The effect of chymotrypsin inhibitors on the intestinal absorption of insulin was investigated in conscious and unrestrained rabbits. Insulin at a dose of 25U/kg with 10mg/kg of chymotrypsin inhibitors was administered intraduodenally via an indwelled catheter. The blood glucose level was decreased following an administration of insulin in combination with each chymotrypsin inhibitor. The order of the effect on the intestinal absorption was related with the order of chymotrypsin inhibitory activity. Maximal decrease was observed when insulin was administered with such a strong inhibitor as FK 448, [4-(4-isopropylpipe-radinocarbonyl) phenyl 1, 2, 3, 4-tetrahydro-1-naphthoate methanesulfonate], whose IC50 value was 7×10-7M, and the decrease of the glucose level was 25%, compared with the level before administration. The effect of inhibitors which inhibit both chymotrypsin and trypsin was almost the same with that of chymotrypsin specific inhibitors.
Elastase activity in human serum was exerted in two different modes when it was determined by a fruorometric method in time-course manner: that is, when the elastase activity was higher in some patients, the enzyme activity showed no lag phase, while when it was lower in other patients, it showed some lag phase in the enzyme activity expression. Addition of exogeneous elastase in vitro to human serum showed the increase in elastase activity in the serum as the lag phase manner. The relation between the elastase activity and the α1-antitrypsin (the inhibitor of the enzyme) level in pancreatomized patients was examined, and it was found that serum elastase once increased after the pancreatomizing operation was lowered as the α1-antitrypsin level increased. The relation between the elastase activity and the inhibitor (α1-antitrypsin and α2-macroglobulin) levels was examined in connection with aging. In contrast with above pancreatomized and pancreatitic cases, both the elastase activity and the inhibitor levels were lower in the aged persons. It can be assumed that elastase activity in human serum is present as inactive form with the inhibitor, which is of regulating the elastase activity in serum and maintaining the activity at a certain level for physiological requirements.
Female rats of Lewis/sea strain were sensitized with a mixture of the homogenate of brain or spinal cord of guinea pigs and Freund's complete adjuvant in order to evoke experimental allergic encephalomyelitis (EAE), and change in the lysosomal enzyme activities, such as those of β-glucuronidase (β-G) and arylsulfatase-A and -B (AS-A and -B), in the brain was investigated. It was found in the present study that I) the elevation of exogenious lysosomal AS and /β-G activities in rat brain increased as EAE developed, II) significant change in both enzyme activities within the lysosome was not observed, III) increase of AS-B activity was remarkable, while AS-A activity did not change in the brain, IV) cerebral AS and β-G activities increased significantly in the EAE-induced rat, and V) at the time of EAE onset, elevation of enzyme activity and cerebral lesion were seen in parallel manner. Thus, possibility of significant change of the lysosomal enzyme activity in EAE-induced animals was suggested as EAE developed. Increase in total AS activity during the development of EAE might be attributable to a particular increase of AS-B activity, suggesting that the elevation of AS-B activity correlated to abnormal metabolism of the sulfate-containing glycosaminoglycan occuring in the myelin in EAE.
An enzyme immunoassay (EIA) method for determination of human urinary kallikrein has been established. At least 5ng of kallikrein per ml urine could be determined, and reliabilty of this method was confirmed by the reproducibility, the dilution test and the recovery test. Several properties of the urinary kallikrein were shown by this method. When the urine was treated with trypsin, the amount of kallikrein increased by about 2-to 5-fold. With both control and the trypsin-treated urine, a linear relationship was obtained between the amount of kallikrein and kallikrein activity, and the slope of the line with control urine was less than that with the trypsin-treated urine. Kallikrein, prokallikrein and trypsin-activated prokallikrein formed a smoothly fused precipitin line by the reaction with rabbit antihuman urinary kallikrein antiserum in Ouchterlony's double diffusion experiment. These results indicate that only the active form of kallikrein is detectable by the EIA method, although both kallikrein and prokallikrein are reactive toward the anti-kallikrein antiserum. It is also indicated that about 50-75% of kallikrein exists as prokallikrein in the urine.
The uptake mechanisms of indocyanine green (ICG) were studied with rat native hepatocytes and denatured ones which were treated at 56° for 10 minutes. The addition of several receptor blockers except aggregated IgG to the reaction system was not able to impair the uptake. Although metabolic inhibitors had no affect on the uptake, it was drastically reduced at low temperature. The denatured hepatocytes also eliminated ICG from reaction system rapidly rather than the native cells and it was inhibited at low temperature. The kinetic analysis of the ICG uptake by double reciprocal plots of Lineweaver-Burk showed the 4.76nmol/min/106 cells of Vmax value and 0.15mM of Km value for native hepatocytes but the line of denatured cells crossed at the origin, i. e., no Vmax and Km value was given as the simple chemical binding of ICG. Subcellular fractionation of both the native and denatured hepatocytes after incubation with ICG indicated that ICG was distributed from membrane to lysosomal fraction in native cells but the most ICG was found in the membrane fraction in the case of denatured cells. Thus the uptake by denatured hepatocytes was not true one but the binding of ICG to the cell membrane.
A simple, rapid and sensitive method for the determination of valproate in serum by isotachophores is (IP) has been established using a potential gradient detector and an isotachophoretic migration tube consisting of a pre-separation tube (6cm×1.0mm i. d.) and a separation capillary tube (15cm×0.5mm i. d.). The leading electrolyte was hydrochloric acid (5mM) adjusted to pH 4.3 with 6-amino-n-caproic acid. To the leading electrolyte was added 0.1% hydroxypropylmethylcellulose to reduce electroendosmosis. 2-(N-morpholino) ethanesulphonic acid served as the terminating electrolyte. Valproate was quantitativery separated without interference by such serum constituents as lactate and phosphate. Ten microliters of serum was injected directly into the apparatus. The analysis time was dramatically reduced by removing chloride ions from the serum sample in the pretreatment procedure with silver carbonate. The calibration curves (zone length-concentration plottings) for valproate in serum were found to be linear and to pass the origin in a concentration range of 1-300μg/ml. The present method was successfully applied to routine measurement of valproate quantity in serum and the results were found to agree fairly well with those obtained with GLC and HPLC.
We have developed an improved method of Hunninghake for the determination of carbamylated plasma protein (CPP) in patients with renal failure. According to our method, CPP values were significantly higher in patients with renal failure than in normal subjects. In patients with renal failure, significant correlations were found between CPP levels and BUN. Thus, CPP appears to be a new indicator of the status of renal failure not only in nondialysis patients but also in dialysis patients.
Changes in the arylsulfatase (AS) activity in the brain were detected in the experimental allergic encephalomyelitis (EAE) using ascorbate-2-sulfate (AAS) as a natural substrate for AS-A. Catecholamine (CA) levels in the brain in EAE rats were also detected. It was found as the results that 1) AS-A hydrolyzing a natural substrate AAS, was not different in the EAE rats from the normal ones. 2) Total AS hydrolyzing a synthetic substrate, 4-methylumbelliferyl sulfate (4-MUS), increased after EAE indction. 3) Only norepinephrine (NE) level among CA levels of the brain in the EAE rats was specifically lower. 4) It may be assumed that only AS-B incrases in the EAE rats, and the disturbance of nor-adrenargic nervous functions can be ascribable to these phenomena.