We hereby would like to describe a highly sensitive chemiluminescence method used for the determination of 3-hydroxybutylate in serum using 3-hydroxybutyric dehydrogenase/isoluminol· micro peroxidase (3-HBDH/isoluminol·micro peroxidase) a procedure for the preparation of protein-free supernatant (Somogyi method).
The method requires only 25μl of pricked ear blood or venous blood and could detect 3-hydroxybutylate ranging from 5-250 pmol/test tube.
The difference values of 3-hydroxybutylate contained in blood have a range of 12-103 (mean 34) μmol/l.
This method is suitable for the efficient screening of the diagnosis and treatment of diabetes.

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We have developed an enzyme-linked immunosorbent assay for glycocholic acid in dried blood spotted on a filter paper.
The linear range of this assay was 1 to 32 nmol/ml and the coefficient of variation of within and between the assay range was 2.6-8.9% and 3.4-8.2%, respectively.
The average concentration of the glycocholic acid value in normal newborn babies was 8.25 nmol/ml, and that in babies with congenital biliary atresia (CBA) was 49.26nmol/ml (n=24).
This method will be applicable in mass screening for CBA.

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The activity of cholinesterase in human serum was determined amperometrically using a dialysis membrane-covered electrode without any interference from such compounds in serum as uric acid and proteins.
The anodic current due to the S-butyrylthiocholine iodide, the substrate, appeared in the potential range more positive than+350mV, while that due to thiocholine, the product, appeared more positive than+100mV and reached a limit of approximately 400mV at pH 7.3. The current derived from a solution containing butyrylthiocholine and cholinesterase at 300 mV and pH 7.3 increased linearly with time and the increase in current per min was proportional to the amount of cholinesterase. On the basis of this result, the activity of cholinesterase in serum was determined in a similar manner as above. Relative standard deviation (RSD) for this measurement (n=5) was 2.65%. The activity of cholinesterase measured by this method agreed well those made by a photometric method using cholineoxidase (r=0.998).

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Neonatal mass-screening for the diagnosis of 21-hydroxylase deficiency has been carried out by measuring 17-hydroxyprogesterone (17-0H-P). It is well recognized that results of the direct measurement of 17-OH-P are much higher than the corresponding results following solvent extraction, and this discrepancy is most marked in premature babies. It is considered that the cause of this discrepancy is because of existance of cross-reactive substance (s) with antibody in serum of premature babies. To investigate the cross-reactive substance (s), serum from premature baby was extracted and applied to high-performance liquid chromatography (HPLC). Eluates were collected and assayed by 17-OH-P enzyme immunoassay (EIA) to investigate fraction (s) which was reacted with the antibody used in the EIA. The results revealed that 17- hydroxypregnenolone, 17-hydroxypregnenolone 3-sulfate and pregnenolone 3-sulfate were crossreactive substances in the EIA. In the above experiment, an unknown cross-reactive fraction was observed. To investigate this substance, the fraction was applied to GC/MS analysis. As a result, 16-dehydropregnenolone 3-sulfate was identified by the GC/MS analysis. To our knowledge, this is the first report to identify 16- dehydropregnenolone 3-sulfate in human serum.
In conclusion, cross-reactive substances in our EIA were 17-hydroxypregnenolone, 17-hydroxypregnenolone 3-sulfate, pregnenolone 3-sulfate and 16-pregnenolone 3-sulfate.

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A second antibody solid phase enzyme immunoassay for dexamethasone was established. Second antibody was immobilized onto microtiter plate by physical adsorption method. Second antibody immobilized plates were stable at least for one month at 4°. Anti-dexamethasone antiserum was obtained by immunizing rabbits with 4-(carboxymethylthio) dexamethasone-bovine serum albumin conjugate. Alkaline phosphatase, a labeling enzyme, was conjugated with 4-(carboxymethylthio) dexamethasone. Cross reactivity for cortisol which is considered as a candidate of interfering substance in serum with the antibody was 0.3%. Intraand inter-assay coefficients of variation for dexamethasone in human serum were 1.2-3.6% and 2.6-9.6%, respectively. Minimum amount of dexamethasone detected was 1.55pg/well and measurable range was 1.55pg-10ng/10μl serum. Chronological changes of serum dexamethasone in 4 normal subjects after an oral administration of 1mg of dexamethasone are also reported.

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A highly sensitive and selective method using high-performance liquid chromatography for simultaneous mesurement of aluminum and iron in human serum of patients on chronic hemodialysis is described. Serum aluminum and iron are liberated by acid hydrolysis followed by deproteinization, and the chelates of the metal ions with 2, 2'-dihydroxyazobenzene are formed. A 100μl of the solution is injected into the HPLC column. The metal chelates are separated on an ODS column in an ion-pair reversed-phase mode and detected by the spectrophotometer. The proposed method enables sufficient recovery and reproducibility, and consists of a simple, sensitive and selective method for serum aluminum and iron measurements. A good agreement exists between this method and conventional method, based on graphite furnace atomic absorption spectrophotometry for aluminum and spectrophotometry for iron, respectively. It was confirmed that patients on chronic hemodialysis are prone to hyperaluminemia and hypoferrism. Further more, a negative correlation was observed between the contents of aluminum and iron in their sera.

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A new method for the determination of catecholamine metabolites in body fluids by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection is described. Plasma and cerebrospinal fluid were deproteinized with perchloric acid, and urine was diluted with 50 mmol/l phosphate buffer. Fifty-microliter portions of the resulting test solutions were then routinely injected directly onto the HPLC column. In addition, the gradient elution mode was used in order to except the organic solvent extraction of the metabolites. The method used was therefore very simple and accurate.
Some of the urine sample metabolites in the diluted test solution proved to be unstable under acidic conditions at room temperature; optimization of various analytical parameters was therefore required.
This simple and precise method for the determination of catecholamine metabolites in body fluids should prove to be of considerable use clinically beyond its application to screening for the extreme abnormal high value case such as neuroblastoma.

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The activity of alkaline phosphatase in serum was determined by measuring the increase in current for a given time due to the oxidation of phenol, as enzymatically released from phenyl phosphate, with a dialysis membrane-covered glassy-carbon electrode. The activities determined amperometrically for thirty serum samples were in excellent agreement with those determined using a colorimetric method (correlation coefficient of 0.996). The electrode is free of deactivation of electrode surface by the protein adsorption typically encountered when an electrode without a dialysis membrane is employed. The electrode can be used repeatedly without appreciable degradation of its sensitivity. The present method is applicable to samples containing significant amounts of oxidizable substances.

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We determined levels of squamous cell carcinoma (SCC) antigen in sera obtained from three patients with sustained high levels but without evidence of carcinoma. High-performance liquid chromatography (HPLC) revealed two to three SCC antigen peaks, one of which eluted at the authentic molecular weight, and the others at a larger one. The sera were not adsorbed by antibodies to IgG, IgA or IgM, and were not inactivated by heating at 56° for 30 min. We demonstrated that the rise in serum SCC antigen in patients without carcinoma could be attributed to the larger molecular weight forms.

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Screening for congenital adrenal hyperplasia due to 21-hydroxylase deficincy in newborns is commonly accomplished in Japan by measuring 17 a-hydroxyprogesterone (17-OH-Δ⁴P) levels in blood impregnated on filter paper with enzymelinked immunosorbent assay (ELISA).
We found that some of the screening tests for 17-OH-Δ⁴P used have been influenced by the temperature at which the immuno-reaction of ELISA is performed. When filter paper blood specimens obtained from neonates were tested at lower immuno-reaction temperatures. significantly increased mean 17-OH-Δ⁴P values were obtained. This phenomenon was observed for screening tests using two commercially available kits and also for an assay system in which antiserum raised in a rabbit against 17-OH-Δ⁴P -bovine serum albumin (BSA) was used.
17 α-hydroxypregnenolone-3-sulfate (17-OH-Δ⁵P-S) is known to be abundant in the blood of newborns, and the antisera employed in the ELISAs noted above have weak affinity for this steroid. Therefore, cross-reactivity of 17-OH-Δ⁵P-S was determined at various immuno-reaction temperatures using 17-OH-⁵P-S standard filter paper specimens prepared from steroid-free blood.
The correlation between 17-OH-Δ⁴Plevels obtained by ELISAs for neonates specimens and immuno-reaction temperatures used may be attributable to variation in cross-reactivity of the 17-OH-Δ⁵P-S contained in the blood of neonates to antibodies used in the ELISAs.

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The apoB gene is the principal structural protein of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) and correspondingly plays an important role in the metabolism of cholesterol and triglyceride. The 3'-flanking region of the apolipoprotein B (apoB) gene contains a hypervariable region comprising a variable number (29-57) of tandemly repeated units. Each unit comprises 14-16 bp. The polymorphic region was amplified by the polymerase chain reaction (PCR) using primers flanking this region. The lengths of products were visualized as bands by ethidium bromide staining. Each band showed polymorphic alleles.
We have typed alleles from patients with hypercholesterolemia, hypertriglyceridemia and hyper-LDL cholesterolemia. The distribution of alleles in these patients is not different from that in a control population. This result suggests that the apoB 3'-flanking region does not contribute significantly to control of serum lipid level.

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Dibucaine- and fluoride-inhibition on serum cholinesterase (ChE: EC 3.1.1.8) have been given with several substrates. When using DMBT (2, 3-dimethoxy thiobenzoyl choline) as substrates, inhibition by dibucaine seemed to be less effective. TIPA (tetraisopropylpyrophosphoramide) which is known as one of the most potent inhibitors does inhibit cholinesterase activities severely and yet presents, some residual activities under DMBT conditions. These conditions suggests that DMBT may serve as another unknown esterase substrate.

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