A simplified colorimetric assay of pancreas-specific lipase in plasma as well as duodenal juice using α-naphthyl palmitate as substrate was developed for the clinical use. The method described by Whitaker was revised by adding deproteinization step, using standardized Fast violet A and increasing alkaline amount in the color development. By sephadex G-200 column chromatography of plasma and duodenal samples, the lipase activity measured by the present method was approximately parallel to that determined by C14-triolein as substrate, which should express pancreas-specific lipase activity. Lipoprotein lipase and trypsin did not show any activity by the described method. Only minor activity was noted for the commertial pseudocholine esterase and liver esterase. However, in hepatitis with marked elevation of plasma GOT or GP r, normal level of plasma lipase was observed, and no elevation of lipase activity was found in plasma with elevated pseudocholine esterase activity. In acute pancreatitis, plasma lipase activity was elevated by 7-30 fold and the elevation seemed to be greater than that of amylase. In subjects with hyperamylasemia above 300 Somogyi U/dl, abnormally elevated lipase activity was found in 87% witti significant correlation (r=0.54) between the two enzyme activities. These data indicate that the described method for lipase determination is simple and expresses pancreas-specific lipase activity in plasma as well as in duodenal samples. These determinations may be useful to evaluate and clinically follow the exocrine-pancreatic function.
A simplified method for the determination of serum dehydroepiandrosterone sulfate (DHEA-S) by acid fluorescence reaction is described. DHEA or DHEA-S produces strong fluorescence by treatment with acetic acid-sulfuric acid (4: 6v/v) at 100° for 10 minutes. The serum sample was deproteinized with 1.5 M perchioric acid and the supernatant was heated at 100° for 40 minutes. The hydrolyzed steroids were extracted with methylene chloride, washed with sodium hydroxide and evaporated to dryness. The residue was submitted to DHEA determination by acid fluorescence reaction. Correlation between urinary 17-KS and serum DHEA-S was studied and high correlation was observed (r=0.745). Serum DHEA-S can be an indicator of androgenic steroid metabolism as a substitute of urinary 17-KS.
Recently, serum bile acid determination was proposed as one of the highly sensitive tests of liver function. In our study, fasting serum bile acid (FSBA) measurement by enzymatic and fluorimetric method was performed on 12 normal subjects and 134 patients with various hepatobiliary diseases. A Correspondence was made between FSBA and other conventional “liver function” tests, in which contain mithochondrial GOT (m-GOT), ornithincarbamyl transferase (OCT), s-GOT, s-GPT, γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (Al-P) and serum total bilirubin (T-Bil). FSBA levels were elevated in almost all patients with repatobiliary disease and most significantly correlated with serum total bilirubin. On the clinical course of acute viral hepatitis, FSBA level was shown a more sensitive index than other “liver function” tests such as s-GOT or s-GPT for liver dysfunction. Other major advantage of FSBA determination was found in several hepatobiliary diseases. Namely, in the case of chronic hepatitis with active type FSBA level was higher than that of inactive type. However, FSBA levels in both types were lower than that of liver cirrhosis. And also, FSBA levels were elevated in both patients with intra- and extra-hepatic cholestasis, but the level of m-GOT, OCT, r-GTP and Al-P were higher in the patients with intrahepatic cholestasis than those of other chronic liver diseases. It indicates that the grade of hepatic cell injury of chronic intrahepatic cholestasis was impaired markedly. To estimate not only FSBA but also m-GOT, OCT, γ-GTP, Al-P and serumbilirubin seems to be helpful for differencial diagnosis of various hepatobiliary diseases. Furthermore, it is worthy of notice that FSBA levels were slightly elevated even in the cases of asymptomatic gallstone and fatty liver without cirrhosis.
Quantitation of the concentration of retinol-binding protein (RBP) and prealbumin in plasma and ascites from patients with cancers, malignant tumors and some diseases has been determined by the method of single radial immunodiffusion (SRID). The results showed that the levels were significantly different in those patients with cancer, malignant tumor compared with those with other benign tumors and diseases. The concentrations of plasma RBP and prealbumin in cancers and malignant tumors were shown to be significantly lower compared with those with other benign tumors, diseases and normal subjects. In addition, the plasma RBP and prealbumin are present in higher concentrations in patients with human malignant glial tumors (such as glioblastoma multiforme) and Cushing's syndrome in comparison to those concentrations with other benign brain tumors (such as meningioma), brain diseases and normal subjects. The levels of RBP and prealbumin in carcinomatous ascitic fluid (cytology-positive) were shown to be significantly higher compared with those with other carcinomatous ascitic fluid (cytology-negative). In addition, the following results were obtained in a analysis of the correlation of plasma sialic acid, C-reactive protein (CRP), 3. 5. 3.'-triiodo thyronin (T3) and carcinoembryonic antigen (CEA) on the plasma RBP, prealbumin levels in patients with cancers and malignant tumors. In cancers and malignant tumors, the plasma levels of sialic acid, CRP, and CEA incerased by the according to decrease in levels of RBP and prealbumin, and plasma T3 levels correlate with concentrations of RBP and prealbumin.
Incubation of fresh human erythrocytes with adenine and inosine in isotonic NaCI-phosphate buffer (PBS) containing glucose at 37° for 3-4h, yielded the high-ATP cells with 2-3 fold increase of ATP contents, while that of the erythrocytes with inosine and pyruvate under the same condition as above yielded the high-2, 3-diphosphoglycerate (DPG) cells with 4-7 fold increase of DPG contents. Similar Incubation with all these constituents together gave the high-ATP-DPG cells with similar increase of both ATP and DPG contents. The shape of these treated cells observed under scanning electron microscope was quite normal. The raised ATP level of high-ATP and high-DPG cells was maintained almost constant upon incubation at 37° in glucose-PBS for 4 h and decreased moderately upon preservation at 4° for 4 weeks. In contrast, the raised DPG level was decreased upon incubation or preservation at the normal or higher rate, although higher-than-normal amount of DPG was still kept in the cells stored at 4° for 4 weeks, due to the very high initial DPG level. The rates of decrease of ATP and DPG in the high-ATP-DPG cells were almost the same as the ATP decrease of the high-ATP cells and the DPG decrease of the high-DPG cells, respectively, indicating that the increased ATP and DPG in the high-ATP-DPG cells were metabolized independently without interfering each other. The amount of lactate produced and the medium pH drop during the preservation of these treated cells were similar to those of the untreated cells. The diminished DPG level in the ATP- and DPG-depleted erythrocytes, prepared from fresh cells by incubating them in PBS at 37° for 36 h, was improved by the treatment of the cells with adenine, inosine and pyruvate in glucose-PBS. Such a rejuvenizing treatment, however, failed to improve the decreased ATP level and osmotic resistance, and the sphero-echinocytic shape in the case of the cells extremely depleted of ATP. These evidences suggest that initial treatment of the fresh cells to raise the ATP and DPG level may be more effective in keeping the freshness of the cells in the course of the long period of the storage than the rejuvenizing treatment of the stored cells already extremely depleted of these components.
The Iterative Truncation Method, a generalization of Hoffmann's method, has been widely applied to determine the normal range, but it has some defects; (1) danger of eliminating normal data,(2) smaller estimation of standard deviation,(3) little consideration on the types of distribution. Supposing theoretical normal distribution, it is made clear mathematically on the estimation of standard deviation that the bias is so large and k (truncation limit factor) smaller than √3 leads to zero estimation.
Norepinephrine (NE) and dopamine (DM) in night and daytime urine of normal subjects (n=45 for men and n=22 for women) were measured simultaneously by the fluorescamine method using HPLC. The excretion rates of NE in the nitghttime were 14.1±6.9 ng/min for men and 11.1±6.5 ng/min for women without sex difference. The values in the daytime were two or three times as high as those in the nighttime. Men excreted more DM in the daytime than in the night while women excreted less DM in the daytime. The excretion rates of DM in the nighttime were 181.0±94.4 ng/min for men and 166.3±70.9 ng/min for women, which correlated well with those of creatinine (γ=0.671 for nighttime and 0.797 for daytime). The nighttime urine was found to be useful for the clinical diagnosis.
We have established a new method for the determination of sialic acid using sialidase combined with NANA-aldolase. Sialic acid was cleaved to N-acetyl-D-mannosamine and pyruvic acid, following pyruvate was measured by means of LDH with NADH as a change of absorbance at 366 nm. Sialic acid concentration was determined with N-acetyl-neuranimic acid as a standard. Sensitivity and precision are excellent, linearity extend to 200mg/dl of sialic acid concentration in serum. The result of this method were paralleled with those of manual sialidase-thiobarbituric acid procedure. Serum sialic acid concentrations in various diseases were observed. In the patient with acute myocardial infarction, high levels of sialic acid in sera had kept until 34 days after the attack. These data clearly indicate that the following determination of sialic acid provides a more sensitive index of the presence of histologically acute myocardial infarction than the other biochemical tests such as CPK and LDH.
In a cause of studies on constituents in humann serum, the spots of the corresponding cholesterol (FC) and cholesterol ester (CE) in thin-layer chromatography for the quantitative analysis by dual wave-length thin-layer chromatography-densitometry have been studied. The human sera were treated to separate into individual components by means of column chromatography, thin layer chromatography and preparative gas chromatography. The structure determination of components were performed on the ground of chemical and spectraum evidence. The chemical treatment was shown in the text. The reduction products of cholesterol ester (CE) with lithium alumium hydride (LAH) were easily analyzed by gas chromatography.
Enzymatic determination of serum uric acid using uricase (EC. 1. 7. 3. 3) and NADH-peroxidase (EC. 1. 11. 1. 1.) is described. The principle of this method is as follows: Uric acid is degradated by uricase to produce allantoin and H2O2, and the H2O2 produced is coupled with NADH-peroxidase. The change in absorbance at 340 nm is measured. Standard curve was linear up to, at least, 20mg/dl in concentration. Correlation between present method and UV method (assay at 291 nm) was compared, and the correlation coefficent value was 0.998 and regression line was y=1.00x-0.08 (y: present method).
Some problems on the determination of lipoperoxides in serum by TBA reaction were described. Reproducibility on this method with 10 samples was good, as CV=3.4%. The relationship between 1, 1, 3, 3 TEP cocentration and fluorescence at 553 nm (Em) was linear up to at least n mole of TEP. However, in serum sample, it was linear up to a serum volume of 50μl. Deproteinization on this method was almost complete. To rinse the precipitant, water and 1/12 N H2SO4 were suitable and one rinse was enough. 0.34% TBA in 50% acetic acid showed maximum fluorescence on TBA reaction. On the timecourse of TBA reaction, 1, 1, 3, 3 TEP and serum showed different response. Stability of lipoperoxides in serum did not change up to 6 days at 4°. New peak by bilirubin was found at the wavelength 580 nm (Em) after TBA reaction. We found the same peak by Bilirubin Control as serum. As the wavelengthis very closed to 553 nm, fluorescence spectra should be checked when the bilirubin level in serum is high. Lipoperoxides values and GOT, GPT, Al-P, γ-GTP, cholesterol, Triglycerides values in various sera were determined. In sera of hyperlipidemia, high lipoperoxides levels were observed though their enzyme levels were low. Significant correlation was observed between lipoperoxides values and cholesterol values (n=50, r=0.83) than that of TG.
Clinical evaluation of urinary dehydroepiandrosterone using 3β-hydroxysteroidoxidase is described. Simplicity of the method than other column method makes it possible to treat manysamples without use of radioactive compounds. Patients with adrenal tumor and other adrenogenital syndrome and some other diseaseare tested with present method. Results of comparison with other method are also good, and this method seems to begood diagnostic tool for some adrenal disease.