The preparation and antigenic properties of 2-hydroxyestrone-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through the 15α position are described. The antiserum raised against this antigen in the rabbit showed a high affinity (K_a=1.3×10⁹M^-1) and excellent specificity to 2-hydroxyestrone, exhibiting little or no cross-reactivities for related steroids with an exception of 2-hydroxyestradiol (14.3%).

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Anti-insulin monoclonal antibody (MoAb) is considered to be useful in terms of its specificity and stable supply for insulin assay when produced by hybridoma method. The spleen cells of male BALB/c mice immunized with monocomponent porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The resulting anti-insuln antibdy (Ab) was purified and characterized with radioimmunoassay (RIA) using ¹²⁵I-porcine insulin and enzyme immunoassay (EIA) of sandwich method using ¹²⁵I-Ab-conjugated beads and β-galactosidase. As reference, anti-insulin polyclonal antibody raised in guineapig (PoAb) was used.
The subclass of this Ab was identified to be IgG 1 by Ouchterlony technique. The EIA using this Ab revealed no enzyme reaction with porcine insulin, in the range of 0 and 12.5 ng/ml, suggesting that the present Ab was MoAb, while dose-dependent increase of the enzyme reaction was obtained using PoAb. In the RIA, this Ab did not cross-react with glucagon, somatostatin, and pancreatic polypeptide. The suitable concentration of this MoAb for RIA was 1: 1,500,000 and the minimum detection level of porcine insulin in RIA using this Ab was 0.5 ng/ml. They were similar to those obtained by application of the PoAb. The binding activity of this MoAb to human insulin was quite similar to porcine insulin. The insulin determined by RIA using this MoAb and PoAb was in good correlation.

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We previously reported a method of fluorometric determination of elastase activity after separating other protein and enzymatic reaction products by trichioroacetic acid.
On the other hand, Substrate of soluble elastin (α-, β-elastin and elastase solubilized elastin) and other protein was not separated by the deproteinizing procedure. We improved above method separating substrate and enzymatic reaction products by dialysis method.
As a result, we could get Km values of porcine pancreatic elastase, human pancreatic elastases 1 and 2 toward α-elastin, which were 0.51, 2, 84, 2.38 respectively and those toward β-elastin were 3.70, 2.24, 2.35 respectively. It may be suggested that porcine pancreatic elastase is tend to digest α-elastin rather β-elastin, but human pancreatic elastases 1 and 2 tend to digest similarly both elastins. Furthermore, we could find that elastase solubilized elastin was also digested by trypsin, chymotrypsin, collagenase and pronase. But insoluble elastin and α-, β-elastin as the substrate were not digested by the above enzymes.
It has been suggested that insoluble elastin is primarily solubilized by elastase and then soluble elastin fragment is digested by other proteolytic enzymes.
The elastase assay presented here should be usefull for the study of elastin metabolism under normal and various disease states.

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An advanced-stage product in the Maillard reaction was determined by fluorometry using high-performance liquid chromatography (HPLC). A peak which could be determined by fluorescence HPLC appeared with a retention time of 12min, increased in height with increasing glucose concentration and incubation time when albumin and glucose were incubated for 4 weeks at 37°C, and did not disappear on pretreatment with NaBH₄ before acid-hydrolysis. Therefore, this peak was considered as an advanced-stage product of the Maillard reaction.

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Many cell types have reportedly been shown to respond to extracellular stimulation with a rapid increase in phosphatidylinositol turnover. However, there has been a discrepancy concerning the relationship between catecholamine secretion and the phosphatidylinositol turnover. The effect of phospholipase C inhibitors including 2-nitro-4-carboxyphenyl N, N'-diphenylcarbamate, spermine and spermidine on acetylcholine-stimulated catecholamine secretion was studied in perifused pig adrenal medulla. Pretreatment of pig adrenal medulla with all the inhibitors employed in the present study blocked the increase of catecholamine output induced by acetylcholine. On the other hand, diacyiglycerol itself was found to release catecholamine from the perifused pig adrenal medulla. These results support the idea that there is a close relationship between phosphatidylinositol turnover and catecholamine secretion.

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Previous investigations from other laboratories have indicated that arachidonic acid stimulates a rapid, dose-dependent release of hormones such as hPL and LH. To investigate the involvement of arachidonic acid in catecholamine secretion, the effect of p-bromophenacyl bromide, a phospholipase A₂ inhibitor, on acetylcholine-stimulated catecholamine secretion was studied in perif used pig adrenal medulla. Such effect was blocked by pretreatment of the tissue with the phospholipase A₂ inhibitor, although arachidonic acid itself caused catecholamine release from the perifused pig adrenal medulla. Exogenous arachidonic acid also induced a release of catecholamine from adrenal medullary cells in culture. Pretreatment of perifused adrenal medulla with nordihydroguaiaretic acid, a lipoxygenase inhibitor, also inhibited the increase of catecholamine output induced by acetylcholine. These results suggest that a certain arachidonic acid metabolite evokes the catecholamine release.

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A high-performance liquid chromatographic method was established for simultaneous determination of zinc-protoporphyrin (ZPP) and free protoporphyrin (FEP) in erythrocytes. These porphyrin were extracted with recoveries about 85% each, completely separated by a reversed ODS column within 10 minutes. The detection limits for ZPP and protoporphyrin (pp) of standards were 0.49 and 0.15 ng, respectively. The normal ranges were estimated to be 13.1-54.0 μg/100 ml RBC for ZPP and 3.9-20.1 μg/100 ml RBC for FEP. The rapidity and simplicity of the method allow its application to the routine analysis of erythrocyte porphyrins in the clinical laboratory.

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