Simultaneous determination of adreno-ovarian steroids from a single aliquot

A standardized technique for simultaneous fractionation and estimation of samples of progesterone, estrone, 17alpha-estradiol, 17beta-estradiol, estriol, corticosterone, cortisone, and cortisol obtained from guinea-pigs during mid-pregnancy is presented. The hormones were separated on a single TLC plate using a single solvent system, and were measured on a single biphasic column on the basis of a single aliquot. The technique affords considerable utility in the inves tigation of the complexities of the adreno-genital syndrome. Progesterone content in the ovaries and plasma showed an increase of 77% during pregnancy, while plasma levels of estrone were increased by 34%. Urinary estradiol levels were also markedly increased. Plasma estradiol and estriol and placental estriol could not be detected by the technique. Adrenal cortisone levels and plasma cortisone and cortisol were considerably higher during pregnancy. An initial overlap was found between cortisol and estriol on the TLC plate, though subsequent estimation by GLC overcome the problem.     

Effect of sample volume and column length on transcortin determination by sephadex G-25 gel filtration.

When Sephadex G-25 columns are used to separate transcortin-bound corticosterone from free corticosterone, there is a non-linear increase of total binding with increasing plasma volume filtered. However, the specific binding (total binding - unspecific binding) shows a linear relationship with the plasma volume filtered. Furthermore, the specific binding decreases with increasing Sephadex column length and absolute binding values are found by extrapolating to a zero length column.     

A radioimmunoassay of plasma unconjugated and conjugated estetrol.

A radioimmunoassay for the measurement of both unconjugated and conjugaged estetrol in plasma has been developed. The antiserum obtained after 6 months of immunization with 6-oxoestetrol-6-(O-carboxy-methyl)oxim-BSA was used at a final dilution of 1:90,000 and showed almost no cross reaction with other steroids except for estriol at 1.24%. Esterol-glucosiduronate was synthesized by incubating with adrenalectomized rat liver homogenate and uridine diphosphoglucuronic acid. Then, plasma estetrol-glucosiduronate was measured in the same manner for unconjugated estetrol after hydrolysis with beta-glucuronidase. Sephadex LH-20 column chromatography (7X110 mm, benzene:methanol, 85:15) was employed for accurate assessment. The sensitivity was 10 pg and the smallest amount measurable was 40 pg/sample. The method bland was consistently negligible. The intra and inter assay precision was 11.8% and 14.2% for unconjugated estetrol and that for estetrol-glucosiduronate was 13.5% and 17.1%.     

An enzyme-immunoassay for estriol.

A practical method was developed for enzyme-immunoassay of serum estriol, with alkaline-phosphatase as a marker enzyme. Alkaline-phosphatase was conjugated with estriol-6-(O-carboxymethyl) oxime using water soluble carbodiimide. The estriol-alkaline-phosphatase complex, which has both enzyme activity and capacity to bind anti-estriol serum, was obtained by Sephadex G-200 gel filtration. This complex, which was stable for at least 3 months at 4 degrees C, was used as enzyme-labelled estriol. Anti-estriol serum raised against estriol-6-(O-carboxymethyl) oxime bovine serum albumin was employed. "Bound and free" estriol were separated by the double antibody method. A linear relation was obtained between estriol concentration and antibody-bound alkaline-phosphatase activity in the range of 0.2-100 ng estriol/ml. In this assay system, cross-reactivity with other steroids was negligible under physiological conditions, and endogenous alkaline-phosphatase, which increases during the late pregnancy, caused no interference. The coefficients of variation were 3.3-14.2% (within assays), and less than 22% (between assays), and the mean recovery rate was 77.5%. Serum estriol values determined by the present method correlated well with those determined by radioimmunoassay (r=0.90 for total estriol; r=0.98 for free estriol). The present method of enzyme-immunoassay is suitable for measurement of serum estriol during pregnancy.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

Post-thaw plasma membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column

Previous experiments have established that filtration of bovine semen through a Sephadex ion-exchange column improves its quality before and after freezing. The present study was conducted to determine the post-thaw membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column and to determine the kind of protection to spermatozoa is provided by glycerol during freezing and thawing. Semen from Holstein bulls diluted in TEST-yolk extender (with and without glycerol) was filtered through a Sephadex ion-exchange column and frozen in liquid nitrogen (-196 degrees C). After thawing, there were more normal acrosomes in filtered spermatozoa than nonfiltered (P < 0.01). Post-thaw plasma membrane integrity and swelling ability in a hypoosmotic solution revealed that the filtered spermatozoa had a stronger (P < 0.005) plasma membranes than the nonfiltered. Filtered spermatozoa demonstrated higher zona-free hamster oocyte penetration than the nonfiltered (30.5 vs 11.5%; P < 0.0005). Spermatozoa extended in TEST-yolk without glycerol had the lowest (P < 0.001) normal acrosomes, intact plasma membranes and swelling ability. Plasma membrane over the post-acrosomal region of the head and post-midpiece region of the tail was more sensitive to damages caused by freezing and thawing than acrosomal and midpiece regions of spermatozoa. Glycerol in the extender provided significant (P < 0.05) protection to the sensitive regions of filtered and nonfiltered spermatozoa during freezing and thawing. Filtered plus glycerolated spermatozoa had the highest (P < 0.01) normal acrosomes, intact plasma membranes and swelling ability. In conclusion, the pre-freezing filtration of bovine semen harvested the spermatozoa possessing stronger plasma membranes which enabled them to endure freezing and thawing stresses. The addition of glycerol to the extender protected the post-acrosomal region of the head and post-midpiece region of the tail of spermatozoa from freezing and thawing shocks.     

Role of thymic hormone (THF) and of a thymic plasma recirculating factor (TPRF) in the modulation of human lymphocyte response to PHA and Con A.

The results presented here point to the possibility that calf thymus extracts contain, in addition to the thymic hormone (THF), a second component: thymic plasma recirculating factor (TPRF). THF, which is involved in the process of T cell maturation and has been characterized as a protein of m.w. 3000 eluted in the void volume of a G-10 Sephadex column (G-10-I), caused an increased level of intracellular cAMP in umbilical cord blood lymphocytes (UCBL). This is in agreement with our previous observation that THF plays a major role in the differentiation of T cells. The second active material, TPRF, also isolated from thymic extract, is of a molecular size below 500 and was eluted in a G-10 Sephadex column at the fourth protein peak; it seems to circulate in the blood. Previously, we had observed in impaired response of UCBL to PHA and Con A stimulation in the presence of dialyzed human plasma (DHP). Our present results indicate that this impaired response is restored exclusively by TPRF. A factor with TPRF-like activity was also isolated from the plasma of normal donors; yet it was not detected in the plasma of thymectomized patients suffering from myasthenia gravis (MG). This suggests that TPRF from plasma is thymus dependent. TPRF does not affect the level of intracellular cAMP in UCBL.     

A rapid specific radioimmunoassay for unconjugated estriol in plasma.

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.     

Chemical forms of selenium in selenium containing proteins from human plasma

The chemical forms of selenium (Se) were determined in human plasma fractions. Human plasma was subjected to gel filtration using Sephadex G-150, and the first Se peak from this column was subsequently chromatographed on DEAE-Sephacel. The form of Se in the Se peak which eluted from this column was shown to be selenocysteine (SeCys). In a second approach human plasma was again subjected to gel filtration and the first Se peak was chromatographed on Affigel blue. SeCys was shown to be the form of Se in both the retained and unretained Se on this column. The second gel filtration Se peak was also chromatographed on Reactive Blue 2-Sepharose CL-6B and the form of Se which was not retained was also shown to be SeCys. However, the form which was retained was shown to be selenomethionine. Evidence is presented that there are three Se containing proteins in human plasma, which are selenoprotein P, glutathione peroxidase, and albumin.     

Insulin-like substance and insulin-degrading complex in hemolysates of human erythrocytes

Human erythrocyte lysate was fractionated on various gel filtration media and immunoreactive insulin, insulinase and the influence of individual fractions of the insulin-degrading activity were determined. The hemolysate was shown to contain a complex of substances including an insulin-like substance, insulinase, protease inhibitor and insulinase activator. The insulin-like substance eluted from a Sephadex G-50 column in the same manner as native insulin, and its concentration exceeded the plasma level. Insulinase (Mr 100,000) degraded insulin to the trichloroacetic acid soluble fragments but did not degrade protein or glycoprotein hormones from human pituitaries. Insulinase was inhibited by low temperature, aprotinin and by a newly discovered protease inhibitor from erythrocytes which also inhibits serine proteases--trypsin and chymotrypsin. Another newly discovered substance eluted from a Sephadex G-100 column in the region of low molecular weight substances and showed an insulinase activating activity. The elution patterns of the protease inhibitor and insulinase activator suggest the possibility of the presence of more than one inhibiting and activating factor. The experimental results suggest that the insulin-degrading complex plays a role of a regulator of plasma insulin level. The nonpancreatic origin of the insulin-like substance is also possible.     

Some observations on the determination of cortisol in human plasma by radioimmunoassay using antisera against cortisol-3-BSA.

Antisera were obtained from rabbits immunised against cortisol-3-BSA with a view to examining their application in a radioimmunoassay of the steroid. One such antiserum was studied in detail; cross-reactivity with other C21 steroids normally present in human plasma was negligible and it proved possible to establish a radioimmunoassay which satisfied all criteria of reliability. The specificity of the cortisol determination achieved in human plasma was examined by performing measurements with and without including an initial Sephadex LH-20 column chromatographic purification step; the values obtained were in excellent agreement both for normal plasma and for that obtained following adrenal stimulation with ACTH.     

The presence of an inhibitor of benzylamine oxidase in human blood plasma.

The presence of an inhibitor of benzylamine oxidase in human blood plasma was observed. It may be removed by use of either a DEAE-cellulose column or a Sephadex G-200 column.     

Isolation and characterization of a protein C activator from the moccasin snake venom

The protein C activator detectable in the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) was isolated by a combination of chromatofocusing on PBE-94 in the range pH9-7 and gel-filtration on Sephadex G-100 column. The peak protein C activator from Sephadex G-100 column appeared as double diffuse bands with apparent molecular weight of 37,700 and 31,400 after electrophoresis in the presence of sodium dodecylsulfate and 2-mercaptoethanol. The isolated enzyme does not clot human fibrinogen and when mixed with normal plasma generates activity of Protein C. It can be used for the measurement of protein C functional activity.     

Purification and properties of rat stomach kallikrein

Kallikrein (EC 3.4.21.8) was purified from rat stomach by column chromatography on p-aminobenzamidine-Sepharose, DEAE-Sephadex A-50 and Sephadex G-150 and by isoelectric focusing, measuring its activities to hydrolyse L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide and to release kinin from heat-treated rat plasma. the purified stomach kallikrein showed a single band on polyacrylamide gel electrophoresis at pH 7.0. Its molecular weight was calculated to be 29 000 by gel-filtration on a column of Sephadex G-50. The kallikrein was stable between pH 6-11 and hydrolyzed L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide optimally at pH 11.0. The L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide hydrolyzing activity of rat stomach kallikrein was inhibited by diisopropyl fluorophosphate and Trasylol, but not by trypsin inhibitors from soybean, lima bean and ovomucoid. These properties of rat stomach kallikrein are different from those of partially purified rat plasma kallikrein, but similar to those of glandular kallikreins from other species. From these results, it was concluded that kallikrein is present in rat stomach and that it can be classified as a glandular kallikrein.     

Estrogen determination using liquid chromatography with precolumn fluorescence labeling

A liquid chromatography procedure is described for the determination of some estrogens using fluorescence detection. The estrogens are labeled by precolumn derivatization with 5-dimethylaminonaphthalene-1-sulfonylchloride (dansyl chloride) and chromatographed on a reversed-phase, C-18 column with a mobile phase consisting of methanol, water, and acetic acid. The eluted analytes are measured with a fluorescence detector using excitation and emission wavelengths of 350 and 540 nm, respectively. The chief advantage of this new procedure is its sensitivity, requiring smaller amounts of sample to detect and quantitate estrogens in biological materials. We could detect less than 400 pg of estriol. With our procedure, this corresponded to about 25 ng in the final reaction mixture, before derivatization. The use of smaller sample volumes could improve this limit. Linearity for dansylated estriol, estrone, and estradiol was excellent over the estrogen range below 100 μg in the sample. This corresponds to approximately 1.7 μg on the column. Within-run precision was better than 5% for the full extraction and derivatization procedure for estriol from pregnancy urine samples. Chromatography is complete within 10 min for dansylated estriol, estrone, and estradiol.     

Estriol concentrations in plasma of normal, non-pregnant women.

Using a rabbit antisera directed against estriol-3-0-carboxy methyl ether complexed to BSA, an immunoassay for estriol (1) was developed. The mean plus or minus SE concentration of estriol in 18 women in days 5-7 of their cycle was 7.9 plus or minus 0.6 pg/ml which was significantly (P less than 0.01) less than the mean value of 11.1 plus or minus 0.8 pg/ml in 15 women in days 20-22 of the cycle. In 3 of 6 women in whom plasma samples were drawn frequently during their cycle, an estriol peak occurred coincident with the estradiol peak. In 3 women from whom plasma was obtained several times during the course of a day estriol levels did not appear to vary significantly. In 8 women who were on oral contraceptives the mean level of estriol was 7.6 plus or minus 1.5 pg/ml. In 8 post-menopausal women the mean level was 6.0 plus or minus 1.2 pg/ml which is significantly (P less than 0.01) less than the mean luteal phase value but not less (P greater than 0.1) than the follicular phase or oral contraceptive user values. We conclude that some of the circulating estriol is directly secreted by the ovary of normal women.     

A novel anticoagulant protein from Scapharca broughtonii

An anticoagulant protein was purified from the edible portion of a blood ark shell, Scapharca broughtonii, by ammonium sulfate precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G- 75, DEAE-Sephacel, and Biogel P-100. In vitro assays with human plasma, the anticoagulant from S. broughtonii, prolonged the activated partial thromboplastin time (APTT) and inhibited the factor IX in the intrinsic pathway of the blood coagulation cascade. But, the fibrin plate assay did not show that the anticoagulant is a fibrinolytic protease. The molecular mass of the purified S. broughtonii anticoagulant was measured to be about 26.0 kDa by gel filtration on a Sephadex G-75 column and SDSPAGE under denaturing conditions. The optimum activity in the APTT assay was exhibited at pH 7.0-7.5 and 40-45 degrees C in the presence of Ca(2+).     

16-sulfates of estriol in body fluids of human pregnancy at term.

A method was developed for the assay of estriol-16-sulfate (E3-16S) and estriol-3, 16-disulfate (E3-3,16-diS) in maternal serum, cord serum and amniotic fluid at delivery in human pregnancy. Tritiated E3-16S and E3-3,16-diS are added to the fluid being analyzed. The conjugates are separated and purified by sequential chromatography on alumina, Celite and Sephadex LH-20. Each conjugate is hydrolyzed with Glusulase and the released estriol is quantified by radioimmmunoassay. E3-3,16-diS was found in each fluid, most concentrated in the cord serum. Small amounts of E3-16S were found in some amniotic fluids, and this conjugate was virtually absent from the sera. These new estriol conjugates comprise less than 1 percent of total, estriol, apparently too low to be of diagnostic value in human pregnancy.     

Purification of an anticoagulant from the body fluid of Ascaris suum

An anticoagulant has been purified from the body fluid of Ascaris suum by sequential passage through Sephadex G-50, CM-cellulose and Sephadex G-25 columns then treated with 2 M NaCl, passaged through a Sephadex G-25 column, separated from the phosphate buffer by precipitation of the latter with the CaCl2, then passaged through a Sephadex G-10 column in water. In the body fluid of the worm, the anticoagulant is ionically-bound to a carrier substance. The complex can be split by treatment with 2 M NaCl. The molecular weight of the anticoagulant is slightly less than 1400.     

Purification and characterization of human plasma Zn-alpha 2-glycoprotein

Zn-alpha 2-glycoprotein (Zn alpha 2gp) was purified from fresh human plasma approximately 670-fold in a yield of 18% over the fractions from DEAE-Sephadex A-50 column chromatography. The purified protein was a glycoprotein with molecular weights of 56,000 and 57,000 on Superose and Sephadex G-150 column chromatographies and of 41,000 and 42,000 on nonreduced SDS-PAGE. Characterization, which included a determination of molecular weight, amino acid composition, amino terminus, and antigenicity, correlated well with known values previously reported for human Zn alpha 2gp.