A characteristic kidney disorder is commonly seen in Itai-itai disease in addition to the severe bone damage. Hence, urinary proteins of Itai-itai disease patients were analyzed by disc electrophoresis in order to diagnose this disease early and to distinguish it from other kidney diseases.
The results are summarized as follows.
1) Five different bands, designated as B-1 to 5 according to the decreasing order of the anodic mobility, were commonly detected in urinary proteins of Itai-itai disease. This pattern was specific for the disease and was provisionally named as I (Itai-itai disease) type.
2) Three different bands, one corresponding in mobility to serum albumin and two other slower migrating bands, were seen in urinary proteins in other kidney diseases, while the urinary protein from healthy adults was composed solely of albumin. These patterns were provisionally named as K (kidney disease) and N (normal) type respectively.
3) Among the five bands seen in the I-type pattern, B-3 and 4 specifically appeared in Itai-itai disease and seemed to be important in distinguishing I-type from K or N-type.
4) Six protein fractions, prealbumin, albumin, α₁, α₂, β, and γ-globulins were prepared by cellulose acetate electrophoresis from a Itai-itai disease patient's urine and were utilized in disc electrophoresis to identify the above described five bands. It was revealed that B-1 was composed of α₁-globulin, B-2 of albumin, B-3 of α₁ and α₂-globulins, and B-4 and B-5 of α₂ and β-globulins, respectively.
These results indicate that the disc electrophoretic analysis of urinary proteins is a useful screening test in the early diagnosis of Itai-itai disease. It also provides a clear distinction of this disease from other kidney diseases.

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This investigation has been performed to detect the individual variances in protein components of the ascitic or pleural fluids obtained from 151 cases of various disorders.
Quantitative determination of fibrinogen, α₁-acid glycoprotein and IgG in the fluids was made by the single radial immunodiffusion method. Fibrinogen and its degradation product increased in parallel with the proliferation of cancer cells in the serous cavities, on the other hand, the increase of α₁-acid glycoprotein reflected the histopathological classification of cancers.
The immunoelectrophoretic analysis performed with the specific anti-human fibrinogen antiserum demonstrated the increase of fibrinogen degradation products in the ascitic and pleural fluids. The reaction patterns of fibrinogen and its derivatives were classified into five types and their relation to the morphological findings of cellular components was investigated. The majority of cancerous fluids was involved in Type 4 and 5 which revealed two distinct lines closer to the cathode. The cancer cell-negative transudates belonged to Type 1 or 2, forming very weak patterns and most of inflammatory effusions to Type 3.
Fibrinolytic activity determined by the fibrin plate method showed a marked variety in each fluid. Based on the comparison of cytological pictures in active and in negative cases of fibrinolysis, it was suggested that the proliferation of macrophages correlated with the increase of fibrinolytic activity.
It may be assumed that the immunological analysis and estimation of several protein components in the ascitic and pleural fluids are of help to the differentiation of cancerous and other disorders.

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Although the thin layer polyacrylamide gel electrophoresis is a highly suitable and satisfactory method for the analysis of serum proteins, the procedure is not so simple as that of cellulose acetate membrane electrophoresis.
The authors have been studying on the micro-electrophoresis of proteins and enzymes in serum and have accomplished a procedure for “Simple polyacrylamide gel film electrophoresis” of high resolution. The procedure and some of the results were reported.
It is based on thin layer electrophoresis on a sheet coated by the thin layer of polyacrylamide gel (1mm or 2mm thick), which has been previously prepared and kept in moist condition. Therefore, preparation of thin layer could be omitted from the procedure.
As the sheets, cellophane of a quality comparable to dialysis tubing, 124 PD cellophane (Du Pont Co.), was selected. A polyester film used for drawing with a smooth surface and a rough back was also found suitable in regard to the firm adhesion of the gel layer to the sheet, since the gel layer could be formed on the rough side.
Various buffer conditions of gel layer could be obtained by using monomer buffer mixture solution which was prepared by dissolving 9.5g of acrylamide and 0.5g of BIS (N, N'-methylenebisacrylamide) in 200ml of 10% glycerol buffer solution. The gelation was performed by adding catalysts to monomer buffer mixture solution.
Sera were applied without dilution into the sample grooves previously prepared on the gel layer surface. 3μl or 10-15μl of serum was sufficient for each groove on the gel layer with 1mm or 2mm thickness, respectively.
The gel layer on a supporting glass plate bridged between the two electrode compartments and the ends of the gel were connected by wet filter papers to the buffer solution in the compartments.
To prevent over-heating, the electrophoresis was carried out at 5°C to 10°C. The voltage was adjusted to deliver a constant current of 0.5 to 1.0mA per cm width of the gel layer and the current was applied for 120min.
Various staining methods for enzyme and protein components could be applied after electrophoresis.
The stained gel layer was inserted between the sheets of cellophane, and the gel layer was plastized by leaving at room temperature for two days.
The procedure was also found to be a useful tool for the estimation of molecular weights of polypeptide chains by using the gel layer containing sodium dodecyl sulfate (SDS).

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Using a simple thin layer polyacrylamide gel electrophoresis, human serum alkaline phosphatase could be separated into up to 5 isozyme bands; 2 or 3 isozyme bands in normal adult's sera and 5 bands in patient's sera. They were numbered as Alp I, II, III, IV and 0 in order of decreasing anodic mobility. It was found that Alp I to IV corresponded in mobility to the major component of the respective tissue extracts, i, e, Alp I to hepatobiliary, Alp II to bone, Alp III to placental and Alp IV to intestinal alkaline phosphatase. Alp 0 was the activity remaining at the origin and was supposed to be polymers or complexes located on large particles, such as cell debris, etc.
Alp III from pregnant sera could be devided into 3 subgroups, i. e., fast (Alp III_F), intermediate (Alp III_I) and slow (Alp III_S), according to its mobility.
The activity of Alp I, sometimes with that of Alp IV, rose mainly in hepatobiliary disease, while activities of Alp II rose in bone and parathyroidal disease. Increase in activity of Alp III and 0 occurred mainly in pregnancy and malignant disease, respectively. The changes of alkaline phosphatase zymograms in pathological states gave us the key to differential diagnosis.
It was shown that the appearance of Alp IV in normal adult's serum was related to the blood group B or O with salivary secretor type.
Thus, the electrophoretic separation of alkaline phosphatase of human serum by using the simple thin layer polyacrylamide gel plate as the supporting medium was shown to give us many useful informations about clinical diagnosis. Treatments of serum before electrophoresis, such as diluting, freezing and thawing, heating and adding surface active agents, enzymes, lectins, etc., gave much more informations for the identification of alkaline phosphatase isozymes.
It was indicated from these data that the migration of alkaline phosphatase isozyme bands was strongly affected by the structure of their end polysaccharide chains.

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Although Bence-Jones proteins are characterized by the property of precipitating on heating at 44-60°C and of redissolving on boiling, the factors governing this unusual solubility behavior have never been defined quantitatively, nor has the phenomenon been satisfactorily explained.
Measurements of the property of precipitating phenomena at high temperatures have been performed by Putnam with various optical instruments.
An ultracentrifugal study of the molecular size and shape changes occurring in Bence-Jones proteins in the temperatures ranging 30-100°C has become possible only with a reliable high temperature accessory to the analytical ultracentrifuge. This report describes the ultracentrifugal analysis of heat denaturations of Bence-Jones protein in solution at 30-100°C. The results obtained are as follows.
1) The Bence-Jones protein forms unstable rapidly sedimenting boundaries above a critical temperature of 60°C, and the sedimentation coefficient could not be obtained.
2) Molecular aggregation occurred when the solution of Bence-Jones protein was heated above 60°C and various types of aggregation products were suspected.
3) The method was found to be not satisfactory for the study of heat denaturation of the urmary Bence-Jones protein in this temperature range.
4) The technical problems associated with this study were discusssed.

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