A SENSITIVE AND SPECIFIC IN VITRO BIOASSAY METHOD FOR THE MEASUREMENT OF FOLLICLE-STIMULATING HORMONE ACTIVITY

1979 ◽  
Vol 91 (2) ◽  
pp. 224-237 ◽  
Author(s):  
M.-P. Van Damme ◽  
D. M. Robertson ◽  
R. Marana ◽  
E. M. Ritzén ◽  
E. Diczfalusy
Keyword(s):  
Human Growth ◽  
Assay Method ◽  
Human Pituitary ◽  
Bioassay Method ◽  
Specific Radioimmunoassay ◽  
Reference Preparation ◽  
Low Levels ◽  
Lh Rh ◽  
Dose Levels

ABSTRACT An in vitro bioassay method for hFSH is presented. The method is based on the principles previously described by Dorrington et al. (1976b) and involves the assay of oestradiol produced from 19-hydroxyandrostenedione by dispersed Sertoli cells of 10-day old rats when cultured in the presence of graded doses of FSH. Using the 1st International Reference Preparation for human pituitary gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104) as standard, the useful range of the method is from 0.5 to 32 mIU/chamber (2 to 128 mIU/ml). The sensitivity of the method is 0.5 mIU/chamber. The mean index of precision <UNK> obtained from 16 multiple assays over 2 or 3 dose levels was 0.084. Parallelism was obtained between the 69/104 preparation and all preparations under study. The practicability of the proposed assay method is such that 15 preparations at 3 dose levels can be assayed by one person in 3 days. The specificity of the assay was investigated by determining the FSH activity in the following preparations: hFSHa-α and β-subunits, hLH, hCG, hTSH, ACTH, human growth hormone (hGH) human prolactin (hPRL) and luteinizing hormone-releasing hormone (LH-RH). The ACTH, hGH, hPRL and LH-RH preparations studied showed no detectable FSH activity in the assay. In the remaining preparations very low levels of FSH activity were found, corresponding to 0.004 to 0.6 % of the weight of these preparations when compared with a highly purified hFSH preparation, suggesting that the method is specific for FSH. The possible synergistic or antagonistic influence of the above preparations when assayed in the presence of the 69/104 preparation was also assessed. No evidence of a synergistic or antogonistic effect was found. The assay of the hFSH potencies of a limited number of hFSH preparations of varying purity by the proposed in vitro bioassay, an hFSH radioreceptor method and an hFSH specific radioimmunoassay technique revealed that – although the relationship of the various potencies obtained with each method showed a close agreement – the bioassays yielded the highest potency estimates, and the radioimmunoassays the lowest ones. Since the proposed bioassay method is sensitive and considered to be specific for hFSH activity, it provides a suitable basis for the assessment of the specificity of other in vitro methods (radioreceptor and radioimmunoassay) currently used for detecting low levels of FSH activity.

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

BIOLOGICALLY ACTIVE LUTEINIZING HORMONE (LH) IN PLASMA

1979 ◽  
Vol 90 (3) ◽  
pp. 585-598 ◽  
Author(s):  
M. Rajalakshmi ◽  
D. M. Robertson ◽  
S. K. Choi ◽  
E. Diczfalusy
Keyword(s):  
Interstitial Cells ◽  
Biologically Active ◽  
Assay Method ◽  
Human Pituitary ◽  
Ether Extraction ◽  
Reference Preparation ◽  
Pre Treatment ◽  
High Concentrations ◽  
Treatment Step

ABSTRACT An in vitro bioassay method for measuring LH activity was applied to male plasma. This method is based on the specific testosterone response to LH activity by interstitial cells from mouse testes. In contrast to assays conducted on female plasma, non-parallel response lines were obtained between serial dilutions of untreated male plasma and the International Reference Preparation for Human Pituitary Gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104). In an attempt to eliminate this source of error, which would invalidate the assays, plasma was subjected to either ether extraction or charcoal adsorption prior to assay. While ether extraction was ineffective, charcoal treatment eliminated the source of non-parallelism. Evidence is presented indicating that the inclusion of a charcoal pre-treatment step provides an assay method for LH which fulfils the recognized criteria of reliability when applied to male plasma. An investigation of the likely causes of non-parallelism was undertaken by incubating mouse interstitial cells with various steroids and steroid sulphates at concentrations likely to be present in plasma. While most of the presumed precursors of testosterone were converted to testosterone, steroid sulphates (dehydroepiandrosterone sulphate and pregnenolone sulphate) at high concentrations as present in male plasma were the most active compounds in forming testosterone. However, the amount of testosterone produced from these precursors under controlled conditions was insufficient to account entirely for the deviation from parallelism observed with male plasma. Hence, the non-parallelism observed with untreated plasma samples cannot be entirely explained by the presence of steroidal testosterone precursors in male plasma.

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.

Influence of the assay method used on the selection of the most active forms of FSH from the human pituitary

1986 ◽  
Vol 113 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Leif Wide ◽  
Bruce Hobson
Keyword(s):  
Biological Properties ◽  
Dominant Factor ◽  
Assay Method ◽  
Major Influence ◽  
Human Pituitary ◽  
Vitro Assay ◽  
Molecular Charge ◽  
In Vivo Bioassay

Abstract. The biological properties of different forms of human pituitary FSH, varying in their molecular charge, were investigated. FSH in two individual human pituitaries and a pool of 30 human pituitaries was extracted and subjected to electrophoresis. From each electrophoresis 14 consecutive fractions with the highest RIA activity were examined with in vitro and in vivo bioassays. The in vitro assay was based upon the estimation of oestradiol produced by cultured Sertoli cells from 10 day old rats. The in vivo bioassay was an hCG augmented test using immature female mice injected on 3 consecutive days. The increase in ovarian weight was the index of response. Both in the individual and in the pooled pituitary material the less negatively charged forms had the highest activity in the in vitro bioassay. In contrast, the more negatively charged forms had the highest activity in the in vivo bioassay. Forms of FSH from each of the two individual pituitary extracts were pooled according to their migration rate and injected iv into mice. The amount of FSH remaining in the circulation of the mouse after 1 h was related to the molecular charge. The highest value was obtained with the pool containing the more negatively charged forms of the hormone. The results indicate that the disappearance rate of the FSH molecule is a dominant factor in the in vivo bioassay. A consequence of these observations will be that the assay method chosen to monitor the purification of FSH will have a major influence on the biological properties of the final preparation.

THE INTERNATIONAL REFERENCE PREPARATION OF HUMAN PITUITARY LUTEINIZING HORMONE, FOR IMMUNOASSAY

1978 ◽  
Vol 88 (2) ◽  
pp. 250-259 ◽  
Author(s):  
P. L. Storring ◽  
D. R. Bangham ◽  
P. Mary Cotes ◽  
Rose E. Gaines ◽  
S. L. Jeffcoate
Keyword(s):  
Luteinizing Hormone ◽  
Reproductive Organ ◽  
Expert Committee ◽  
Confidence Limits ◽  
Binding Assays ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Standardization ◽  
Reference Preparation

ABSTRACT The preparation and nature of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay are described. A collaborative assay of this material (coded 68/40) in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH (ICSH)), for Bioassay (coded 69/104) was carried out by twelve laboratories in seven different countries, using different bioassay and immunoassay methods. The weighted combined potency estimate (with 95 % confidence limits) was 52.1 (46.3–58.7) IU/ampoule with male accessory reproductive organ weight gain assays; 80.2 (73.2–87.9) IU/ampoule with ovarian ascorbate depletion assays; 127 (124–129) IU/ampoule with in vitro Leydig cell testosterone production assays; and 124 (121–126) IU/ampoule with testis receptor binding assays. Immunoassay estimates in terms of the same standard were heterogeneous and gave an unweighted mean potency estimate of 33.2 IU with 95% confidence limits of 14.8– 74.4 IU/ampoule. Estimates from different methods gave significantly different results, and the reasons for this are discussed in terms of the differences between the materials being compared and the methods used in the comparison. These data illustrate the conceptual difficulties involved in comparing hetero geneous reference preparations, especially by both bioassay and immunoassay, and some of the causes of inevitable discontinuity of assay results, as described in the 26th Report of the WHO Expert Committee on Biological Standardization. On the basis of these results, and in the interest of maintaining continuity of its unitage, the International Reference Preparation has been allocated a potency of 77 IU/ampoule.

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.

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.

ABSENCE OF AN IMMUNOLOGICALLY DISTINCT FOLLICLE-STIMULATING HORMONE RELEASING FACTOR IN RAT STALK MEDIAN EMINENCE EXTRACTS

1976 ◽  
Vol 68 (1) ◽  
pp. 89-94 ◽  
Author(s):  
M. SHAHMANESH ◽  
S. L. JEFFCOATE
Keyword(s):  
Median Eminence ◽  
Male Rats ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Pituitary Tissue ◽  
Rat Pituitary ◽  
Pre Treatment ◽  
Lh Rh ◽  
Dose Levels

SUMMARY Rat stalk median eminence (SME) extract was incubated with various quantities of an antiserum raised against synthetic luteinizing hormone releasing hormone (LH-RH) and the resulting material was used at two or more dose levels to stimulate release of LH and FSH from anterior pituitary tissue, obtained from male rats, incubated in vitro. The resulting dose–response lines were used to obtain the LH- and FSH-releasing potency of the material, expressed as a percentage of SME extract pre-incubated with normal rabbit serum. Treatment with various doses of antiserum reduced LH-releasing and FSH-releasing potencies to a similar extent. Stalk median eminence extract and synthetic LH-RH were incubated with antiserum and directly compared in the same experiment in vitro. The changes in LH and FSH release caused by pre-treatment with antibody were similar for SME extract and synthetic LH-RH. In a final experiment, SME extract was passed through a column of Sepharose 2B particles to which was coupled anti-LH-RH antibody. The resulting material, when mixed with synthetic LH-RH and used to stimulate rat pituitary tissue in vitro caused a similar increase in the LH- and FSH-releasing potencies of the synthetic decapeptide. It is concluded that rat SME extract contains a single releasing factor for LH and FSH immunologically indistinguishable from synthetic LH-RH.

The stability in vitro of bioactive and immunoreactive LH in human blood and plasma

1988 ◽  
Vol 117 (1) ◽  
pp. 139-145 ◽  
Author(s):  
A. Tsatsoulis ◽  
K. Mavroudis ◽  
J. Frost ◽  
A. Lambert ◽  
S. M. Shalet ◽  
...  
Keyword(s):  
Human Blood ◽  
Prolonged Storage ◽  
Human Blood Plasma ◽  
Immunological Activity ◽  
Human Pituitary ◽  
Reference Preparation ◽  
Degree Of Stability ◽  
The Stability ◽  
Mouse Leydig Cell

ABSTRACT The degree of stability in vitro of bioactive and immunoreactive LH in human blood, plasma and serum was examined. Bioactivity and immunoreactivity of LH were assayed by the dispersed mouse Leydig cell assay and by standard radioimmunoassay respectively, using the same reference preparation (first international reference preparation for human pituitary LH 68/40 for immunoassay). Bioactive and immunoreactive LH were stable in blood and plasma at 22 °C for up to 4 and 24 h respectively, and in blood at 4 °C for up to 24 h. There was no loss of biological or immunological LH activity in plasma which had been either snap-frozen and stored at −70 °C, allowed to freeze at −20 °C and stored at that temperature or kept at 4 °C for 24 h and then stored at − 70 °C. Likewise, the levels of LH in plasma and serum which had been stored at either − 20 or − 70 °C and then thawed and refrozen up to four times remained unchanged. In addition, the biological and immunological activity of LH was not affected after vortexing samples of plasma or serum for up to 60 s. Bioactive LH was also stable in plasma samples after prolonged storage (up to 9 months) at either −70 or −20 °C. We conclude that LH bioactivity and immunoreactivity are stable in blood and plasma following a variety of treatments commonly experienced during normal handling of a blood sample after venepuncture. J. Endocr. (1988) 117, 139–145

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