To find a method for separately extracting sulfates and glucuronides conjugates of 17-oxosteroid (17OS) by ion pair extraction, we made 17OS conjugates react with six quarternaryammonium ions at pH 9, 0, and extracted the resulting ion pairs of 17OS sulfates or 17OS glucuronides with quaternaryammonium ions. As a result, we obtained a method that causes 17OS sulfates and 17OS glucuronides in sample to react with tetrapentylammonium ions (TPA), extracts the produced ion pairs of 17OS sulfates and TPA with benzene, then addes sodium sulfate to the remaining sample, and extracts the ion pairs of 17OS glucuronides and TPA with dichloromethane.
To determine 170S sulfates and glucuronides, which separately extracted by ion pair extraction as described in the previous report, by high-performance liquid chromatography using a reversed phase Capcell-Pak C8 column, we conducted experiments on labeling condition of the steroids with dansylhydrazine and on chromatographic condition for the separation of the labeled steroids. As a result, we found a method that obtained much more labeled steroids under lower temperature than already reported methods, and obtained three mobile phase solutions that can separate individual steroid. The labeled 170S sulfates were perfectly separated by using methanol-0.5% (w/v) sodium acetate-50% (v/v) acetic acid (57: 42: 1, v/v) at 30°C. The labeled 170S glucuronides were also clearly separated by using methanol-0.03%acetic acid (v/v)(46: 54, v/v) and methanol-0.01% acetic acid (v/v)(56: 44, v/v) at 55°C.
An automated method for the determination of pranoprofen, an anti-inflammatory drug, in human serum has been developed by means of column-switching high-performance liquid chromatography with ultraviolet detection (247 nm).A bovine serum albumin-coated ODS column (MK precolumn BSA-ODS) and a reversed phase column (TSK gel ODS-80TM) were used as the sample preparation column and analytical column, respectively. A twotimes diluted serum sample (20 μl) was applied onto the sample preparation column. The detection limit of pranoprofen is 0.05?Eg/ml serum. The recovery of pranoprofen added to serum (2.0-8.0 μg/ml) was approximately 100% with a standard deviation of 3% or less.
A sandwich ELISA system was developed using two monoclonal antibodies (JK- 199 and JK-132) against type IV collagen. The type IV collagen was determined in sera from liver diseases and healthy volunteers to assess relation of the protein level with clinical manifestations. Type IV collagen concentration in sera of healthy volunteers (n=100) was 0-65 ng/ml, but was elevated up to 480 ng/ml in sera of patients with liver cirrhosis. Type IV collagen concentration in sera of patients with liver disease showed no correlation with the serum concentrations of GOT, GPT, Γ-GTP and PTV, indicators of liver diseases. Either type IV collagen or PIIIP level was increased in sera from all the cases of hepatic carcinoma and/or liver cirrhosis compared to normal cases.
A simple and sensitive high performance liquid chromatographic determination method for free polyamines and their acetylated derivatives was developed. Polyamines were separated on an octadecyl polymer column with isocratic elution. The separated polyamines were introduced into an enzyme reactor containing immobilized acylpolyamine amidohydrolase, amine oxidase (flavin-containing) and putrescine oxidase, in which they were deacetylated and oxidizld to generate hydrogen peroxide. The amount of hydrogen peroxide generated was then determined with an electrochemical detector. When a mixture of nine pure polyamines, i.e., putrescine, acetylputrescine, cadaverine, acetylcadaverine, spermidine, N1-acetylspermidine, N8-acetylspermidine, spermine and acetylspermine, was analyzed with this method, a linear dose response curve up to 1 nmol was obtained for each polyamine except for spermine, which showed linearity in the range of 50 pmol to 1 nmol. The detection limit (S/N>5) for six of polyamines was 1 pmol, but for N8-acetylspermidine and acetylspermine was 10 μmol and for spermine was 30 pmol, respectively. The coefficients of variation were in the range of 1-3%, except those for spermine and N8-acetylspermidine, which were 6.5% and 8.7%, respectively. This method can be applied to the quantitative determination of individual polyamines in clinical specimens such as urine
The superoxide generated from neutrophils in peripheral blood and synovial fluid of rheumatoid arthritis (RA) was measured by flow cytometry and compared with those of healthy controls. Fluorescence intensity of resting neutrophils in peripheral blood and synovial fluid of RA was significantly higher than that in healthy controls. Phorbol myristate acetate (PMA) stimulated neutrophils from the healthy controls seemed to produce the same degrees of superoxide to those from RA. The superoxide generation from normal neutrophils was stimulated by synovial fluid of RA. It suggests that neutrophils of RA were continuously stimulated by same factors in periphral blood and synovial fluid and generate superoxide.
Serum from a male patient gave a unreliable result in two-site immunoenzymometric assay (EMA) kit, Enzymun Test CEA (Boehringer Mannheim) in which mouse monoclonal antibody for specific carcinoembryonic antigen (CEA) was used. Using other kits employing the same method using mouse monoclonal IgG also showed the simillar results; however, normal value of 2.1 ng/ml was obtained by conventional radioimmunoassay for CEA. The interference was eliminated by either heating (70, 15min), or by adding mouse IgG, it's fragment Fab or Fc in reaction buffer. Futhermore, patient's serum treated with antibody against the human IgM or treated with protein A gave decreased content of CEA. In fractionation of patient's serum by Sephacryl S-300 gel chromatography, the CEA activity was detected on the first peak which contained predominantly IgM. In addition, mixed type cryoglobulins containing IgG and IgM were separated from the serum and CEA activity was also detected in it. It is concluded that patient's serum IgM binds to mouse IgG and have the capability of interfering in immunoenzymo (radio) metric assay using mouse monoclonal IgG.