Interference of peptide binding antibiotics in urinary 17-hydroxycorticosteroids (17-OHCS) assay is described. In the urine of subjects given an oral dose of cephalexin and kefglycine, elevated optical density (O. D) has been observed. These drugs, followed by the procedure of 17-OHCS assay show a maximum absorption at 380-390nm with porter-silber reagent both color and blank.
The result by means of thin-layer chromatography showed that the metabolites of these drugs cause the interference in determination and these drugs themselves make little interference.
These drugs, as a result, show an error even with subtraction of blank reagent O. D and since these drugs ofen show an elevated O. D over 1.000, it is impossible to correct the value in practice except to quit the administration of these drugs before the test.

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There are at least two types in double albuminemia. The one group named “Fast type” has higher mobility than normal albumin, and the other group named “Slow type” has the lower mobility in electrophoretic pattern.
In this paper, a family of the double albuminemia was reported.
Two years old boy who visited the 2 nd Dept. of Surgery of Nigata University Hospital, his mother and his uncle of mother side showed “Fast type” of double albuminemia.
The comparative studies on the “Fast type” albumin to normal person showed no significant difference in their immunogenecity.
It was observed that the molecular weight of “Fast type” albumin showed similar to that of normal albumin by SDS-Disc electrophoresis. The amino acids analysis of “Fast type” albumin was performed, and the composition of the “Fast type” albumin was compared with that of normal albumin and the pre-albumin of human serum to find rather low percentage in Lysin portion.

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The relationships between the serum Carboxypeptidase A activity (CPase) and the results of exocrine function tests of the pancreas were studied. The following results were obtained.
(1) Concerning the relationships between the CPase and three factors in pancreozymin-secretin (PS) test, significant correlation was observed between CPase and total amylase output (r=0.571, p<0.005) or maximum bicarbonate concentration (r=0.415, p<0.05), but no significant correlation was observed between CPase and volume flow per kilogram.
(2) While 80% of patients with normal level of CPase was also within normal ranges in PS test, about 70% of patients with abnormal low level of CPase activity, we found any degree of dysfunction in PS test.
(3) No significant correlation was found between CPase and the results of ¹³¹l-triolein test.
(4) Low levels of CPase were observed in patients whose pancreatic scanning showed abnormality. It seems to be significantly correlated between the CPase activity in serum and the results of the exocrine function tests of pancreas.

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Cholesterol contents of four subcellular fractions of the rat liver and intestine and the distributions of radioactive cholesterol to them were estimated after an oral administrations of labeled cholesterol. The change of the distribution of labeled cholesterol to these hepatic subcellular fractions was also examined in the rats fed with a high cholesterol diet.
The ratios of cholesterol to protein contents in every four subcellular fractions of intestine were higher than those in the corresponding fractions of the liver. But the percentages of ester form to total cholesterol were almost the same in the corresponding fractions both in the liver and intestine.
Percent distribution of the ingested labeled cholesterol to the particle fractions was identical in both liver and intestine, though the distribution rate to the cytosol in the intestine was lesser than those in the liver. The ratio of ester form in labeled cholesterol was larger in cytosol fraction than in other particle fractions in both liver and intestine.
Percentage of distributed radioactive cholesterol to hepatic microsomes was decreased and that to cytosol was increased after the cholesterol administration. The ratio of ester fraction in labeled cholesterol was increased in the cytosol and decreased in the microsomal fraction.
In conclusion, the difference of negative feedback control between liver and intestine in cholesterol synthesis by oral administration of cholesterol can not be accounted by subcellular distribution of exogenous cholesterol, and the change of subcellular distribution in the liver after cholesterol feeding might be related to the metabolic changes of cholesterol.

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A new method was developed to improve the linearity of plasma LCAT reaction by adding synthetic dipalmitoyl lecithin sol in incubation medium and subsequently measuring the change of free cholesterol contents after incubation by combined enzymatic method using cholesterol oxidase and peroxidase.
The optimum concentration of dipalmitoyl lecithin in sol which was added into the fresh plasma was found in the range of 1.0-3.0mg/ml.
The time course of the plasma LCAT reaction in this system proceeded almost linearly until two hours' incubation when plasma containing less than 120 n moles/ml/hr of their LCAT activities were used.
The following additional data were obtained R=78 n moles/ml/hr, S. D.=4.73 n moles/ ml/hr and C. V.=6.0%. The correlation coefficient of the present method to GLC method was 0.83.
The present method was found to be simpler than other techniques such as GLC or isotope methods and therefore may be suitable for the routine assays of plasma LCAT activities in the clinical laboratories.

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A simple and sensitive assay system was presented for the determination of total bile acids in serum. One tenth ml of serum in tube (A) and the same amount of distilled water in tube (C) were added with Tris-aminomethane hydrochloric buffer and inactivated at 67° for 20 min. After heating 3α-hydroxysteroid: NAD oxydoreductase (EC. 1.1.1.50, 3α-HSD), NAD, diaphorase (EC. 1.6.4.3.), and resazurin were added to this solution. This mixture was incubated at 20° for 40 min. As blank another 0.1ml of the same serum in tube (B) and the same amount of distilled water in tube (D) were made after the same treatment in the same assay system without 3α-HSD as tubes (A) and (C). The fluorescence of resorfin which was converted from resazurin in these tubes were measured at 580 nm with the excitation at 560nm. ΔF was calculated as follows and the amount of bile acids was obtained according to the standard curve. ΔF=fluorescence intensity in tube (A)-(C)-[(B)-(D)] Relationship between the amount of standard bile acid and the fluorescence intensity was linear in the range of 0 to 50uM.
About ninety percent of recovery was gained irrespective of the species and forms of conjugation of bile acids when added to serum. Reproducibility in a serum was 6.5uM with standard deviation of±0.20 and C. V. of 3.1%.
Normal value in fasting serum was found in the range of 4.5uM to 9.2uM with mean of 6.4uM in male (n=9) and 3.8 to 6.5uM with mean of 4.8uM in female (n=5). Constant values were obtained in sera of a female which were taken successively for five days in the fasting state.
Serum bile acids level was elevated after eating egg yolk in all eight normal subjects tested.
Described method is an improved one which was reported in detail in “Clinica Chimica Acta” (in press, 1976)

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This is a summary of our studies on the determination of urinary 17-hydroxycorticosteroids (Porter-Silber Chromogens).
The conjugated steroid is hydrolysed with bovine liver β-glucuronidase in the presence of Na₂SO₄ and NaHSO₃. Na₂SO₄ almost entirely inhibits the formation of electrostatic complexes between the enzyme and its urinary endogenous high molecular inhibitors, which play the main role in inhibiting the activity of β-glucuronidase. NaHSO₃ maximizes the enzyme hydrolysis in the presence of Na₂SO₄. Probably NaHSO₃ reduces some urinary endogenous thermostable low molecular inhibitors to inactive substances.
The addition of Na₂SO₄ to urine is highly effective for the extraction of the steroids with methylene chloride: Na₂SO₄ increases the recoveries of THF_K and Allo-THF_K, two of the three major cortisol metabolites possessing the dihydroxyacetone side chain, which are relatively insoluble in the solvent.
After the enzyme hydrolysis the urine isisaturated with NaHSO₃, followed by extraction with methylene chloride. The NaHSO₃ forms addition compounds with Δ⁴-3-keto-steroids, ketonic and ketogenic medications interfering with the Porter-Silber reaction: These addition compounds are hardly soluble in methylene chloride. With NaHSO₃ the blank values are two thirds of those without NaHSO₃.
In order to increase the specificity in routine analysis, a procedure based on a study by Allen is often conducted. But this procedure can not always be used with urines from patients receiving drugs, because the absorption curves of the drugs and their metabolites in the Porter-Silber reaction are often strongly convex or concave over the range of wave lengths studied.
The Porter-Silber reagent has been improved in order to obtain satisfactory colour intensities for THF_K and Allo-THF_K.
Twenty four hr urine samples are collected with NaHSO₄, resulting in the urine being kept acidic to inhibit the growth of urinary microorganisms including bacteria: Thus the steroids remain unchanged at 20-30° in a dark place for five days.

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Creatinine amidohydrolase was used to determine creatinine in a totally enzymatic procedure. Creatine was produced by hydrolysis of creatinine, and coupled with creatine kinase, pyruvate kinase and lactate dehydrogenase to result in a change in absorbance at 340 nm. Results of studies on each reaction rate in the enzymatic reaction sequence were discussed. The relationship between creatinine concentration and absorbance at 340 nm was linear up to a creatinine concentration of at least 20mg/dl.
Glucose, uric acid, urea, bilirubin and arginine were without significant effects on the accurate determination of creatinine at the concentrations employed. The addition of significantly higher concentrations of ascorbic acid and hemoglobin slightly inhibited the activity of the reaction sequence.
When results of this procedure and of the standard direct Jaffe test were compared using seru mand urine samples, the correlation coefficient was 0.984, but the former were slightly higher. Unlike the Jaffe method, the present method of determining creatinine is rapid, subject to few interfering substances, and requires no serum deproteinization.

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