Changes in lactate dehydrogenase activity in cultured rat aortic smooth muscle cells with the growth and effect of iron on these changes were investigated.
When smooth muscle cells were grown in the medium which was changed on days 3, 5 and 7 after plating, LDH activity increased with the growth. But LDH activity decreased when the medium was not changed for more than two days.
Maximum proliferation occurred when iron concentration was physiological, whereas increase in LDH activity occurred at about 10% of physiological concentrations and the activity decreased with increasing concentrations of iron.

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Phosphatases in rat thoracic aorta and cultured smooth muscle cells derived from the aorta have been investigated. In the present study, changes in the phosphatase activity of cultured smooth muscle cells with the passage and with the growth and the effect of change of medium on acid phosphatase activity were investigated.
Acid phosphatase activities of rat thoracic aorta and cultured smooth muscle cells in early passages (4-30) increased at pH 5.5, while those of cells in late passages (more than 100) increased at pH 3.5. This suggests that there may be two types of acid phosphatase. Although alkaline phosphatase activity of the intact aorta increased at pH 10.5, that of cultured cells was significantly weak at any alkaline pH.
Acid phosphatase activity expressed on the basis of protein content increased with the growth, while that expressed on the basis of cell number remained nearly constant.
Acid phosphatase activities of cells cultured for 1-2 days after change of medium were lower than those of cells cultured for the same period without change of medium. Of components present in the medium, serum was most effective for this reduction of enzyme activity.

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In order to examine the effect of dietary iron on the atherosclerosis, male rabbits were fed with diet containing 1.3g/kg iron as experimental group and 0.3g/kg iron as the control group for 18 weeks. At 14 weeks, noradrenalin was injected for 14 days and after a cessation of 14 days they were sacrificed.
The level of plasma essential metals (calcium, magnesium, iron, zinc and copper) and lipids (triglyceride, total-cholesterol, phospholipid and lipoperoxide) were analyzed and the pathological severity of atherosclerotic lesions were observed.
The results were obtained as follow:
1. Grossly visible atherosclerotic lesions were seen 3 out of 4 animals in the experimental group and 3 out of 5 animals in the control group.
2. The level of plasma iron in the experimental group were not significantly different from that of the control group. The level of iron in the liver was significantly increased (p<0.05), and in the aorta tended to decrease in the experimental group, respectively.
3. Plasma lipoperoxide, triglyceride and totalcholesterol were not significantly different between in the two groups by feeding.
4. The contents of triglyceride and calcium in the thoracic aorta from rabbits with grossly visible atherosclerotic lesions were remarkably increased. The level of totalcholesterol in the abdominal aorta in the experimental group were significantly increased (p<0.05) and the level of triglyceride in the liver in the experimental group were increased.
5. As pathological finding, a severe split and stretch of elastic fiber caused by calcification were diffusely observed in the thoracic aorta of the experimental group.
6. From the above results, it was concluded that intaked iron may play an important role indirectly in the pathogenesis of atherosclerosis, because the increased content of iron in the liver damaged the function of reticuloendotherial system-cells.

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Sixteen human dissected aorta were classified into three histological types: Normal, fatty streak or fibrous plaque and atheroma.
The contents of lipids (total-cholesterol, esterified-cholesterol, free-cholesterol, triglyceride and phospholipid) and essential metals (calcium, magnesium, iron and zinc) of each specimen of the aorta were determined.
The results were as follow:
The concentrations of total-cholesterol, esterified-cholesterol, free-cholesterol, triglyceride and phospholipid were increased significantly with the progression of atherosclerosis and with age (p<0.05-0.01). Especially, esterified-cholesterol had markedly increased in atheroma. It was assumed that esterified-cholesterol was important for the pathogenesis of atherosclerosis.
The contents of essential metals such as magnesium and zinc increased significantly in fatty streak or fibrous plaque with the advance of atherosclerosis and with age (p<0.01, p<0.05), respectively.
On the contrary, calcium and iron were not significant. Calcium to magnesium ratio had decreased in fatty streak or fibrous plaque, and had again increased in atheroma, since the degree of magnesium content was greater than that of calcium.
There was significantly negative correlationship between iron and triglyceride at normal site (p<0.01) and significant positive correlationship between iron and free-cholesterol or phospholipid in fatty streak or fibrous plaque (p<0.05, p<0.01), respectively. Wherease no relationship was observed between essential metals and lipids in atheroma.
From the above results, it was suggested that iron might participate in the synthesis of triglyceride at the normal site and in the synthesis of free-cholesterol or phospholipid at the sites of fatty streak or fibrous plaque, and other metals (zinc, especially magnesium) might be anti-atherogenic.

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It was previously reported that excess iron accelerated the progress of noradrenalin-induced atherosclerosis. In the present experiment, influence of addition of beef tallow to the diet on atherosclerosis was examined.
Of 14 male Japanese white rabbits, five were fed on an iron-adequate diet with vegetable oil (I-group), four fed on an excess-iron diet with same oil (II-group) and five fed on the same diet as in the II-group to which beef tallow was added (III-group).
All of the rabbits were fed for 18 weeks. They were loaded by drip infusion of noradrenalin from the 14th week to the 16 week of the feeding period.
The results were as follow:
1) The incidence rate of grossly visible atherosclerosis was 60% in I-group, 75% in II-group and 20% in III-group.
2) The level of total-iron in liver was significantly higher (p<0.001) in II-group than in I- and III-groups and that in III-group was between those in I- and II-groups. The level in small intestine was lowest in I-group and highest (p<0.025) in III-group.
3) The level of calcium in the thoracic aorta was extremely high in II-group and that in III-group was lower than in I-group.
4) The level of total cholesterol in the abdominal aorta was significantly higher (p<0.05) in II-group than in I- and III-groups.
5) The level of triglyceride in the thoracic aorta was higher in II-group and that in III-group was lower than in I-group.
6) The severity of atherosclerosis in the thoracic aorta was parallel with both calcium and triglyceride contents.
From the above results, it was assumed that beef tallow might have inhibited development of atherosclerosis due to excess iron, because P/S ratio in the diet influenced on permiability of the membrane of cells, and as a result of that also on passing of iron through the membrane.

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We investigated the influence of lanthanum (La³⁺) as a channel blocker of calcium ion on the experimental atherosclerosis in rabbit.
Twenty male rabbits were divided into three groups (A-, B- and C-groups). A- and C-groups were fed on a basal diet and B group were fed on a basal diet plus 0.12% LaCl₃ through experimental period. A- and B-groups (experimental group) were injected with nor-epinephrine by drip infusion from 14 to 16 weeks. At 18 weeks, they were autopsied.
1) Increases of body weight did not show significant difference among three groups.
2) Incidence of grossly visivle atherosclerotic rabbits were found 0% in C-group, 57% (4 out of 7 rabbits) in A-group and 37.5% (3 out of 8 rabbits) in B-group.
3) There were no differences in serum total cholesterol, triglyceride and phospholipid among three groups. On the other hand, HDL cholesterol was significantly lowered in B-group than in A-group (p<0.05).
4) There were no differences in serum calcium, magnesium, sodium and potassium among three groups, while inorganic phosphorus in A-group was significantly higher than that in C-group (p<0.05).
5) Significant differences were obtained that total cholesterol in the kidney was higher in A-group than in B-group (p<0.01) and in the thoracic aorta higher in B-group (p<0.05).
6) Significant differences were found that calcium content in the thoracic aorta was higher in B-group than in C-group (p<0.05) and magnesium in the myocardium was higher in B-group than in C-group (p<0.01), and sodium in the liver were higher in B-group than in C-group (p<0.05) and higher in A-group than in B-group (p<0.05), and sodium in the adrenal were higher in B-group than in C-group (p<0.01) and higher in A-group than in B-group (p<0.01). Potassium in the liver was higher in B-group than in A-group (p<0.05).
From the above results, it was suggested that lanthanum chloride might have suppressive effect on the incidence of significant atherosclerosis induced by nor-epinephrine. Nevertheless, inhibition effect on the calcification of thoracic aorta were not at all observed.

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The changes in the lactate dehydrogenase (LDH) isozyme pattern of small pieces (1mm²) of rat thoracic aorta in primary culture and isolated smooth muscle cells from enzyme dispersed aorta in primary culture and in subculture have been investigated using cellogel electrophoresis.
Cultured pieces of aorta exhibited a progressive change. In the intact uncultured tissues, LDH-3 was the predominant isozyme, but the relative amount of LDH-5 increased when cells started to grow out of these pieces and after the outgrowth of cells, LDH-5 became predominant.
Isolated smooth muscle cells from the aorta in primary culture underwent a change in LDH isozyme pattern from the isozymes rich in H subunits to the isozymes rich in M subunits and showed virtually the same characteristic patterns as subcultured smooth muscle cells: the most prominent isozyme was LDH-5, next in intensity was LDH-4, then LDH-3, then LDH-2 and finally very faint traces of LDH-1 occured.
LDH isozyme patterns of smooth muscle cells in culture showed differences between exponential and stationary phases of cell growth. The relative amount of LDH-5 decreased and that of LDH-4, LDH-3 and LDH-2 increased with change from exponential phase to stationary phase.

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The activities of phosphatase have been reported to increase with the development of atherosclerosis. This increase in phosphatase activity during atherogenesis suggests that phosphatases may be associated with the development of atherosclerosis. For this reason, phosphatases of the cultured rat aortic smooth muscle cells have been investigated. The alkaline phosphatase activity in cultured smooth muscle cells decreased drastically within primary culture and decreased gradually with subculture, whereas the acid phosphatase activity increased. In the arterial wall several alkaline phosphatases may change their activities with the development of atherosclerosis. Therefore, in order to know the type of alkaline phosphatase of the aorta, properties of that of the aorta were compared with those of the liver and intestine. Unlike human alkaline phosphatases, rat alkaline phosphatases of the aorta, liver and intestine showed no distinct differences in property. Acid phosphatase activities of smooth muscle cells cultured in the medium with various concentrations of Mg were virtually the same at any concentration of Mg. However, those in the medium with the physiological concentration of P and without P were high and enzyme activities were increased by 10% of the physiological concentration of Ca, Ca antagonists and chelating agents. Enzyme activities were also increased by the addition of chloroquine and sucrose to the medium, suggesting that these enzymes may be associated with lysosomes. These results suggest that low concentrations of extracellular Ca may enhance lysosomal function.

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Rat aortic smooth muscle cells in culture were exposed to various concentrations of iron and magnesium in order to assess their effects on cell proliferation, glucose consumption and lactate production.
Glucose consumption and lactate production were enhanced in the early stage of both exponential and stationary phases of cell growth.
Iron at physiological concentrations (10^-5M), or more, was effective to promote the proliferation of cells and to increase glucose consumption and lactate production.
The maximal stimulation to cell proliferation and glucose consumption by magnesium was observed at approximately 10% of physiological concentrations, whereas lactate production was most increased at 10 times the physiological concentrations.

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The studies on Chilaiditi's syndrome reported in Japan have been mostly case reports on one or two cases. There have been studies on 356 cases in all. We have selected 262 cases with detailed data takes from the above 356 cases, together with 116 further cases of Chilaiditi's syndrome that we have encountered up to 1990, i. e. since our previous report in 1965. We also investigated roentgenograms, complications, interpositions and age distribution.
The syndrome was mostly found in middle-aged and elderly patients of 50 years or older. More male patients were involved than female, however this is not necessarily true if the total number of patients under study is taken into account. Among the complications, gastric and duodenal diseases (ulcer and carcinoma) were most frequently found, accounting for 20.6% of the total reported cases in Japan. In our own study, the percentage was as high as 33.7% because radiography with contrast medium was positively conducted on the digestive tract. Of the interpositions, the colon accounted for 93.1% of our cases, and 72.9% of the total reported cases in Japan. The involved sites were mostly the right hepatic flexure of the colon and the transverse colon. From the reports by other researchers in the past and our data from 1965, 1975 and 1977, it appears that the incidence is about 0.003-0.03%.
There were no subjective symptom specific to Chlaiditi's syndrome, and there may be various factors accounting for the occurrence of the syndrome. The symptoms were primarily based on the underlying diseases, and Chilaiditi's syndrome merely indicates the condition where the digestive tract interposes between the diaphragm and liver. Therefore, we regard this as a radiographical sign rather than a syndrome, and we propose that this should be called “Chilaiditi's sign”. We also emphasize that the digestive tract must be strictly examined if this sign is to be noted.

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