A correction of temperature coefficient for lactate dehydrogenase (LDH) activity against human sera from healthy, myocardial infarction and hepatitis patients, was discribed earlier2).
When pure forms of LDH isozyme, H₄ or M₄, were used as enzyme sources, it was the highest sensitivity by temperature variation of all samples in this work.
An Arrhenius plot of all samples gave two phase line that it was increased sharply from 25°to 37°C, and more slowly up to 40° as like as in previous report2), but the break occurs around 37°C. From these observation, it is pressumed that the enzyme molecule occurs a conformational change and a variation of heat stability depending upon pH change.
In other hand, percentage of subunits constitution calculated from the ratio of LDH activities using NAD in the presence of 450mM lactate and thionicotinamide hypoxanthine dinucleotide.(TNXD) in the presence of 15mM lactate were identical with that values calculated from enzymograms.
When percentage of subunits constitution was taken into account, it was postulated that the difference of temperature coefficient was reflecting the amount of its constitution precisely.

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A new enzymatic method is described for determination of free fatty acid (FFA) by using acyl CoA synthetase (6.2.1.3).
The principle of this method is as follows: FFA is activated by acyl CoA synthetase in the presence of ATP and CoA, producing acyl CoA, PPi and AMP. AMP thus produced was determined by coupling the reaction in which NADH was oxidized to NAD in the presence of myokinase (2.7.4.3), pyruvate kinase (2.7.1.40) and lactate dehydrogenase (1.1.1.27). The amount of NADH was measured by optical density at 340 nm.
Under these conditions 2 mmoles of NADH are oxidized to NAD when 1 mmol of FFA is activated.
In view of the results so far investigated, 30 mg/l ml of ATP, 6 mg/l ml of CoA and 50μl of the acyl CoA synthetase preparation obtained from rat liver according to the method of Bar-Tana, appeared to be an optimal condition for the reaction.
The standard curve was linear within the concentration of FFA rang from 200μEg/l to 1, 000μEg/l and recovery of FFA from serum specimen was satisfactory when the proposed method was employed.
Since the enzymatic method is specific and simple in comparison with the chemical method, an application of this method for measurment of FFA in a clinical laboratory would be promising.

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Studies are reported here of a colorimetric microdetermination of cholinesterase activity in serum. Acetate hydrolyzed from a substrate by cholinesterase, which was coupled with ATP, acetate kinase, phosphoenolpyruvate and pyruvate kinase, was stoichiometrically converted to pyruvate. This pyruvate produced was colored with 2, 4-dinitrophenylhydrazine in the alkaline solution. The activity of cholinesterase was linear until approximately 20 ul of serum. The relationship between this assay and a phenol red method was close (r=0.88). A normal value of the cholinesterase activity was 0.9±0.18 units by the present assay.

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Concentrations of adenine nucleotides in rat brain and liver have been determined spectrofluorometrically by the coupled enzymatic reactions and the value of energy charge ([ATP]+0.5[ADP]/[ATP]+[ADP]+[AMP]) has been calculatated. The value of energy charge of normal rat brain and liver was 0.89 and 0.85, respectively.
When rat was inhaled with 100% nitrogen gas, the value of energy charge of the brain decreased rapidly to 0.48 at 2 min after the inhalation. This was due to a rapid decrease in ATP concentration in the brain and a concomitant increase in AMP concentration. On the other hand, that of the liver was reduced slowly to 0.69 at 2 min under the same conditions.
The results indicate clearly a higher affinity of the brain tissue to oxygen than the liver.

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In the urine of subjects given an oral dose of spironolactone, several metabolites have been found. Metabolites which interfere with the estimation of 17-oxosteroids (17-KS) by the zimmermann reaction and 17-hydroxycorticosteroids (17-OHCS) by the Porter- Silber reaction were analyzed by thin-layer chromatography, and the interference of each metabolite with the estimations of 17-KS and 17-OHCS was investigated. In the estimation of 17-KS, the metabolite of spironolactone had a maximum absorption at 610nm. The values obtained by the formula of Fraser give positive errors, but the values are corrected by using the reference absorption. In the measurement of 17-OHCS, since the interference is caused by the reaction of the metabolites with sulfuric acid, the value is corrected by subtracting the blank value.

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The galactose excretions in urine during oral galactose tolerance test were examined in cases of 17 normal, 31 diabetes and 32 liver diseases.
Galactose excretions in urine for 2 hours in diabetes and various liver diseases were remarkable as compared with normal subjects, especially liver cirrhosis showed the most conspicuous excretion of galactose in urine.
There was no significant difference among normal, diabetes and various liver disease, in excreating amounts of urine for 2 hours. The changes of urinary protein, ketone bodies, urobilinogen and glucose during oral galactose tolerance test were semi-quantitatively examined. Protein test was unchanged and ketone bodies test turned to positive only in each 1 case in diabetes and liver diseases. Urobilinogen test generally turned to negative after galactose loading except 2 cases in liver diseases, it should be considered to depend on dilution of urine concentration after loading. Glucose test in urine after loading turned to positive in 7 cases out of 17 cases in normal subjects. This positive grade was higher percentage in diabetes and liver diseases than in normal. On the urinary excreting amount of glucose and galactose, slightly positive correlation was observed in urine after galactose loading.

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Oral galactose tolerance test was performed on the cases of 17 normal subjects, 28 diabetes and 32 liver diseases, and then blood glucose and galactose levels were examined on the positive and negative group in urinary glucose test after loading. The change of blood glucose level in normal cases during the galactose tolerance test was recognized no significant difference between negative and positive group of urinary glucose reaction. The similar tendencies were observed in the cases of diabetes and liver diseases. It was found that urinary glucose excretion during galactose tolerance test was not concerned to elevation of blood glucose level.
Fositive group on urinary glucose during galactose loading showed more significant increasing of blood galactose level as compared with negative group. Especially in absorption period, 30 and 60 minutes after loading, it was remarkable in all cases. Therefore it was showed that glucose excretion in urine during galactose tolerance test had some relations for the elevation of blood galactose level.
The relationship between the increasing of blood glucose-Fgalactose levels and glucose excretion in urine during galactose tolerance test was significantly recognized in 30 and 60 minutes after loading on the cases of normal and diabetes.
Glucose excretion in urine during galactose tolerance test, was mainly affected by the elevation of blood galactose level, but the elevation of glucose + galactose levels had also some participations.

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In order to avoid the interference of aldoses and ketoses in gas chromatographic analysis of sugar-alcohols in urine, a sample preparation was developed using a Dowex 1×8 (OH-) column.
Urinary excretion rate of sugar-alcohols in man was measured by the method after fasting and taking glucose, glucuronolactone or Sake. Whereas the excretion of xylitol was incresed remarkably after administration of glucuronolactone, a slight increase of sugar-alcohols except xylitol was seen, but not in all subjects. The influence of taking Sake was observed on the excretion of erythritol, arabinitol, xylitol and mannitol.

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