Cytotoxicity of docosahexaenoic acid and eicosapentaenoic acid in tumor cells and the dependence on binding to serum proteins and incorporation into intracellular lipids.

Docosahexaenoic acid (DHA) inhibited the DNA synthesis in Ehrlich ascites tumor cells more markedly than eicosapentaenoic acid (EPA), which was more inhibitory than oleic-, linoleic-, linolenic-, and arachidonic acids. Their activities augmented according to the increase of number of double bonds in the molecule. To correlate the cytotoxicity with lipid syntheses in the cells, distribution of EPA and DHA incorporated into cellular lipids was assessed. EPA was incorporated into triglycerides (TG) and DHA into phosphatidylcholine (PC). These synthesis into TG and PC etc., which shattered from cytotoxicity, may be involved in tumor-cellular protecting actions against EPA or DHA. EPA and DHA involved in cytotoxicity exhibition are their free acid forms. Thus, as an anticancer reaction, intracellular accumulation in the free acid form of DHA was more marked than that of EPA.

Abnormal cell morphology and cytotoxic effect are induced by 6-0-palmitol-ascorbate-2-0-phosphate, but not by ascorbic acid or hyperthermia alone.

A new derivative of ascorbic acid (AsA), 6-0-palmitoyl-ascorbate 2-0-phosphate (Asc2P6Pal) was developed to enhance the antitumor activity of AsA. When Ehrlich ascites tumor cells were treated with 50 microM Asc2P6Pal at 37 degrees C or 42 degrees C for 1 h and then cultured for 20 h, most of cells exhibited some morphological abnormalities, including exudation of intracellular granules together with other contents on the cell membrane surface, resulting in cell fragmentation. The abnormal features were further enhanced by a long term culture for 96 h and heat treatment at 42 degrees C. In contrast, no abnormality was detected for untreated cells or cells treated with AsA (free acid) at 37 degrees C or 42 degrees C. Cells cultured for 96 h after the treatment suffered from inhibition of DNA synthesis and proliferation. This inhibition was markedly enhanced by combination with the hyperthermic treatment at 42 degrees C, but not for a short-term culture of 20 h after the treatment. No effects were seen upon similar treatment with AsA. The abnormal cells produced during culture for 20 h after the treatment were evaluated to be viable, because they failed to be stained with trypan blue and retained most of the DNA synthesizing ability of Asc2P6Pal-untreated cells. However, they appeared and died after a continuous 76 h of culture (96 h).

Studies on in vitro effect of free fatty acids on water content and osmotic fragility of rabbit (Lepus cuniculus) erythrocytes.

The effects of free fatty acids in the blood on osmotic fragility and water content of erythrocytes when combined with hyperthermia were investigated. The isotonic buffer, containing the fatty acid, was added to the erythrocyte suspension to a final concentration of 200 microM/L. The samples were kept for one hour in an incubator at 37 degrees C or 42 degrees C. The osmotic fragility of erythrocytes was determined by coil planet centrifuge system and intracellular water content was measured by gas-liquid chromatography. A high concentration of unsaturated fatty acid and hyperthermia caused the increase in intracellular water and the decrease in osmotic resistance of the red blood cells compared to saturated fatty acid and the control. The present experiment demonstrated that unsaturated fatty acid was one of the principal chemical and metabolic factors which caused sports anemia.

[Osmotic fragility test of erythrocytes with a coil planet centrifuge]. / Mesure de la fragilité osmotique des érythrocytes avec une centrifugeuse planétaire à serpentins.

The measurement of osmotic fragility of erythrocytes has been applied to the diagnosis of hemolytic diseases, studies of membrane permeability and alternations leading to destruction of erythrocytes. Almost 30 years have gone by since the coil planet centrifuge system was devised for measuring the osmotic fragility of erythrocytes. Many excellent studies by means of this centrifuge system have been published. Prominent investigations are reviewed as follows: in relation to the osmotic fragility, various liver diseases, angina pectoris, trapping the aged erythrocytes, tumor, lactic acid, unsaturated fatty acids, free cholesterol, exercise and lead exposure level were examined.

Effect of lactic acid on water content and osmotic fragility of erythrocytes in vivo.

OBJECTIVE: A coll planet centrifuge is an apparatus for the dynamic measurement of erythrocyte osmotic fragility, and it was applied to the observation of altered erythrocyte osmotic fragility induced by the lactic acid. Changes in intracellular water content of red cells were also measured according to the method, based on gas-liquid chromatography. EXPERIMENTAL DESIGN: Blood was withdrawn from the animal by cardiac puncture before and after lactic acid injection. The lactic acid was injected to rabbit through the auricular vein till the final concentration for the circulating blood was 0.7 mg/ml. RESULTS: The water content in the red cells before the lactic acid injection was between 71.37% and 73.33%. It increased after the lactic acid injection and reached the maximum of 102% of the original content. Hemolysis of erythrocytes before the injection of lactic acid began at 108.3 to 110.3 mOsm and ended at 77.0 to 81.0 mOsm, and after the injection it began at 117.0 to 120.7 mOsm and ended at 84.5 to 87.3 mOsm. CONCLUSIONS: In the present experiment the decrease in pH of the blood in lactic acid administered rabbits was too small to cause any changes in the osmotic fragility, suggesting that changes of the erythrocyte membrane properties, owing to the presence of lactic acid in the blood had occurred.

[In vitro effects of free fatty acids on water content and osmotic fragility of erythrocytes in rabbits]. / Effets in vitro d'acides gras libres sur la teneur en eau et la fragilité osmotique d'érythrocytes de lapin.

The effects of free fatty acids in the blood on osmotic fragility and water content of erythrocytes when combined with hyperthermia were investigated. The isotonic buffer, containing the fatty acid, was added to the erythrocyte suspension to a final concentration of 200 microM/l. The samples were kept for one hour in an incubator at 37 or 42 degrees C. The osmotic fragility of erythrocytes was determined by Coil Planet Centrifuge System and intracellular water content was measured by gas-liquid chromatography. A high concentration of linoleic acid and hyperthermia caused the increase in intracellular water and the decrease in osmotic resistance of the red blood cells. The present experiment demonstrated that unsaturated fatty acid was one of the principal chemical and metabolic factors which cause sports anemia.

Application of high-performance liquid chromatography/mass spectrometry to drug screening.

High-performance liquid chromatography/mass spectrometry enabled us not only to measure bromvalerylurea rapidly, < or = 2 h following accumulation of human bone marrow, but also to improve sensitivity without interference. The method can be widely and effectively used in the fields of pharmacy and forensic sciences.

Effect of lactic acid on water content and osmotic fragility of erythrocytes in vitro.

Effects of lactic acid in red blood cells on osmotic fragility and water content of erythrocytes after hyperthermia were investigated. The osmotic fragility of erythrocytes increased following one-hour incubation with the addition of lactic acid at both 37 degrees C and 42 degrees C and that also increased after heating in vitro at 42 degrees C compared with those incubated at 37 degrees C, whether the lactic acid was added or not. The water content increased with the addition of lactic acid after heating in vitro at 42 degrees C. A high concentration of lactic acid and hyperthermia seem to cause the increase of intracellular water and the decrease of osmotic resistance of the red blood cells.

DNA synthesis inhibition and transmembrane permeation into tumor cells by various dialkyl phthalates upon hyperthermia.

Diisobutyl-, dibutyl-, and dioctyl phthalates, at a concentration of 50 microM, markedly inhibited the incorporation of [3H]thymidine into DNA in tumor cells and also cell proliferation. The inhibitions were synergistically enhanced by hyperthermia at 42 degrees C for 1 h. In contrast, dimethyl-, and diethyl phthalates induced almost no inhibition. The effective phthalates were detected in the cells by gas-liquid chromatography, whereas the non-effective drugs were not or were little detected, suggesting involvement of permeability through cell membrane in cytotoxic effects. Combination of the cytotoxic phthalic acid esters and hyperthermia is suggested to be an advantageous treatment for cancer.

Rapid and simple gas chromatographic measurement of lactic acid in red blood cells, plasma, and tumor cells after hyperthermia.

The content of lactic acid in red blood cells, plasma, and Ehrlich ascites tumor cells were measured by a gas-liquid chromatography using a column with a terephtalic acid support coated with polyethylene glycol-6000. The lactic acid contents were directly determined in aqueous samples, because they were converted to a volatile derivative in the column. The method was rapid and simple, compared with previous methods which need time-consuming conversion of lactic acid to volatile derivatives. Our measurements showed the increase in the contents of intra- and extracellular lactic acid after hyperthermia.

The maximum and minimum water content and cell volume of human erythrocytes in vitro.

The maximum and minimum water contents of human erythrocytes were measured after exposure to various osmotic pressures. Within a range of osmolarities, at which no haemolysis occurred, the water content reached its maximum, 78.1%, at 180 mosM and its minimum, 54.8%, at 800 mosM. Simultaneously, the mean cell volume increased to 98.5 microns 3 at 180 mosM and decreased to 77.2 microns 3 at 800 mosM.