High-permeability alternatives to current dialyzers performing both high-flux hemodialysis and postdilution online hemodiafiltration.

Most high-flux dialyzers can be used in both hemodialysis (HD) and online hemodiafiltration (OL-HDF). However, some of these dialyzers have higher permeability and should not be prescribed for OL-HDF to avoid high albumin losses. The aim of this study was to compare the safety and efficacy of a currently used dialyzer in HD and OL-HDF with those of several other high permeability dialyzers which should only be used in HD. A prospective, single-center study was carried out in 21 patients. Each patient underwent 5 dialysis sessions with routine dialysis parameters: 2 sessions with Helixone (HD and postdilution OL-HDF) and 1 session each with steam sterilized polyphenylene, polymethylmethacrylate (PMMA), and medium cut-off (MCO) dialyzers in HD treatment. The removal ratios (RR) of urea, creatinine, ß2 -microglobulin, myoglobin, prolactin, α1 -microglobulin, α1 -acid glycoprotein, and albumin were compared intraindividually. A proportional part of the dialysate was collected to quantify the loss of various solutes, including albumin. Urea and creatinine RRs with the Helixone-HDF and MCO dialyzers were higher than with the other 3 dialyzers in HD. The ß2 -microglobulin, myoglobin and prolactin RRs with Helixone-HDF treatment were significantly higher than those obtained with all 4 dialyzers in HD treatment. The ß2 -microglobulin value obtained with the MCO dialyzer was also higher than that obtained with the other 3 dialyzers in HD treatment. The myoglobin RR with MCO was higher than those obtained with Helixone and PMMA in HD treatment. The prolactin RR with Helixone-HD was significantly lower than those obtained in the other 4 study sessions. The α1 -microglobulin and α1 - acid glycoprotein RRs with Helixone-HDF were significantly higher than those obtained with Helixone and PMMA in HD treatment. The albumin loss varied from 0.54 g with Helixone-HD to 3.3 g with polyphenylene. The global removal score values ((UreaRR + ß2 -microglobulinRR + myoglobinRR + prolactinRR + α1 -microglobulinRR + α1 -acid glycoproteinRR - albuminRR )/6) were 43.7% with Helixone-HD, 47.7% with PMMA, 54% with polyphenylene, 54.8% with MCO and 59.6% with Helixone-HDF, with significant differences. In conclusion, this study confirms the superiority of OL-HDF over HD with the high-flux dialyzers that allow both treatments. Although new dialyzers with high permeability can only be used in HD, they are in an intermediate position and some are very close to OL-HDF.

High production and optimization of the method for obtaining pure recombinant human prolactin.

Prolactin is a pituitary hormone that is involved diverse physiological functions, such as lactation, reproduction, metabolism, osmoregulation, immunoregulation, and behavior. Its level of glycosylation is low in vivo, which favors its expression in bacterial systems. In the present work recombinant human prolactin (rec-hPRL) was expressed from the p1813-hPRL vector in Escherichia coli strain in inclusion bodies with 530.67 mg of rec-hPRL per liter of induced bacterial culture. The solubilization and renaturation of rec-hPRL followed by two methods described in the literature for this protein: one with detergent and basic pH, and other urea and dialyses was done by studying. The protocol with detergent/basic pH was not successful, whereas protocol with urea/dialyses was obtained pure protein and this was optimized. Rec-hPRL was obtained in a soluble, pure and active form, when the sample was 8-fold concentrated in the solubilization phase, allowing 33% recovery, 3-fold more that the original method. The pure protein was obtained with 38.37 i. u./mg activity, which is three times greater than that of the PRL standard from the WHO. In conclusion, this work obtained the highest production of rec-hPRL, and concentrating the sample eight times in the solubilization stage was decisive for obtaining a highly concentrated, active protein for future work.

New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin.

Recombinant chicken prolactin (chPRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16 h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins.

A 60 kDa prolactin variant secreted by cervical cancer cells modulates apoptosis and cytokine production.

Prolactin (PRL) is associated with different types of cancer, such as cervical cancer. Recombinant PRL has antiapoptotic effect on cervical cancer cells, and it can also induce cytokine production on macrophages. A 60 kDa variant of PRL is produced by cervical cancer cells. The aim of the present study was to evaluate this variant's bioactivity, to test its effect on cervical cancer cell apoptosis, and to assess its ability to induce cytokine production on THP-1 macrophages. First, 60 kDa PRL was isolated and used to stimulate Nb2 cells. Later, apoptosis was measured after exposure to 60 kDa PRL. Finally, cytokines were measured on THP-1 stimulated supernatants. Our results show that 60 kDa PRL increased Nb2 cell proliferation. Apoptosis was decreased after stimuli with 60 kDa PRL in cervical cancer cells. IL-1ß and TNF-α are produced by THP-1 macrophages after stimuli. These results suggest that 60 kDa PRL produced by cervical cancer cells is able to reduce apoptosis in HeLa, SiHa and C-33A cells and induce IL-1ß and TNF-α production by THP-1 macrophages.

Serum prolactin revisited: parametric reference intervals and cross platform evaluation of polyethylene glycol precipitation-based methods for discrimination between hyperprolactinemia and macroprolactinemia.

BACKGROUND: Hyperprolactinemia diagnosis and treatment is often compromised by the presence of biologically inactive and clinically irrelevant higher-molecular-weight complexes of prolactin, macroprolactin. The objective of this study was to evaluate the performance of two macroprolactin screening regimes across commonly used automated immunoassay platforms. METHODS: Parametric total and monomeric gender-specific reference intervals were determined for six immunoassay methods using female (n=96) and male sera (n=127) from healthy donors. The reference intervals were validated using 27 hyperprolactinemic and macroprolactinemic sera, whose presence of monomeric and macroforms of prolactin were determined using gel filtration chromatography (GFC). RESULTS: Normative data for six prolactin assays included the range of values (2.5th-97.5th percentiles). Validation sera (hyperprolactinemic and macroprolactinemic; n=27) showed higher discordant classification [mean=2.8; 95% confidence interval (CI) 1.2-4.4] for the monomer reference interval method compared to the post-polyethylene glycol (PEG) recovery cutoff method (mean=1.8; 95% CI 0.8-2.8). The two monomer/macroprolactin discrimination methods did not differ significantly (p=0.089). Among macroprolactinemic sera evaluated by both discrimination methods, the Cobas and Architect/Kryptor prolactin assays showed the lowest and the highest number of misclassifications, respectively. CONCLUSIONS: Current automated immunoassays for prolactin testing require macroprolactin screening methods based on PEG precipitation in order to discriminate truly from falsely elevated serum prolactin. While the recovery cutoff and monomeric reference interval macroprolactin screening methods demonstrate similar discriminative ability, the latter method also provides the clinician with an easy interpretable monomeric prolactin concentration along with a monomeric reference interval.

Evaluation of hyperprolactinaemia with the use of the intervals for prolactin after macroforms separation.

Macroprolactin (MaPRL) - a complex of monomeric prolactin (PRL) with immunoglobulin G, may be a cause of laboratory diagnosed hyperprolactinaemia. To quantify MaPRL, a precipitation with polyethylene glycol may be performed. This method involves calculating of recovery ratio but the cut-off value is not precisely determined. Moreover, it is proposed that the assessment of macroprolactinaemia should include also the evaluation of real PRL concentration which means the level of the hormone after macroforms separation. The study included 245 patients with hyperprolactinaemia, in whom precipitation was performed. A recovery ratio ≤40% indicated macroprolactinaemia. The real PRL concentrations of the studied subjects were compared with reference ranges suggested by the assay manufacturer and with new intervals for PRL after macroforms separation. On the base of the recovery ratio after the precipitation, macroprolactinaemia was detected in 21 persons. In these patients true hyperprolactinaemia (elevation of real PRL concentration above manufacturer's reference ranges) was noted in 9 cases. Among 224 patients with a recovery >40%, real PRL concentration turned out to be within the manufacturer's reference range (pseudohyperprolactinaemia) in 36 persons. The new intervals for PRL after macroforms separation were about 20% lower than the manufacturer's reference ranges. After applying new ranges in patients with macroprolactinaemia, true hyperprolactinaemia was observed in 14 persons, while in the group without MaPRL dominance, pseudohyperprolactinaemia was noted in 5 patients. The use of the recovery ratio only to recognize macroprolactinaemia may lead in some subjects to the misclassification of the results. For that reason the assessment of the PRL concentration after macroforms separation that can help to distinguish true hyperprolactinaemia and pseudohyperprolactinaemia, seems to be reasonable. To evaluate the real PRL concentration, the reference intervals suggested by the manufacturer of immunoassay might be used. However, possibly better means to diagnose patients with hyperprolactinaemia accurately is using an appropriate range for the concentration of PRL after macroforms separation.

Label-free detection of ovarian cancer biomarkers using whispering gallery mode imaging.

Small optical microresonators that support whispering gallery mode (WGM) resonances are emerging as powerful new platforms for biosensing. These resonators respond to changes in refractive index and potentially offer many advantages for label-free sensing. Recently we reported an approach for detecting WGM resonances based on fluorescence imaging and demonstrated its utility by quantifying the ovarian cancer marker CA-125 in buffer. Here we extend those measurements by reporting a simplified approach for launching WGM resonances using excitation light coupled into a Dove prism. The enhanced phase matching enables significant improvements in signal-to-noise, revealing the mode structure present in each resonator. As with all label-free biosensing techniques, non-specific interactions can be limiting. Here we show that standard blocking protocols reduce non-specific interactions sufficiently to enable CA-125 quantification in serum samples. Finally, fluorescence imaging of WGM resonances offers the potential for large scale multiplexed detection which is demonstrated here by simultaneously exciting and imaging over 120 microsphere resonators. For multiplexed applications, analyte identity can be encoded in the resonator size and/or location. By encoding analyte identity into microresonator size, we simultaneously quantify the putative ovarian cancer markers osteopontin (38 µm diameter sphere), CA-125 (53 µm diameter sphere), and prolactin (63 µm diameter sphere) in a single PBS assay. Together, these results show that fluorescence imaging of WGM resonances offers a promising new approach for the highly multiplexed detection of biomarkers in complex biological fluids.

Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space.

Prolactin (PRL) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. Recombinant mouse prolactin (r-mPRL), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hPRL) is usually employed in a heterologous mode. Synthesis of r-mPRL was carried out here via secretion in Escherichia coli periplasmic space using a plasmid containing mPRL cDNA joined to the DsbA signal peptide sequence under the control of a constitutive major leftward promoter of the bacteriophage λ (λPL). Fermentation in a pilot bioreactor was carried out at 30°C, with 6 H of induction at 37°C, reaching an optical density of 23 A600 units, a specific yield of 0.06-0.1 µg mPRL/(mL A600), and a concentration of up to 2.2 µg/mL. Even with such a low yield and a poor mass fraction, r-mPRL was purified via a three-step laboratory process based on hydrophobic chromatography, reversed-phase high-performance liquid chromatography, and high-performance size-exclusion chromatography (HPSEC). The purified hormone was then characterized using SDS-PAGE, Western blotting, and HPSEC and showed, by Nb2 rat lymphoma cell proliferation assay, a bioactivity of 39.5 IU/mg, determined against the International Standard of recombinant hPRL [World Health Organization (WHO)-97/714].

Diagnóstico bioquímico de la hiperprolactinemia monometrica / Biochemical diagnosis of monomeric hyperprolactinemia

Fundamento. La prolactina se puede presentar bajo varias formas moleculares siendo la forma monométrica (PRLm) la biológicamente activa. La presencia de macroprolactina (MPRL) puede originar un falso diagnóstico de hiperprolactinemia debido a la interferencia en el procedimiento de medida. El objetivo ha sido desarrollar un protocolo que permita diagnosticar la hiperprolactinemia monométrica, que además sea complementario al procedimiento que detecta MPRL. Material y métodos. La población de referencia para PRLm estaba formada por 122 mujeres y 140 hombres aparentemente sanos a los que se les extrajo sangre para la cuantificación de PRL. Además, se recogieron49 sueros (33 mujeres y 16 hombres) hiperprolactinémicos. Se cuantificó PRL en todas las muestras en un Immulite 2000. La detección de MPRL y de PRLm se realiza tras precipitación con polietilenglicol. Se confirmó el resultado por cromatografía de filtración en gel. Para la obtención de los valores de referencia se siguieron las indicaciones del Panel de Expertos de la IFCC. Resultados. Los valores de referencia de PRLm fueron 3,4-26,6 ¦Ìg/L y 4,6-16,4 ¦Ìg/L en mujeres y varones, respectivamente. De los 49 pacientes hiperprolactinémicos, en el57 % la concentración de PRLm tras PEG se encontraba fuera del intervalo de referencia previamente obtenido, confirmándosela presencia de hiperprolactinemia monométrica. Conclusiones. Se ha desarrollado e implantado un protocolo para la cuantificación de PRLm. La obtención del os valores de referencia de PRLm permite el diagnóstico de la hiperprolactinemia monométrica o activa de forma complementaria a la identificación de MPRL (AU)

Background. Prolactin can take several molecular forms of which the most biologically active is the monomericform (PRLm). The presence of macroprolactin (MPRL) can give rise to a false diagnosis of hyperprolactinemia due to interference in the measuring procedure. The aim was to develop a protocol that enables diagnosis of monomeric hyperprolactinemia, which should also be complementary to the procedure for detecting MPRL. Material and methods. The reference population for PRLm was made up of 122 healthy women and 140healthy men, from whom blood was extracted for PRL quantification. Additionally, 49 hyperprolactinemic serums (33 women and 16 men) were collected. PRL was quantified in all the samples in an Immulite 2000.The detection of MPRL and PRLm was carried out following precipitation with polyetylenglicol (PEG). The result was confirmed by gelatin filtration chromatography. The reference values were obtained following theindications of the Expert Panel of the IFCC. Results. The PRLm reference values were 3,4-26,6¦Ìg/L and 4,6-16,4 ¦Ìg/L in women and men, respectively. In 57% of the 49 hyperprolactinemic patients the concentration of PRL m following PEG fell outside the previously obtained reference interval, confirming the presence of monomeric hyperprolactinemia. Conclusions. A protocol for quantifying PRL m has been developed and implemented. Obtaining PRLm reference values makes it possible to diagnose monomeric oractive hyperprolactinemia in a complementary form to the identification of MPRL(AU)

Inhibitory activity of the peptides derived from buffalo prolactin on angiogenesis.

The peptide fragments obtained by cathepsin digestion of purified buffalo prolactin (buPRL) monomer have been characterized using SDS-PAGE and FPLC with regard to size and pI. Their antiangiogenic activity was tested in chick embryo chorioallantoic membrane (CAM) assay and the human endothelial cells wound healing assay. Antiangiogenic activity was observed in cathepsin-cleaved fragments from buPRL. Further, a peptide sequence 45A- 46Q-47G-48K-49G-50F-51I-52T-53M-54A-55L-56N-57S-58C, which matched with human somatostatin (hSST), a known antiangiogenic factor, was located in the second loop between the first and second alpha-helices in the three dimensional structure of buPRL, obtained by homology modelling. The synthetic peptide matching with SST sequence was found to exhibit antiangiogenic activity in both in vitro and ex vivo assays. It was also observed that all the peptides related to buPRL could antagonize the vascular endothelial growth factor (VEGF) and bradykinin (BK)- dependent production of endothelial nitric oxide (NO), which is a pre-requisite for endothelial tube formation. It is concluded therefore that an internal sequence in buPRL and peptide fragments derived from cathepsin-digested buPRL exhibit antiangiogenic activities.

Purification and biological characterization of bacterially expressed recombinant buffalo prolactin.

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.

Enhancement of human prolactin synthesis by sodium butyrate addition to serum-free CHO cell culture.

Sodium butyrate (NaBu) has been used as a productivity enhancer for the synthesis of recombinant proteins in Chinese hamster ovary (CHO) cells. Thus, the influence of NaBu on the production of recombinant human prolactin (hPRL) from CHO cells was investigated for the first time. CHO cell cultures were submitted to a treatment with different concentrations of NaBu (0.25 to 4 mM). Quantitative and qualitative analyses by reverse-phase high-performance liquid chromatography (RP-HPLC) and Western blot or SDS-PAGE, carried out directly on CHO-conditioned medium, showed that the highest hPRL expression was obtained with 1 mM NaBu. In vitro biological assays based on noble rat lymphoma (Nb2) and mouse pro-B lymphoma (Ba/F3-LLP) cells were carried out on purified hPRL. Its bioactivity in the presence of NaBu was not apparently different from that of the First International Reference Reagent of recombinant hPRL (WHO 97/714). Our results show that NaBu increased the synthesis of recombinant hPRL in CHO cells, apparently without compromising either its structure or function.

Thermal degradation of polyethylene glycol 6000 and its effect on the assay of macroprolactin.

OBJECTIVES: To study the effectiveness of partially degraded polyethylene glycol 6000 (PEG) as a precipitant for macroprolactin. DESIGN AND METHODS: PEG was heated to 63 degrees C in air for up to 20 days and its effectiveness assessed as a precipitant for sera containing normal prolactin or macroprolactin. Decomposition was studied chemically and with NMR spectroscopy. RESULTS: Thermal degradation was similar to what had occurred over several years of natural degradation. Initially PEG degraded 2-5 days caused excess precipitation of monomeric prolactin (false-positive macroprolactinemia). Samples degraded 18-20 days failed to precipitate macroprolactin, giving false negative results. Two 1H NMR peaks at 4-4.5 ppm were not detectable in undegraded PEG but were after 1 day. Their relative integral increased to 20 days. CONCLUSIONS: Aging of PEG can be accelerated by heating. The suitability of PEG for use in macroprolactin assays can be assessed by the absence of peaks at 4-4.5 ppm by 1H NMR.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells.

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

Production, characterization, and applications of mouse monoclonal antibodies against gonadotropin, somatolactin, and prolactin from common carp (Cyprinus carpio).

The gonadotropin alpha subunit (cGTH alpha), gonadotropin II beta subunit (cGTHII beta), somatolactin (cSL), and prolactin (cPRL) were isolated from the pituitaries of common carps, purified by traditional chromatographic analysis, identified by mass-chromatographic analysis, and used as immunogens in the B-lymphocyte hybridoma technique. Totally, 7, 11, 17, and 8 hybridoma cell lines were established, which were able to stably secrete monoclonal antibodies (mAbs) against cGTH alpha, cGTHII beta, cSL, and cPRL, and designated as FMU-cGTH alpha 1-7, FMU-cGTHII beta 1-11, FMU-cSL 1-17, and FMU-cPRL 1-8, respectively. The isotype, titer, and specificity were identified by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical staining, respectively, and application of these mAbs in the aforementioned tests has been proved. Furthermore, sensitive sandwich-ELISA systems for quantitative detection of the hormones mentioned above were also developed.

Purification of monomeric prolactin charge isoform from buffalo pituitaries.

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S-200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti-buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.

Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields.

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).

Anti-prolactin autoantibodies in pregnant women with systemic lupus erythematosus: maternal and fetal outcome.

The aim of this study was to determine in pregnant women with systemic lupus erythematosus (SLE) the frequency of anti-prolactin autoantibodies and to compare the outcome of pregnancy in SLE women with and without anti-prolactin autoantibodies. Ninety-nine consecutive SLE pregnant women and 151 healthy pregnant women were studied prospectively. Patients with or without anti-prolactin autoantibodies were identified by gel filtration chromatography and affinity chromatography for IgG. Serum total and free prolactin (PRL) levels and molecular heterogeneity of PRL at each trimester of pregnancy were determined. The frequency of anti-PRL autoantibodies in SLE pregnant women was 13.1%. Serum total PRL levels were significantly higher in women with anti-PRL autoantibodies compared with SLE women without anti-PRL autoantibodies and in healthy pregnant women; and serum free PRL levels were lower in the third trimester in women with anti-PRL autoantibodies than in healthy pregnant women. In contrast, serum total and free PRL levels were significantly lower in the second and third trimester in SLE pregnant women without anti-PRL autoantibodies compared with healthy pregnant women. All adverse outcomes of pregnancy studied were more frequent in SLE women without anti-PRL autoantibodies than anti-PRL autoantibody-positive SLE women. Moreover, both maternal and fetal main complications were significantly higher in SLE women without anti-PRL autoantibodies than anti-PRL autoantibody-positive SLE women (P

Detection of molecular variants of prolactin in human serum, evaluation of a method based on ultrafiltration.

BACKGROUND: In human blood, there are several molecular variants of prolactin with different biological effects. There is a need for new methods to detect and quantify these variants in order to fully understand the pathophysiological role of prolactin. METHODS: A method based on ultrafiltration was optimized, validated and compared to PEG precipitation. Serum samples from 84 patients were analyzed before and after pre treatment on two immunoassays, Elecsys (Roche) and Access (Beckman). Protein G precipitation was used to confirm presence of macroprolactin. RESULTS: The recovery of prolactin after ultrafiltration was lower than after PEG precipitation. A limit of 40% recovery after PEG precipitation corresponded to 27% recovery after ultrafiltration. Using these limits there were total agreement regarding detection of macroprolactin (r(s)=0.96). In contrast, recovery of prolactin in samples without macroprolactin showed a considerable disagreement between ultrafiltration and PEG precipitation (r(s)=0.48). Within-run CV was 4% for the ultrafiltration method. The correlation coefficient (r) between the immunoassays was 0.96 after ultrafiltration. CONCLUSIONS: Ultrafiltration can be used to compare different prolactin immunoassays and to detect macroprolactin in assays with interference from PEG. For samples without macroprolactin ultrafiltration may give additional information reflecting individual variations of other molecular variants of prolactin.

Isolation of Atlantic halibut pituitary hormones by continuous-elution electrophoresis followed by fingerprint identification, and assessment of growth hormone content during larval development.

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.