First-trimester hyperglycosylated human chorionic gonadotropin and development of hypertension.

OBJECTIVE: This study aimed to determine whether urine levels of hyperglycosylated human chorionic gonadotropin (HhCG) in the first trimester are predictive of subsequent development of hypertension during pregnancy METHOD: This prospective cohort study consisted of women seeking care before 12 weeks gestation. A clean catch urine was obtained at the first prenatal visit and tested for HhCG and creatinine levels. The median HhCG levels and multiples of the median (MoM) by gestational age were compared between the groups that either developed hypertension or did not. RESULTS: Urine HhCG were determined for 204 women between 4 weeks 4 days to 11 weeks 6 days. The median HhCG of those who developed gestational hypertension (n = 7) or preeclampsia (n = 15) did not differ from the group that did not (median: 284 ng/mg creatinine vs 365 ng/mg; p = 0.55). If the MoM of HhCG for the no hypertension group was 1.00, the MoM of HhCG for the hypertension group was 0.93 (p = 0.93). A possible association was observed after 10 weeks between low HhCG levels and the development of late-onset hypertension (≥34 weeks). CONCLUSIONS: Prenatal screening for subsequent hypertension is unreliable with a single measurement of maternal urine HhCG at 10 weeks or less.

Hyperglycosylated hCG.

Hyperglycosylated hCG (hCG-H) is a glycosylation variant of the hormone hCG. Here we review all that is known about this independently functioning molecule. As discussed, it is a very different molecule to the hormone hCG. First, hCG-H is produced by cytotrophoblast cells while regular hCG is made in syncytiotrophoblast cell. Second, it is an autocrine acting directly on the cells which produce it, while regular hCG is an endocrine acting on maternal corpus luteal cells. Third, hCG-H has minimal biological activity in promoting progesterone production compared to regular hCG. Fourth, hCG-H functions unlike regular hCG as an invasion promoter, whether invasion as in choriocarcinoma and testicular germ cell malignancies, or as in implantation of pregnancy. These functions seemingly occur through action on cytotrophoblast cell TGFbeta receptors. Fifth, hCG-H is an essential component for successful human implantation to prevent early pregnancy loss and spontaneous abortion. Sixth, hCG-H is critical for promoting the midtrimester hemochorial implantation, and for preventing preeclampsia. Seventh, measurements of hCG-H have advantages over measurements of regular hCG or total hCG, in detecting pregnancy, pregnancy outcome (failing or term pregnancy), predicting preeclampsia in pregnancy, or as a tumor marker for gestational trophoblastic diseases.

Mice devoid of fer protein-tyrosine kinase activity are viable and fertile but display reduced cortactin phosphorylation.

The ubiquitous Fer protein-tyrosine kinase has been proposed to regulate diverse processes such as cell growth, cell adhesion, and neurite outgrowth. To gain insight into the biological function of Fer, we have targeted the fer locus with a kinase-inactivating missense mutation (fer(D743R)). Mice homozygous for this mutation develop normally, have no overt phenotypic differences from wild-type mice, and are fertile. Since these mice lack both Fer and the testis-specific FerT kinase activities, these proteins are clearly not essential for development and survival. No differences were observed in overall cellularity of bone marrow, spleen, or thymus in the absence of Fer activity. While most platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation was unchanged in fer(D743R) homozygous embryonic fibroblasts, cortactin phosphorylation was reduced. However, Fer kinase activity was not required for PDGF-induced Stat3, p120(ctn), or epidermal growth factor (EGF)-induced beta-catenin phosphorylation. Also, no defects were observed in changes to the actin cytoskeleton, adherens junctions, or focal adhesions in PDGF- or EGF-stimulated fer(D743R) homozygous embryonic fibroblasts. Therefore, Fer likely serves a redundant role in regulating cell growth, cell adhesion, retinal development, and spermatogenesis but is required for efficient phosphorylation of cortactin.

Discordant synthesis and secretion of human chorionic gonadotropin and subunits by cervical carcinoma cells.

Epidermoid carcinoma of the cervix cell lines DoT and CaSki were examined for the production of human chorionic gonadotropin (HCG) and its subunits (HCG-alpha and HCG-beta). Additionally, the effect of butyrate and other regulatory agents on synthesis and secretion was investigated. Media and cell lysates were analyzed by radioimmunoassay for HCG and HCG-beta and by radioreceptor assay for intact HCG. Although DoT and CaSki cells secreted more HCG-beta than HCG, the cell lysates contained only one-half as much HCG-beta as HCG. This raises the possibility that HCG-alpha synthesis may be limiting to the formation of intact HCG in DoT and CaSki cell lines. Our results also indicate discordance in HCG and HCG-beta production in response to butyrate. Whereas the production of HCG-beta was increased by 5 mM sodium butyrate, the synthesis and secretion of intact hCG were decreased. These results support the concept of independent regulation of alpha- and beta-subunit synthesis and secretion.

Urinary human chorionic gonadotropin free beta-subunit and beta-core fragment: a new marker of gynecological cancers.

Many investigators have shown that a small proportion (13-36%) of subjects with nontrophoblastic gynecological cancers have elevated serum levels of human chorionic gonadotropin (hCG). The low proportion with detectable levels and the accompanying low titers have limited the use of hCG as a tumor marker. hCG is a glycoprotein composed of two noncovalently linked subunits (alpha and beta), which are the products of separate genes. With the intent of expanding the use of hCG as a tumor marker we investigated levels of hCG free beta-subunit and asialo free beta-subunit and its core glycopeptide (composed of beta-subunit residues 6-40 disulfide-linked to 55-92), collectively called urinary gonadotropin fragments (UGF), in healthy and cancer patients. An immunoradiometric assay was developed, using the core glycopeptide-directed antibody B204, that similarly measures the hCG free beta-subunit and the asialo free beta-subunit and its core glycopeptide. Parallel urine and serum samples were collected from 87 women with active gynecological cancer and hCG and UGF were measured. Just 18% of the women tested had detectable serum levels of hCG (greater than 0.2 ng/ml); none had elevated serum levels in the UGF assay (greater than 0.2 ng/ml). Of the same group, 32% had detectable urine hCG levels (mean titer, 0.50 ng/ml) and 74% exhibited elevated urinary levels in the UGF assay (mean titer, 2.0 ng/ml). In a control group (urines from 50 nonpregnant healthy women), 47 negative and three borderline positive results (0.30, 0.35, and 0.48 ng/ml) were observed in the UGF assay. These results suggested a sensitivity of 74% and specificity of 92% for the UGF test for gynecological cancers. By disease, 70% of those with cervical, 73% of those with ovarian, and 77% of those with endometrial cancers had detectable UGF levels (greater than 0.2 ng/ml). By stage, 50, 62, 75, 86, and 100% of those with stage 1, 2, 3, 4, or recurrent disease, respectively, had positive results. UGF is a promising new marker of gynecological malignancies.

A distinctive form of human chorionic gonadotropin beta-subunit-like material produced by cervical carcinoma cells.

The DoT and CaSki human cervical carcinoma cell lines ectopically produce material immunologically similar to the beta-subunit of human chorionic gonadotropin (hCG beta). Culture fluids were analyzed by gel filtration chromatography and radioimmunoassay (RIA) using (a) antiserum directed to conformation-specific (core-directed) determinants not involving the carboxyl-terminal peptide (CTP) in hCG beta purified from urinary hCG (i.e., standard hCG beta) or (b) antiserum directed to the CTP in standard hCG beta. CTP-directed RIA recognized a peak of hCG beta-like immunoreactive material that eluted in the same position as standard hCG beta. However, core-directed RIA recognized additional hCG beta-like material (i.e., ectopic beta-II), most of which eluted before standard hCG beta. CaSki cells were incubated with [3H]mannose, [3H]proline, and [3H] leucine, and the spent medium was immunoprecipitated and analyzed by gel electrophoresis. Several labeled peaks were detected in the lane from the anti-hCG beta X Sepharose immunoprecipitate, one of which corresponded in mobility to standard hCG beta, with two more intense components migrating at higher apparent molecular weights. Carboxypeptidase Y digestion released only 0.2 mol equivalents each of [3H]proline and [3H]leucine from the labeled CaSki material immunoprecipitated with anti-hCG beta X Sepharose, compared to 1 mol equivalent each in similar analysis of standard hCG beta. These findings were consistent with the absence of the 4-carboxy-terminal amino acids from 80% of the hCG beta-like immunoreactive material secreted by CaSki cells. The affinity purified ectopic beta-II failed to combine with standard hCG alpha under conditions in which combination of standard hCG beta with standard hCG alpha was essentially complete. Neither aggregation nor proteolytic degradation was the cause of failure of ectopic beta-II to combine with hCG alpha. We conclude that both the DoT and CaSki cervical carcinoma cell lines secrete a distinctive form of hCG beta-like material, ectopic beta-II. Lack of recognition by CTP-directed antisera and amino acid analysis suggest that ectopic beta-II may lack the CTP, despite its apparent larger size relative to standard hCG beta.

Origin and occurrence of human chorionic gonadotropin beta-subunit core fragment.

Human chorionic gonadotropin (hCG) beta-subunit core fragment (beta-fragment) is present in the urine of pregnant individuals as well as those with trophoblast disease and certain other cancers at concentrations 0.8 (early pregnancy) to 7 (second trimester pregnancy)-fold greater than that of hCG. The core fragment may be directly secreted by trophoblast tissue into the circulation or possibly originates from peripheral degradation of circulating hormone by the kidney. We examined the former hypothesis. We examined 24-h organ cultures of trophoblast tissue from first, second, and third trimester pregnancy. The media from this tissue contained hCG, free beta-subunit, and beta-fragment. The amount of beta-fragment present exceeded that of hCG, as was observed in second and third trimester pregnancy urine. The beta-fragment immunoreactive material produced by trophoblast tissue was compared to a standard preparation of urinary beta-fragment. The material in medium was identical to the standard beta-fragment in its elution pattern from a gel filtration column, from a reverse-phase HPLC column, from an ion-exchange gel, and from an immobilized lectin affinity column, and also by electrophoresis and immunoblotting with fragment-reactive monoclonal antibodies. We conclude that beta-fragment can also originate directly from trophoblast tissue, and could be the principal hCG beta-immunoreactive molecule secreted.

Significant steroidogenic activity of luteinizing hormone is maintained after enzymatic removal of oligosaccharides.

Several reports have described the destruction of the N-linked oligosaccharides on glycoprotein hormones by hydrogen fluoride treatment and have noted the accompanying marked reduction, or complete loss, in biological activity. This has led to the concept that the oligosaccharides have an obligatory role in glycoprotein hormone steroidogenic function. Using a less radical and more complete method for removing sugar units, endoglycosidase treatment and ovine LH (oLH) and human LH (hLH) as examples, we examined the role of oligosaccharides in hormone function. Ovine LH and hLH were digested with endoglycosidase F. After treatment cleavage of oligosaccharides was demonstrated by compositional studies, greater than 87% cleavage was demonstrated and only N-acetylglucosamine or N-acetylglucosamine-Fucose shown to remain attached to the peptide, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate size change) and by chromatofocusing (appearance of a single basic peak). Biological activities and relative potencies of preparations were then assessed in an in vitro assay, in which the ability of samples to promote testosterone production by testicular interstitial cells was measured. Although endoglycosidase F treatment reduced relative potencies 2- to 3-fold in the bioassay, (possibly in part due to subunit dissociation) it did not lessen abilities to induce maximal testosterone response (that of native hLH and oLH). These findings contrast with those obtained from hydrogen fluoride studies and indicate that the oligosaccharides, per se, do not play an obligatory role in the steroidogenic activity of LH.

Use of glycosidase digested human chorionic gonadotropin beta-subunit to explain the partial binding of ectopic glycoprotein hormones to Con A.

Recent studies have demonstrated that ectopic glycoprotein hormones only partially bind Con A. To investigate the basis for these findings, the Con A binding of ectopic hCG beta from DoT cervical carcinoma cells was examined after digestion with various glycosidases. Ectopic hCG beta only partially bound Con A, as was the case after digestion with neuraminidase and beta-galactosidase. However, subsequent digestion with N-acetylhexosaminidase increased Con A binding to 96%. It is apparent that Con A binding of ectopic hCG can be inhibited by a residue removed by N-acetylhexosaminidase, probably extra beta-N-acetylglucosamine linked to beta-mannose on N-linked oligosaccharides. The method used involved the glycosidase digestion of glycoprotein alkylated under denaturing conditions and was first validated with milligram amounts of standard hCG beta.

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.

The heterogeneity of human chorionic gonadotropin (hCG). I. Characterization of peptide heterogeneity in 13 individual preparations of hCG.

Peptide variations in the alpha-subunit (molecules starting at alpha 3 and alpha 4) and beta-subunit (missing linkages at beta 44-45 and beta 47-48) of hCG have been reported by several investigators. Studies, however, have been limited to standard hCG preparations (purified from large pools of urine) and other hCG samples from mixed urines. In this study we used chromatographic procedures to purify the total hCG content of 13 individual urines, 6 from patients with pregnancy and 7 from those with trophoblast disease (no hCG-containing fractions were excluded). Then, we examined for the first time the peptide variability among individual samples of hCG. We report 1) that individual hCG preparations have nicks (missing linkages) in the beta-subunit, primarily between residue 47-48 (11 of 13 samples) and, less commonly, at the linkage 44-45 or 46-47 (3 of 13 samples); 2) the extent of nicking varies greatly between individual preparations (range, 0-100% of molecules); 3) varying alpha-subunit N-terminal heterogeneity (N-terminus starting at alpha 3 or alpha 4) was also present (range, 0-28% of molecules), but was confined to preparations from individuals with trophoblast disease (6 of 7 samples from trophoblast disease urine, 0 of 6 from pregnancy urine); 4) hCG missing the beta-subunit C-terminal region was also detected (2 of 13 hCG preparations); and 5) 1 of 13 preparations was nicked on the hCG alpha-subunit, between residues 70 and 71. Thus, 12 of 13 individual hCG samples demonstrated at least 1 of 4 different forms of peptide heterogeneity. We conclude that individual hCG samples vary widely in the type and extent of peptide heterogeneity, an observation that is not appreciated when pools of hCG are studied.

Absence of the COOH-terminal peptide on ectopic human chorionic gonadotropin beta-subunit (hCG beta).

A comparison of the physical properties and immunoreactivity of ectopic hCG beta from cultured cervical cancer cells and standard hCG beta was performed. The heterogeneity of the preparations was examined by ion-exchange chromatography, and the molecular size by analytical gel filtration. Two distinct forms of ectopic hCG beta were identified. One form was indistinguishable from standard hCG beta, while the other, although larger, lacked the characteristic COOH-terminal peptide (CTP). This was shown by the failure of antisera specific for determinants on the cTP to recognize this molecule, and by the apparent absence of the 0-linked oligosaccharides and thermolysin cleavage site normally found in this region.

Detection of the free beta subunit of human chorionic gonadotropin (HCG) in cultures of normal and malignant trophoblast cells, pregnancy sera, and sera of patients with choriocarcinoma.

Immunoaffinity adsorption techniques, utilizing specific antisera for hCG and its subunits bound to Sepharose 4B, have been employed to separate hCG alpha beta dimer and free subunits of hCG. As previously reported by this and a number of other laboratories, trophoblast cells (in vivo and in vitro) produce free alpha subunit in addition to hCG dimer. We have now shown that cultured JAr choriocarcinoma cells also secrete free beta subunit: 37% of the total beta subunit (combined and free) secreted by JAr cells is in the free form. Moreover, in pooled sera from choriocarcinoma patients 15% of total beta subunit is free, and in media from placental explant cultures and in pooled first trimester pregnancy sera 11% and 6.5%, respectively, of total beta subunit are in the free form. The free beta s are all of similar mol wt to the combined forms, and associate with urinary hCG alpha to form hCG. Free alpha s, which are larger than the combined forms, are unable to associate with urinary hCG beta to form hCG. We propose that the supply of combinable alpha subunit, rather than beta, limits dimer formation.

The heterogeneity of human chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nicked hCG.

Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of the human chorionic gonadotropin beta-subunit fragment from pregnancy urine.

A major portion of the hCG immunoreactivity detectable in pregnancy urine is derived from a fragment of hCG beta. This lacks the COOH-terminal portion of hCG beta, but retains immunoreactivity with most antibodies raised against the beta-subunit of hCG. To improve clinical measurements of hCG and assess the importance of such fragments in human urine, we have isolated and determined the structure of this molecule. The hCG beta fragment was isolated from a partially purified commercial preparation of hCG (Organon) by gel filtration and immunoaffinity chromatography using monoclonal antibodies. It was found to consist of two polypeptide chains composed of residues beta-(6-40) disulfide-bridged to residues beta-(55-92). It also differs from the beta-subunit of hCG in its carbohydrate structure, lacking sialic acid and having a low but variable amount of galactose. A beta-fragment containing the same two NH2-terminal sequences was also isolated from a single pregnant woman's urine. The two major polypeptides comprising the beta-fragment contain a total of nine half-cystine residues, raising the possibility that a free thiol may exist or that a third undetected disulfide-bridged peptide is present in the intact fragment. However, tests for the presence of a free thiol have been negative. Another intrinsic characteristic of the beta-fragment is the formation of a variable amount of dimer in solutions of neutral pH. beta-fragment will not combine with intact alpha-subunit. Despite the absence of regions beta-(1-5), beta-(41-54), and beta-(93-145), the beta fragment is recognized by the SB-6 antibody and most monoclonal antibodies elicited to the beta-subunit, thus excluding half of the amino acids of the beta-subunit from the epitope(s) where these antibodies bind.

The O-linked oligosaccharide structures are striking different on pregnancy and choriocarcinoma HCG.

hCG is a glycoprotein hormone which is detected in the serum and urine of pregnant women and of patients with hydatidiform mole and choriocarcinoma. The molecule contains 4 O-linked sugar chains. In an effort to identify cancer markers, the structures of these sugar units on the hCG produced in pregnancy and choriocarcinoma were compared. hCG molecules in patient urines were purified by immuno-affinity chromatography and gel filtration. beta-elimination was used to cleave the O-linked sugar units, radioactive sodium borohydride to label them, and gel filtration on Bio-Gel P4 to size them and compare their elution volumes with those of standard oligosaccharides of known structure. A trisaccharide, NeuAc alpha 2-3Gal beta 1-3GalNAc-, was found to be the principal unit attached to urinary hCG from pregnant women (10 samples). A hexasaccharide, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-6)GalNAc-, which accounted for just 6% (mean, range 0-14%) of the O-linked sugar units on pregnancy hCG, was the principal unit (mean 52% of total, range 50-56%) attached to the hCG from choriocarcinoma patient urines (3 samples). These results indicate that hexasaccharide-abundant hCG is an indicator of choriocarcinoma.

Serum HCG beta-core fragment is masked by associated macromolecules.

To determine if beta-core fragment is present in serum from individuals with pregnancy and choriocarcinoma, samples were analyzed by gel filtration and fractions assessed by immunoassays (Mr = 15,000). In agreement with published reports, only minute amounts of beta-core fragment were detected (less than 0.1% of hCG level). A small amount of beta-core fragment activity was identified near the void volume of the column (Mr greater than 60,000). This high molecular weight material was pooled and dissociated with 3M ammonium thiocyanate, and gel filtration repeated. A much more significant beta-core fragment peak was then detected (Mr = 15,000). After dissociation, beta-core fragment was 18% (mol/mol) of the hCG level in early pregnancy serum, 91% in mid-pregnancy serum, 50% in term pregnancy serum, but only 1.3% in choriocarcinoma serum, pre-therapy. We conclude that the major form of beta-core fragment found in serum is associated with other macromolecules, which mask the beta-core fragment epitope to existing hCG beta antibodies. Measurements made on the dissociated beta-core fragment complex are much more in keeping with pregnancy levels found in urine.

Human chorionic gonadotropin beta-subunit nicking enzymes in pregnancy and cancer patient serum.

hCG is a dimer composed of an alpha- and a beta-subunit, joined noncovalently. In addition to the hCG dimer, uncombined alpha- and beta-subunits (free beta) and nicked hCG and free beta molecules (cleaved at 44-45 or 47-48) can be detected in the circulation. Of these circulating molecules, only the intact hCG dimer fully expresses biological activity. The pathways that dissociate, nick, and degrade hCG and beta-subunit molecules in pregnancy are unknown and could have a major role in regulating hormone levels. Immunoassays for intact (nonnicked) hCG and intact (beta-subunit (with < 1% detection of nicked molecules) and a subtractive immunoassay system for measuring nicked hCG levels have been described previously. A multiantibody scavenger assay is described here for measuring nicked beta-subunit levels (< 6% detection of intact beta-subunit). In this report we use these four assays to assess conversion of intact hCG or beta-subunit to nicked forms over time (nicking enzyme activities) in control (healthy nonpregnant), pregnant, and cancer patient serum samples. Pools of pregnancy and control sera were supplemented with intact hCG and its dissociated beta-subunit and incubated at 37 C. Intact and nicked molecule measurements were made between 0-48 h. In two different pools of control sera, no loss of intact hCG or intact beta-subunit and no significant gain in nicked hCG or nicked beta-subunit were detected over 48 h. This indicated a lack of nicking enzyme activity in control serum. In two different pools of first trimester pregnancy sera, we found no obvious loss of intact hCG or gain of nicked hCG levels over 48 h. However, we found 70% and 62% losses (pools 1 and 2) of intact beta-subunit and 51% and 39% gains of nicked beta-subunit over the same time period. We inferred that an uncombined or free beta-subunit-modifying activity was present in pregnancy serum. We repeated the pregnancy serum experiment with six different concentrations of beta-subunit (0.62-29 mg/L). A linear relationship, percent nicking against time, existed for the six concentrations for up to 6 h at 37 C (r = 0.97); after that, the rate of nicking declined. A plot of rate against concentration against revealed a classical Michaelis-Menten enzyme relationship (logarithmic regression, r = 0.96). The pregnancy serum beta-subunit nicking activity was partially purified by gel filtration. A single peak of activity emerged, eluting between the 150,000-443,000 mol wt standards.(ABSTRACT TRUNCATED AT 400 WORDS)

Urine hCG beta-subunit core fragment, a sensitive test for ectopic pregnancy.

hCG is a glycoprotein hormone composed of 2 dissimilar subunits, alpha and beta, joined non-covalently. hCG and its free beta-subunit are the principal hCG beta immunoreactivities in pregnancy serum samples, and the same plus beta-core fragment (beta-subunit residues 6-40 disulfide-linked to residues 55-92) in urine samples. Ectopic or tubal pregnancy is difficult to diagnose in emergency rooms. With the objective of finding better hCG-related assays for differentiating tubal and normal pregnancies, we tested 2 hCG, 1 free beta-subunit and 2 beta-core fragment immunoassays. Twelve urine samples were collected in the emergency room from women later shown by surgery to have tubal pregnancy. All were 38 to 80 days since last period. A further 36 urine samples were collected from the same period from those with normal intrauterine pregnancies. Using the 2 hCG assays the median level in tubal pregnancy samples was 1/38th and 1/48th of normal pregnancy concentrations. With the free beta-subunit assay tubal pregnancy levels were 1/28th of normal levels. Using 2 beta-core fragment assays (Ciba-Triton UGP kit and B204-FBT11 scavenger test), however, tubal levels were most different from intrauterine pregnancy, 1/149th and 1/800th of normal levels. A cut-off level of 100 micrograms/L was considered for the B204-FBT11 beta-core fragment test, at which a predictive value of > 98% was suggested for ectopic pregnancy. In an additional patient, levels were measured 15 days prior to the diagnosis of tubal pregnancy. At this time, results from the 2 hCG tests were 1/97th and 1/126th, from the free beta-subunit test was 1/8th and the 2 beta-core assays were 1/413th and 1/240th of median normal intrauterine pregnancy levels. While hCG levels are reduced in tubal pregnancy, beta-core fragment are reduced much further. beta-core fragment measurements may offer a major improvement over hCG in diagnosing tubal pregnancy in the Emergency Room, and in screening for this life threatening disease.

The deactivation of hCG by nicking and dissociation.

Human chorionic gonadotropin (hCG) is composed of an alpha- and a beta-subunit, joined noncovalently. A proportion of hCG molecules in pregnancy serum and urine samples have nicks or a missing peptide linkage between either beta-subunit residues 44 and 45 or beta-subunit residues 47 and 48. These nicks ablate the steroidogenic activity of hCG. We examined the source of nicking, and the occurrence and stability of nicked hCG molecules produced during pregnancy. We investigated the source of nicking. Standard hCG was added to three samples of whole blood, and incubated 18 h at 37 C. No change in extent of nicking was detected. However, nicking of hCG beta-subunit was detected by gel electrophoresis (bands at M(r) = 17,000 and M(r) = 22,000, corresponding to the peptides beta 1-47(44) and beta 48(45)-145, respectively) in culture fluids from first trimester placental explants and from JAr malignant trophoblast cells. We inferred that nicking occurs before or immediately upon secretion by trophoblast tissue. We examined the occurrence of nicking. Levels of total hCG (nicked+nonnicked) and intact hCG (nonnicked) were determined in 233 serum and 168 urine samples from 4-40 weeks of pregnancy. From the two measurements the extent of nicking was estimated. A linear relationship was indicated between advancing weeks of gestation and increasing extent of nicking (regression analysis, months vs. percent nicked, 95% correlation). Minimum nicking was observed in serum from the first 2 months of pregnancy (mean = 9% of hCG molecules), increased nicking in the months after, and maximum nicking in samples from the last 2 months of pregnancy (mean = 21% of hCG molecules, t test first 2 months vs. last 2 months, P < 0.00005). Similar results were observed with urine samples (first 2 months mean = 9%, last 2 months = 27%, t test P < 0.00005). We concluded that nicking is more prevalent after the hCG peak (after 2 months of pregnancy). Finally, we examined the stabilities of nicked and intact hCG molecules. Standard hCG (batch CR127, 20% nicked) and hCG preparation C5 (100% nicked) were incubated for varying times in whole blood. C5 hCG dissociated rapidly into free alpha- and beta-subunits (dissociation half-life 22 +/- 5.2 h), over 30 times faster than standard hCG (dissociation half-life 700 h).(ABSTRACT TRUNCATED AT 400 WORDS)