Ml Of Human Serum Research Articles

Determination of Two Antiandrogenic Anthrasteroids in Human Serum by Column Switching High-Performance Liquid Chromatography with Electrochemical Detection

A column switching high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of two modified steroidal antiandrogen anthrasteroids: 3aβ, 6-dimethyl- 2, 3, 3a, 4, 5, 8, 9, 10, 10aβ, 11, 11aβ, 11bα-dodecahydro-1H-cyclopenta[α]anthracene-3, 8-dione (I) and its 3-hydroxy derivative (II) in human serum. After solid phase extraction from serum, the anthrasteroids were derivatized with 2, 5-dihydroxybenzohydrazide during the evaporation process of the reaction mixture at 20°C for 25min. The derivatives were detected electrochemically at +0.45V vs. Ag/AgCl. The detection limits (S/N=3) for compounds I and II in 0.5ml of human serum were 0.5 and 1ng ml-1, respectively. Calibration curves for both compounds were linear over concentration ranges of 2-20ng ml-1 and 20-200 ng ml-1, respectively.

Direct Radioimmunoassay for Haloperidol in Human Serum

A direct radioimmunoassay for the accurate determination of haloperidol in human serum has been developed. Based on recent information about the metabolism of haloperidol, a new haloperidol hapten, in which a (3-carboxypropionyl)methylamino group was attached as a bridge in the place of fluorine atom, was synthesized and coupled to bovine serum albumin through the bridge to provide a new immunogen. Guinea pigs were used for the immunization. Since the antisera obtained by the new immunogen still cross reacted >10% with reduced haloperidol, the immunological tolerance to reduced haloperidol was induced by administration of a copolymer of D-glutamic acid and D-lysine linked with reduced haloperidol. This gave an antiserum in guinea pigs which was highly specific for unchanged haloperidol with negligible cross reactivity (≤1.0%) to any haloperidol metabolites including the newly found ones. With the newly developed antiserum and [3H]haloperidol, serum haloperidol levels can be determined over the concentration range from 0.3 to 20 ng/mL, using 0.1mL of human serum, without an extraction procedure.

Determination of oxytetracycline in urine and human serum by differential pulse polarography

A differential pulse polarographic method for the determination of oxytetracycline in urine and human serum in acid media (HClO4 of pH 2) is proposed. The effects of the amount of sample taken and the concentration of HClO4 present were investigated. The detection limit was 5.5×10−6 mol/l. The standard deviation of the determination of 5.5×10−5 mol/l of oxytetracycline in 2 ml of urine was 1.7×10−6 mol/l and that of the determination of 5.5×10−5 mol/l of oxytetracycline in 2 ml of human serum was 1.9×10−6 mol/l.

Determination of orotic acid (vitamin B13) in human serum and urine by differential-pulse polarography.

A differential-pulse polarographic method for the determination of orotic acid in urine and human serum is proposed. The effects of the amount of sample taken and the concentration of HClO4 present were investigated. The detection limit was 1.54 µg ml–1. The standard deviation of the determination of 15.4 µg ml–1 of orotic acid in 5 ml of urine was 0.50 µg ml–1 and that of the determination of 15.4 µg of orotic acid in 1 ml of human serum was 0.28 µg ml–1.

Liquid-chromatographic determination of five orally active cephalosporins--cefixime, cefaclor, cefadroxil, cephalexin, and cephradine--in human serum.

We report an isocratic "high-performance" liquid-chromatographic (HPLC) procedure for measurement of five orally administered cephalosporins (cefixime, cefaclor, cefadroxil, cephalexin, and cephradine) in 0.1 mL of human serum. Serum protein is precipitated with acetonitrile, the sample is centrifuged, and the supernate is evaporated under nitrogen. The residue is reconstituted in 0.1 mL of mobile phase, and 50 to 80 microL of this is injected onto a reversed-phase Altex Ultrasphere Octyl (C8) column. The five cephalosporins are resolved by elution with a pH 2.6 mobile phase of methanol/monobasic phosphate buffer (20/80) by vol), flow rate 2 mL/min. The column effluent is monitored at 240 nm. Cefixime serves as the internal standard for the analysis of the four other compounds, cephalexin as the internal standard for cefixime. We used two standard curves for all compounds: a low-range curve for concentrations commonly observed clinically and a higher-range curve for higher concentrations. The former were linear from 1.0 to 10 mg/L for cefaclor, cefadroxil, cephalexin, and cephradine and from 0.1 to 1 mg/L for cefixime. The high-concentration curves were linear from 1 to 10 mg/L for cefixime and from 10 to 100 mg/L for the other compounds. The detection limits were 0.1 mg/L for cefixime, 1 mg/L for the other cephalosporins. Mean within-run and day-to-day CVs were always less than 15% for all compounds studied.

Development of enzyme imnmnoassay for serum 13,14-dihydro-15-ketoprostaglandin F 2α

An enzyme immunoassay was developed for a convenient and sensitive assay of 13,14-dihydro-15-ketoprostaglandin F 2α, a metabolite of prostaglandin F 2α appearing in human blood. The compound was chemically conjugated to β-galactosidase from Escherichia coli. The enzyme-labeled antigen was mixed with a sample containing 13,14-dihydro-15-ketoprostaglandin F 2α, and the mixture was allowed to react competitively with the antibody immobilized in a polystyrene tube. The activity of β-galactosidase bound to the antibody was assayed by fluorometry. The enzyme activity was plotted against the amount of authentic 13,14-dihydro-15-ketoprostaglandin F 2α to obtain a calibration curve, and the compound was detectable over a range of 10 fmol to 10 pmol. Prostaglandins were extracted from human serum by the use of an octadecylsilyl silica column, and the extract gave an abnormally high level of 13,14-dihydro-15-ketoprostaglandin F 2α by enzyme immunoassay due to the presence of unidentified interfering substance(s), which was removed by high-performance liquid chromatography (HPLC). The purified material gave a value in the order of 0.1 pmol per ml of human serum. Validity of the enzyme immunoassay was confirmed by radioimmunoassay and gas chromatography/mass spectrometry (GC-MS) of a methyl ester n-butoximedimethylisopropylsilyl ether derivative.

Enzyme immunoassay of 6-ketoprostaglandin F 1α in a solid phase

A solid-phase enzyme immunoassay of 6-ketoprostaglandin F 1α (a stable degradation product of prostaglandin I 2) was developed in which the hapten molecule was labeled with β-galactosidase. The antiserum was bound to a polystyrene tube, and the enzyme-labeled and unlabeled 6-ketoprostaglandin F 1/ga were allowed to react in a competitive manner with the immobilized antibody. The activity of the immunologically bound β-galactosidase was assayed by fluorometry, and correlated with the amount of unlabeled 6-ketoprostaglandin F 1α According to a calibration curve 6-ketoprostaglandin F 1α was detectable in a range of 30 fmol-10 pmol. When 6-ketoprostaglandin F 1α was extracted from human serum by using an octadecylsilyl silica column (Sep-Pak C 18) and the crude extract was subjected to the enzyme immunoassay, the content of 6-ketoprostagland F 1α was 50–60 pmol / ml of human serum. An endogenous substance(s) which disturbed the immunoreaction and gave such an apparently high concentration of 6-ketoprostaglandin f 1α was separated from the endogenous 6-ketoprostaglandin F 1α by HPLC. With the purified 6-ketoprostaglandin F 1α fraction there was a good correlation ( r = 0.994) between enzyme immunoassay and radioimmunoassay. The validity of the enzyme immunoassay was confirmed by gas chromatography-selected ion monitoring.

Comparison of a cytosol radioreceptor assay with a radioimmunoassay for 1,25-dihydroxyvitamin D in serum or plasma

Human plasma and serum levels for lα, 25-dihydroxyvitamin D were determined by a cytosol radioreceptor assay (RRA) and a radioimmunoassay (RIA). For both assays, 1.5 ml of human serum or plasma is used. Prior to RRA or RIA, extraction with benzene is performed followed by ‘high-performance’ liquid chromatography (HPLC) on a silica column (25 × 0.46 cm) with hexane isopropanol (9/1 by vol), to isolate 1α,25-dihydroxyvitamin D from the other vitamin D metabolites. The cytosol receptor was isolated from the intestine of healthy chickens. The antisera were raised in rabbits to 1α,25-dihydroxyvitamin D 3-3-hemisuccinate coupled to bovine serum albumin. The standard curves for RRA and RIA are prepared with 1α,25-dihydroxyvitamin D 3. 1α,25-dihydroxy[ 3H]vitamin D 3 of high spec act (158 kCi/mol) is used as tracer. The reactants are incubated for 16 h at 4°C. Then, bound and free ligand are separated after the addition of dextran-coated charcoal. Both assays have a sensitivity of 2 pg/tube. The cytosol receptor and the antibodies have about the same absolute affinity for 1α,25-dihydroxyvitamin D 3 but the cytosol receptor has a higher relative affinity for 1α,25-dihydroxyvitamin D 3 (compared with other vitamin D metabolites). Reproducibility and precision are better for the RIA. The between-and within-assay CVs are 16.0% ( x = 58.7 ng/l , n = 16) and 11.2% ( x = 52.1 ng/l , n = 15), respectively, for RRA and 12.6% ( x = 61.8 ng/l , n = 27) and 7.4% ( x = 61.8 ng/l , n = 15), respectively using RIA. Reference values obtained by both assays on healthy males and healthy premenopausal females are the same for both sexes; 53.9 ±31.0 ng/l ( n = 46) using RRA and 51.8 ± 30.2 ng/l ( n = 91) for RIA (mean ± 2 sd).

Simple method for preparing and quantifying very-low-density lipoprotein.

A micromethod is described for isolating and quantifying human very-low-density lipoprotein (VLDL) from 0.1 mL of human serum, by which more than 100 samples can be processed in half a day with a standard ultracentrifuge. Replicate analysis of 10 normal and hyperlipemic samples gave median coefficients of variation of 11.7% for VLDL cholesterol, 5.1% for VLDL triglycerides. Results obtained by micro-ultracentrifugation and enzymic cholesterol measurement agreed closely with those measured with an electrophoretic method (Pearson's r = 0.98). Compared with the established method, on the other hand, lower values were found for both VLDL cholesterol (r = 0.89) and triglycerides (r = 0.97).

Quantitative determination of praziquantel in serum by high-performance liquid chromatography

A simple method for the quantitation of praziquantel by high-performance liquid chromatography is described. This method has a lower limit of sensitivity of 2.5 ng of drug per ml of human serum and a relative standard deviation of 2.6% at concentrations of 5 ng/ml.

High-performance liquid chromatographic determination of free fatty acids with 1-naphthylamine

A 1% benzene solution of oxalyl chloride was added to saturated and unsaturated fatty acids and the mixture was allowed to react at 70° C for 30 min; by this procedure, each fatty acid was converted into its acid chloride in a considerably quantitative manner. By reacting this acid chloride with 1-naphthylamine at 30° C for 15 min, naphtylamine derivatives were produced, which showed strong ultraviolet absorption around 280–290 nm. Experiments were made on the recovery of the fatty acids added to 0.5 ml of human serum, and the recovery was found to fall in the range of 94–106% (coefficient of variation = 0.5–4.1%) when the following amounts of six fatty acids were added: C 14 :0, 2 μg; C 16 :0, 20 μg; C 16 :1, 5 μg; C 18 :0, 4 μg; C 18 :1, 20 μg; C 18 :2, 10 μg.

A simple micromethod for determining human serum cortisol by high-pressure liquid chromatography using 0.1 ml serum

A simple method of high-pressure liquid chromatography for the determination of cortisol in 0.1 ml of human serum is described. Multiple steps of the previous procedures can be simplified by shaking a glass rod for effective extraction of hormones and by performing all procedures on a microscale. The recovery rate was better than that of the previous method and the coefficient of variation was 5.3%. The present method which has a limit of detection of 1–30 ng is particularly useful for the assay of serum cortisol in infantile patients.

Progressive inhibition of human glandular (urinary) kallikrein by human serum and identification of the progressive antikallikrein as alpha 1-antitrypsin (alpha 1-protease inhibitor).

Human urinary kallikrein was inhibited by human alpha 1-antitrypsin (alpha 1-protease inhibitor) in a similar way as by equivalent amounts of human serum. The inhibitor present in the kallikrein-inhibitor complex formed was identified as alpha 1-antitrypsin by two-dimensional immunoelectrophoresis. Under the experimental conditions applied, 90 mIU (= 185 microgram) alpha 1-antitrypsin inhibits about 9 microgram of human urinary kallikrein in 24 h at 37 degrees C, 1 ml of human serum, containing 2-4 mg alpha 1-antitrypsin, inhibits about 70 microgram kallikrein. At an incubation temperature of 25 degrees C, the rate of inactivation is significantly lower than at 37 degrees C. No inhibition was observed at 0 degrees C or when alpha 1-antitrypsin was presaturated with trypsin.

Quantitation of the main metabolites of vitamin D in a single serum sample II. Determination by uv-absorption and competitive protein binding assays

The present report, together with extraction, separation and purification procedures described previously [1], constitutes a sensitive method for determination of vitamin D and its main metabolites in 2 ml of human serum. By omitting vitamin D itself, 25-OHD, 24,25-(OH) 2D, 25,26-(OH) 2D and 1,25-(OH) 2D could be determined in less than 0.5 ml serum. The quantitation methods described have detection limits of 1.8 pmol for vitamin D, 20 fmol for 25-OHD, 24,25-(OH) 2D and 25,26-(OH) 2D, and 1 fmol for 1,25-(OH) 2D.

Isolation and some of the physicochemical and immunologic properties of a platelet adhesion inhibitor from human serum

An inhibitor of platelet adhesion to glass has been isolated from delipidated human serum by a combination of: 1) ammonium sulfate fractionation, 2) affinity chromatography on Affi-gel Blue, and 3) chromatography on Sephadex G-100 and Sepharose-6B. The average yield of platelet adhesion inhibitor from 100 ml of human serum was approximately 10 mg. The platelet adhesion inhibitor at a protein concentration of 5.6 mg/ml inhibited platelet adhesion by 87%. It migrated in the α 1– α 2 region in Microzone electrophoresis and gave a UV-absorption spectrum characteristic of proteins with λmax at 278 nm at pH 7.0 and E 1% 1cm value of 6.25. The platelet adhesion inhibitor is antigenic in rabbits, and antisera to it produced two bands in immunodiffusion and two peaks of immunologic identity in crossed immunoelectrophoresis. Immunologically, the preparations of the inhibitor appear to contain IgG and albumin perhaps in an organized molecular complex. However, IgG alone or in mixtures with albumin over a wide range of concentration and combinations does not inhibit platelet adhesion to glass. Albumin alone in concentrations ranging from 25 to 100 mg/ml inhibits platelet adhesion by about 20%.

Interaction between human pancreatic elastase and plasma protease inhibitors.

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.

Determination of haloperidol in human serum by radioimmunoassay.

A radioimmunoassay has been developed which enables accurate determination of haloperidol in human serum. Antiserum was prepared by immunizing guinea pigs with haloperidol (O-carboxymethyl)oxime derivative (III) coupled with bovine serum albumin. With the antiserum, 3H-haloperidol and dextran-coated charcoal, the assay of haloperidol in serum was possible over a concentration range of 1 to 50 ng/ml, using 0.1 ml of human serum without the need of an extraction procedure. Data obtained by radioimmunoassay are in good agreement with those obtained by gas chromatography. No appreciable cross-reactivity was observed neither with haloperidol metabolites nor with other butyrophenone neuroleptics. Serum levels of haloperidol in schizophrenic patients receiving single oral dosing (6 mg/subject) have also been determined.

Immunoglobulin G independent activation of the classical complement pathway by monosodium urate crystals.

10 mg of monosodium urate crystals reduced the CH50 of 1 ml of human serum by 57% after 30 min at 37 degrees C. C1, C4, and C3 depletion of 52, 68, and 46% were typical of classical pathway activation. C1 binding and activation occurred when urate crystals were incubated with isolated precursor C1, and required the intact macromolecule, C1qrs. Activation of isolated C1 by urate crystals was not diminished by F(ab')2 anti-Fc under conditions in which C1 activation by aggregated immunoglobulin (G) was blocked by the F(ab')2 antibody.

Purification of alpha-2 macroglobulin by immunoadsorbent chromatography

A method for preparation of alpha-2 macroglobulin (α2M) by immunoadsorbent chromatography utilizing rabbit-anti-human α2M-conjugated Sepharose 4B is described. This procedure offers a rapid, simple and inexpensive method for purification of α2M from as little as 2.0 ml of human serum. By multiple applications to the absorbent column, as much as 10 mg of purified α2M may be easily prepared in one day. This technique is especially useful for isolation of α2M from individual sera and for studies of cultured cell supernatants containing human α2M.

High-performance liquid chromatographic assay for 25-hydroxyvitamin D3 in serum.

We describe a precise and specific method for measuring 25-hydroxyvitamin D3 in 1 ml of human serum. Extraction with chloroform/methanol followed by chromatography on a 0.5 X 2-cm silica gel column yields a sample that is sufficiently free of extraneous material for high-performance liquid chromatography on a column of microporous silica gel (10 micron average particle diameter). The measurement is not influenced by vitamins D2 or D3, 25-hydroxyvitamin D2, or any of the more hydroxylated metabolites of the vitamin D group. Results by this method correlate well with a competitive protein-binding assay (r = 0.961), but with a negative bias of 6.9 +/- 3.3 microgram/liter. We measured concentrations of 25-hydroxyvitamin D3 in serum drawn during February from 24 persons who were judged normal by physical examinations. The range was 5.5-23.8 microgram/liter (mean, 15.0 +/- 5.2 microgram/liter). The day-to-day CV for the assay was 5.46%.