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Biomedical subjects

D M Robertson

Publications and source records attributed to D M Robertson.

At least 109 records · Page 6Linked to original sources
PubMed1995

Inhibin forms in human plasma.

In order to identify the molecular weight forms of bioactive and immunoactive inhibin in human plasma, plasma/serum was sequentially fractionated by immunoaffinity chromatography (using immobilised inhibin alpha subunit antiserum), reversed phase HPLC and preparative SDS-PAGE. The electroeluted gel fractions were assayed for inhibin in vitro bioactivity and immunoactivity, the latter by RIA. Initial experiments examined human follicular fluid as an inhibin-rich source. Bioactive and immunoactive fractions of 30, 35, 53, 65 and approximately 120 kDa were identified in addition to bio-inactive, immunoactive fractions of 26 kDa and 32 kDa. These molecular weights correspond to those of known inhibin forms and are attributed to differing degrees of glycosylation of the inhibin alpha subunit and variable processing of the alpha and beta inhibin subunits. Fractionation of male plasma pools revealed the presence of higher molecular weight immunoactive forms (55-120 kDa) as well as 28-31 kDa forms although the molecular weight distribution of activity between pools varied. To assess if the molecular weight pattern was modified by storage and/or subsequent fractionation, protease inhibitors were added initially to plasma and fractionated as above. The molecular weight distribution of immunoactivity was largely unaffected by the treatment, indicating that minimal processing had occurred. Postmenopausal serum itself showed low to undetectable activity. The addition of recombinant human 31 kDa inhibin to postmenopausal serum resulted in a molecular weight profile of inhibin immunoactivity consistent with the presence of 31 kDa inhibin. Fractionation of a serum pool from women undergoing gonadotrophin stimulation, in which inhibin levels were elevated, showed a range of bioactive and immunoactive inhibin forms over the 30-120 kDa range.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Affinity↗
PubMed1994

Ancillary testing for metastasis in patients with newly diagnosed retinoblastoma.

PURPOSE: The primary objective of this study was to determine whether ancillary testing for metastasis is justified in the routine examination of patients with newly diagnosed retinoblastoma. METHODS: The records of all patients with retinoblastoma diagnosed at the Mayo Clinic from 1952 through 1989 or examined within two months of diagnosis elsewhere were reviewed. The ancillary tests used to detect metastatic disease, available histopathologic specimens, and five-year follow-up were recorded on all patients. RESULTS: We identified 100 patients, with newly diagnosed retinoblastoma, who were ultimately followed up for at least five years after diagnosis or until death. Of the 100 patients, 35 underwent bone marrow aspiration, lumbar puncture, or both, within one month of diagnosis. Results of all ancillary tests performed in these 35 patients were negative for metastatic disease. Nine of the 35 patients had choroidal extension and two had retrolaminar involvement. Although one patient developed orbital recurrence that was successfully treated with exenteration and chemotherapy, none of the 35 patients died of metastatic retinoblastoma in a mean follow-up period of 9 1/2 years. The overall survival free of metastasis from retinoblastoma in these 100 patients was 97% at five years (95% confidence interval: 94% to 100%). CONCLUSIONS: The routine use of bone marrow and lumbar puncture studies in the examination of all patients with newly diagnosed retinoblastoma cannot be supported by this study.

Bone Marrow Examination↗
PubMed1994

Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome.

We investigated the role of vascular endothelial growth factor (VEGF) in ovarian hyperstimulation syndrome (OHSS). Two similar peaks of permeability activity were seen in OHSS ascites and liver ascites spiked with recombinant human VEGF (rhVEGF); no activity was seen in control liver ascites. Incubation with rhVEGF antiserum decreased activity in the two OHSS peaks by 79% and 65% and the two spiked liver peaks by 49% and 50%. Control serum produced 24% and 27%, and 17% and 0% reductions, respectively. This is evidence that the major capillary permeability agent in OHSS ascites fluid is VEGF.

Animals↗
PubMed1994

Image analysis quantification of the Miles assay.

The Miles assay for vascular permeability has high intra- and interassay coefficients of variation (CVs). Quantification, usually by dye extraction and spectrophotometry, is time consuming. In this study, quantification by this means was compared with image analysis using the Olympus CUE-2 Image Analyzer (version 4). The test substance was recombinant human vascular permeability factor (rhVPF). The quantification process took approximately 10 min with image analysis. Formamide extraction and spectrophotometry required 1 hr of preparation, 4-6 days of incubation, and 1 hr for filtration and spectrophotometry. Between assay CVs ranged from 0 to 30% for spectrophotometry, but were all < 10% for image analysis. The sensitivity (2SD above the negative control mean) of the image analysis approach was 64 +/- 25 ng/mL, whereas for spectrophotometry it was 65 +/- 29 ng/mL. Interanimal CVs for rhVPF at 200 and 1000 ng/mL were 15% and 26% when assessed by spectrophotometry and 7% and 22%, respectively, by image analysis. The R2 value for the correlation of image analysis with spectrophotometry was 81.4%. Test substances injected close to the spine evoked a greater permeability response than those injected laterally: at 200 ng/mL p = 0.005, at 1000 ng/mL p = 0.1 (unpaired t tests).

Animals↗
PubMed1994

Testosterone effects on spermatogenesis in the gonadotropin-releasing hormone-immunized rat.

Active immunization of adult rats with a GnRH fusion protein was used to inhibit gonadotropin secretion and to establish an in vivo model for studying the hormonal control of spermatogenesis. The model was characterized in terms of the efficacy of the immunogen as well as the time course and nature of the immunological and biological responses. To study the reinitiation of spermatogenesis, testosterone (T) was chosen for this initial study as it is known to restore spermatogenesis in gonadotropin-deficient rats. Adult Sprague-Dawley rats were actively immunized with a proprietory GnRH immunogen (BA-11, 100 micrograms s.c.) every 4 wk. After 12 wk, 58% of animals showed markedly suppressed testicular size, as assessed by scrotal palpation, and were classed as responders. Serum LH, FSH, and T as well as the testicular elongated spermatid count (ESC) and epididymal sperm were undetectable in responding animals. Marked reductions in testicular (29% of control), prostatic (8% of control), and epididymal (32% of control) weights were seen. Spermatogenesis was severely disrupted with no evidence of progression beyond round spermatids. To study the action of T in GnRH-immunized animals, T (defined by lengths of s.c. silastic implant, T3-T24 cm) was given to responding animals. Animals were killed 2, 8, and 12 wk after T24 administration. In response to T24, serum T levels increased to 4 times control levels, serum FSH levels were restored to 65% of control levels by 2 wk, and serum LH remained undetectable. Testicular weight increased to 80% of control levels at 12 wk (p < 0.05 vs. control). Epididymal and prostatic weights were normalized by T. ESC increased to 82% of control values at 12 wk (110 +/- 10 vs. control 134 +/- 8 million/testis, p = 0.001). Spermatogenesis was histologically normal after 8 wk of T24 treatment. To study the time course and dose response of T action, animals were immunized with another GnRH immunogen (BA-17), which yielded an 87% response rate at 12 wk. Testicular weight increased by Day 5 of T24 treatment, and a clear dose-response effect was apparent. The restoration of ESC was delayed compared to that of testicular weight (no restoration at 2 wk) and required > or = T6 treatment. Rats immunized for 20 wk and then given T24 treatment showed a similar pattern of restoration in testicular weight and ESC. Serum FSH was normalized by Day 2 of T treatment by doses > or = 3 cm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Effects of testosterone on spermatogenic cell populations in the adult rat.

The aim of this study was to investigate the progression of germ cell populations through the rat spermatogenic cycle when spermatogenesis was suppressed by LH withdrawal through the use of a combination of testosterone (T) and estradiol (E) and then reinitiated by the administration of high doses of T. Adult Sprague-Dawley rats received 3-cm T and 0.4-cm E silastic implants for 6, 8, or 12 wk to suppress spermatogenesis followed by high-dose T (24-cm implants) for up to 12 wk to reinitiate spermatogenesis. The number of spermatogonia, primary spermatocytes, and round spermatids per testis was established by stereological techniques, and the elongated spermatid number was determined by the testicular content of nuclei resistant to homogenization in Triton X-100. Suppression for 6-12 wk resulted in moderate and significant (p < 0.05) reductions in the numbers of type A and type B spermatogonia (to 44-59% of control levels), preleptotene (68-72% of control), and leptotene/zygotene spermatocytes (62-79% of control) as well as in the numbers of stage I-VII (56-69% of control) and stage VIII-XIV (35-43% of control) pachytene spermatocytes. Round spermatids were suppressed to 29-45% of control levels (p < 0.05) while elongated spermatids were undetectable. The hourly production rates of germ cells (calculated using published time divisors) were used to study the cellular conversions through spermatogenesis (based on the ratios of the hourly production rates) and revealed that T withdrawal consistently abolished the conversion of round to elongated spermatids. The duration of suppression (6, 8, or 12 wk) had no effect on the degree to which germ cell populations or conversions were reduced. In response to high-dose T administration, spermatogonial and spermatocyte numbers and production rates (up to stage I-VII pachytene) remained suppressed, while stage VIII-XIV pachytene spermatocytes showed an increase of borderline significance. On the other hand, round and elongated spermatid numbers and production rates increased significantly (to 81% and 78% of control, respectively) and their conversion was normalized, i.e., the spermiogenic process was restored to a level consistent with the numbers of earlier germ cells proceeding through the cycle. These data suggest that, in the presence of low T levels, spermatogenesis proceeds at approximately 65% of normal levels between the spermatogonial and round spermatid stages, irrespective of the duration of T-induced suppression. This is followed by a precipitous decline in elongated spermatid number that is attributed to the disappearance of round spermatids.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Development and validation of a radioimmunoassay for follistatin in human serum.

OBJECTIVE: Follistatin (FS/FSH-suppressing protein/activin-binding protein) is a single-chain glycoprotein, structurally distinct from inhibin, that has been shown to have inhibin-like activity in suppressing FSH secretion both in vivo and in vitro. The aim of these studies was to develop and validate a radioimmunoassay (RIA) for FS in human serum, and to describe the physiological variations of serum FS in humans. PATIENTS: Serum was collected from normal men, and normal women in the follicular and luteal phases of the menstrual cycle. Clinical samples were also collected from male patients with hypogonadism, post-menopausal women and pregnant women in the first, second and third trimesters. MEASUREMENTS: A RIA for FS in human serum was developed using antisera raised against purified bovine FS and using bovine FS as tracer and standard. Serial dilutions of serum were non-parallel to purified bovine FS standard in the RIA. The addition of sodium dodecyl sulphate (SDS 0.05%) to the assay buffer resolved the non-parallelism suggesting that the interference of serum in the RIA was an assay matrix effect. To characterize the serum FS immunoactivity further, serum was fractionated by gel filtration on Sephadex G-100. At neutral pH, FS immunoactivity eluted as a major peak in the molecular weight range > 200 kDa. In 0.1 M HCl, a second peak of FS immunoactivity eluted in the molecular weight range 30-60 kDa consistent with the known sizes of FS. This suggested that FS was dissociating from a larger complex. Fractions taken from the low molecular weight region diluted in parallel with bovine FS in contrast with fractions from the high molecular weight region which were non-parallel. It is concluded that the non-parallelism of human serum in the FS RIA is due to the binding of serum FS to an unknown high molecular weight factor. This interference is eliminated by the inclusion of 0.05% SDS in the assay buffer. The RIA in 0.05% SDS has been applied to the description of the normal and pathophysiological variations of FS in human serum. RESULTS: There were no significant differences between FS levels in serum from hypogonadal men, normal women in the follicular phase of the menstrual cycle, post-menopausal women and pregnant women from the first, second and third trimesters. FS levels in serum of women in the luteal phase of the menstrual cycle were significantly lower than all other groups. FS levels in normal men were significantly higher than those in both luteal phase women and women in the first trimester of pregnancy (P < 0.05). CONCLUSIONS: These findings argue against a role for circulating follistatin in the control of gonadotrophin secretion and suggest that the gonads and/or conceptus are not the primary source of follistatin immunoactivity in serum. The lower follistatin levels in the luteal phase of the menstrual cycle remain unexplained.

Chromatography, Affinity↗
PubMed1994

Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle.

Several spermatogenic cell types have been reported to be responsive to testosterone (T) in vivo. We have proposed that the principal action of T is to facilitate the maturation of round to elongated spermatids during spermiogenesis. To identify T-dependent cell types during spermiogenesis, round spermatid populations were counted using stereological techniques in adult rats after T withdrawal and replacement. The number of round spermatids per testis in stages I-III, IV-VI, VII, and VIII of the spermatogenic cycle were determined and, based on the known duration of each stage, the hourly production rates of round spermatid populations calculated. Sprague-Dawley rats received 3 cm T and 0.4 cm estradiol implants (TE treatment) for 11 weeks to suppress LH, testicular T levels, and spermatogenesis. To restore sperm production, high-dose T (24 cm) implants were then given, and animals were perfused 0, 2, 4, and 7 days later. Total testicular T levels were suppressed to 2.9% of control levels by TE treatment and significantly increased (P < 0.05) after 2 days of high-dose T, to remain between 7-12% of control. The hourly production rates of round spermatids between stages I and VII were suppressed to 29-35% of control by TE treatment and remained unchanged by high-dose T administration. Stage VIII round spermatid hourly production rates however, were markedly reduced to 5% of control by TE treatment and increased significantly (P < 0.05) to 27% of control after 4 days of high-dose T. The efficiency of conversion of spermatids through spermiogenesis was estimated from the ratios of the hourly production rates of successive spermatid groups. The conversion of spermatids between stages I-VII of the cycle did not differ from control regardless of the treatment. However, the conversion of round spermatids between stages VII and VIII was markedly suppressed to 16% of control by TE treatment and was then normalized after 4 days of high-dose T administration. We conclude that the conversion of round spermatids between stages VII and VIII is a highly T-dependent step during spermiogenesis.

Animals↗
PubMed1994

Partial characterization of inhibin, activin, and follistatin in the term human placenta.

To determine whether the human term placenta contains inhibin, activin, and follistatin, placental homogenates from normal placentae were subjected to several fractionation procedures: 1) dye affinity chromatography; 2) hydrophobic interaction chromatography using phenyl sepharose; 3) gel filtration under acid conditions; 4) reversed phase-high pressure liquid chromatography (RP-HPLC); and 5) preparative polyacrylamide gel electrophoresis and electroelution. Purification was followed by RIA for inhibin, activin, and follistatin as well as in vitro bioassay based on the FSH cell content of rat anterior pituitary cells in culture. Two peaks of immunoactive and bioactive inhibin with differing elution patterns on RP-HPLC were shown to have molecular weights of 33K and 32K on polyacrylamide gel electrophoresis. These peaks may represent inhibin A and B. Three peaks of immunoactive activin on RP-HPLC had molecular weights of 26.5K, 25.5K, and 27K and may represent activin A, AB, and B. Immunoactive follistatin eluted on RP-HPLC as a broad peak, which on polyacrylamide gel electrophoresis consisted of three main activities consistent with molecular weights of 31K, 35K, and 38K. These studies demonstrate that the term placenta contains inhibin, activin, and follistatin, which have been partially characterized. The presence of these substances within the placenta raises the possibility of their function in local paracrine interactions.

Activins↗
PubMed1994

Specific roles for the asparagine-linked carbohydrate residues of recombinant human follicle stimulating hormone in receptor binding and signal transduction.

FSH comprises two distinct subunits, both of which contain asparagine-linked carbohydrate residues, located at positions 52 and 78 on the alpha-subunit and positions 7 and 24 on the beta-subunit. These carbohydrate chains have been shown to regulate the biological activity of FSH, including signal transduction and receptor binding. However, the specific roles of the individual carbohydrate chains have been poorly defined. Using site-directed mutagenesis we disrupted the consensus sequences for glycosylation and expressed the mutated cDNAs in Chinese Hamster Ovary cells. Specifically deglycosylated FSH variants were secreted from all clonal cell lines expressing the mutated FSH cDNAs except for the cell line that lacked all four glycosylation sites. Analysis of the singly or doubly deglycosylated FSH mutants revealed that removal of the carbohydrate residue at position 78 on the alpha-subunit significantly increased the receptor binding affinity of human FSH by 72%. Removal of the other carbohydrate residues had no significant effect on receptor binding. The carbohydrate residue at position 52 on the alpha-subunit was found to play an essential role in signal transduction as its removal resulted in a significant decrease in potency to 26% of wild type levels. The other individual carbohydrate residues appear to play a minor role in signal transduction, although removal of each residue results in reduced maximal response. The removal of both alpha-subunit carbohydrates resulted in a significant decrease in biopotency, to 41% of wild type levels; whereas, the removal of both beta-subunit carbohydrate chains resulted in a significant increase in biopotency, to 216% of wild type levels. These studies have allowed the identification of site-specific roles for the carbohydrate residues of human FSH. Our data suggest that the carbohydrate residues play a greater role in determining the biological activity of FSH than has been suggested in similar studies of other glycoprotein hormones.

Animals↗
PubMed1993

Serum immunoactive inhibin levels in early pregnancy after in vitro fertilization and embryo transfer.

OBJECTIVE: To determine the maternal serum concentrations of inhibin, E2, P, and hCG in early pregnancies arising from IVF and ET or GIFT and to assess the value of these hormone measurements in determining outcome of pregnancy. DESIGN: Serum immunoactive inhibin, E2, P, and hCG levels were measured in the first trimester of pregnancies after IVF-ET and GIFT procedures. SETTING: In vitro fertilization and ET or GIFT was undertaken at Monash IVF, Melbourne, Victoria, Australia. PATIENTS: At least two blood samples were collected from 117 women between 4 and 11 weeks of gestation. MAIN OUTCOME MEASURES: The hormone concentrations in the IVF-ET and GIFT pregnancies were compared with those in pregnancies and related to outcome of pregnancy. RESULTS: Serum inhibin levels in singleton pregnancies were significantly higher than in comparable normal pregnancies. In contrast to normal conceptions in which inhibin concentrations rose to peak at 11 weeks, the levels found in IVF-ET and GIFT singleton pregnancies were high at 5 weeks' gestation and declined subsequently. In twin pregnancies, the inhibin levels were significantly greater than those in singleton pregnancies. In biochemical pregnancies diagnosed by increasing hCG concentrations in the absence of an embryonic sac, inhibin levels were significantly lower than those found in singleton pregnancy, as were E2, P, and hCG levels. In anembryonic pregnancies, diagnosed by the confirmation of an intrauterine gestation sac with no evidence of a fetal complex, inhibin concentrations were highest at week 4 and declined, being significantly lower at all stages of gestation. In ectopic pregnancy, serum inhibin levels were lower at all stages of gestation, whereas E2 concentrations were not lower until 6 weeks and P levels until week 5. Serum hCG levels were significantly lower at all stages of gestation. In women with spontaneous abortions, inhibin levels were lower than singleton pregnancies at 7 weeks. CONCLUSIONS: Serum inhibin concentrations are elevated in pregnancies arising from ovarian hyperstimulation in the first trimester when compared with those in normal pregnancy, probably as a result of the presence of multiple corpora lutea resulting from ovarian hyperstimulation. Serum inhibin, E2, P, and hCG are useful markers of abnormal pregnancy outcome.

Chorionic Gonadotropin↗
PubMed1993

A syndrome of acute severe muscle necrosis in intensive care unit patients.

Four septic patients and one asthmatic patient are described who developed a severe paralytic disorder in an intensive care unit (ICU), associated with a rise in serum creatine kinase and a severe necrotizing myopathy. All cases had received non-depolarizing muscle blocking agents and large intravenous doses of glucocorticoids. Three patients developed myoglobinuria. No improvement or very little improvement in muscle function was noted in the four fatal cases. The single survivor recovered his strength after 6 months. This syndrome ("necrotizing myopathy of intensive care") provides one of the differential diagnoses for ICU-acquired weakness. The myopathy appears to have several interdependent causes and it is proposed that these should be classified as myonecrosis "priming" factors (glucocorticoids, myotropic infections, sepsis) and "triggering" factors (non-depolarizing muscle blocking agents).

Acute Disease↗
PubMed1993

Isolation of FSH from bovine pituitary glands.

An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5.7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay. SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19.5 kDa and 15.8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with alpha- and beta-subunits, and a third minor (< 20%) sequence consistent with the alpha-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators.

Animals↗
PubMed1993

Radioimmunoassay of FSH-suppressing protein in the ewe: concentrations during the oestrous cycle and following ovariectomy.

A sensitive and specific heterologous radioimmunoassay for FSH-suppressing protein (FSP or follistatin) was applied to ovine plasma. Following a logit-log dose transformation, parallel dose-response lines were observed between purified bovine 35 kDa FSP used as standard and serial dilutions of ewe plasma. Activin-A, inhibin-A and a range of other proteins showed low (< 0.5%) cross-reactivity in the assay. Daily variations in the peripheral concentrations of FSP were measured across the ovine oestrous cycle. The peripheral concentrations of plasma FSP in adult ewes revealed a significant (P < 0.01) increase (33%) during the luteal phase above follicular phase levels, peaking 10 days after the LH surge. FSP concentrations were determined in arterial and venous plasma from the ovary, head, kidney and liver. A significant (P < 0.05) increase across the ovary was detected with no significant differences across the head, liver and kidney. To investigate the relationship between gonadal FSP and the pituitary, ewes underwent ovariectomy and hypophysectomy. FSP levels rose (100-110%, P < 0.01) during the period of surgery for both bilateral ovariectomy and sham ovariectomy, and then decreased significantly (37-44%) at 4-6 h after surgery. A further rise in plasma FSP (180-200% increase above pretreatment levels, P < 0.001) was observed 10-12 h after ovariectomy and sham ovariectomy. FSP levels then returned to preoperative levels during the following 26 h. Plasma FSP levels in long-term ovariectomized and hypophysectomized ewes were not significantly different from preoperative levels. To determine whether the pattern of plasma FSP seen during the ovariectomy study was due to the effect of the induction of anaesthesia, ewes were treated with sodium thiopentone and halothane or 0.9% (w/v) NaCl by procedures of similar duration to that used during surgery. Both treatments resulted in an elevation of FSP levels (33-62%) over pretreatment values only at the time of induction of anaesthesia. To examine further whether this rise in plasma FSP observed after anaesthesia was due to a stress response and therefore under the control of the pituitary-adrenal axis, ewes were treated with ACTH, dexamethasone or saline only. A further group of sheep were exposed to a barking dog for 10 min. No change in FSP levels compared with pretreatment levels or saline-treated controls were noted following any of these treatments.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗
PubMed1993

Isolation and characterization of human LH isoforms.

Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (> 90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38,650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were < 0.5% and < 7.8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23.5-24.5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21.1-22.4 kDa and 18.0-20.5 kDa, probably corresponding to the alpha and beta subunits of hLH respectively. The remaining three less pure isoform preparations (70-90% pure) contained additional bands of 16 kDa and 26.3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11-14 kDa which was < 7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two alpha and two beta hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of alpha and beta subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species.

Amino Acid Sequence↗
PubMed1993

Immunological activities of highly purified isoforms of human FSH correlate with in vitro bioactivities.

In a recent study, a five- to eightfold range in human FSH radioreceptor activity (RRA) was documented for highly purified isoforms of FSH when the data were expressed on an FSH protein content basis as determined by amino acid analysis. This study examined the FSH in vitro bioactivity and immunoactivity of these preparations. FSH in vitro biological activity showed a five- to eightfold range in activity with a high correlation with the RRA values (r = 0.82). A similar five- to eightfold range of values was obtained with a specific FSH radioimmunoassay and an FSH two-site immunoassay with high correlations again observed between each other, between each immunoassay and with either the in vitro bioassay or the RRA method (r = 0.77-0.995). Although there was overall a close correlation between these assays, significant differences in ratios of activities between the in vitro bioassay and other methods were observed with highly purified FSH isoform preparations from different pI regions. The high correlation between in vitro bioassay/RRA methods and immunoassay methods over a wide range of isoform specific activities suggests that these methods are detecting similar structural features on each isoform. It is thus concluded that these immunoassays are not solely measuring hormone mass based entirely on amino acid composition. This conclusion raises questions about ratio measurements of FSH, where immunoassay methods are presumed to measure total protein content, and their application in physiological situations and clinical practice.

Biological Assay↗