The International Standard for Pituitary FSH: Collaborative study of the Standard and of four other purified human FSH preparations of differing molecular composition by bioassays, receptor assays and different immunoassay systems

1989 ◽  
Vol 123 (2) ◽  
pp. 275-293 ◽  
Author(s):  
P. L. Storring ◽  
Gaines Das R. E.
Keyword(s):  
Geometric Mean ◽  
In Vitro Bioassay ◽  
International Standard ◽  
Receptor Assay ◽  
Specific Activities ◽  
In Vivo Bioassay ◽  
Ranking Order ◽  
Receptor Assays

ABSTRACT The International Standard for Pituitary FSH (IS; in ampoules coded 83/575) was assayed in terms of the Second International Reference Preparation of Human Pituitary FSH and LH for Bioassay (IRP 78/549) by 27 laboratories in 13 countries using bioassays, receptor assays and immunoassays. Estimates of the FSH content of the IS by in-vivo bioassay were homogeneous both within and between laboratories and gave a combined geometric mean (with 95% fiducial limits) of 79·9 (74·6–85·4) i.u./ampoule. Estimates by different in-vitro bioassays and receptor assays were also homogeneous between assays and laboratories, and gave a combined geometric mean (with 95% fiducial limits) of 31·2 (28·8–33·9) i.u./ampoule. However, estimates by the 19 different immunoassay systems were heterogeneous and varied between 5 and 31 i.u./ampoule. The material in ampoules coded 83/575 was established by the World Health Organization as the International Standard for Pituitary FSH. It was assigned a unitage of 80 i.u./ampoule on the basis of its calibration by in-vivo bioassay, because this assay best identifies and defines the hormone. However, the introduction of the new IS will necessitate the recalibration of immunoassay kits. FSH 84/530, prepared in the same way as the IS from the same FSH preparation, did not differ significantly from the IS in any of the assay systems studied and appeared to be equally suitable as a standard. Four highly purified preparations of human FSH (FSH A–D), differing in their isoform compositions and in their in-vivo: in-vitro bioactivity ratios, were also studied. The ranking order of the specific activities of FSH A–D by in-vitro bioassays paralleled their order by receptor assays and the order of their content of FSH isoforms with isoelectric points > 4·5. (Potency estimates of FSH B and C in terms of the IS were greater by receptor assay than by in-vitro bioassay.) The overall ranking order of the specific activities of FSH A–D by immunoassays was different. Contrary to expectation, estimates in terms of the IS of specific activities by immunoassay differed more between preparations than those by in-vitro bioassay or receptor assay. Differences in specificity between immunoassay systems were demonstrated not only in the calibration of the IS in terms of the crude FSH of IRP 78/549 but also in the comparisons of the highly purified FSH in the IS and FSH A–D. The differences in the immunoreactivities and bioactivities of FSH preparations differing in their isoform compositions greatly complicate the standardization of assays for FSH. Journal of Endocrinology (1989) 123, 275–293

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.

A comparison of preparations of highly purified human pituitary luteinizing hormone: differences in the luteinizing hormone potencies as determined by in vivo bioassays, in vitro bioassay and immunoassay

1982 ◽  
Vol 101 (3) ◽  
pp. 339-347 ◽  
Author(s):  
P. L. Storring ◽  
A. A. Zaidi ◽  
Y. G. Mistry ◽  
Monica Lindberg ◽  
Bridget E. Stenning ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
In Vitro Bioactivity ◽  
In Vitro Bioassay ◽  
Response Curves ◽  
Human Pituitary ◽  
International Reference ◽  
Reference Preparation ◽  
In Vivo Bioassay

Abstract. The LH potencies of 12 preparations of highly purified human pituitary LH, from 6 laboratories, were estimated by 2 in vivo bioassays and an in vitro bioassay in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (coded 69/104); and by immunoassay in terms of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay (IRP; coded 68/40). The LH potencies varied between preparations, including the IRP (68/40), from 864 to 5740 IU/mg by seminal vesicle weight gain (SVW) assay; from 1510 to 11500 IU/mg by ovarian ascorbate depletion (OAAD) assay; from 4490 to 14500 IU/mg by in vitro (testicular interstitial-cell testosterone production) bioassay; and from 2030 to 9180 IU/mg by immunoassay. Estimates of protein content were based on the assumption that the absorbance of LH at 280 nm (A 1% 1 cm) was 6.0. The LH potency of most preparations was highest by in vitro bioassay and lowest by SVW assay. The correlation between activities determined by SVW and OAAD assays was more marked than that between estimates by OAAD assay and in vitro bioassay; there was no correlation between estimates by SVW assay and in vitro bioassay. The slopes of the log dose-response curves of preparations in the OAAD assay were positively correlated with their potencies by OAAD assay and negatively correlated with the slopes of their log dose-response curves in the SVW assay. The qualitative differences between preparations are considered to be a reflection of the heterogeneity of LH and of its modification by different purification procedures. The present data, together with the different patterns of heterogeneity found in some of these preparations by isoelectric focusing in a separate study, suggest that the more basic molecular forms of LH, which are preferentially purified during the isolation of LH free from FSH and TSH, have shorter plasma survival times than the more acidic forms. The LH immunoreactivities of all preparations were significantly correlated with their potencies estimated by each of the in vivo bioassays but not with those estimated by in vitro bioassay. The ratios of in vitro bioactivity (in terms of IRP (68/40)): immunoreactivity varied between preparations from 0.53–1.5. The FSH content of each preparation was less than 2% (w/w) by bioassay and immunoassay. Most preparations were more potent by in vitro bioassay than by in vivo bioassay, which contrasted with, and complemented, findings for purified FSH preparations. This indicated that, as in the case of LH, the more basic molecular species of FSH are associated with lower ratios of in vivo: in vitro bioactivity than are the more acidic species. This study provides the most comprehensive comparison available of the activities of purified preparations of LH isolated from frozen and acetone-dried human pituitary glands in different experienced laboratories. These data are needed for selecting material for an international reference preparation of LH for immunoassay on the basis of high LH potency by in vivo bioassay, recommended by the WHO as a criterion for the identity of the hormone and for its freedom from contaminants. The consequences of the heterogeneity of LH are considered for the purification of the reference material and for the suitability of the latter for the various types of specimens which require LH assays.

512. COMMERCIAL EQUINE CHORIONIC GONADOTROPHIN ISOFORM COMPOSITION, IMMUNOACTIVITY, AND BIOACTIVITY IN A MURINE MODEL USING OVARIAN AUGMENTATION AND TESTICULAR INTERSTITIAL CELL BIOASSAYS

2009 ◽  
Vol 21 (9) ◽  
pp. 111 ◽  
Author(s):  
U. A. Ciller ◽  
J. D. McFarlane ◽  
J. R. McFarlane
Keyword(s):  
Interstitial Cell ◽  
Chorionic Gonadotrophin ◽  
In Vitro Bioassay ◽  
Glycoprotein Hormone ◽  
Immature Female ◽  
Future Studies ◽  
In Vivo Bioassay

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein hormone that is harvested from the plasma of pregnant mares for the formulation of commercial products which are used in a variety of assisted reproductive procedures in livestock. It is well documented that the bioactivity of gonadotrophin products is highly variable. The aim of this study was therefore to determine how different eCG products affected target tissues and cells. Isoforms of eCG were separated with iso-electric focusing and immunoactivity measured with RIA. For in vivo bioassay, dose-response eCG treatments were administered subcutaneously to immature female mice with follow-up treatment on day 2. On day 3 the mice were asphyxiated and ovaries dissected and weighed relative to standard. For in vitro bioassay, adult male mice were asphyxiated, testes removed, decapsulated, dispersed, strained and washed with DMEM:F12 0.1% BSA. The cell stock was counted and diluted for culture at 20,000 cells/well. Cell viability was determined using trypan blue. Five doses were tested for each eCG product. The cells were incubated at 32ºC for 3 hours in 5.0% CO2 in humidified conditions. Media was collected after 3 hours and immediately assayed for testosterone with RIA. Product eCG with ~90% isoforms with pH 3.0–3.7 and ~10% with pH 3.8–4.4, showed 29% greater biologically activity in the ovarian augmentation assay and 44.8% more immunoactivity then stated product bioactivity. Product eCG with ~70% isoforms with pH 3.0–3.7 and ~30% with pH 3.8–7.4 was 3.0% less effective in vivo and had 7.9% less immunoactivity then stated bioactivity, however, in vitro testosterone production was more effectively stimulated then with the previous more acidic eCG product. Our study shows a selective difference in commercial eCG biological activity that appears to depend on subtle isoform heterogeneity. Future studies aim to determine the specific actions of eCG isoforms in target tissues.

Synthesis and Fungicidal Activity of (E)-5-[1-(4-Phenyl-2-oxo-1-oxaspiro[4.5]dec-3-en-3-yl)ethylidene]-2-aminoimidazolin-4-one Derivatives

Synthesis ◽  
2017 ◽  
Vol 49 (20) ◽  
pp. 4663-4669
Author(s):  
Zhao Yu ◽  
Tang Bo ◽  
Guan Aiying ◽  
Wang Weiwei ◽  
Zhang Zhenhua ◽  
...  
Keyword(s):  
1H Nmr ◽  
Fungicidal Activity ◽  
In Vitro Bioassay ◽  
In Vivo Bioassay

(E)-5-[1-(4-Phenyl-2-oxo-1-oxaspiro[4.5]dec-3-en-3-yl)eth­yl­idene]-2-aminoimidazolin-4-one derivatives, as novel fungicidal agents, are designed and synthesized in moderate to excellent yields in four steps from (1-hydroxycyclohexyl)(phenyl)methanone and diketene as the starting materials. The products are characterized by 1H NMR and HRMS (ESI) analysis. An in vivo bioassay shows that some of the products exhibit good to excellent inhibition against P. cubensis and C. lagenarium, whilst up to 94.7% inhibition against P. capsici and up to 78.1% inhibition against B. cinerea is demonstrated in the in vitro bioassay. EC50 values of 3.40 and 5.86 μg/mL are demonstrated against P. capsici and B. cinerea.

The second International Standard for Human Pituitary LH: its collaborative study by bioassays and immunoassays

1993 ◽  
Vol 138 (2) ◽  
pp. 345-359 ◽  
Author(s):  
P. L. Storring ◽  
R. E. Gaines Das
Keyword(s):  
World Health ◽  
Expert Committee ◽  
Human Pituitary ◽  
International Standard ◽  
Receptor Assay ◽  
Wide Range ◽  
In Vitro Bioassays ◽  
Second International

ABSTRACT The second International Standard for Human Pituitary LH (in ampoules coded 80/552; 2nd IS) and LH 81/535 (prepared in the same way as the 2nd IS from the same LH preparation) were compared with the International Reference Preparation of Human Pituitary LH for Immunoassay (IRP 68/40) by 19 laboratories in 11 countries, using in-vivo and in-vitro bioassays, a receptor assay and immunoassays. Geometric mean estimates of the LH content of the 2nd IS (with 95% fiducial limits) in terms of IRP 68/40 were: 34·6 (29·1–41·0) IU/ampoule by in-vivo bioassays; 35·8 (27·0–47·4) IU/ampoule by in-vitro bioassays; 58·6 IU/ampoule by one receptor assay; and 36·8 (35·5–38·1) IU/ampoule by immunoassays. The close agreement between the relative activities of the 2nd IS and IRP 68/40 in the wide range of assay systems studied appears to reflect the fact that both standards contain highly purified LH with similar isoform compositions as judged by isoelectric focusing. Estimates of the LH content of LH 81/535 in terms of IRP 68/40 and in terms of the 2nd IS tended to be lower than those for the 2nd IS across all methods, but the differences were not statistically significant. The 2nd IS was found to be as suitable as IRP 68/40 as a standard for the in-vitro bioassay and immunoassay of LH in the two serum samples studied. However, the mean estimates of serum LH in terms of either of these standards were more than 150% larger by in-vitro bioassays than by immunoassays and more than 50% larger by one-site than by two-site immunoassays. This may be a reflection of the differences in the isoform composition of the highly purified LH of the 2nd IS and IRP 68/40 and that of the LH in the sera. The significant intra- and inter-laboratory variability observed in this study for LH estimates, especially by in-vitro bioassays but also by immunoassays, is very pertinent to the interpretation of published comparisons of LH bioactivity and immunoreactivity. Estimates of the LH content of ampoules of the 2nd IS and LH 81/535 kept at elevated temperatures showed that both the 2nd IS and LH 81/535 appeared to be adequately stable under normal storage conditions; the in-vivo and in-vitro bioactivities and receptor binding activities of LH were more sensitive than its immunoreactivities to thermal degradation of the LH structure. On the basis of these results, the World Health Organization Expert Committee on Biological Standardization established the preparation in ampoules coded 80/552 as the second International Standard for Human Pituitary Luteinizing Hormone, and assigned an activity of 35 International Units of human pituitary LH to the contents of each ampoule. Journal of Endocrinology (1993) 138, 345–359

Lethality of a tritiated amino acid in early mouse embryos

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 209-217
Author(s):  
Janet L. Wiebold ◽  
Gary B. Anderson
Keyword(s):  
Specific Activity ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Radiation Induced ◽  
Specific Activities ◽  
Tritiated Amino Acids ◽  
Early Mouse

2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.

scholarly journals Platelet Alterations Caused by Antiplatelet Antibodies in the Circulation

1977 ◽  
Author(s):  
T. Nagasawa ◽  
B.K. Kim ◽  
M.G. Baldini
Keyword(s):  
Platelet Count ◽  
Specific Activity ◽  
Rabbit Model ◽  
Antiplatelet Antibodies ◽  
Large Numbers ◽  
Severe Reduction ◽  
Series Of Experiments ◽  
Specific Activities

It is known that antiplatelet antibodies cause loss of platelet cytoplasmic and granular contents in vitro. It is, however, unknown whether similar platelet changes occur in vivo, in the circulation, leading to destruction and phagocytosis of platelets in the R.E. system. To study this possibility a rabbit model was devised. Severe and stable thrombocytopenia was first produced in rabbits by one intravenous injection of Adriamycin. Large numbers of allogenic platelets labeled in vitro with 51Cr and 14C-serotonin were then infused to raise the circulating platelet count to 180-250 × 103/mm3. A dilute heteroimmune antiplatelet serum prepared in the guinea pig was infused intravenously and platelet samples were collected four times during the subsequent 30 minutes to 24 hours. Platelet hexokinase and β-glucuronidase, 14C-serotonin and 51Cr were measured. Within the first 60 min the specific activity of 51Cr in platelets decreased by 21%, 14C-serotonin declined by 30%, hexokinase by 5% and β-glucuronidase by 29%. During the subsequent 24 hours only 51Cr and hexokinase registered a mild decrease but 51C-serotonin and β-glucuronidase remained essentially unchanged. In a second series of experiments the effect of platelet alloantibodies was studied in rabbits previously immunized with allogenic platelets. The decline in the specific activities of the enzymes and 14C-serotonin was similar to that observed in animals treated with heteroimmune sera but loss of 51Cr was more severe. These results demonstrate that the platelets remaining in the circulation after the disappearance of the immediate effect of hetero- or alloantibodies were qualitatively altered with a severe reduction of their granular and cytoplasmic contents.

Effect of Sodium-Transport Inhibitors on Bronchial Reactivity in Vivo

1990 ◽  
Vol 79 (4) ◽  
pp. 325-330 ◽  
Author(s):  
Alan J. Knox ◽  
John R. Britton ◽  
Anne E. Tattersfield
Keyword(s):  
Confidence Intervals ◽  
Sodium Transport ◽  
Geometric Mean ◽  
Normal Subjects ◽  
Bronchial Reactivity ◽  
Mean Values ◽  
Doubling Dose ◽  
The Difference

1. We have recently shown that ouabain, an inhibitor of Na+/K+-adenosine triphosphatase, causes contraction of bovine and human airways in vitro, and that amiloride causes relaxation and inhibits receptor-operated contraction in bovine trachealis. 2. To determine whether such drugs alter bronchial reactivity in vivo, we have studied the effect of oral digoxin (an inhibitor of Na+/K+-adenosine triphosphatase) and oral and inhaled amiloride on bronchial reactivity to histamine in three double-blind, placebo-controlled studies. 3. Histamine reactivity was measured as the provocative dose causing a 20% reduction in the forced expiratory volume in 1 s (PD20FEV1) or, when normal subjects were included, the provocative dose causing a 35% reduction in the specific airways conductance (PD35sGaw); the results are given as geometric mean values. 4. In study 1, 13 atopic asthmatic subjects were given 20 mg of oral amiloride or placebo on separate days. Two hours after the drug, the geometric mean PD20FEV1 for histamine was 0.43 μmol after amiloride and 0.54 μmol after placebo (95% confidence intervals for the difference: 0.9 to −0.2 doubling doses of histamine; P = 0.2). 5. In study 2, six normal and 24 atopic asthmatic men inhaled 10 ml of 10−2 mol/l amiloride or diluent control in a crossover study. The mean values of PD35sGaw for histamine immediately after inhalation of amiloride and placebo were 3.0 μmol and 4.3 μmol, respectively, in the normal subjects (95% confidence intervals for the difference: −0.53 to 1.52 doubling doses, P = 0.2), and 0.33 μmol and 0.29 μmol in the asthmatic subjects (95% confidence intervals for the difference: −0.95 to 0.57 doubling doses; P = 0.6). 6. In study 3, 24 atopic asthmatic men were treated for 7 days with placebo or oral digoxin (1.5 mg loading dose plus 0.25 mg twice daily for 6 days). The PD20FEV1 for histamine was measured before, 12 h after the loading dose and on day 7 of treatment. The change in PD20FEV1 did not differ significantly after digoxin and placebo, after either 1 day's treatment [mean (95% confidence intervals) difference: 0.56 doubling dose (−0.37 to 1.5 doubling dose)] or 7 day's treatment [mean (95% confidence intervals) difference: 0.3 doubling dose (−1.23 to 1.8 doubling doses)]. 7. Although our work in vitro has suggested that membrane sodium transport may play an important role in determining airway smooth muscle contractility, we have been unable to demonstrate any effect of the sodium-transport inhibitors amiloride and digoxin on histamine reactivity in these studies.

Proteinsynthese in grünen Blättern

1964 ◽  
Vol 19 (3) ◽  
pp. 235-248 ◽  
Author(s):  
Benno Parthier
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Subcellular Fractions ◽  
Mechanical Destruction ◽  
Cell Organization ◽  
Specific Activities ◽  
Short Time ◽  
Natural Cell

In the green leaves of Nicotiana rustica, protein synthesis of various subcellular fractions has been investigated in vivo after 14CO2-photosynthesis and also in vitro by incorporation of radioactive amino acids. Following photosynthesis, homogenization of the tissues, and differential centrifugation of the homogenates, the results show that all structural particles of the cell are able to use photosynthetically formed amino acids for the incorporation into their proteins. The proteins with the highest specific activities are found in the mitochondria-rich fractions, and with the lowest in the soluble cytoplasma supernatant. High specific activities are also observed in the ribosomal-rich fraction in short-time experiments, and also in the chloroplasts after exposure of the leaves to light. After an osmotic-mechanical destruction of the isolated 14C-labelled chloroplasts, the specific activities of lamellar proteins exceed the colourless soluble proteins of the chloroplasts. A green fraction, sedimented at 1,000 g, and perhaps mainly consisting of broken and leached chloroplasts, shows the highest specific activity of all chloroplast fractions. Obviously, due to the destruction of the natural cell organization, in vitro experiments give not only drastically decreased specific activities but also another distribution of the incorporated amino acids between the subcellular fractions, compared with experiments in vivo.

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