Enzymatic method for assaying calcium in serum with Ca++ -ATPase

A kinetic assay for total calcium in serum was developed which is based on the activation of Ca(++)-ATPase by free Ca(++) [Ca(++)](f) maintained by EGTA in the reaction mixture. The concentration of Ca(++)(f) was dependent on total reference calcium added or serum calcium. Ca(++)-ATPase activity was coupled to the reduction of NADH by pyruvate kinase (PK) and lactate dehydrogenase (LDH) and monitored by change in absorbance at 340 nm. The calcium in normal serum was 10.08 +/- 0.24 mg/dl (n = 35) by our method while with o-cresolphthalein complexone (CPC) method, the total calcium in the same 35 serum samples was 10.14 +/- 0.54 mg/dl. The range of within-run coefficient of variations (CVs) by this method was 0.9-2.87% at 8-12 mg/dl and day-to-day CVs were 0.72-3.17%. The presence of other ions and standard clinical interfering agents did not affect this assay system. The correlation between values obtained with our method (y) and CPC method (x) for normal serum was: y = 1.064x-0.580 mg/dl (r = 0.912, n = 59).     

The measurement of serum human parathyroid hormone (h-PTH53-84) and effect of exercise on calcium metabolism

This study was focussed our attention on the measurement within the upper physiological level of human serum parathyroid hormone (PTH), using kits of human PTH 53-84. This assay kit was able to detect serum PTH in sera with subtle changes of serum calcium concentrations before and after short term exercise. These serum PTH levels before and after exercise seemed to be changed within the upper physiological levels of PTH. Thus, this study suggested that the assay kit was likely to become a useful tool of the measurement of the physiological level of serum PTH in humans.     

Influences of serum magnesium on measurements of serum parathyroid hormone

The amino sequence recognizable parathyroid hormones (PTH) in lower and non-lower magnesemic groups in randomized samples were measured by using various kinds of the well established PTH kits. The levels of carboxy terminal, mid-region and intact PTH in lower magnesemic group were more decreased than these in non-lower magnesemic group. It is likely that the shortage of magnesium in serum makes a suppression of PTH secretion.     

Analysis of polyunsaturated fatty acids in blood serum after fish oil administration.

Free and total fatty acids in the blood serum of patients with hyperlipoproteinemia have been analysed as their methyl esters by capillary gas chromatography using an FFAP column. In one-step reactions the free fatty acids in serum react with methanol-acetyl chloride (50:1, v/v) at 25 degrees C, the total fatty acids (free plus esterified) are transesterified with methanol-toluene-acetyl chloride (8:2:1, v/v) at 100 degrees C. The quantification of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is based on an internal standard (13,16,19-docosatrienoic acid) and on calibration standards. Under normal diet the concentrations of EPA and DHA are as follows (mean +/- S.D., n = 27): free EPA, 0.2 +/- 0.1 mg/dl; free DHA, 0.6 +/- 0.2 mg/dl; total EPA, 3.6 +/- 2.1 mg/dl; total DHA 11.4 +/- 3.1 mg/dl. Under a fish oil intake of 9 g per day, free and total EPA concentrations rise by ca. five- to six-fold, and free and total DHA concentrations by ca. two-fold.     

Basic studies on an immunoradiometric assay system for human parathyrin (intact PTH1-84)

Two-site immunoradiometric assay for human parathyrin (PTH1-84) is specific for the intact, secreted, biologically active 84 amino peptide. This system incorporates two-different polyclonal antibodies to human intact PTH and has several technical advantages for use. This assay could detect a wide range of PTH in patients with hypo-, hyperparathyroidism, chronic renal failure and hypercalcemia with malignancy, especially distinguishing the level of human intact PTH in hypoparathyroidism from in normal.     

Flow-injection analysis of serum urea using o-phthalaldehyde and naphthylethylenediamine

An analytical procedure is developed for the determination of urea using flow injection analysis. The methodology is based on the color that develops (lambda(max), 517 nm) when urea reacts with o-phthalaldehyde in the presence of naphthylethylenediamine under acidic conditions. Calibration curves are found to be linear up to 51 mg urea N/dl when the FIA manifold is operated at 42 degrees , utilizing 90-mul samples and a flow-rate of 0.44 ml/min. By manual injection up to 40 samples can be analysed per hour. Recovery yields varied between 95-99%. The within day CV was 0.5-2.2% whereas the day to day CV was 2.1-3.9%. When applied to the analysis of urea in serum samples, excellent correlations (0.998) are obtained when the FIA results are compared with those obtained for the same serum samples analysed by the free urease/Berthelot's and the hospital method (employing the Abbot Bichromatic Analyser). Interferences are observed with sulphur compounds such as sulphanilamide (0.76 mg/dl) as well as with many amino acids but at relatively high concentrations (21 mg/dl).     

The correlation of serum parathyroid hormones among patients with dementia of vascular type, hemodialysis, and bone-joint disorders

PTH levels were analysed in sera of patients receiving maintenance hemodialysis, with the vascular type of dementia and orthopedic patients with bone-joint disorders, using PTH assay kits. There were significant interrelationship among all kinds of PTH levels in the hemodialysis patients, but not in patients with the vascular type of dementia and the bone-joint disorders. Renal functions might be responsible for fragmentation of PTH in blood stream. These results suggested that the measurement of PTH levels is needed to examine various kinds of amino-sequence of PTH levels in keeping normal function of kidney.     

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

Summary A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).     

The measurements of parathyroid hormones in sera of elderly osteoporotic patients with serum high alkaline phosphatase activity

This study was carried out to investigate effects of drugs treated in the elderly osteoporotic patients on measurements of carboxy terminal, mid-region and intact PTH, by using the various kinds of PTH kits. The drugs such as 1 alpha(OH)D3, calcitonin and so on, in the treatment of osteoporotic patients, was brought about no significant inter-relationships among carboxy terminal, mid-region and intact PTH in sera of the elderly osteoporotic patients with serum high alkaline phosphatase activity.     

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose,Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract

A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.     

Determination of serum cortisol by reversed-phase liquid chromatography using precolumn sulphuric acid-ethanol fluorescence derivatization and column switching.

An assay method for serum cortisol, using precolumn sulphuric acid-ethanol fluorescence derivatization and reversed-phase liquid chromatography with a column-switching technique, has been developed. The crude precolumn fluorescence cortisol derivative was prepared by the addition of sulphuric acid to serum deproteinized with ethanol, and directly injected onto an octadecylsilane-bonded silica gel (ODS) precolumn for concentration and purification. After switching columns the samples were separated using an ODS analytical column and monitored fluorimetrically. When the pH of the mobile phase in the analytical separator decreased to 1.85, the emission wavelength of the cortisol derivative changed to 520 nm (excitation of 365 nm) and the fluorescence intensity increased. Among the sulphuric acid-ethanol derivatives of various steroids, cortisol, corticosterone and testosterone emitted fluorescence. However, their retention times differed from those of the cortisol derivatives (12.5 min). The detection limit of cortisol was 0.3 micrograms/dl (signal-to-noise ratio of 3). Use of the fully automated column-switching system contributed to good reproducibility and recovery.     

Investigation of a capillary electrophoretic approach for direct quantification of apolipoprotein A-I in serum

In the present study a rapid, reproducible and robust capillary electrophoresis (CE) procedure for the quantification of apolipoprotein A-I (Apo A-I) in serum without pretreatment has been developed (total run time, 11 min). The coefficients of variation (CV; n = 10) for the relative peak area are 1.8% at a concentration of 145 mg/dL and 1.6% at 196 mg/dL; and for the inter-assay 8.9% at 161 mg/dL (10 consecutive days), i.e., similar to the CVs of a high-throughput immunonephelometric routine assay. The CV for the migration time is 0.4% (n = 20). The robustness of the CE approach was tested in patient samples with hemolysis, hyperbilirubinemia and hyperlipidemia. A comparison of 99 Apo A-I serum values with results of a fixed-time immunonephelometric routine assay showed a positive constant bias of 60% (mean) for the immunonephelometric values, no deviation from linearity, but significant deviations in several samples. Investigations on interferences in the CE analyses gave no evidence that CE failed. Our study shows that CE is amenable to a fast analysis and a reproducible and reliable quantification of Apo A-I level in sera of various clinical samples.     

Factors affecting the serum thyroid hormone concentration during fasting in rats--with special reference to the relation with temperature

The present studies examine the effect of starvation together with cold or hot exposure on thyroid hormone levels in rats. At 23 degrees C starved for 5 days, serum thyroid hormone levels decreased significantly compared with fed rats, averaging 3.6 +/- 0.5 micrograms/dl of thyroxine (T4), 47 +/- 11 ng/dl of triiodothyronine (T3), 1.4 +/- 0.3 ng/dl of free T4 and 39.6 +/- 5.1 pg/ml of reverse T3, respectively. At 15 degrees C rats starved for 5 days, serum free T4 level significantly more increased than that of 23 degrees C starved rats, while serum T4 level and T3 did not increase significantly. At 30 degrees C rats whether concomitant starvation or not, serum thyroid hormone levels of both group markedly more decreased than control rats. These experiment provide additional evidence that thyroid gland and the peripheral metabolism of thyroid hormone respond to variety situations such as cold or hot exposure together with starvation or not.     

Clinical significance of serum bile acid radioimmunoassay in hepatobiliary diseases--with special reference to the CG/SLCG ratio (author's transl)

Both serum cholylglycine (CG) and sulfolithocholylglycine (SLCG) levels were radioimmunoassayed by PEG method in 209 samples (204 hepatobiliary diseases, 5 normal controls). 1) The results revealed that serum bile acid levels were excellent indicators for hepatic dysfunction in comparison with the conventional liver function tests. 2) Means of 19.4 +/- 9.3 microgram/dl for CG and 21.7 +/- 6.7 microgram/dl for SLCG were obtained in controls. Most hepatobiliary diseases demonstrated abnormally high bile acid levels, with extremely high CG values in conditions with bile stasis. 3) To differentiate various hepatobiliary diseases more clearly, the ratio of the primary and secondary bile acids (CG/SLCG ratio) was introduced (1.0 +/- 0.6 for controls). In cases of bile stasis, CG/SLCG ratios ranged from 7.8 +/- 4.8 for intrahepatic cholestasis to 34.8 +/- 27.6 for congenital biliary atresia, while other hepatic disorders demonstrated relatively low values. We conclude that the CG/SLCG ratio is a useful index for cholestasis. Diagnosis of the congenital biliary atresia could be possible.     

毛细管胶束电动色谱法分析PTH氨基酸

建立了用毛细管胶束电动色谱法（MEKC）分离19种PTH氨基酸的方法,并探讨了电压、pH值、温度、胶束浓度对氨基酸迁移时间的影响。方法具有速度快、灵敏度高、样品用量少的优点。     

Analysis of micafungin in serum by capillary zone electrophoresis

A method is developed for measuring the concentration of micafungin, an echinocandin antifungal agent, in serum, using capillary zone electrophoresis. With a 0.1M borate buffer (pH 10.0) as the run buffer, detection is carried out at 200 nm. Pretreatment is performed by adding acetonitrile to serum (1:2) for deproteinization, allowing the supernatant fluid to be taken for measurement. The detection limit of the assay is 0.5 mg/L at a signal-to-noise ratio of 3.0. Coefficients of variation for intra- and inter-assay precision are 3.45-4.47% and 2.61-7.15%, respectively, at a nominal concentration of 5.0-25.0 mg/L. Their recovery rates are 92-110%. It is convenient for the monitoring of micafungin therapy in patients contracting clinically important deep mycoses.     

Improved method for the immunological detection of malondialdehyde-modified low-density lipoproteins in human serum

Oxidized low-density lipoproteins (ox-LDL) such as malondialdehyde-modified LDL (MDA-LDL) play an important role in the pathogenesis of atherosclerosis. This study is aimed to establish a standard enzyme-linked immunosorbent assay (ELISA) for measuring serum MDA-LDL, and evaluate its usefulness by analyzing serum samples of diabetes mellitus (DM) patients. Serum sample stability, analytical sensitivity, intra- and inter-assay precision, dilution linearity, supplementation and recovery, and interfering substances were examined. Normal reference levels in 86 healthy subjects (33 males and 53 females; average age 34.4±7.8 years) with normal lipid profiles were also determined. MDA-LDL levels in blood and serum were unstable and gradually increased during storage. However, it could be stabilized by the addition of a reagent, which included sucrose. The detection limit of the assay was 6.3 U/l. Intra- and inter-assay imprecisions were <5.6 and <9.4%, respectively. Excellent dilution linearity was observed, and the average recovery was 105%. None of the test substances, for example, hemoglobin, lipid and bilirubin interfered with the assay. The normal reference levels of MDA-LDL and MDA-LDL/LDL-C in the 86 subjects with normal lipid levels were calculated as 58.8±17.9 U/l and 60.4±11.9 mU/mg, respectively. The both serum levels of MDA-LDL (122±62.8 U/l) and MDA-LDL/LDL-C (96.8±48.5 mU/mg) in the DM patients were significantly higher than those of controls (P<0.0001). Moreover, DM patients with atherosclerosis complications showed significantly higher MDA-LDL and MDA-LDL/LDL-C levels than those without complications, 174±81.3 versus 101±39.2 U/l, (P<0.01) and 138±59.0 versus 80.3±32.1 mU/mg, (P<0.005), respectively. These results suggest that the ELISA method for MDA-LDL described in this study fulfilled the sensitivity, reproducibility, and accuracy requirements for routine clinical assays and might therefore be a useful tool for evaluating atherogenicity.     

A radioimmunoassay for serum estradiol

A radioimmunoassay procedure to measure estradiol levels in human serum was developed using¹²⁵I labeled estradiol and antiestradiol antibody raised in-house using the estradiol-6-CMO-BSA as the immunogen. Controlled process serum was used to maintain the serum matrix and sample-standard identity. The assay was validated by analysing several (n=40) samples and comparing with established commercial assays. The assay had a sensitivity of 35 pg/mL and a range of 50 pg/mL to 5 ng/mL. The interassay C.V. were between 2 to 10% while the intraassay C.V. were between 2 and 8%. This assay is sensitive enough for monitoring ovulation and hypersecretions in ovarian tumours and was primarily developed to be used as an in-house assay in the local hospitals.     

Application of the vanadium(V)-xylenol orange reagent to the assay of serum glucose

The VXO reagent (mixture of V(V) and xylenol orange) provided in our earlier studies is found very useful for the colorimetric determination of traces of hydrogen peroxide. In this paper the reagent is successfully extended to the assay of glucose in serum by the combined use of glucose oxidase.The VXO reagent exhibited a characteristic absorption maximum at 582 nm, and the presence of glucose together with glucose oxidase led to a significant decrease in the absorbance of the reagent. A linear relation was found between the magnitude of the decrease and glucose concentration in the range of 5 to 400 mg/100 ml. The average recovery of glucose added in serum was 98.6% and the data were reliable with a coefficient of variation below 3.0%. A good correlation to the method by 4-aminoantipyrine-phenol was found: r = 0.970. Ascorbic acid and uric acid did not interfere with the assay.     

新生儿尿钙测定及临床意义(附38例测定分析)

通过测定新生儿随意尿的钙来替代血钙的测定,并探讨其临床意义,用尿钙检测试剂盒对３８例新生儿婴儿的尿钙进行了检测,结果表明,随意尿钙值可以反映血钙的水平,检测尿钙对诊断新生儿低钙血症有一定意义;对新生儿惊厥的鉴别诊断有一定的意义。