Studies on the biological and immunological properties of human follitropin: profiles of two international reference preparations and of an aqueous extract of pituitary glands after electrofocusing

1981 ◽  
Vol 97 (2) ◽  
pp. 157-165 ◽  
Author(s):  
A. A. Zaidi ◽  
D. M. Robertson ◽  
E. Diczfalusy
Keyword(s):  
Biological Activity ◽  
Aqueous Extract ◽  
Receptor Binding ◽  
Immunological Reactivity ◽  
Human Pituitary ◽  
International Reference ◽  
Pituitary Glands ◽  
The Mean ◽  
Ph Range

Abstract. Two international reference preparations, the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH/ICSH) for Bioassay (identified hereafter by its code no.: 69/104) and the First International Standard of Urinary FSH and LH (ICSH) for Bioassay (hereafter: hMG 1st IS) and an aqueous extract of human pituitary glands (hPE) were fractionated in triplicate by isoelectric focusing on sucrose density gradient (Ampholine, pH range 2.5–10.0). The FSH activity was monitored in each fraction by an in vitro bioassay, a radioimmunoassay and a receptor binding assay. Unfractionated 69/104 was used as standard in each assay system. Compared with the hPE, the activity profiles of the 69/104 and hMG reference preparations were spread over a significantly wider pH range; the biological activity eluted in the pH range of 3.5–5.0 was of the order of 90% for hPE, 72% for 69/104 and less than 60% for hMG. Major variations in biological to immunological (B/I) and biological to receptor binding (B/R) activity ratios were observed in the individual electrofocusing fractions. The B/I ratios ranged from 0.8 to 2.2 (69/104), 1.0–5.7 (hMG) and 1.3–6.0 (hPE), respectively. The variation in the corresponding B/R ratios was: 0.5–1.7 (69/104), 0.58–4.0 (hMG) and 0.55–1.5 (hPE). When equal aliquots from each of the electrofocusing fractions were combined and this 're-constituted' pool was compared with the starting material, significant differences were observed in the B/I ratios: 1.63 instead of 1.0 (69/104), 2.58 vs 2.34 (hMG) and 2.72 vs 1.32 (hPE). There was no significant change in the mean B/R ratios before and after electrofocusing: 1.0 vs 1.02 (69/104), 1.09 vs 1.02 (hMG) and 1.05 vs 1.04 (hPE). The mean recovery of biological activity in each of the three reconstituted pools of the three preparations was 97% for 69/104, 88% for hMG and 98% for hPE, and the corresponding recoveries of receptor binding activities were 95%, 94% and 98%, respectively. In contrast, the mean recovery of immunological reactivity was only 55% (69/104), 80% (hMG) and 47% (hPE), respectively. The reduction in the immunological reactivity of the 69/104 preparation without any apparent loss of biological or receptor binding activities following electrofocusing indicates that its immunoreactivity is unstable under mild experimental conditions, which do not influence its biological and receptor binding activities. Hence, whereas this preparation might possibly be suitable as a standard for in vitro bioassays and receptor binding assays, it is unsuitable as a standard for the radioimmunoassay of hFSH.

Molecular composition of human luteinizing hormone: biological and immunological profiles of highly purified preparations after electrofocusing

1982 ◽  
Vol 94 (1) ◽  
pp. 29-36 ◽  
Author(s):  
A. A. Zaidi ◽  
M. H. Qazi ◽  
E. Diczfalusy
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Sucrose Density Gradient ◽  
Molecular Composition ◽  
Immunological Reactivity ◽  
Human Pituitary ◽  
Before And After ◽  
Pituitary Glands ◽  
Ph Range

Five highly purified human pituitary LH preparations (candidate preparations for a new International Reference Preparation (IRP) and coded A to E) and three highly purified commercial preparations (referred to as Kabi I, II and III) were fractionated by electrofocusing on a sucrose density gradient with ampholytes in the pH range of 3·5–10·0 The LH activity was monitored in each of the eluted fractions by an in-vitro bioassay and a radioimmunoassay procedure and the profiles of biological activity and immunoreactivity were compared with those of the first IRP of Human Pituitary Luteinizing Hormone for Immunoassay (code no. 68/40). The overall recovery of the biological activity after electrofocusing of the nine preparations ranged between 75 and 92% (mean 86%) and was higher (P < 0·05) than that of the immunological reactivity which varied between 71 and 94% (mean 79%). The profiles of the biological activity and immunological reactivity were in close agreement with each other in all preparations. Significant differences were, however, observed in the distribution of various molecular species between the currently used standard and the eight other highly purified preparations. The ratios of biological activity (B) to immunoreactivity (I) of preparation B and of the three Kabi preparations were significantly higher than those of the other five preparations, both before and after electrofocusing. After electrofocusing, a slight but significant (P < 0·05) rise in the B/I ratios was observed, suggesting that some biologically inactive immunoreactive material was removed during fractionation. All purified preparations lacked the characteristic 'acidic' human LH species which accompanies FSH and which is abundant in the IRP (69/104) and is also present in aqueous extracts of postmenopausal pituitary glands. A comparison of the electrofocusing profiles of the nine highly purified preparations with those of human LH from plasma and individual pituitary glands revealed marked differences, suggesting that the methods used for the purification of the hormone significantly altered its molecular composition.

A COMPARISON OF PREPARATIONS OF HIGHLY PURIFIED HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE: DIFFERENCES IN THE FOLLICLE-STIMULATING HORMONE POTENCIES AS DETERMINED BY IN-VIVO BIOASSAY, IN-VITRO BIOASSAY AND IMMUNOASSAY

1981 ◽  
Vol 91 (2) ◽  
pp. 353-362 ◽  
Author(s):  
P. L. STORRING ◽  
A. A. ZAIDI ◽  
Y. G. MISTRY ◽  
BERIT FRÖYSA ◽  
BRIDGET E. STENNING ◽  
...  
Keyword(s):  
Biological Activity ◽  
Follicle Stimulating Hormone ◽  
In Vitro Bioassay ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Potency ◽  
Reference Preparation ◽  
In Vivo Bioassay

The FSH potencies of 12 preparations of highly purified human pituitary FSH, originating from six different laboratories, were determined by in-vivo and in-vitro bioassays and by immunoassay in terms of the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (IRP; coded 69/104). The contamination of these FSH preparations with LH was also determined. Estimates of protein content were based on the absorbance at 280 nm of solutions of the preparations, assuming that A1%1 cm 280 = 10. The FSH potencies varied between different preparations from 827 i.u./mg to 13 100 i.u./mg by in-vivo bioassay; from 2930 to 14 600 i.u./mg by in-vitro bioassay and from 1680 to 5690 i.u./mg by immunoassay. The ratios of in-vivo biological activity relative to in-vitro biological activity and to immunoreactivity respectively varied between preparations from 0·06 to 2·3 and from 0·15 to 4·1, and there was a significant positive correlation between each of these ratios and the in-vivo biological potency of the preparations; such differences could be due to varying degrees of sialylation between preparations. On the other hand the ratios of in-vitro biological activity to immunoreactivity between preparations were fairly constant (approx. 2). The excess biological activity relative to immunoreactivity observed, in terms of the IRP, in all these materials is consistent with recent findings of some immunoreactive FSH in the IRP unassociated with biological activity. These data did not demonstrate any significant advantage, in terms of FSH in-vivo biological potency, from the use of fresh-frozen rather than acetone-dried pituitary glands for the isolation of FSH. Contamination of all these preparations with LH appeared to be less than 3% (w/w), as determined by in-vitro bioassay and by immunoassay. The results of this study are discussed in relation to the selection of material for an international reference preparation for immunoassay and attention is drawn to the value of high in-vivo biological FSH potency as a criterion of the identity of a preparation as well as of its freedom from contaminants without FSH biological activity.

BIOLOGICAL AND IMMUNOLOGICAL PROPERTIES OF DIFFERENT MOLECULAR SPECIES OF HUMAN FOLLICLE-STIMULATING HORMONE: ELECTROFOCUSING PROFILES OF EIGHT HIGHLY PURIFIED PREPARATIONS

1982 ◽  
Vol 92 (2) ◽  
pp. 195-204 ◽  
Author(s):  
A. A. ZAIDI ◽  
B. FRÖYSA ◽  
E. DICZFALUSY
Keyword(s):  
Biological Activity ◽  
Sucrose Density Gradient ◽  
Relative Distribution ◽  
Human Pituitary ◽  
Sialic Acid Content ◽  
Reference Preparation ◽  
Proportional Increase ◽  
Α And Β Subunits ◽  
Ph Range

Eight highly purified human pituitary FSH preparations and purified preparations of the α-and β-subunits of FSH were fractionated by an electrofocusing technique in the pH range of 2·5–10·0 on a sucrose density gradient. The human (h) FSH activity in each of the eluted fractions was monitored by an in-vitro bioassay and a radioimmunoassay procedure. After electrofocusing, the overall recovery of the biological activity of the eight preparations was between 80 and 94% (mean 88%). On the other hand, the recovery of immunoreactivity ranged between 30 and 84% (mean 71%). A loss of over 85% of hFSH immunoreactivity was observed when the α- and β-subunits of hFSH were fractionated by the same procedure. The specific loss of varying amounts of immunoreactivity in all preparations during electrofocusing was also reflected by a proportional increase in the ratios of biological activity (B) to immunoreactivity (I); preparation A, which exhibited a loss of 70% of the immunoreactivity, had a threefold increase in its B/I ratio after electrofocusing. Significant differences were observed in the electrofocusing profiles of the eight preparations both in terms of their pI values and of their spread. The disparity in the relative distribution of hFSH activities in different pH regions suggested major differences in the carbohydrate moieties (sialic acid content) of the preparations studied, probably as a result of the chemical manipulations involved in the purification of the hormone. It is suggested that a combination of several (but certainly not all) of the preparations might serve as a provisional International Reference Preparation for hFSH radioimmunoassays.

EFFECT OF STORAGE ON THE BIOACTIVITY OF TWO INTERNATIONAL REFERENCE PREPARATIONS OF HUMAN PITUITARY LUTEINIZING HORMONE IN VITRO

1979 ◽  
Vol 81 (1) ◽  
pp. 153-155 ◽  
Author(s):  
D. M. ROBERTSON ◽  
BERIT FRÖYSA
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Interstitial Cell ◽  
Saline Solution ◽  
Plasma Albumin ◽  
Human Pituitary ◽  
International Reference ◽  
Bovine Plasma ◽  
Reference Preparation

There was no loss of biological activity in vitro when the 1st International Reference Preparation (IRP) for human pituitary gonadotrophins [FSH and LH/interstitial cell stimulating hormone (ICSH) for bioassay] code no. 69/104 and the 1st IRP for human pituitary LH/ICSH [for immunoassay] code no. 68/40 were stored for 1 year at −70 °C in a buffered 0·8% saline solution containing 1% bovine plasma albumin (BPA). However, storage of the 69/104 preparation at −20 °C in either 0·1 or 1% BPA, or at −70 °C in the presence of 0·1% BPA showed a small but significant decrease (∼ 10%) in activity over the same period. It is, therefore, advantageous to store these reference preparations at −70 °C in the presence of 1% BPA.

Polymorphism of human pituitary FSH: analysis of immunoreactivity and in vitro bioactivity of different molecular species

1994 ◽  
Vol 141 (2) ◽  
pp. 359-367 ◽  
Author(s):  
M Simoni ◽  
F Jockenhövel ◽  
E Nieschlag
Keyword(s):  
Molecular Species ◽  
Assay Method ◽  
Aromatase Activity ◽  
Human Pituitary ◽  
Pituitary Glands ◽  
Reference Preparation ◽  
Commercial Kits ◽  
Ph Range ◽  
The Individual

Abstract Follicle-stimulating hormone is known to be highly heterogeneous in serum and in the pituitary. In the present study, we have partially separated different molecular species of human pituitary FSH and characterized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n=15) and female (n=9) human pituitary glands were chromatographed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4–6. FSH was measured in the individual fractions by an in vitro bioassay, based on the FSH-dependent aromatase activity of immature rat Sertoli cells, and by the following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA). In each assay, the kit standard, calibrated against the 2nd International Reference Preparation (IRP) 78/549, and the International Standard (IS) 83/575 were run in parallel. The relative potencies of the kit standards in terms of IS 83/575 were: IFMA 3·08, IRMA 1·62, RIA 2·42, IEMA 1·45 and bioassay 1·14. After chromatofocusing, pituitary FSH eluted mostly in fractions with pH≈4·5, without sex-related differences. In both sexes ≈25% of bioactive material showed a pI<4 and eluted with 1 m NaCl. Although the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest intermethod variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the assay method and the standard. In terms of 2nd IRP 78/549 the activity profiles were similar but not identical to those obtained in terms of IS 83/575 and the ratios between the two values (IS:IRP ratios) were significantly different among the methods. No significant correlation was found between IS:IRP ratios and FSH concentrations or pH. These data suggest that: (1) all the methods have different affinities for standard and unknown and/or for different molecular isoforms of FSH; (2) overall, they perform more accurately when assaying female pituitary FSH, perhaps because of a closer resemblance between standard and unknown; (3) the variability of the IS:IRP ratios indicates a different affinity of the antibodies for IS 83/575 and the kit standard, highlighting the importance of the molecular composition of the reference preparations for the final result; and (4) the results do not support the commonly accepted concept of a higher in vitro biopotency of less acidic species of pituitary FSH. Journal of Endocrinology (1994) 141, 359–367

THE EXTRACTION OF PROLACTIN FROM HUMAN PITUITARY GLANDS

1965 ◽  
Vol 49 (1) ◽  
pp. 1-16 ◽  
Author(s):  
M. Apostolakis
Keyword(s):  
Growth Hormone ◽  
Pituitary Gland ◽  
List Type ◽  
Distilled Water ◽  
Hormone Activity ◽  
Isoelectric Precipitation ◽  
Human Pituitary ◽  
Pituitary Glands ◽  
Prolactin Content ◽  
The Mean

ABSTRACT A method for the extraction of prolactin from human pituitary glands is described. It is based on acetone drying, distilled water extraction, acetone and isoelectric precipitation. Two main products are obtained: Fraction R8 with a mean prolactin activity of 12.2 IU/mg and fraction U8 with a mean prolactin activity of 8.6 IU/mg. The former fraction does not contain any significant gonadotrophin activity and the latter contains on an average 50 HMG U/mg. In both cases contamination with ACTH and MSH is minimal. The growth hormone activity of both these fractions is low. It is postulated that in man too, prolactin and growth hormone are two distinct hormones. A total of 1250 human pituitary glands have been processed by this method. The mean prolactin content per pituitary gland has been found to be 73 IU.

scholarly journals Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

2005 ◽  
Vol 49 (8) ◽  
pp. 3341-3346 ◽  
Author(s):  
D. T. A. te Dorsthorst ◽  
P. E. Verweij ◽  
J. F. G. M. Meis ◽  
J. W. Mouton
Keyword(s):  
Amphotericin B ◽  
Phosphate Buffer ◽  
Vitro Activity ◽  
Nonlinear Relationship ◽  
In Vitro Activity ◽  
The Mean ◽  
Ph Range ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

ABSTRACT In this study, we investigated the pH dependency of the in vitro activities of amphotericin B (AMB) and flucytosine (5FC) against Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, Rhizopus spp., and Scedosporium prolificans in RPMI 1640 buffered with citrate buffer (pH 4.0, 5.0, 5.4, and 6.0), citrate-phosphate buffer (pH 5.4, 6.0, 6.4, and 7.0), and 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 6.4, 7.0, 7.4, and 7.9). For 5FC, no significant differences were found between MICs obtained with the different buffers, while for AMB, significant differences were found. The MICs obtained with citrate-phosphate buffer were approximately 1 twofold-dilution step higher than the MICs obtained with MOPS. We demonstrated that the in vitro activities of AMB and 5FC against yeast and mold isolates were pH dependent. The in vitro activity of AMB decreased when the pH was lowered, while the in vitro activity of 5FC increased. The effect of the pH on the in vitro activities was dependent not only on the antifungal agent tested but also on the microorganism. For AMB, there was a nonlinear relationship (median r 2, 0.864) for Candida spp., C. neoformans, A. fumigatus, and Rhizopus spp. over the pH range tested. The mean MICs ranged from 0.5 to 2.52 μg/ml at pH 7.0 and from 20.16 to 32 μg/ml at pH 5.0. For S. prolificans, there was no relationship. For 5FC, there was a linear relationship for Candida spp. (median r 2, 0.767) and a nonlinear relationship for C. neoformans and A. fumigatus (median r 2, 0.882) over the pH range tested. The mean MIC values ranged from 0.125 to 1,024 μg/ml at pH 7.0 and from 0.02 to 4 μg/ml at pH 5.0. For Rhizopus spp. and S. prolificans, the relationship could not be determined, since the MIC was >1,024 μg/ml over a pH range of 4.0 to 7.9.

Calcitonin-Like Immunoreactivity in Rat and Human Pituitary Glands: Histochemical, in Vitro, and in Vivo Studies*

Endocrinology ◽  
1980 ◽  
Vol 107 (1) ◽  
pp. 98-107 ◽  
Author(s):  
CARY W. COOPER ◽  
TAI-CHAN PENG ◽  
JOHNNY F. OBIE ◽  
SANFORD C. GARNER
Keyword(s):  
In Vivo Studies ◽  
Human Pituitary ◽  
Pituitary Glands

EVALUATION OF A DEHYDROGENASE ASSAY BASED ON TETRAZOLIUM REDUCTION FOR RAPID IN VITRO ESTIMATION OF FERMENTATION ACTIVITY IN RUMEN CONTENTS

1978 ◽  
Vol 58 (3) ◽  
pp. 501-511 ◽  
Author(s):  
G. A. JONES ◽  
B. A. HUMPHREY
Keyword(s):  
Dehydrogenase Activity ◽  
Rumen Fluid ◽  
Stomach Tube ◽  
Triphenyltetrazolium Chloride ◽  
Activity Maximum ◽  
Alfalfa Hay ◽  
The Mean ◽  
Ph Range ◽  
Second Peak

Rumen contents obtained from fistulated steers fed brome–alfalfa hay was used to evaluate a dehydrogenase assay based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) for rapid in vitro estimation of fermentation activity. Maximum absorbance of TPF occurred at 485 nm over the pH range 3–10. TPF formed from TTC was stable to atmospheric O2. Absorbance decreased if TPF was exposed to light. In the presence of excess TTC, production of TPF in strained rumen fluid (SRF) at 39 °C was linear with elapsed incubation times up to at least 5 min. Dehydrogenase activity was abolished in the presence of air. It was significantly increased if the CO2 gas phase above SRF was replaced with N2 or H2, owing, at least in part, to an increase in the pH of the fermentation. The dehydrogenase activity of SRF was maximal at pH 7.8–8.0. Fractionation of SRF by differential centrifugation showed that about 50% of the dehydrogenase activity was associated with a protozoal fraction sedimenting at 188 × g, and 42% with a bacterial fraction sedimenting at 1,475 × g. Neither the cell-free supernatant obtained by centrifuging SRF at 21,500 × g, nor boiled SRF, nor the hay fed to the animals showed dehydrogenase activity. Whole rumen contents (WRC) showed a mean dehydrogenase activity that was 58% higher than that of SRF. The dehydrogenase activity of SRF was increased in the presence of substrates representing major plant carbohydrates, the greatest stimulatory effect being shown by ground brome–alfalfa hay. When rumen contents were sampled at the same time by stomach tube and through the fistula, the mean dehydrogenase activity of SRF prepared from the stomach tube samples was 137% higher than that of SRF prepared from the fistula samples. In an animal fed at 12-h intervals, the dehydrogenase activity of SRF rose within 2 h after feeding, declined for the next 6 h, and showed a second peak at 9 h. Activity then declined to the pre-feeding level. The dehydrogenase activity of WRC showed a similar pattern but the peaks of activity were less pronounced. It was concluded that dehydrogenase activity based on TTC reduction provides a simple and rapid means of estimating the fermentation activity of rumen contents which may reflect the availability of energy substrates to the microbiota.

Divergence of the in vitro biological activity and receptor binding affinity of a synthetic insulin analog. [21-Asparaginamide-A]insulin

Biochemistry ◽  
1980 ◽  
Vol 19 (20) ◽  
pp. 4547-4556 ◽  
Author(s):  
G. Thompson Burke ◽  
Jacob D. Chanley ◽  
Yoshio Okada ◽  
Alexandros Cosmatos ◽  
Nicolaos Ferderigos ◽  
...  
Keyword(s):  
Biological Activity ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Insulin Analog ◽  
Receptor Binding Affinity
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