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.

beta-Core chemical and clinical properties.

beta-Core is the most abundant hCG-related molecule in pregnancy urine. The structure of beta-core as well as aspects of its metabolic clearance suggest that beta-core is a metabolic fragment of the hCG-beta subunit. The occurrence of beta-core in the urine of patients with a broad spectrum of malignancies imparts an important role to beta-core as a tumor marker. The recent development of antisera with enhanced specificity and sensitivity for beta-core will facilitate further studies on the clinical significance of this molecule.

Human chorionic gonadotrophin-like and beta-core-like materials in postmenopausal urine.

In addition to human chorionic gonadotrophin (hCG), the urine of pregnant women contains a small molecular weight form of the hCG-beta subunit known as beta-core. Human CG-like material has been described in tissues, serum and urine of normal man, particularly in postmenopausal women. We examined different urine preparations from postmenopausal women to determine whether beta-core-like material, as well as hCG-like material, could be detected. We studied an acetone extract of a pool of 11 litres of postmenopausal urine, three different commercial preparations of human menopausal gonadotrophins and two commercial preparations of 'pure' FSH. After Sephadex G-100 chromatography of these various postmenopausal urine extracts, fractions were assayed using four assay systems to detect hCG, beta-core, LH and FSH immunoreactivities. Human CG immunoreactivity was readily detected in all urinary extracts and it eluted in a position indistinguishable from that of purified hCG. In addition to this hCG-like material, all urinary extracts, except the commercial 'pure' FSH preparations, contained material which reacted in the beta-core radioimmunoassay. This beta-core immunoreactive material eluted from Sephadex G-100 in a position corresponding to that of purified pregnancy-derived beta-core. We conclude that the urine of postmenopausal women contains material resembling the beta-core molecule found in pregnancy urine. The origin of this beta-core-like material remains to be determined, and its presence will have an impact on the application of urinary beta-core as a tumour marker.

Disparity between beta-core levels in pregnancy urine and serum: implications for the origin of urinary beta-core.

We used a highly purified preparation of the naturally occurring core fragment of hCG beta (beta-core) and a new RIA for beta-core to investigate the concentrations and behavior of beta-core in serum and urine. We collected serum and 24-h urine specimens from healthy pregnant women during the first trimester of pregnancy. The concentrations of beta-core in serum were determined by analysis of fractions eluted from Sephadex G-100. Serum concentrations of beta-core immunoreactivity were very low (0.13-1.25 micrograms/L), while large amounts of beta-core were excreted in urine during pregnancy (as much as 4-5 mg/day). Interference with measurement by serum factors did not account for the low levels of beta-core immunoreactivity in pregnancy serum. Based on the known urinary clearance rate of beta-core in healthy nonpregnant subjects, we calculated that urinary clearance of serum beta-core accounts for only about 1% of the beta-core in pregnancy urine. We conclude that during pregnancy, the concentrations of beta-core in plasma are measurable, but extremely low, and that most of the beta-core in urine is derived by mechanisms other than urinary clearance of serum beta-core; most likely, these mechanisms involve nephrogenous production of beta-core from precursor molecules such as hCG and hCG beta.

Reduced metabolic clearance of acidic variants of human choriogonadotropin from patients with testicular cancer.

We investigated the plasma disappearance of purified urinary human choriogonadotropin (hCG) and acidic variants of hCG (AV-hCG) that are excreted in the urine of patients with testicular cancer to determine if there are differences in their disappearance rates. All cancer patients had relatively increased fractions (10% to 46%) of AV-hCG in their serum when compared with hCG from pregnant control subjects (less than 4% present as AV-hCG). After initiation of therapy in 12 patients with nonseminomatous testicular tumors, the proportion of hCG present as acidic variants in serum rose despite lower serum total hCG concentrations. Samples of highly purified AV-hCG (pI, 3.3 to 4.0) obtained from three patients by concentration from urine and isoelectric focusing were evaluated for their disappearance from serum after injection into rats. All AV-hCG samples had decreased clearance rates when compared with that of highly purified hCG (CR123). These data indicate that acidic forms of hCG have a delayed clearance that contributes to the increased proportion of AV-hCG in the serum of patients with hCG-producing neoplasia after therapy.

Carbohydrate composition of beta-core.

beta-Core is a major component of the hCG-related molecules found in pregnancy urine. We previously have purified the beta-core molecule and have deduced portions of its carbohydrate structure based on lectin binding data. In the present study we used recently developed technology to determine the carbohydrate composition of beta-core and hCG beta (CR119). For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both beta-core and hCG beta. hCG beta contained additional sugars consistent with the presence of four O-linked oligosaccharides. Compared to hCG beta, beta-core contained negligible sialic acid, galactose, or N-acetylgalactosamine. The compositional data suggest that beta-core does not contain N-acetylglucosamine at the nonreducing end of the molecule, whereas the trimannosyl-chitobiose core is apparently intact at both glycosylation sites, consistent with the ability of the molecule to bind to Concanavalin-A. Comparable fucose contents and abilities of beta-core and hCG beta to bind to Lens culinaris indicate a similar extent of fucosylation on the internal N-acetylglucosamine in both molecules. We propose that the N-linked oligosaccharides on beta-core closely resemble the underlying N-linked structures of hCG beta with the antennary sialic acid, galactose, and N-acetylglucosamine removed.

Metabolic fate of human choriogonadotropin.

Review of the literature reveals a number of recent insights concerning the metabolic fate of human choriogonadotropin (hCG). In man, only a fraction (21.7%) of the circulating hCG molecules is excreted in urine. Results from animal studies indicate that the retained hCG is taken up by various tissues, principally kidney, liver, and ovary, where degradation occurs. Ovarian uptake is receptor mediated and saturable. Hepatic uptake of hCG is not preceded by desialylation, and blockade of hepatic receptors for galactose-terminated glycoproteins does not impair hepatic accumulation of hCG. Kidney uptake is quantitatively the most important; parenchymal metabolism, as well as excretion in urine constitute major renal components of hCG disposal. The intracellular products of hCG catabolism in kidney include certain fragments of the hCG molecule that exhibit relative resistance to processing by degradative enzymes. These products are fragments of the hCG beta subunit that lack the hCG beta carboxyterminal antigenic determinant, but retain an hCG beta core antigenic determinant, and, thus, they are designated beta-core molecules. Both kidney from hCG-infused rat and urine from pregnant women contain beta-core molecules in great abundance, in fact, in greater abundance than hCG, itself. Structural characterizations of the beta-core purified from pregnancy urine indicate a mol. wt of about 10,000 and an absence of sialic acid, galactose, and carboxyterminal peptide region, including O-linked oligosaccharides. Human volunteers given purified hCG or hCG beta by infusion excrete beta-core in their urines, which establishes the existence of catabolic pathways in humans for the production of beta-core. Thus, urinary excretion of beta-core may reflect an important fate for the metabolic products of hCG degradation.

Metabolic clearance rate and urinary clearance of purified beta-core.

We injected a highly purified preparation of the beta-core molecule, a fragment of hCG beta excreted in pregnancy urine, into five men and three women to determine its kinetic parameters, MCR, and urinary clearance. The beta-core molecule was distributed in an initial volume [1950 +/- 156 (mean +/- SEM) mL/m2 body surface area] approximately equal to the estimated plasma volume. Its disappearance was multiexponential on a semilogarithmic plot, with a rapid phase t1/2 of 3.5 +/- 0.7 min and a slow phase t1/2 of 22.4 +/- 4.2 min. The transit time (the mean time spent by a molecule of beta-core in transit) was 20.6 +/- 2.1 min. The MCR was 192.0 +/- 8.0 mL/min.m2 body surface area. About 5% of the injected dose of beta-core was excreted into the urine in the first 30 min after injection, and low levels of excretion persisted for up to 7 days. The urinary clearance rate of beta-core was 13.7 +/- 1.4 mL/min.m2, accounting for about 8% of the elimination of beta-core from the plasma. The beta-core immunoreactivity in serum and urine was characterized by gel filtration and three independent RIA systems to show that its properties were indistinguishable from those of the injected beta-core. Serum levels of beta-core in pregnant women were less than 0.2 ng/mL, while the amounts excreted in their urine were as much as 5 mg/day. Based on these clearance parameters of beta-core in normal subjects, less than 0.2% of the beta-core excreted in pregnancy urine is derived by urinary clearance of plasma beta-core. Therefore, more than 99% of the beta-core excreted in pregnancy urine is derived from beta-core in a compartment separate from plasma. In particular, these data indicate that there is relatively little placental secretion of beta-core into plasma and that placental secretion does not account for the vast majority of beta-core in pregnancy urine. These findings are consistent with previous data that point to renal parenchymal degradation of hCG and hCG beta as the major source of urinary beta-core in pregnancy.

Structure-kinetic relationships of choriogonadotropin and related molecules.

To assess how profound differences in carbohydrate and/or polypeptide structures affect parameters of plasma disappearance of glycoprotein hormones, we calculated and compared the initial volume of distribution, rate constants, and metabolic clearance rates of several highly purified human choriogonadotropin (hCG) analogues in monkeys. hCG, deglycosylated hCG, desialylated hCG, or core fragment of hCG-beta purified from pregnancy urine (beta-core) was administered as a rapid intravenous injection to adult male cynomolgus monkeys (n = 3/group). The metabolic clearance rates of deglycosylated hCG, beta-core fragment, and desialylated hCG were increased 15-, 47-, and 152-fold, respectively, over that of hCG. Their corresponding initial volumes of distribution, however, remained essentially unchanged compared with that of hCG and approximated the estimated plasma volume. In contrast, the fast and slow rate constants of plasma disappearance of the hCG analogues were increased as much as 18- and 23-fold, respectively, relative to those of hCG. These studies of structure-kinetic relationships in primates show that major carbohydrate and polypeptide modifications of a glycoprotein hormone cause profound changes in the rate constants of the disappearance curves without changes in the initial volume of distribution.

Stimulation of testosterone production in the cynomolgus monkey in vivo by deglycosylated and desialylated human choriogonadotropin.

Modifications of carbohydrate structures of hCG, such as deglycosylation or desialylation, have been shown to reduce the biological activity of the hormone derivatives in vivo. We posed the question of whether deglycosylated hCG (dg-hCG) and desialylated hCG (ds-hCG) would behave as agonists at the LH/CG receptor in the primate in vivo, as this would bear on their potential clinical utility as LH/CG agonists or antagonists. Thus, we administered large doses (approximately 3 nmol) of highly purified dg-hCG, ds-hCG, hCG, or normal saline as a rapid iv injection to adult male cynomolgus monkeys (n = 3/group). Mean areas under the curves of plasma T over the first 6 h achieved with dg-hCG and ds-hCG were about 5-fold, significantly (P less than 0.05) greater than that in the saline controls and not significantly (P greater than 0.05) different from that in hCG-injected animals. Despite comparable plasma T responses in the first 6 h, mean plasma concentrations of ds-hCG, dg-hCG, and hCG differed dramatically among the groups. Plasma ds-hCG and dg-hCG levels were undetectable by 15 and 180 min, respectively, while the mean plasma hCG level was more than 2.10 nmol/L at 360 min. These data indicate that 1) dg-hCG is a full agonist at the LH/CG receptor in the primate in vivo, despite having minimal intrinsic activity in the rat Leydig cell adenyl cyclase assay and being able to near-completely antagonize hCG action therein; and 2) ds-hCG is a full agonist in the monkey in vivo, capable of stimulating a full testicular response over 6 h, despite being cleared from the circulation in 15 min. We conclude that the signal transduction system at the monkey LH/CG receptor is capable of achieving full steroidogenesis despite dramatically shortened exposure to stimulus or exposure to a stimulus with markedly reduced adenyl cyclase-stimulating activity in vitro.

Beta-core fragments are contaminants of the World Health Organization Reference Preparations of human choriogonadotrophin and its alpha-subunit.

Highly purified preparations of human choriogonadotrophin (hCG), hCG alpha and hCG beta, including those preparations which are being distributed by the World Health Organization as International Standards, cross-reacted in a new radioimmunoassay with increased relative specificity for the beta-core fragment of hCG. A major portion of the beta-core immunoreactivity in the hCG and hCG alpha preparations eluted from Sephadex G-100 in a position (approximate apparent molecular size 15,000-18,000) corresponding to that of purified beta-core fragment prepared from pregnancy urine. However, in the case of hCG beta-subunit preparations, virtually all of the beta-core cross-reacting material eluted from Sephadex G-100 in the same fractions as the native hCG beta-subunit. Quantitatively, the cross-reacting beta-core material accounts for less than 1% (w/w) of the total hCG or subunit immunoreactivity, as measured by conventional radioimmunoassays. The presence of the beta-core fragments as discrete molecular components of the hCG and hCG alpha preparations should be borne in mind when these preparations are used to calibrate new radioimmunoassays for hCG-related molecules.

A radioimmunoassay for the core fragment of the human chorionic gonadotropin beta-subunit.

We developed a RIA for the beta-core fragment of hCG that is excreted in the urine of pregnant women and some patients with cancer. We purified beta-core from crude commercial preparations of hCG (of which beta-core is a major constituent) derived from pregnancy urine and used this purified beta-core material to immunize rabbits. One antiserum (RW25) was particularly useful in that a RIA with purified beta-core as both radioligand and reference preparation had high sensitivity for beta-core detection and low cross-reactivity with other hCG-related molecules and glycoprotein hormones. The cross-reactivities of purified hCG (CR125), hCG beta (CR125), and hCG alpha (CR125) preparations were in each instance less than 3 x 10(-3) (wt/wt). The cross-reactivities of purified pituitary glycoprotein hormones were in each instance less than 2 x 10(-4) (wt/wt). Using the RW25 RIA, virtually all beta-core immunoreactivity in pregnancy urine eluted from Sephadex G-100 in a position coincident with that of purified beta-core. Urine from men and nonpregnant women contained very low levels of beta-core immunoreactivity (less than 6.5 micrograms/L), while urine from pregnant women and patients with testicular cancer or other neoplasms had levels of beta-core immunoreactivity ranging as high as 26,000 micrograms/L. We conclude that the improved specificity of our beta-core RIA will facilitate studies of the physiology and cancer biology of beta-core molecules.

Serum testosterone response to desialylated human choriogonadotropin in man.

To assess the in vivo steroidogenic activity of desialylated human choriogonadotropin (hCG) in man, highly purified desialylated hCG was administered as a constant intravenous infusion over 6 hours to four normal men at a rate sufficient to maintain substantial levels of desialylated hCG in the serum. The mean percent change of serum testosterone from baseline during the first 6 hours in men given an infusion of desialylated hCG was compared to that in saline-infused controls and that in men given highly purified intact hCG. The mean change of serum testosterone at 6 hours in the group infused with desialylated hCG (129% of baseline) was significantly greater than that of the controls (69% of baseline). Furthermore, the response to desialylated hCG could not be distinguished from that of hCG (125% of baseline). It was concluded that desialylated hCG, when given as a constant intravenous infusion, can elicit a substantial serum testosterone response comparable to that seen with purified hCG, and thus, that desialylated hCG behaves as an agonist of the LH/CG receptors on human Leydig cells.

Purification of beta-core fragment from pregnancy urine and demonstration that its carbohydrate moieties differ from those of native human chorionic gonadotropin-beta.

Pregnancy urine contains large quantities of hCG, free beta-subunit, free alpha-subunit, and a population of fragments of beta-subunit known as beta-core. This beta-core population, which can account for as much as 70% of the total beta-immunoreactivity in pregnancy urine, is of interest as both a normal metabolite of pregnancy and a potential marker for malignancy. We have purified the beta-core fragment from pregnancy urine (P-core) and have characterized it with respect to size and carbohydrate composition. P-Core was purified by chromatography on Sephadex G-100, Concanavalin A (Con A)-Sepharose, DEAE-Sephacel, and Sephadex G-75 (superfine). The purified P-core has an apparent mol wt of 17,500 and 17,000, as determined by gel filtration on Sephadex G-75 (superfine) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, respectively. The sialic acid content of P-core was assayed chemically and was less than 0.07 mumol sialic acid/mumole P-core. For comparison to P-core, we have prepared a trypsin fragment of beta-subunit that retains the beta-core immunological determinant recognized by SB6 antiserum and lacks the carboxy-terminal immunological determinant. We have designated this beta-core molecule as T-core (tryptic fragment of beta-subunit) to distinguish it from the beta-core molecule that we have purified from pregnancy urine (i.e. P-core). Most of the P-core and T-core molecules bind to Con A (84% and 86%, respectively). The Con A-bound material was used for subsequent characterizations. Neither P-core nor T-core binds to DEAE using conditions under which intact hCG beta binds to DEAE. A variety of agarose-bound lectins were used to further investigate the carbohydrate nature of the Con A-bound P-core and T-core molecules. The lectin binding data indicate that the antennae on P-core do not contain appreciable sialic acid or galactose, in contrast to the antennae on T-core, which contain both. We conclude that P-core, the naturally occurring beta-core fragment in pregnancy, has been processed to a form in which nearly all of the sialic acid and galactose residues are removed, but the Con A-binding site (consisting of the core sugars) and most of the core fucose are retained.

The use of biochemical assays in epidemiologic studies of reproduction.

Recent progress in the assay of urinary hormones has opened new opportunities for epidemiologists to study hormones and health outcomes. This is especially true for studies of female reproduction. The cyclic nature of female reproduction can be fully described only by continuous frequent measurements that, in order to be practical, require easily collected biological specimens. We describe our experience in collecting and analyzing daily urine specimens from 301 healthy women. We conclude that this approach is not only feasible but potentially of great value to epidemiologists for studying fertility, early pregnancy, the effects of toxic exposures on reproduction, and the relationships between reproduction and later risk of chronic diseases.

Basal serum prolactin levels and prolactin responses to constant infusions of thyrotropin releasing hormone in healthy aging men.

We measured serum prolactin (PRL) levels by RIA before and during a 240-min constant infusion of TRH (0.4 microgram/min iv) in three similarly sized groups of healthy aging men 30 to 49, 50 to 69, and 70 to 96 years. Basal data were evaluated by analysis of variance with Duncan's multiple range test and regression analysis. Mean basal serum PRL level was elevated (p less than .05) in the oldest group, attributable to PRL elevations (between 20 and 40 ng/ml) in 4 men over 75 years. Serum PRL levels decreased (p less than .001) from -30 min to 0 min before TRH infusion in all groups, but there was no age-dependent difference (p greater than .3) in the magnitude of the reduction. Repeated measures analysis of variance showed increased serum PRL levels (p less than .001) during TRH infusion in all age groups, and an age-dependent increase (p less than .05) in magnitude of peak PRL response. This significant difference was between the two oldest age groups early in the infusion. Chi-square analysis revealed an increased (p less than .05) frequency of early (less than 120 min) peak responses in the oldest age group. The present data suggest that basal and TRH-stimulated PRL secretion may be augmented in some healthy older men.

Measuring early pregnancy loss: laboratory and field methods.

We intensively studied 30 women attempting pregnancy in order to lay groundwork for larger studies of early pregnancy loss. These women collected first morning urine specimens for up to 6 months after discontinuing use of birth control. Urine specimens were successfully collected for 98% of the woman-days in the study. Three assays for human chorionic gonadotropin (hCG) were performed on each urine specimen. An immunoradiometric assay (IRMA) specific to the carboxyterminal peptide of the hCG beta-chain proved to be more sensitive and more specific than two radioimmunoassays (RIAs). Using the IRMA, we found four cases in which hCG rose and fell over successive days, consistent with early pregnancy loss. For three of these four cases, the level of hCG was too low to be detectable with the RIAs. Among the control group of five women with tubal ligations, there was no detectable hCG above threshold with the IRMA. Thus, the enhanced sensitivity and specificity of the IRMA allows very early pregnancy losses to be identified that would otherwise be undetectable. Furthermore, its effectiveness with small quantities of first morning urine makes the IRMA a useful tool for epidemiologic studies.

Suppression of thyrotropin in the low-thyroxine state of severe nonthyroidal illness.

In a prospective study, we assessed the role of thyrotropin in the development of the low-thyroxine state that is associated with severe illness. We measured the serum thyrotropin and thyroid hormone concentrations longitudinally in 35 patients with hematopoietic cancer or aplastic anemia who were treated by bone-marrow transplantation. In 19 patients thyroxine declined sharply after bone-marrow transplantation and was associated with a reduction of the serum thyrotropin in the 17 patients tested, often to levels below the normal range. The serum triiodothyronine level, free thyroxine index, and free thyroxine level also declined in these patients. In the patients who recovered, clinical improvement was accompanied by the return of thyrotropin and thyroid hormone concentrations to their pretreatment ranges. These and related findings suggest that the low-thyroxine state of severe illness is the result of several events, one of which is failure of the normal negative-feedback control of the pituitary-thyroid axis due to illness-associated, decreased secretion of thyrotropin. The notion that such patients are "euthyroid" must be questioned, but the possible value of thyroid hormone-replacement therapy in these circumstances remains to be determined.

Estimation of a physiologic replacement dose of levothyroxine in elderly patients with hypothyroidism.

We gave graded doses of levothyroxine sodium to 11 elderly hypothyroid subjects (mean age, 66.1 years). The daily levothyroxine sodium dose was initially 75 micrograms or less, and was increased by 25 micrograms every six weeks. Serum total thyroxine, total triiodothyronine, and basal thyrotropin levels were measured at the start of the study and at the end of each six-week dose period. A protirelin (thyrotropin-releasing hormone) test was performed when the thyrotropin level returned to normal. Mean daily levothyroxine sodium doses that normalized serum thyrotropin levels and protirelin test were 110 +/- 8 micrograms and 113 +/- 9 micrograms, respectively. Serial basal thyrotropin determinations during stepwise increments in levothyroxine dose indicated physiologic hormone replacement. As determined in our elderly patients, levothyroxine replacement dose was a third less than that formerly recommended.