The relationship between fat and progesterone, estradiol, and chorionic gonadotropin levels in Quebec cow's milk.

PURPOSE: The majority of milk in industrialized countries is obtained from pregnant cows, which contains increased levels of estrogen and progesterone compared to non-pregnant cows. The aim of this study was to quantify the amount of hormones present in milk with different fat content because previous studies on humans have shown potential effects of increased milk consumption on serum and urine hormone levels as well as on sperm parameters. However, it is unclear whether consumption of milk at the currently recommended levels would lead to systemic effects. METHODS: Samples of cow's milk of varying fat concentrations (0, 1, 2, 3.25, 10, and 35%) were analyzed via competitive ELISA assays. RESULTS: Progesterone concentrations were significantly correlated to increasing fat content of milk (r = 0.8251, p = 0.04). CONCLUSIONS: Research on conditions in which additional progesterone may have an effect on human health should consider inclusion of limitation of milk intake and its effects. Further studies are needed to determine the concentration of progesterone in milk of different fat content in other regions and countries and to quantify the potential pathophysiologic role.

Effects of a urinary factor from women in early pregnancy on HIV-1, SIV and associated disease.

The effects of clinical grade crude preparations of human chorionic gonadotropin (hCG) on Kaposi's sarcoma, HIV, SIV and hematopoiesis were examined in vitro and in vivo. In contrast to previous studies, we report that the antiviral activity of hCG associated factors is not due to the native hCG heterodimer, including its purified subunits or its major degradation product, the beta-core. Using gel permeation chromatography of the clinical grade hCG and urine concentrates from pregnant women, we demonstrate that an as yet unidentified hCG associated factor (HAF) with anti-HIV, anti-SIV, anti-KS and pro-hematopoietic activities elutes as two peaks corresponding to 15-30 kDa and 2-4 kDa.

Simplified purification of equine chorionic gonadotropin (eCG)--an example of the use of magnetic microsorbents for the isolation of glycoproteins from serum.

Classical purification of the glycoprotein equine chorionic gonadotropin (eCG) from serum includes pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. A simplified process requiring only 1/3 of the solvent and improving the yield from 53 to 65% has been developed. The process comprises an ultra-/diafiltration step after the first ethanol precipitation, directly followed by an adsorption/desorption procedure based on magnetic microadsorbents with N,N-diethyl-ammonium functionalization. The process reaches an overall purification factor of eCG of more than 1800 and an average product activity of 1300 IU(ELISA)/mg. After adapting the parameters of the fractionation and the type of magnetic microadsorbents, the new concept is likely to be transferable to other serum proteins.

Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography.

Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficient affinity chromatographic matrix for eCG purification directly from plasma was developed. The matrix consisted of chitosan mini-spheres with immobilized wheat germ agglutinin (WGA). The matrix allowed 98% adsorption of eCG directly from plasma without any pre-treatment with an overall yield of around 60%. The matrix chosen was able to maintain the efficient performance of the purification process for three consecutive cycles. Also, the process was scaled-up 500 times in volume and tested over seven consecutive cycles maintaining its chromatographic performance. The results presented here suggest the potential application of this matrix to one-step purification of eCG from plasma.

Purified human chorionic gonadotropin induces apoptosis in breast cancer.

Agents that induce apoptosis in breast cancer cells have great potential to facilitate chemotherapeutic intervention and improve patient outcomes. In this study, the effects of injecting purified human chorionic gonadotropin (hCG) directly into human breast cancer xenografts grown in nude mice were examined. It was shown that intratumoral injection of purified hCG increased the apoptotic index in breast cancer xenografts. These results were supported by the findings that exposure of breast cancer cells to purified hCG decreased cell viability in five different breast cancer cell lines. In some of these cell lines, the effects of hCG in cell viability appear to correlate with activation/expression of the hCG/luteinizing hormone receptor. Preoperative apoptotic induction by factors such as purified hCG may improve local control or work synergistically with neoadjuvant chemotherapy to improve complete pathologic response of locally advanced breast cancer.

Ultrasensitive biosensor based on magnetic microspheres enhanced microfiber interferometer.

A critical barrier for the successful development of fiber sensors for bio-chemical processes is their limitedly improved sensitivity, restricted by the sensor structural design. To solve this, in this paper, a novel concept was proposed using functionalised modified magnetic microspheres (MMSs) to "amplify" the effect of target bio-chemical analytes to significantly improve the fiber sensor's sensitivity, which has been demonstrated using human chorionic gonadotropin (hCG) as an example. Two types of antibody hCG, (ß and α, both can specifically bind with hCG), were adhered on the surface of fibre sensor and MMSs respectively. Both hCG and MMSs will be specifically captured by the fibre sensor, where MMSs act as an "amplifier" to improve the sensor sensitivity. Experimentally immunomagnetic detection limit of 0.0001 mIU/mL has been achieved, which is the highest reported so far. This newly developed methodology opens a new direction for sensitivity improvement and could be further explored to applications require ultrahigh sensitivity detections such as earlier medical diagnostics.

Carbon-protein covalent conjugates in non-instrumental immunodiagnostic systems.

An original technique for obtaining stable conjugates using colloid carbon particles as indicator labels has been developed. The reliability and stability of the diagnostic reagents obtained is provided by covalent binding of various affine compounds on the surface of carbon particles. The stability of the reagents has been studied under various storage conditions for 3-10 years. It was shown that even when storage conditions were outside the optimal range, some conjugates preserved their analytical characteristics for 10 years. A number of systems for analytical detection of various ligands have been designed based on the carbon-protein conjugates synthesized. The major characteristics of these systems are their high sensitivity and specificity, reliability, and reproducibility, simplicity in operation, and quick results.

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction-tandem mass spectrometry to detect hCG use.

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti-Doping Agency (WADA) list of prohibited substances. The majority of WADA-accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study we measured the concentration of intact hCG, free ß-subunit (hCGß) and ß-subunit core fragment (hCGßcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGß and hCGßcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGß and hCGßcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.

Differences between purified ectopic and normal alpha subnits of human glycoprotein hormones.

"Ectopic" proteins, not distinguished immunologically from the common alpha subunit of the human glycoprotein hormones, were purified approximately 10,000-fold from a gastric carcinoid tumor (A.L.-alpha) and from tissue culture medium of bronchogenic carcinoma cell lines (ChaGo-alpha). The purified A.L.-alpha was homogeneous by sodium dodecyl sulfate (SDS) gel electrophoresis while the purified ChaGo-alpha showed multiple components, some of which represented aggregated species. In SDS gel electrophoresis, the apparent molecular weights of A.L.-alpha (15,000) and dithioerythritol-reduced ChaGo-alpha (13,000) were significantly lower than those of the alpha subunits of human chorionic gonadotropin (hCG-alpha), luteinizing hormone, follicle-stimulating hormone, or thyroid-stimulating hormone (22,000-23,000). Binding experiments with [35S]-SDS suggested that these apparent differences in molecular weight resulted, at least in part, from diminished binding of the SDS by the normal compared to the ectopic alpha subunits. In gel chromatography, the apparent molecular weights of A.L.-alpha (27,000) and ChaGo-alpha (30,000) were slightly higher than those of normal alpha subunits (23,000-24,000). Both A.L.-alpha and ChaGo-alpha were not distinguished from hCG-alpha in ion-exchange chromatography. The composition of A.L.-alpha was similar to that of hCG-alpha in 13 amino acids but showed decreased phenylalanine and increased valine; glucosamine was identified in both A.L.-alpha and hCG-alpha. Under conditions in which hCG-alpha combined with the hCG beta subunit (hCG-beta) to produce 95% of the expected gonadotropin-binding activity in a rat testis radioreceptor-assay, A.L.-alpha incubation with hCG-beta resulted in only 2% of the expected activity, and ChaGo-alpha incubation with hCG-beta produced no detectable activity. These characteristics of ectopic alpha subunits may reflect abnormalities of neoplastic protein synthesis or carbohydrate attachment which result in polypeptides with chemical and immunologic similarity to normal subunits but with differences in physical and combining properties; alternatively, the ectopic subunits may represent as yet unrecognized alpha precursor forms.

Ectopic production of the isolated beta subunit of human chorionic gonadotropin.

A material similar to the beta subunit of human chorionic gonadotropin (hCG-beta) was detected in serum (300 ng/ml) and tumor extract from a 75-yr-old man with pancreatic adenosquamous carcinoma. This material was indistinguishable from hCG-beta in three different types of radioimmunoassay that displayed widely varying reactions with glycoprotein trophic hormones and their subunits. In gel chromatography there appeared to be heterogeneity of the serum beta-like immunoactivity, including one component that coeluted with standard hCG-beta tracer and another immunologically indistinguishable component that displayed a slightly lower elution volume. Neither complete human chorionic gonadotropin (hCG) nor its alpha subunit was detected in radioimmunoassays of serum, before or after fractionation, or in tumor extract. The absence of complete hCG was confirmed in a gonadotropin bioassay sensitive to 15 ng of hCG, which showed no bioactivity in serum or tumor extract containing 450 and 90 ng of hCG-beta, respectively. This case probably represents the first demonstration of isolated polypeptide subunit production of ectopic origin and suggests that hCG-beta, as well as other subunits, may prove useful as cancer markers.

Thyroid-stimulating activity and chorionic gonadotropin.

The nature of the substance with thyroid-stimulating activity (TSA) present in human chorionic gonadotropin (hCG) prepared from pregnancy urine was investigated. In the mouse thyrotropin bioassay, the characteristic maximum of blood radioactivity obtained with the TSA in hCG preparations occurred after that obtained with pituitary thyrotropin (hTSH) but before that obtained with long-acting thyroid stimulator. Antiserum to the alpha subunit of hCG produced significant neutralization of the TSA in hCG. Significant antagonism of hTSH biologic activity was achieved with certain doses of hCG, suggesting that the TSA in hCG was a partial agonist of hTSH. This antagonism was neutralized by antiserum to the beta subunit of hCG. These immunologic results suggest that the substance with TSA in hCG preparations contains antigenic determinants similar to those of both the alpha and the beta subunit of hCG. Amounts of highly purified hCG and crude commercial hCG of equal immunologic activity were biologically indistinguishable in the bioassay for TSA. Both hCG immunoreactivity and the TSA in hCG adsorbed to concanavalin A and eluted with 0.2 M methyl alpha-D-glucopyranoside. These results are consistent with the hypothesis that TSA is an intrinsic property of hCG or of a glycoprotein molecule physicochemically, biologically, and immunologically similar to hCG.

A Label-Free and Ultrasensitive Immunosensor for Detection of Human Chorionic Gonadotrophin Based on Graphene FETs.

We report on a label-free immunosensor based on graphene field effect transistors (G-FETs) for the ultrasensitive detection of Human Chorionic Gonadotrophin (hCG), as an indicator of pregnancy and related disorders, such as actopic pregnancy, choriocarcinoma and orchic teratoma. Pyrene based bioactive ester was non-covalently anchored onto the graphene channel in order to retain the sp² lattice. The G-FET transfer characteristics showed repeatable and reliable responses in all surface modifying steps using a direct current (DC) readout system. The hCG concentration gradient showed a detection limit of ~1 pg·mL-1. The proposed method facilitates the cost-effective and viable production of graphene point-of-care devices for clinical diagnosis.

Ultra-high sensitivity of the non-immunological affinity of graphene oxide-peptide-based surface plasmon resonance biosensors to detect human chorionic gonadotropin.

Specific peptide aptamers can be used in place of expensive antibody proteins, and they are gaining increasing importance as sensing probes due to their potential in the development of non-immunological assays with high sensitivity, affinity and specificity for human chorionic gonadotropin (hCG) protein. We combined graphene oxide (GO) sheets with a specific peptide aptamer to create a novel, simple and label-free tool to detect abnormalities at an early stage of pregnancy, a GO-peptide-based surface plasmon resonance (SPR) biosensor. This is the first binding interface experiment to successfully demonstrate binding specificity in kinetic analysis biomechanics in peptide aptamers and GO sheets. In addition to the improved affinity offered by the high compatibility with the target hCG protein, the major advantage of GO-peptide-based SPR sensors was their reduced nonspecific adsorption and enhanced sensitivity. The calculation of total electric field intensity (ΔE) in the GO-based sensing interfaces was significantly enhanced by up to 1.2 times that of a conventional SPR chip. The GO-peptide-based chip (1mM) had a high affinity (KA) of 6.37×1012M-1, limit of detection of 0.065nM and ultra-high sensitivity of 16 times that of a conventional SPR chip. The sensitivity of the slope ratio of the low concentration hCG protein assay in linear regression analysis was GO-peptide (1mM): GO-peptide (0.1mM): conventional chip (8-mercaptooctanoic acid)-peptide (0.1mM)=8.6: 3.3: 1. In summary, the excellent binding affinity, low detection limit, high sensitivity, good stability and specificity suggest the potential of this GO-peptide-based SPR chip detection method in clinical application. The development of real-time whole blood analytic and diagnostic tools to detect abnormalities at an early stage of pregnancy is a promising technique for future clinical application.

Absence of TGF-ß Receptor Activation by Highly Purified hCG Preparations.

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-ß receptor (TGFßR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-ß1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFßR in mink lung epithelial cells transfected with a reporter gene for TGFßR activation. No such activation was found with highly purified hCG or its free ß-subunit (hCGß), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFßR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation.

Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed-bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed 'rotor-stator' type High Gradient Magnetic Fishing (HGMF) system for large-scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single-cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively.

Sequential Versus Continual Purified Urinary FSH/hCG in Men With Idiopathic Hypogonadotropic Hypogonadism.

CONTEXT: Gonadotropin therapy using a human chorionic gonadotropin (hCG) and FSH preparation is an effective regimen in inducing masculinization and spermatogenesis in men with idiopathic hypogonadotropic hypogonadism (IHH). However, the high cost of medication and frequent injections affect compliance. OBJECTIVE: The aim of this study was to determine the efficacy of sequential use of highly purified urinary FSH (uFSH)/hCG in men with IHH. DESIGN AND SETTING: A randomized, open-label, prospective, controlled noninferiority trial with an 18-month follow-up was conducted in 9 tertiary hospitals. PATIENTS AND INTERVENTION: A total of 67 Chinese men with IHH were randomly allocated into group A receiving continual uFSH (75 U, 3 times a week) and hCG (2000 U, twice a week) injection and group B receiving sequential uFSH (75 U, 3 times a week every other 3 months) and hCG (2000 U, twice a week) injection. MAIN OUTCOME MEASURE: The primary outcome was the proportion of subjects with a sperm concentration of ≥ 1.0 × 10(6)/mL during the 18 months. The efficacy between groups A and B was compared for noninferiority. RESULTS: Of the patients, 17/33 (51.5%) receiving continual uFSH/hCG and 19/34 (55.9%) receiving sequential uFSH/hCG achieved sperm concentrations of ≥ 1.0 × 10(6)/mL. The efficacy in the sequential uFSH/hCG group was not inferior to that in the continual uFSH/hCG group (noninferiority, P = .008) by intention-to-treat analysis. CONCLUSIONS: The efficacy of the sequential uFSH/hCG regimen is not inferior to that of the continual uFSH/hCG regimen in inducing spermatogenesis and masculinization of patients with IHH.

Physical, biological, and immunological characterization of highly purified urinary human chorionic gonadotropin components separated by gel electrofocusing.

A highly purified urinary hCG preparation was subjected to electrofocusing on polyacrylamide gel. It was shown to fractionate into at least 11 components that can be detected by coomassie blue staining, a radioligand-receptor assay (RRA), and four hCG RIAs using antisera against the purified hCG, its alpha- and beta-subunits, and the hCG beta carboxyl-terminal peptide. Refocusing shows that these components are not artifacts of the electrofocusing technique. Six of the major components were isolated, and their pIs were shown to correlate with their sialic acid content. When these components were assayed by radioligand-radioreceptor assay, the four hCG RIAs, and a rat uterine weight assay, they showed 1) similar in vitro biological activities, 2) identical immunological activities that were indistinguishable from the in vitro biological activities, 3) hCG-like and not hLH-like immunological characteristics, and 4) different in vivo biological activities which increased with sialic acid content. Thus, sialic acid alone and/or differences in subunit assembly seem to be responsible for the electrophoretic heterogeneity of highly purified hCG.

The heterogeneity of human chorionic gonadotropin (hCG). II. Characteristics and origins of nicks in hCG reference standards.

hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG.