Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGß), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women. An amide column (150 × 2.1 mm, 2.6 µm, 150 Å), a mobile phase composed of acetonitrile and water both containing 0.1% of trifluoroacetic acid, and a temperature of 60 °C were used. The gradient was from 85 to 40% ACN in 30 min. The use of TFA that had been shown to be necessary for the separation of glycoforms caused, as expected, an ion suppression effect in MS that was partially overcome by increasing the amount of protein injected (2 µL at 1 mg mL-1) and reducing the detection m/z range (from 1500 to 300). These conditions allowed the detection of different glycoforms of hCGα. The performance of the HILIC-HRMS method was compared with that previously obtained in RPLC-HRMS in terms of the number of detected glycoforms, selectivity, and sensitivity. The complementarity and orthogonality of the HILIC and RP modes for the analysis of hCG at the intact level were demonstrated.

Differences in recognition of the 1st WHO international reference reagents for hCG-related isoforms by diagnostic immunoassays for human chorionic gonadotropin.

BACKGROUND: The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental elucidation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes. METHODS: Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free beta-subunit (hCGbeta), and 6 recognized hCG, hCGbeta, and the beta core fragment. RESULTS: Differences in calibration of current hCG assays are moderate. Mean recovery of the current International Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%-164%). Between-method variation for the latter (CV 12.3%) was also greater than for IS 75/589 (CV 8.8%). Recognition of hCGbeta varied markedly (CV 37%). Most assays overestimated it, but 2 RIAs produced results that were slight underestimations. Recognition of the beta core fragment was even more variable (CV 57%) and was closest to equimolarity for the RIAs. CONCLUSIONS: Assays for hCG show considerable variation in their recognition of various forms of hCG, and this variability is the most important cause of method-related differences in hCG results in serum and an even more important cause of method-related differences in urine measurements. Equimolar recognition of the major hCG isoforms is essential if between-method comparability for hCG is to be improved.

Can glycans unveil the origin of glycoprotein hormones? - human chorionic gonadotrophin as an example -.

Doping with (glyco)protein hormones represent an extremely challenging, analytical problem as nearly all are constitutively present at low concentrations that fluctuate according to circadian or alternative periodical, or external stimuli. Thus the mere concentration in a biological sample is only resolutive when this surpasses extreme values. As the vast majority of these molecules are produced by recombinant DNA technology it is believed that the exogenous molecules could bear the signature of the host cell. In particular, these could comprise structural differences originated from co or post-translational differences. In this study we have employed both proteomics and glycomics strategies to compare recombinant and urinary human chorionic gonadotrophin in order to evaluate this hypothesis. As anticipated the recombinant hormone could be shown to contain N-glycolyl neuraminic acid, a sialic acid that cannot be produced by humans. Furthermore, differences were observed in the overall glycosylation, in particular the presence of abundant hybrid-type glycans that were much less pronounced in the recombinant species. These differences were determined to occur predominantly in the alpha-subunit for which antidoping strategies focussed on these elements could be used for both chorionic gonadotrophin and lutrophin as they share the same alpha-subunit.

[Ovulation induction in high risk IVF patients: GnRH-agonist, diminished dose of urinary or recombinant chorion gonadotropin].

In high risk IVF patients the ovulation triggering was done with agonist (0.2 mg), 1.gr., Pregnyl 5000 IE, 2. and 4.gr., Ovitrelle 0.250 mg, 3.gr., and Pregnyl 10000 IE in the fifth--non-risk group. The protocol of the first and fourth group included antagonist, the second and third group was with short and the fifth group with long agonist protocol. There was no grave OHSS in the first group, one case in each second, third and fourth group and 4 cases in the fifth group, as a whole 7 patients (3.3%). In all of them an abdominal drainage lasting 4 to 30 days was performed and all pregnancies were preserved. The average success rate was 50%, 71.4% in the fourth and 43.1% in the fifth group. A protocol with antagonist and ovulation triggering with agonist or reduced dose ChG in order to diminish OHSS in high risk IVF patients is recommended.

Human chorionic gonadotropin (hCG) in the male reproductive tract.

Normal hypothalamic-pituitary testicular and prostatic functions are essential for maintenance of male fertility, whereby glycoprotein hormones (GPH) as well as androgens are major endocrine and local regulators. We have investigated whether the GPH human chorionic gonadotropin (hCG) and the free alpha and beta subunits thereof are produced in the target organs themselves and potentially act as auto/paracrine modulators of fertility. Immunofluorometric assays (IFMAs) based on our panel of highly selective monoclonal antibodies, immunohistochemistry (IHC), confocal laser scanning microscopy (CLSM) and 1- and 2D gel electrophoreses with subsequent western blotting have been utilized for the detection of hCGalpha, hCGbeta and its metabolite hCGbeta core fragment (cf) in human testis, prostate and seminal plasma. Both organs synthesize hCGalpha and hCGbeta, which are subsequently detectable at high concentrations in seminal plasma of healthy probands (n=17): hCGalpha 2630+/-520 ng/mL (mean+/-S.E.M.), hCGbeta 2+/-0.28 ng/mL, hCGbetacf and hCG 0.19+/-0.039 ng/mL. These parameters significantly exceed physiological values, e.g. ten thousand-fold in the case of hCGalpha, in serum of young men (n=20): hCGalpha 0.142+/-0.054 ng/mL (mean+/-S.E.M.), hCGbeta 0.05 ng/mL and hCG 0.004+/-0.003 ng/mL. Levels of these markers were not correlated with sperm counts. Of all body fluids including those of pregnant women seminal plasma is the richest physiological source for genuine free i.e. non-dissociated GPHalpha (M(r,app) 23k) which may even appear as di- or tetramers. Its concentration is similar to that observed in maternal serum (weeks 10-12 of gestation) and in extra-embryonic coelomic fluid. In contrast to those fluids where ratios of free subunits to hCG are in the range of 1:100 highly inverse ratios in the range of 10.000:1.000:1 were observed for hCGalpha:hCGbeta:hCG in seminal plasma. hCGalpha is not derived from heterodimeric GPH suggesting hCG-independent functions of hCGalpha and hCGbeta in male and female fertility.

Metabolism of gonadotropins: comparisons of the primary structures of the human pituitary and urinary LH beta cores and the chimpanzee CG beta core demonstrate universality of core production.

The gonadotropins are a family of closely related heterodimeric glycoprotein hormones homologous in structure to disulfide-knot growth factors. Metabolic proteolytic processing in vivo of this disulfide cross-linked region results in urinary excretion of a residual highly stable core structure. The primary structure of the pituitary form of the hLH beta core was reported earlier, but it has proved difficult to isolate the urinary core, although antibodies to the pituitary core demonstrated its presence. By conventional and immunoaffinity methods, the urinary core has been isolated and its structure determined by both chemical and mass spectrometric methods. The urinary hLH beta core is the same as the pituitary-extracted hLH beta core, beta 6-40 disulfide bridged to beta 55-93, except that the pituitary core is more heterogeneous containing also beta 49-93. These findings imply a dual origin of urinary cores, both directly from a secreting tissue and by kidney processing of circulating hormone. We also found that pregnant chimpanzees excrete a CG beta core with a primary structure identical to that of the human CG beta core of pregnancy. In conclusion, gonadotropin core generation and urinary excretion of nearly identical gonadotropin metabolites is common among primates. Although possible biological functions of these core fragments remain unproven, they have diagnostic utility because of their stability and abundance.

Developmental changes in the glycosylation of glycoprotein hormone free alpha subunit during pregnancy.

Glycoprotein hormone alpha subunit, in its free form (free alpha), is a major placental product. Its glycosylation was found to change dramatically during the advancement of pregnancy. In this study, we have analyzed these glycosylation changes in five normal pregnancies. Binding to Lens culinaris lectin increased dramatically in all subjects between weeks 14 and 17 from the last menstrual period, indicating more core fucosylation as well as possible changes in branching of glycans. Studies using Datura stramonium agglutinin confirmed that the type of triantennary branching changed in this period of pregnancy. The precise structural nature of these changes was determined by high-pH anion-exchange chromatography and electrospray ionization mass spectrometry. Amounts of core fucosylation and of triantennary glycans increased substantially from early to late second trimester, and a shift was observed from 1-->4/1-->3- toward predominantly 1-->6/1-->6-branched triantennary structures. The glycosylation changes occurred in all five individuals at the same time period in gestation, suggesting developmental regulation of N-acetylglucosaminyltransferases IV and V and alpha6-fucosyltransferase during normal pregnancy. These enzymatic activities also appear to be affected in malignant transformation of the trophoblast. Our findings have important implications for the proposed use of specific forms of glycosylation as markers for cancer, as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.

Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma.

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the "nicking" of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG alpha- and beta-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease. Peptide-bond nicking was restricted to a single linkage (beta 47-48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma beta-subunit. In the carbohydrate moiety, alpha-subunit from normal pregnancy hCG contained nonfucosylated, mono- and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma alpha-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P < 0.0001). The beta-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in beta-subunit from normal pregnancy, disaccharide-core structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in beta-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidiform mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy. Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or "hyperbranched" glycoforms on both alpha- and beta-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, alpha 1,6 fucosyltransferase and N-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

Urinary multiple marker screening for Down's syndrome.

We have examined the possibility of using multiple markers in maternal urine rather than serum in order to screen for Down's syndrome. Urine samples were available from 36 cases (24 Down's syndrome, five Edwards' syndrome, three Turner's syndrome, one Klinefelter's syndrome, one triploidy, one triple-X, one twin discordant for Down's syndrome) and 294 controls, including three twins. Three markers were tested: the beta-core fragment of human chorionic gonadotrophin (hCG), total oestrogen (tE) and the free alpha subunit of hCG. Levels were corrected for creatinine excretion and expressed as multiples of the gestation-specific median (MOM) level from the singleton controls. The median value for the singleton Down's syndrome cases was 6.02, 0.74, and 1.08 MOM for beta-core-hCG, tE, and alpha-hCG, respectively. The increases in beta-core-hCG and the reduction in tE levels were highly significant (P < 0.0001 and 0.005, respectively; Wilcoxon rank sum test) but the increase in free alpha-hCG was not (P = 0.40). On the basis of a mathematical model, the expected detection rate for a 5 per cent false-positive rate was 79.6 per cent for beta-core-hCG alone, which increased to 82.3 per cent when combined with tE. Aneuploidies other than Down's syndrome were characterized by low levels of tE and either low or high beta-core-hCG.

Urinary chorionic gonadotropin subunits and beta-core in nonpregnant women. A study of benign and malignant gynecologic disorders.

BACKGROUND: The presence of urinary excretion products of human chorionic gonadotropin (hCG) has been proposed as a tumor marker. To ascertain the clinical value in gynecologic cancers, the authors studied 612 nonpregnant women. METHODS: Three different assays in four clinical groups were compared: no disease, benign disease, malignant disease, and complete remission of previously treated malignant disease. The assays were for the urinary beta-core, "total" beta-hCG, and free alpha-subunit. RESULTS: Measurement of the alpha-subunit was of no obvious clinical value. In some patients with benign disease, hCG metabolites were elevated. In the 141 patients with active gynecologic malignancy the sensitivity of the total beta-hCG assay was 47% and that of the beta-core assay was 36%. The specificities were 80.3% and 90.4%, respectively. Advanced cancers generally had higher levels of total beta-hCG and beta-core. Squamous cell and poorly differentiated cervical tumors had higher levels of total beta-hCG than did adenocarcinomas and well-differentiated cervical tumors. Invasive, serous, endometrioid, and germ cell ovarian tumors had higher total beta-hCG, beta-core, and alpha-subunit levels than did borderline, mucinous, and clear cell ovarian tumors. Six of 16 patients with disease in complete remission had elevated levels. CONCLUSION: The excretion of hCG and its metabolic fragments is a common event in gynecologic cancer, but sensitivity and specificity are low, and there is little consistent relationship between tumor stage and histologic type.

Glycosylation changes in human chorionic gonadotropin and free alpha subunit as gestation progresses.

Carbohydrate is important to the structure, function, and circulatory survival of the glycoprotein hormones. Human CG (hCG) and the related free alpha-molecule of pregnancy contain four and two asparagine-linked oligosaccharides, respectively. The present study analyzes changes in the glycosylation patterns of hCG and free alpha in early vs. late gestation. Five volunteers provided 24-h urine samples, weekly, throughout their pregnancies. Extracts of early pregnancy (weeks 7-12) and late pregnancy (weeks 28-32) urines were pooled. Early and late samples from each patient were subjected to gel filtration to separate hCG and free alpha, and the populations thus obtained were analyzed by lectin affinity chromatography on Concanavalin A-Sepharose (Con A) and Lens culinaris-agarose (Lch). Using Con A, free alpha and hCG were separated into an unbound fraction (eluted with Con A buffer), a weakly bound fraction (eluted with 10 mmol alpha-methyl-D-glucoside) and a tightly bound fraction (eluted with 500 mmol alpha-methyl-D-mannoside). For free alpha-molecule, a significant decrease in tightly bound Con A forms, was noted from early to late pregnancy with a mean difference of 17.0 +/- 2.4% (P less than 0.01). Concomitantly, in late pregnancy, an increase in Con A unbound forms of free alpha was noted with mean difference of 12.5 +/- 1.7% (P less than 0.01). These changes indicate the presence of more highly branched oligosaccharides on free alpha as gestation advances. No changes were noted in the Con A binding of intact hCG; nearly all hCG bound in both early and late pregnancy. Using Lch, free alpha and hCG were separated into an unbound fraction (eluted with Lch buffer) and a bound fraction (eluted with 500 mmol alpha-methyl-D-mannose). Both free alpha and intact hCG in late pregnancy exhibited increased binding to Lch, with mean differences from early to late pregnancy of 30.2 +/- 4.8% (P less than 0.01) and 11.4 +/- 4.5% (P less than 0.05), respectively. These data indicate increased incorporation of fucose into the carbohydrate moieties in late pregnancy. Taken together, these data derived by analysis using lectin specificity imply the presence of more highly branched, fucosylated oligosaccharides as gestation progresses.

Free alpha molecules from pregnancy stimulate secretion of prolactin from human decidual cells: a novel function for free alpha in pregnancy.

Free alpha molecules isolated from pregnancy, as well as highly purified reference preparations of hCG alpha subunit (CR119 or CR123), stimulated the release of prolactin from human decidual cells in culture. The amount of prolactin secreted during a 24 h incubation was concentration-dependent over a range of increasing doses of alpha from 0.2 to 20 ng/ml with an ED50 of about 1.6 ng/ml. These concentrations are well within the physiologic maternal serum free alpha levels which average 350 ng/ml during the third trimester of pregnancy. Incubation of decidual cells with a reference preparation of intact hCG (CR123) at a concentration of 260 ng/ml resulted in stimulated secretion of prolactin, however, the observed stimulation could be attributed to contamination of the preparation with free alpha or dissociated hCG alpha subunit. Purified hCG beta subunit had no stimulatory activity on the decidual cell culture. The effect of alpha subunit on the stimulated release of prolactin was not due to a generalized stimulation of protein synthesis and secretion since no increase was observed in the release of 35S-labeled proteins compared to controls. In addition, the observed increase in prolactin secretion was not due to a toxic effect of the alpha subunit since there was no visible effect on cell viability, and the cellular enzymes, LDH and alkaline phosphatase, were not detected in the culture medium. Addition of exogenous hCG alpha subunit to primary cultures of human trophoblast cultures did not result in stimulated release of human placental lactogen. We conclude that free alpha molecules of pregnancy stimulate release of prolactin from human decidual cells in culture. These results suggest a novel role for free alpha in the paracrine regulation of decidual prolactin secretion.

Measurement of thyroid stimulating hormone (TSH) in human urine.

Using a highly sensitive and specific immunoradiometric assay kit for human TSH, we measured TSH concentrations in unprocessed urines in normal subjects, in patients with primary hypothyroidism, and patients with renal disease. In five of ten normal subjects TSH was detectable in urine samples (less than 20-69 microU/day). In five patients with hypothyroidism, the urinary TSH excretion was increased. In seven out of ten patients with nephrotic syndrome, eight out of nine patients with chronic renal failure and two patients with tubular dysfunction, the urinary TSH excretion was increased. The urinary TSH excretion correlated significantly with both urinary protein excretion and urinary beta 2-microglobulin excretion. These results suggest that the renal handling of TSH involves both glomerular filtration and tubular re-absorption, and that urinary TSH excretion is increased when serum TSH is increased and either glomerular or tubular function is impaired.