydrolyzed when PC was present.Treatment of brain microsomes with purified phospholipase C reduced membrane phospholipids by 69%. All phospholipids were attacked including PE. PC was reduced to 4% and all other phospholipids to 23–43% of their control levels. Total fatty acid composition of brain microsomes was not affected by phospholipase C action.     

Effects of fat ingestion on high density lipoprotein profiles in human sera

Serum concentrations of triacylglycerol, apolipoprotein A-I, apolipoprotein A-II, HDL2, and HDL3 were determined in sera of nine normolipidemic adult males, just before and 3, 5, and 8 hr after ingestion of 250 ml of cream (100 g of triacylglycerol). In all individuals a rapid hypertriglyceridemic response was observed. Triacylglycerol concentrations increased from 624 +/- 124 mg/liter of serum to 1435 +/- 350 mg/liter of serum 3 hr after cream ingestion. In most individuals the hypertriglyceridemic response was followed by a decline in serum triacylglycerol concentration to below basic levels. As a result of cream ingestion, small but statistically highly significant increases in serum cholesterol and apolipoprotein A-I concentrations were observed that persisted till the end of the observation period. In most individuals a small rise in the apolipoprotein A-II concentration in serum was also present. Marked changes were observed in serum HDL as illustrated in the HDL absorption at 280 nm and cholesterol profiles obtained by single-spin rate-zonal density gradient ultracentrifugation of the sera. Due to a prominent increase in phospholipids (up to about 18%) and a smaller increase in protein (up to about 6%), flotation rates and concentrations of HDL2 as well as HDL3 increased. These changes in HDL subclass flotation characteristics and chemical composition are best explained by uptake of surface material from chylomicrons by existing HDL2 and HDL3 particles. The data do not support a previously proposed concept in which HDL3 is converted into HDL2 by uptake of surface remnants formed during catabolism of triglyceride-rich lipoproteins.     

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.     

Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine

Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PC-specific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 microg, and that for PC was 2.5-20 microg. The mean percentage recovery was 98.0 +/- 5.2% for SM and 96.6 +/- 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 +/- 0.05% for SM and 3.1 +/- 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.     

Ion-pair high-performance liquid chromatographic assay of 4-aminopyridine in serum

An assay for the quantitative estimation of 4-aminopyridine in biological fluids has been developed using 2-aminopyridine as internal standard and ion-pair reversed-phase (C_1$$$) high-performance liquid chromatography with detection at 263 nm. A 7.5% solution of acetonitrile in water containing tetrabutylammonium iodide and sodium heptanesulfonate buffered at pH 3.0 provided excellent separation of the analytes from each other and from an interfering peak that was occasionally observed in the outdated human sera used in these studies. Sensitivity, specificity, precision, accuracy and reproducibility all were judged sufficient for the routine use of this assay for pharmacokinetic and pharmacodynamic studies.     

The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin

Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.     

Dual method for the determination of peroxidase activity and total peroxides-iodide leads to a significant increase of peroxidase activity in human sera

Peroxidases are very important enzymes, e.g., as preventive antioxidants by removing noxious peroxides from the blood. For this reason we evaluated a colorimetric method which detects the activity of endogenous peroxidases by their reaction with hydrogen peroxide, using tetramethylbenzidine as the chromogenic substrate. This assay design can be easily reversed by change of the variable compound to measure also total peroxides in plasma or serum. An increased total antioxidant status was reported previously by the addition of iodide to human serum. In this study iodide activated the endogenous peroxidases significantly in comparison to control sera and isomolar NaCl as well as horseradish peroxidase. Corresponding to the increased peroxidase activity a concomitant decrease of total peroxides occurred in the same samples. This exchangeable assay design is a beneficial opportunity to screen total peroxide levels as well as peroxidase activity in human sera without time-consuming preparations. The method proved to be simple and is favorable due to its specificity, reproducibility, and low costs. Moreover, we were able to find an explanation for the increased total antioxidant status in the presence of iodide, which is presumably an indirect protective effect via an enhanced activity of enzymatic antioxidants, thereby reducing endogenous peroxides.     

Detection and titration of antitetanus antibodies using an automated passive hemagglutination method]

An automated reversed passive haemagglutination technique is described, using human erythrocytes sensitized by the chromic chloride method with higly purified tetanus toxoid. Such erythrocytes are agglutinated by the specific antibodies in a Technicon autoanalyzer. Clots are decanted, supernatant is hemolyzed and optical density is measured at 550 millimicron. Assay standards for comparison, ranging from 5 to 25 UI/ml, are prepared from human antitetanus immunoglobulins titrated by toxin neutralisation assay in mice. This method allows to screen donors' sera for presence of high titers of tetanus antibodies (larger than or equal to 5 UI) suitable for preparation of antitetanus immunoglobulins. 4,4% of donors have circulating antitetanus antibody levels corresponding to 5 UI/ml or more, by this method. The specificity of antibodies has been confirmed by the neutralisation assay in mice. The results obtained among 1.000 donors well agree with those of the counter-immuno-electrophoresis technique (C.E.P.). But, when, compared with C.E.P., the haemagglutination assay appears more objective, more quantitative and sensitive, and allows to get not only a rapid screening test but also a precise titration simultaneously.     

Role of lipids in the assembly of endoplasmic reticulum and dictyosomes in neuronal cells from the cerebral cortex of Yakutian ground squirrel (Citellus undulatus) during hibernation

Phospholipids and cholesterol were assayed in homogenates and microsomal fractions from the cerebral cortex of summer-active, winter-torpid, and winter-active Yakutian ground squirrels (Citellus undulatus). Ultrastructural analysis of both microsomal fraction and intact neurons was performed by serial ultramicrotomy. The levels of sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) were decreased in homogenates from the cerebral cortex of winter ground squirrels compared with the summer-active animals, while the levels of phosphatidylcholine (PC) and cardiolipin (CL) were increased. The level of cholesterol was decreased in the cerebral cortex of winter-torpid animals compared with both winter-active and summer-active animals, and the level of total phospholipids was decreased in comparison to the summer-active animals. Three-dimensional reconstruction of serial membrane profiles displayed the microsomal fraction to be an interconnected system of cisterns and vesicles, which corresponds to endoplasmic reticulum and dictyosomes (Golgi stacks) of intact neurons. In winter the content of PC was increased in the microsomal fraction, while the contents of lysophosphatidylcholine (LPC), PS, phosphatidylinositol (PI), and SM were decreased. In winter-torpid animals compared with the winter-active ones the contents of total phospholipids, PEA, LPC, and cholesterol were decreased. As for the winter-active ground squirrels, their lipid contents did not differ from those in the summer-active animals, but LPC content was decreased. The changes in microsomal lipid contents in intact pyramidal neurons throughout the hibernation were accompanied by disassembly of dictyosomes and endoplasmic reticulum (ER), including the decomposition of polyribosomes to monosomes. The ultrastructural analysis of nucleoli, ER, and dictyosomes of both winter-active and torpid ground squirrels showed a direct correlation between the increasing contents of both cholesterol and total phospholipids (mainly PEA and LPC) in microsomes and the structural recovery of endoplasmic reticulum, Golgi stacks, and nucleoli in intact pyramidal neurons. A role of seasonal variations in lipid contents of brain cells in their adaptation to low temperature is discussed. We also propose an involvement of cholesterol in the activation of protein-synthesizing function of endoplasmic reticulum and Golgi stacks in intact neurons.