Administration of chloroquine or 4, 4'-bis (diethylaminoethoxy) α, β-diethyldiphenylethane (DH) to rats in oral doses of 100mg/kg for 7 days causes phospholipid and cholesteryl ester accumulation in liver. We have isolated and characterized the lipids of subcellular fractions from control rats and rats treated with chloroquine, DH, and Triton WR-1339. The phospholipid content of liver is increased 1.5-fold by chloroquine or DH treatment but is unaffected by Triton WR-1339. Chloroquine and DH cause a shift of acid phosphatase from the light mitochondrial fraction (L) to the heavy mitochondrial fraction (M). Multilamellar bodies, an ultrastructural hallmark of chloroquine and DH-induced lipidosis, were isolated in a highly-purified form from the M fraction of chloroquine-or DH-treated rats. They are highly enriched in acid phosphatase indicating their lysosomal origin. In addition, they contain large amounts of phospholipid, cholesterol, and cholesteryl ester and are the sole site of bis (monoacylglycero) phosphate and the enzyme which catalyzes its synthesis from phosphatidylglycerol. Analysis of the phospholipid content shows that the entire excess phospholipid content of chloroquine-or DH-treated liver can be accounted for by the drug-induced multilamellar bodies. Triton WR-1339-induced lysosomes, which were isolated for comparison, also contain bis (monoacylglycero) phosphate and bis (monoacylglycero) phosphate synthetase. However, they differ from the drug-induced lysosomes in that their sphingomyelin content is much higher and their total phospholipid and phosphatidylinositol content much lower. The multilamellar bodies are the vrincinal intracellular site of accumulation of chloroquine and DH, respectively. Increased delivery of phospholipid to lysosomes and decreased lvsosomal catabolism of phospholipid are the factors which are thought to cause this experimental lipidosis. High levels of phosphatidylinositol in the multilamellar body may be in part responsible for the increased content of bis (monoacylglycero) phosphate since it has been identified as an acyl donor in bis (monoacylglycero) phosphate synthesis.

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When leupeptin, athiolproteaseinhibitorofmicrobialorigin, wasinjectedintorats, the activity of fructose-1, 6-biphosphatase [D-fructose-1, 6-biphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1. 2.13] in the liver decreased to about 70% of that in control rats. Two types of aldolase molecules isolated from the livers of control and leupeptin-treated rats, respectively, were not distinguishable on the basis of their electrophoretic mobilities, molecular weights, immunological properties or behaviours on affinity chromatography. But the specific activity of purified enzyme from livers of leupeptin-treated rats was lower than that from livers of control rat. Amino-terminal analysis of two types of aldolase indicated that decrease of aldolase activity in the liver of leupeptin treated rats is attributable to limited hydrolysis of a peptide linkage near the carboxyterminal of the peptide chain. Injection of leupeptin also caused marked increase in the activities of free lysosomal proteases, such as cathepsin A, cathepsin B, cathepsin L and cathepsin D in the cytosol fraction. After injection of leupeptin was stopped, the decreased aldolase activity and the increased cathepsin B activity in the cytosol fraction returned in parallel to the levels in control rats. A clear inverse relationship between aldolase and cathepsin B activities in the cytosol fraction was demonstrated. When insulin, which is known to stabilize the lysosomal membrane, was injected to rats simultaneously with leupeptin, both increase in free activity of cathepsin L and decrease in aldolase activity were prevented. These findings indicate that modification of aldolase may be due to action of a lysosomal protease (s). The possibility that the decrease of liver aldolase activity on administration of leupeptin to rats was produced during homogenization was excluded by showing that the aldolase activity was not changed by addition of various protease inhibitors to the homogenization medium. Enhanced sensitivity of lysosomes to osmotic shock was demonstrated in the livers of leupeptin-treated rats, suggesting that the lysosomal membrane is labilized by administration of leupeptin. This paper provides a model system to evaluate the stability of lysosomal membrane and proteolytic modification of cytosol enzymes by a released lysosomal protease (s) to the cytosol fraction.

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The relationship between morphology and lipid composition of red blood cells in patients with hepatobiliary disease has been clearly demonstrated by several investigators. Little information is, however, available regarding the mechanism by which erythrocyte membrane abnormalities are induced. In order to clarify such mechanism, biochemical, ultrastructural and physiochemical studies of red blood cell membranes from patients with biliary obstruction and congenital biliary atresia have been carried out.
Scanning electron microscope observation revealed that red blood cells from patients with biliary atresia classified as Group I (red cell lecithin≥41%) are mostly of the “target” type. On the other hand, red blood cells from Group III (red cell lecithin<31%) are almost normal biconcave disks.
Phospholipids and free cholesterol were increased strikingly in red blood cells from Group I, whereas no significant difference was observed between Group III and control. The abnormalities in the lipid profile as well as morphology are dependent upon the degree of biliary obstruction. It is obvious that increase in the phospholipid content is principally due to an increment of lecithin. The analysis of the fatty acid composition of lecithin revealed a similar pattern in red blood cell membranes, plasma and bile. It is of particular interest that the lecithin fatty acid composition of lipoprotein-X (LP-X) was found to be quite similar to that of erythrocyte membranes. On the other hand, in spite of little change in the lipid content of erythrocyte membranes, the lecithin fatty acid composition of erythrocyte membranes and plasma in Group III showed a similar but smaller change compared to that of patients in Group I.
Freeze-fracture electron microscopy showed significant alterations in Group I membranes, such as depressions on the fracture faces and reduced density of membrane particles (3,584→2,432/μm²) on the protoplasmic face.
Electron spin resonance studies on erythrocyte membranes labeled with 5-nitroxide stearate demonstrated that the erythrocyte membranes from Group I are more fluid than the control membranes.
Consequently, two working hypotheses were proposed by which excess membrane may be incorporated into erythrocyte membranes; fusion of LP-X with erythrocyte membranes (Group I) and exchange of lipids between erythrocyte membranes and plasma lipoproteins (Group III).
Red blood cells from patients with congenital biliary atresia exhibited a wide variation in the morphology, such as target, spur and cup formed cells. By freeze-fracture electron microscopy, membrane particle-free areas were prominent in the fracture faces and the density of membrane particles were 20% lower than those of control subjects.
Both free cholesterol and phospholipids were found to be increased in erythrocyte membranes from patients with congenital biliary atresia. In addition, the fatty acid composition of erythrocyte lecithin was similar to that of acquired biliary obstruction.
Fluorescence polarization studies by using fluorescent probe, 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) on red blood cells demonstrated that erythrocyte membrane lipids from patient are less fluid than those of control subject.
From the similarity of lipid alterations to those observed with acquired biliary obstruction and the fact that LP-X was found in all patients examined, it is reasonable to consider that LP-X might be involved in causing erythrocyte membrane abnormalities in congenital biliary atresia.

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Cytoplasmic enzymes from red blood cells are frequently assayed in clinical studies. but the use of membranes is rare. The latter are composed mainly of insoluble proteins, are rich in lipid, and are easily broken. The three ATPase,(Mg),(Na+K+Mg),(Ca+Mg) ATPase are incorporated into the membrane where they related to other components, and constitute a part of the membrane. Usually the active center of these ATPase are masked and show little activity. Depending on the treatment used, the total detectable ATPase activity is variable. Studies of the (Na+K+Mg) ATPase of the red blood cells in muscular dystrophy for example, have produced variable results.
In order to detect the high activities of the ATPase, freezing-thawing treatment was here used. With acetylcholine esterase which is said to be on the outer side of red blood cell membrane and aldolase which is said to be on the inner side, no increase of the activities was found after the treatments. However, the effect of freezing-thawing on the distribution of two membranous enzymes varied. The differences between patients with Duchenne type muscular dystrophy and normals on these activities and distribution of two enzymes could not be caught.
The ATP hydrolysed by the hemolysate appeared to be due to the membranous ATPase because hemolysate and washed membranes showed a similar cation activation profile of ATPase activities. The activities of the hemolysate ATPase from patients with Duchenne type and their mothers were lower than that from their fathers, although the values of their mothers were variable. After the cytoplasma was added to the membrane, the activities of the membranous ATPase from patients showed a smaller increase than that from their mothers.
As a result of it, the red blood cell from patient seems to wear a disorder-a membrane disorder.

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Pathogenesis of Duchenne muscular dystrophy (DMD) has recently been attributed to a plasma membrane defect of muscle. Several studies on erythrocyte membranes from the patients suggest that DMD might be a generalized membrane disorder. However, other reports on the erythrocytes are against this hypothesis.
This investigation was made in an attempt to clarify whether DMD is a systemic membrane disease or not, analysing the adenylate cyclase activities (AC) and phosphatidyl inositol metabolism of erythrocyte membranes.
1) Basal AC of erythrocyte membranes from mothers of DMD were higher than controls (p<0.01), but no significant correlation was obtained among the basal AC, serum creatine kinase activities and carrier states.
2) ³H-inositol was incorporated mainly into diphosphoinositide in human erythrocyte membranes. Its incorporation was markedly inhibited by Ca⁺⁺and facilitated by washing the membranes in the presence of EDTA.
3) No difference was observed in the ³H-inositol incorporation between DMD and controls in the EDTA lysed membranes.
4) Neither AC were different between DMD and controls in the EDTA lysed membranes.
Conditions for the preparation of erythrocyte membranes and age matched study are emphasized to be critical and important.
It was not concluded that DMD might be a systemic membrane disorder from the above data. Possible factors influencing the membrane enzymes were also discussed.

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The erythrocytes of children patients infected with Mycoplasma pneumoniae are very hemolytic and abnormally shaped. It appears that the toxic substances of M. pneumoniae or M. pneumoniae itself effected on the erythrocyte membrane. The activity of Ca++ -ATPases and the contents of total sialic acid of the erythrocyte membrane (ghost) from patients were decreased by about 50%. And also the level of intracellular ATP was declined. In accordance with the recovery from illness, the activity of Ca++ -ATPase, the content of sialic acid and the level of ATP were increased to the level of the control. Whereas in the cases of mixed infection with Staphyro coccus aureus, Streptococcus pyogenes and RS virus, the activity of Ca++ -ATPases and the contents of total sialic acid had been recovered to the level of the control within a week or so, the ATP contents were less than that of the erythrocytes from patients infected with M. pneumoniae alone.
There must be different effects of their toxic substances between M. pneumoniae, bacteria and virus on the erythrocyte membrane.

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We reported previously that the maximal binding sites of insulin receptor on human erythrocyte are significantly increased by diabetes. In the present study, we investigated the possibility that insulin binding may be regulated through the alteration of membrane phosphorylation. The mechanism regulating interaction of insulin with receptors was evaluated by studying the binding of insulin to intact and ATP-phosphorylated membranes and the characteristics of the binding sites for cAMP and cGMP on membrane. Specific insulin binding to membranes reached a maximum after a 2-hr incubation at 0°. Binding sites with relatively high affmity (K₁: 1.2×10⁹M-¹) could be distinguished from those of lower affinity (K₂: 4.2×106M^-1). The phosphorylation of membranes with 1 mM ATP plus 20μM cAMP caused a significant increase in the binding of insulin to lower capacity sites with high affinity (117%), while the affinity constants were unaffected by phosphorylation of membranes. GTP plus cAMP produced an increased effect on specific binding of insulin. The amount of phosphorylation with ATP was estimated as 1.2μmol/g protein/hr and that with GTP as 0.6μmol/g protein/hr. Binding sites for both cAMP and cGMP were composed of more than two types of receptors with different affinity constants. Secondly, we compared kinetic constants of cAMP binding between normal and diabetic subjects. The high affinity constant and the maximal capacity of high affinity binding sites were 4.85±0.66 (×10⁷M^-1) and 11.83±1.39 (pmol/mg protein)(n=33) in normal group and 3.50±037(×10⁷M^-1) and 17.84±1.64 (pmol/mg protein)(n=52) in diabetic group. These results have led us to a conclusion that human erythrocytes contain specific receptors for insulin, cAMP and cGMP, and suggested that the increase of insulin binding in diabetes may be associated with more highly phosphorylated membrane.

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A new variant of glycogen storage disease Type I is described. The clinical symptoms and laboratory findings were consistent those of glucose-6-phosphatase deficiency. The activity of glucose-6-phosphate phosphohydrolase was assayed in a liver specimen biopsied from a patient according to Nordlie and Arion's method. A markedly low activity was found in the absence of detergent but normal activity was obtained by the addition of detergent. These findings suggest a defect in the glucose-6-phosphate transport system in the microsomal membrane of the patient's liver. This may be the first example of a disorder involving the transport system of an intracellular membrane.

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The term “Fanconi's syndrome” describes a disturbance of proximal renal tubular function of multiple causation and comprising generalized hyperaminoaciduria, renal glucosuria, and hyperphosphaturia, as well as renal loss of potassium, bicarbonate and water, and other substances conserved by the proximal tubule. Ultrafiltered solutes as glucose, amino acids, phosphate and low molecular weight proteins are reabsorbed at proximal renal tubule through brush border membrane. It has been suggested that Fanconi's synd-rome is caused by the abnormality of transport carriers of renal brush border membrane. A reversible Fanconi's syndrome often occurs after ingestion of outdated tetracycline. This investigation aimed whether or not renal brush border membrane in Fanconi's syndrome induced by 4-epi-tetracycline (epi-TC) was damaged.
Male albino rabbits, weighing about 2kg, received an intravenous injection of epi-TC (16mg/kg or 100mg/kg). One hour after the injection, renal brush border membrane was isolated by the continuous sucrose density gradient centrifugation method of Sacktor et al. The activities of γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (AlP), trehalase and Na-K ATPase were measured. AlP of brush border membrane was investigated histochemically under electron microscope. SDS disc electrophoresis of brush border membrane proteins was performed.
In control rabbits, the bulk of γ-GTP, AlP and trehalase activities immigrated to higher density fractions by the centrifugation than that of Na-K ATPase activity. On the other hand, in rabbits treated with 16mg/kg of epi-TC, the bulk of γ-GTP, AlP and trehalase activities was in lower density fractions than that of Na-K ATPase activity. In rabbits treated with 100mg/kg of epi-TC, the great bulk of γ-GTP, AlP and trehalase was in further lower density fractions. These data suggest that brush border membrane immigrated to low density fractions by the centrifugation in proportion to the dose of epi-TC.
Electron micrographically, brush border membrane isolated from control rabbits was closed vesicle of membrane, whereas brush border membrane isolated from rabbits treated with 100mg/kg of epi-TC was open sheet of membrane.
An electrophoretic pattern of brush border membrane proteins of rabbits treated with 100mg/kg of epi-TC differed from that of control rabbits. In experiment, the protein whose molecular weight was about 40,000 daltons was observed, not in control.
We interpret these results as follows: One of proteins of brush border membrane was degradated by the injection of epi-TC and separated to subunits whose molecular weight was about 40,000 daltons. The degradation of this protein made brush border membrane fragile and the fragment of brush border membrane derived by the homogenizing operation became to the open sheet of membrane. The sedimentation constant would be decreased by this morphological changes of the fragment.
Consequently, we detected the biochemical and morphological damages of renal brush border membrane in Fanconi's syndrome induced by epi-TC.

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Marked alteration of neutral amino acid levels in the circulating blood was observed in patients with severe liver diseases. Km value and predicted velocity (Vpre) of a specific carriermediated transport system of neutral amino acids across the blood-brain barrier (BBB)(Pardridge; Neurochem., 28, 103, 1977) would be changed in these cases. Greater extent of aminogram abnormality in cerebro-spiral fluid (CSF) as compared to that in the serum indicates that the accerelated transport through BBB may be involved in pathogenesis of hepatic encephalopathy. We tried to perform a quantitative analysis of BBB amino acid transports from a ratio ([CSF]/Vpre) of the measured concentrations in CSF ([CSF]) to calculated Vpre values. An availability of the theoretical application for infering a close participation of abnormal BBB transport of amino acids to hepatic encephalopathy is also evaluated.
Five cases with fulminant hepatitis, 8 with liver cirrhosis without encephalopathy and 7 with encephalopathy were adapted as clinical materials for this study. Samplings of their CSF and sera for amino acid analysis were carried out simultaneously.
Aromatic amino acids (AAA) and methionine increased to a greater extent in CSF of encephalopathic patients, while branched chain amino acids (BCAA) in CSF remained unchanged even with their marked decreases of the serum levels. Ratios of CSF levels to serum concentrations of amino acids ([CSF]/[Serum]) elevated significantly in neutral amino acids such as AAA, methionine and BCAA in comatous cases. Furthermore, a direct correlation between [CSF]. _tyrosine and [Serum] _tyrosinewere not confirmed in encephalopathic patients in contrast to subjects with _tyrosine no neurological abnormality. Total concentrations of three BCAAs and ratios of [CSF] _{phenylalanine}/ [Serum] _{phenylalanine} were, however, well correlated inversely each other, suggesting that serum BCAA can regulate AAA entry into the brain in a fasion of competition at level of BBB. Vpre values of BCAA and AAA were significantly smaller and much larger, respectively, in patients with severe liver diseases; this may directly reflect the deranged serum aminogram. [CSF]/Vpre of all the amino acids including even BCAA increased greatly only in encephalopathic patients, indicating that their transports in coma were accelerated abnormally over their theoretical velocities. Improvement of both [CSF] and Vpre for each amino acid was obtained rapidly following intravenous administration of a BCAA-rich solution. Stable [CSF]/Vpre during this infusion indicated an availability of theoretical application for analyzing BBB function in liver diseases.
Abnormal transport of neutral amino acids through BBB may play an important role on the pathogenesis of hepatic encephalopathy in severe liver diseases. Arousal observed reliablly and rapidly following infusion of a BCAA-rich solution may indicate, at least partly, to be due to marked decreases of [CSF_AAA].

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Cholestasis was induced by administration of ethinylestradiol (E. E.) to rats. E. E. treatment of rats increased phospholipid and cholesterol concentration and alkaline phosphatase activity in their sera. From the livers of E. E. -treated rats, the liver plasma membranes were isolated and fractionated into two subfractions, of which the low density fraction was mainly derived from the bile canalicular surface and the high density one from the other surface areas. Na-K ATPase activity was decreased in both of the subfractions. Mg-ATPase and leucyl-β-naphthylamidase activity were elevated in the heavy fraction. 5'-Nucleotidase activity was decreased in the light fraction. Alkaline phosphatase activity was increased markedly in both of the subfractions. Cholesterol and phospholipid contents were not changed significantly in both of the subfractions. The reduction of Na-K ATPase activity in the bile canalicular surface brought about the decrease of bile acid-independent fraction of bile formation. The reduction of the activity in the basolateral surface caused the loss of the Na⁺- gradient across the plasma membrane and decreased the uptake of bile acids, leading to the reduction of bile formation.

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The new immobilized enzyme membrane for a glucose sensor has been developed and evaluated by biological fluids. Glucose oxidase (GOD) was immobilized in the cellulose acetate asymmetric membrane which consists of a- thin and dense skin layer and a porous sponge-layer. The GOD was impregnated in the holes of the skin-layer and cross-linked by glutaraldehyde.
The glucose sensor was constructed using this GOD membrane and a polarographical H₂ O₂ electrode. The response of this sensor to 5×10^-3 M glucose is only 15s. It shows that this membrane has good diffusionability and permeability.
The sensor fitted with a flow through cell was assembled in a flow analysis system. The linear curve in glucose concentration was obtained in the 0-1,000 mg/dl range when flow rate was 2 ml/min and sample size was 5μl, and only 15s. was required to assay one sample.
The sensor was also evaluated by biological fluids. It was used continually at 37° for a month with almost 3,500 assays of serum. The coefficient of variation was 2.5% in the whole blood assay. Having been compaired the whole blood assay value by the glucose sensor with serum assay value by the colorimetry correlation between methods was 0.991.
These results suggest that the new enzyme membrane is expected for practical use.

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Amounts of reduced hemoglobin, oxygen hemoglobin, carboxyhemoglobin in only 15μl blood were successively determined with oxygen electrode and erythrocytes in 3-4 min. The procedure is composed of measurement of consumption of oxygen with reduced hemoglobin, release of oxygen from oxygen hemoglobin by addition of K₃ Fe(CN)₆, and release of oxygen due to exchange of carbonmonoxide with oxygen of oxyhemoglobin in the erythrocytes.

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The automatic glucose analyzer with new hydrogen peroxide electrode produced by Mitsubishi Chemical Industries, LTD. is described.
The automatic glucose analyzer consists of new hydrogen peroxide electrodes, ionexchange column and immobilized glucose oxidase silicagel column. The merit of this electrode is that the electrode can measure hydrogen peroxide generated from glucose under laminar flow without electrode membrane or inclusion electrolytes.
The tested results of this analyzer for response to variables in clinical use are as followings;
1) Standard curve for glucose concentrations shows linear correlation to electrical current in O to 700 mg/dl of concentration.
2) This electrode reacts slightly to bilirubin, but it has no influence to glucose measurement clinically.
3) This electrode, however, reacts directly to glutathione, EDTA, ascorbic acid andhemoglobin.
The most of these substances are removed easily by using ion-exchange resin except ascorbic acid. As these experiments are carried out under abnormal concentration of these substances, influences of them to glucose value are negligible clinically. 4) Measured values of glucose by this analyzer and other procedures reveal a good correlation between them.
It should be considered that the automatic glucose analyzer with new hydrogen peroxide electrodes is useful to measure glucose in biological materials.

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We have developed a bedside type artificial endocrine pancreas, a closed loop control system, which consisted of a continuous blood glucose monitoring device, a microcomputer system, infusion pumps and a printer.
In the microcomputer system, 4 major programs are compiled, the first is a glucose forecasting program which compensates the time delay of glucose measuring, the second is an insulin infusion program, the third is a glucose infusion program and the last is a self-adaptive control algorithm for adaptive blood glucose control.
The validity of the system has been widely proven in clinical applications.
In the conventional artificial endocrine pancreas system, continuous blood glucose measurement was conducted with a Technicon AutoAnalyzer II system using modified glucose-oxidase method, but its total blood sampling volume is rather large (72 ml/day).
So in this study, in order to make the size of total artificial endocrine pancreas system smaller and to minimize blood sampling volume, we developed glucose sensor system for continuous glucose monitoring in whole blood glucose, by combining the immobilized glucose oxidase membrane with electrode which measures hydrogen peroxide, one of the reaction products, polarographically.
Because the key components in making a low noise glucose sensor with long term stability is its membrane, Cuprophan 100PM ®,(hydrophilic, mean suspected pore size 30Å, membrane breadth 10±0.5 μm) was applied to cover the immobilized glucose oxidase and the hydrogen peroxide electrode was composed of platinum cathode and argent anode, and its diameter is as small as 4 mm.
The efficacy of this glucose sensor system was studied with glucose or insulin challenges in anesthetized dogs.
The characteristics of this glucose sensor were summerized as follows, 1) high specificity for glucose and a quick response to change of glucose concentration, 2) long term stability, low drift and low noise, 3) linearity and accuracy of measurement over full range of interest, 4) easy to operate and maintain, 5) high bioadaptability indicated by no adhesion of reticulocytes or platelets on membrane surface after long-term use.
Although in this glucose sensor system, blood transmission tubing time inside the device was 3 minutes and 40 seconds, and response time in electrode was no less than 20 seconds, this 4 minutes time lag is short enough to detect the change in blood glucose level in order to infuse insulin or glucose timely.
By using our glucose sensor system, the whole size of new bedside type artificial endocrine pancreas could be made as small as 50×51×151 cm, and also be minimized the sampling blood volume as small as 30 ml/day.
The animal experiments and clinical applications also proved the efficacy of the new bedside type artificial endocrine pancreas.

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We present an automated enzymatic method for simple, rapid and sensitive assay of various biological substances in serum by use of H₂O₂ electrode. A flow through chamber was specially designed to connect the H₂O₂ electrode with Technicon Auto Analyzer and to allow the reaction mixture to pass through the surface of the electrode. H ydrogenp eroxidee nzymaticallyp roduced was determined amperometrically.
1) Optimalc onditionsf or measurementso f uric acid, c holesterolt, riglycerides and phospholipids were established. Good results were obtained in a precision study. Turbid or coloured serum specimens did not affect the electrode response. Interference by catalase was avoided by the additiono f NaN3. A scorbateo xidasep rotected the interferenceb y ascorbica cid.
2) The type of buffer solution and pH value had an effect on the electrode response. Each buffer solution had an original basal electric current and showed different response to H₂O₂ added. Phosphatea nd PIPESw ere shown to be more favorablef or the H₂O₂ electrodet han Tris-HCl and other Good's buffers.
3) Effect of various compounds present in reaction mixture or specimens on the electrode response was investigated. Reducing compounds such as ascorbic acid, uric acid, cysteine or glutathione which consume dissolved oxygen was found to increase the electrode response even in the absence of H₂O₂. Pyruvic acid and a-ketoglutaric acid continuously decreased the current level by consuming H₂O₂.

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Low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and choline containing phospholipids in serum are enzymatically determined by use of a polarographic oxygen analyzer with a long life and high sensitive sensor to determine the low concentration of serum lipids.
The final assay system for cholesterol in the serum, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of phosphate buffer (0.1mol/l, pH 7.3), containing 50 μmol of phenol, 0.5% sodium taurocholate as a surfactant, 5U of cholesterol ester hydrolase, 9U of cholesterol oxidase and for phospholipids in serum, consists of 1ml of Trishydrochloride buffer (0.05 mol/l, pH7.6) containing 25μmol of phenol, 0.1 % of Triton X 100 as a surfactant, 5.4 U of phospholipase D and 12 U of cholineoxidase.
Oxygen consumption and the concentration of cholesterol or choline containing phospholipids are lineary related to 2.5 g/l from 0.2 g/l using 50μl of serum within 60 seconds-incubation. Replicated analysis of cholesterol and phospholipids by the present method demonstrated the following inter-run precision; HDL-C, mean (mg/dl)±S. D., 39.0±0.60, c. v., 1.54 %, LDL-C, 116.0±0.70, c. v., 0.60 %, phospholipids, 194.0±6.0, c. v., 3.09.
Bilirubin and ascorbic acid were without effect on the present method unlike the enzymic colorimetric methods.
The correlationship of cholesterol and phospholipids between polarographic method and enzymic colorimetric method was well in normal serum.

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Presently ion sensors are widely utilized in the field of clinical chemistry.
In case of applying ion sensors for clinical chemistry, extremely high accuracy is required in the measurements compared with ordinary analytical chemistry.
Various problems occur in case of clinical applications, related with the high accuracy measurement mentioned above.
The 3 problems listed below are discussed in the present paper.
(1) High accuracy measurement of electrode potential
(2) Contamination of the ion sensor by the sample
(3) Problem of the standard solution

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To determine the intracellular ion activities of potassium and chloride, the present study was aimed to make potassium sensitive and chloride sensitive liquid ion exchanger microelectrodes (K⁺-microelectrode and Cl^--microelectrode, respectively), and to measure the intracellular K⁺ and Cl^-activities in the toad urinary bladder epithelial cells.
The characteristics of K⁺- and Cl^--microelectrodes were examined. The sensitivity slope (sRT/zF In 10) and the standard potential of the K⁺-microelectrode was 59.04±0.03mV/10 fold of ionic activity and 22.2±0.69mV, respectively. The slope and the standard potential of the Cl^--microelectrode was-58.38±0.19 and-42.23±0.90, respectively. The selectivity constant of the K⁺-mircoelectrode was 53.96±4.00 for K⁺/Na⁺ and 3.48±0.15 for K⁺/NH₄⁺. In addition, that of Cl^-- microelectrode was 8.00±0.15 for Cl^-/HCO₃^- and 63.59±1.20 for Cl^-/H₂PO₄^-.
It was suggested that K⁺- and Cl^--microelectrodes could be available for determination of the intracellular K⁺- and Cl^- activities in a biological membrane.
The present authors attempted to determine the intracellular K' and Cr activities in the toad urinary bladder epithelial cells, and to investigate the different forms of these ions in the cells and their transport mechanisms.
The intracellular concentration of free potassium ion could be calculated to be 54.2mM from the obtained aⁱ_K, 41.2mM, and that of free chloride could be calculated to be 65.7mM from the obtaineda aⁱ_K, 49.9mM undert he basalc onditions. By the method of the chemical analysis, the intracellular concentrations of potassium and chloride were reported about 130 mMand 62mM, respectively. So, it was suggested that about one half of the intracellular potassium might be free and another half might be bound or sequestrated, and that the intracellular chloride might be almost free.
Furthermore, it was suggested that potassium ion might be actively intaked into the cell from the serosal medium, probably Na-K exchange system, and that the mechanism of the chloride absorption could not need the active process.

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We evaluated ion-selective electrode instruments on Na, K and Cl, especially for accuracy problems. The instruments used for this study were NOVA I, CLIN-ION AI-3, STAT-ION II and HITACHI 702 models. The results from these instruments were compared with those from IL-443 flame photometer and HIRANUMA chloride meter.
The values of aqueous standard solution, such as NaCl or KC1, obtained from these instruments except NOVA I did not agree with theoretical values, even the values from STAT-ION II and HITACHI 702 which used sample-dilution method were not affected with ionic strength.
The values of commercial control sera obtained from these instruments were different from the stated values. Especially the values of potassium on Versatol A from CLIN-ION AI-3 and STAT-ION II and values of chloride on Hyland from HITACHI 702 differed 10% over from the stated values.
On the sodium levels of 100 patients sera estimated from these instruments, higher values (2mEq/l) by NOVA I and lower values (1mEq/l) by STAT-ION II were obtained respectively. And higher values (2mEq/l) of chloride were obtained by CLIN-ION AI-3, STAT-ION II and HITACHI 702.
We concluded that the variation in among instruments apparently deserve continual attention.

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Abnormally high chloride concentration has been occasionally observed in the sera of patients when examined by a Stat/Ion system in which an ion-selective electrode technique is used. One of the causes is presumably the interference of medicaments on chloride electrode. The purpose of the present communication is to identify the medicaments which may interfere with the determination of chloride levels in the serum.
Aliquots of the samples of the sera from the same patients were examined by the ion-selective electrode technique and by Schales-Schales' method. In ten patients (most of the samples were taken several days after operation when the patients were in a serious condition), a marked discrepancy in the chloride values was observed between the above two methods. The medicaments, which were administered during the period in which the chloride concentration was high, was picked up from chart of the above ten patients. Drugs thus selected were added to the pooled serum and the chloride concentration was measured by the above two methods. It was found that the chloride electrode showed a high response to the following six drugs,(a) timepidium bromide (Sesden),(b) butylscopolamine bromide (Buscopan),(c) neostigmine bromide (Vagostigmin),(d) propanthelin bromide (Methaphyllin),(e) composite preparation of salicylate sodium, calcium bromide and dextrose (Salbro) and (f) thiosulfate sodium.
When three healthy volunteers of 3 groups received (a),(b) or bromhexine-HCl (Bisolvon, having bromine in its aromatic nucleus), respectively, of a regular therapeutic dose, no remarkable changes in serum chloride concentration were observed in any groups.
Animal experiments were then carried out as follows. Rats were received orally a single large dose of 300mg/kg of (a) through a gastric tube under the general anesthesia using ethyl ether. A marked difference in serum chloride concentration was observed in between the values just before and one hour after administration, as determined by the Stat/Ion system.
The ion-selective electrode of silver-chloride is not specific to chloride ion. It responds not only to Cl^-, but also to Br^-, I^-, CN^-, Fe(CN)^3-₆, Fe(CN)^4-₆, SCN^-, S^--, S₂O₃^--, and to the compounds which contain SH group, such as glutathione and L-cysteine. The disturbance of the silver-chloride electrode with the presence of SH group was not confirmed until today.
There will be an increasing demand for the use of the ion-selective electrode technique for thefollowing reasons: 1) there is a necessity of repeated electrolyte determinations in emergency situation such as post-operative stage, 2) this technique is rather simple and 3) this technique causes no environmental pollution.
However it is to be emphasized that the silver-chloride electrode which currently in use be improved or replaced with one of a new type.

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