Sources of errors in quantitative paper electrophoresis of the serum proteins have been investigated, and found that the sources of errors are the so-called trail, difference of dye-combining capacity of each protein fractions, influence of the amount of protein on unit area of the paper to the optical density with white light, and the artifact of oiling process in densitometry. From the experimental results, to obtain reproducible data, following standard procedure is recommended.
1. For accurate quantitation, the electropherogram should be clear-cut, but the distance between each fractions should be reduced as narrow as possible to minimize the trail effect of the migrated protein. For this purpose, barbital buffer of higher ionic strength (0.1) than conventionally used (0.05 to 0.06) should be employed, and migration should be done with low voltage (75V/20cm).
2. Another measure to minimize the protein-trail effect is to select a filter paper such as Whatman No.1 or Toyo No.51 which adsorbs less protein.
3. To eliminate the densitometric artifact, preliminary removal of moisture and air in the stained paper and oil is absolutely necessary.
4. There are certain limitation of protein amount on unit area of the paper to obain linear relationship between protein amount and optical density with ordinary photocell-white light densitometer. In other words, optical density of dense area (e. g. center part of albumin) is not proportionally higher than that of light area (e. g. alpha-, beta-globulin part). To obtain Beer's relationship, the protein amount on the unit area should be below 0.0035mg/mm². In practice, less than 0.01ml of serum diluted with buffer to the total protein concentration of 6.0g/dl should be applied evenly 3cm wide. For this reason, thinner paper is preferable, and it should be avoided to apply repeatedly even with a sample of low protein concentration, and when there is found a point which over 1.0 of optical density in densitometry, it should be re-migrated with less amount of the sample.
5. In the final quantitative analysis of the densitometric curves, the trail effect should be cut as described in the text.
In this experiment, in addition, I have confirmed following facts; that (1) trail is not only originate from albumin, but also from globulins, and its appearance is not additive, and there are certain point of saturation according to the property of paper and serum.(2) The amidoblack 10 B-combining capacity of alpha-globulin is almost same with that of albumin, but beta, and gamma-globulin combine slightly less than albumin.

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The effects of dl-ethionine and acute carbon tetrachloride poisoning on the rabbit serum proteins, and effects of cortisone, estradiol and testosterone on the serum proteins of rabbits which were treated for longer periods with carbon tetrachloride have been observed.
For acute intoxication of ethionine, 10mg of dl-ethionine per kilogram of body weight were injected intraperitoneally daily using 1per cent aqueous solution for 10 days. For acute carbon tetrachloride intoxication, 0.5ml of 20per cent solution in olive oil per kilogram of body weight were injected intramuscularly for consecutive 2 days. For chronic liver damage with carbon tetachloride, 0.1ml of 20per cent solution in olive oil per kilogram of body weight were injected intramuscularly for every 4 to 5 days for 4 to 6 months. During that time, after 2 months of carbon tetrachioridetrezttrnent, hormones were injected simultaneously. Five mg of cortisone acetate per kilogram of body weight, 10mg/kg of testosterone propionate, 0.04mg/kg of estradiol were injected for every 4 to 5 days. However, sometimes hormon or carbon tetrachloride injection was stopped when the general condition of rabbit was dangerous.
From this experimental results, following findings were obtained.
1. Total serum protein concentration was lreduced by ethionine injection, and this decrease was chiefly, due to the decrease of albumin. There were not found any definite changes in beta-globulin fraction, and gamma-globulin decreased slightly. Most characteristic finding was the gradual decrease of alpha-globulin after initial increase. In short, it might be said that by ethionine, synthesis of albumin and alpha-globulin in the liver is impaired.
2. In acute carbon tetrachloride intoxication, all fractions of the serum proteins decreased, but decreese of albumin was most remarkable.
3. From the above findings, it could be said there is no tissue destruction by ethionine administration or acute carbon tetrachloride poisoning, and albumine and alpha-globulin are synthesized chiefly in the liver and part of beta- and gamma-globulin are also synthesized in the liver.
4. In chronic carbon tetrachloride intoxication, all fractions except alpha-globuline were decreased, but that of albumin was most remarkable. The increase of alpha-globuline indicates the presence of tissue destruction in this case, and this might be the cause of fibrosis.
5. Cortisone reduces the chronic injury by carbon tetrachloride regardless of sex.
6. Sex hormone affects slightly the cortisone action on the serum proteins, but hypofunction of sexual organ might have relation with the hypergammaglobulinemia and decrease of alpha- and betaglobulin in chronic liver diseases. There are reports on atrophy of sex organ in liver cirrhosis. Therefore, the degree of hypergammaglobulinemia is a indicator of severe damage of the liver. These gamma-globulin in hypergammaglobulinemia originate from extrahepatic tissues. However, it is doubtful whether these gammaglobulin is benificial to the organism or not, and its function is also unknown.

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The effect of storage of serum upon the paper electrophoretic protein fractions has been studied for 9 days. There were not found any significant differences between the sera stored at room temperature (15°C) and 0°C, if kept aseptically. However, trail on the electopherogram tends to appear more intensely by storage, therefore, in these cases, it is necessary to eliminate the trail effect in the quantative clensitometry as described in the previous paper.

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The serum β-lipoprotein, its bound protein and lipid were assayed by Burstein's method on 91maternal-fetal pairs, and 148 cases of non-pregnant women for comparison. The serum α-and β-lipoprotein cholesterols were also determined in parallel with the serum β-lipoprotein on the 18 meternal-fetal pairs by the combined method of paper electrophoresis and chemical analysis.
The results observed are as follows;
(1) The serum β-lipoprotein, its bound protein and lipid levels of the non-pregnant women are 267.8±94.4mg%. 233.7±7.14mg% and 48.4±15.8mg% respectively.
(2) The β-lipoprotein, its bound protein and lipid levels of the maternal serum are 516.7±99.2mg%, 427.5±83.7mg% and 94.7±19.0mg% respectively, while those values of the fetal serum are 125.3±28.1mg%, 102.6±25.0mg% and 22.5±4.1mg% respectively; the meternal levels being four times as much as the fetal levels.
(3) The total serum cholesterol (α-+β-lipoprotein-cholesterol) level is 195.7±48.2mg% on the maternal side and 68.2±22.4mg% on the fetal side; the former is three times as much as the latter, and the maternal level is almost 1.5 times higher, but the fetal level is less than one half of the normal value.
(4) The maternal and fetal serum α-lipoprotein -cholesterol levels are 44.5±20.7mg% and 20.5±11.5mg% respectively, the former being twice as much as the latter.
(5) The β-lipoprotein-cholesterols of the maternaland fetal serum are 151.5±26.9mg% and 44.7±28.9mg% respectively, the former still being more than three times as much as the latter.
(6)β-/α-lipoprotein-cholesterol ratio is 3.2±0.88 on the maternal side and 2.7±0.59 on the fetal side, the former being 1.2 times as high as the latter.
(7) The serum β-lipoprotein level parallels those of the serum α-and β-lipoprotein-cholesterols both on the maternal and fetal sides.
(8) The possible explanation concerning great difference between maternal and fetal serum βlipoprotein, α-and β-lipoprotein-cholesterol is discussed with regard to their placental transfer.

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Following the previous works, the serum total lipid, lipid phosphorus and beta-lipoprotein levels of 15 maternal-fetal cases and 10 non-pregnant women as control were determined and compared.
The results observed are as follows;
(1) The serum total lipid content.
The serum total lipid content of the non-pregnant women is 650±60.5mg% and those of the maternal and fetal samples are 893.0±130.6 mg%, and 231.0±37.3mg% respectively. The maternal serum level of total lipid is 1.35 times higher than the normal value and the fetal one is only one thirds of it, so the maternal value is almost 3.6 times as much as the fetal one.
(2) Serum lipid phosphorus.
The serum lipid phosphorus content of the normal, maternal and fetal serum are 6.7±2.09, 14.5±2.81, and 4.8±1.84mg% respectively. The maternal serum lipid phosphorus level is 2.2 times higher than the normal one, but the fetal level is lower than the normal, so, the maternal value is three times as much as the fetal one.
(3) The serum beta-lipoprotein level parallels those of the serum lipid phosphorus in control, maternal and fetal groups.
(4) The serum total lipid and lipid phosphorus contents also parallel each other in the maternal and fetal groups.

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The serum glycoprotein (protein-bound hexose) levels of 19 maternal and fetal pairs and 59 nonpregnant women as control were determined. The following results are obtained;
(1) The normal serum glycoprotein (protein-bound hexose) content of the Koreans (non-pregnant women: 88.7±13.8mg%) is much lower than that of the Americans (121.0±21.1mg%).
(2) The glycoprotein (protein-bound hexose) contents of the maternal and fetal serum are 110.4 15.4mg% respectively, the former being twice as much as the latter.
(3) The glycoprotein (protein-bound hexose) content of the maternal serum at the delivery is higher than that of the non-pregnant women.

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The vitamin B₁₂ levels of the maternal and fetal sera on 40 delivery cases, were assayed microbiologically, employing Lactobacillus leichmannii. The serum vitamin B₁₂ content of the 14 non-pregnant women were also determined similarly as control.
It was found that the vitamin B₁₂ level of the maternal serum was considerably lower than that of the non-pregnant women, which is, in turn, still lower than that of the fetal serum.
These findings confirm the previous observations, which suggest that vitamin B₁₂ may be drawn considerably from the maternal circulation to the fetal one.
The possible explanations for this phenomen are discussed.
The authors wish to express their sincere tanks to Prof. KI YUNG LEE for his kind guidance and valuable advice.

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