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Clinical Chemistry 45: 532-538, 1999;
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(Clinical Chemistry. 1999;45:532-538.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Cross-Reaction with Luteinizing Hormone ß-Core Is Responsible for the Age-dependent Increase of Immunoreactive ß-Core Fragment of Human Chorionic Gonadotropin in Women with Nonmalignant Conditions

Ray K. Ilesa, Mohammed K. Javid, Lionel K. Gunn and Tim Chard

a Author for correspondence. Fax 44 0171-600-1439; e-mail R.K.Iles{at}mds


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The ß-core fragment of human chorionic gonadotropin (hCGßcf), also termed "ß-core" and urinary gonadotropin peptide (UGP), has been reported to be present in the urine of healthy women and to increase in concentration after menopause. This could reflect cross-reaction with the equivalent metabolite of luteinizing hormone (LH), the ß-LH-core.

Methods: We measured immunoreactive LH, hCG, free {alpha}-subunit, and free ß-subunit hCG (hCGß), as well as ß-core, using the S504 RIA and Triton UGP enzyme immunoassay in 274 urine samples from women with nonmalignant gynecological conditions. The molar cross-reaction of each assay with purified ß-LH-core was determined.

Results: Cross-reaction with ß-LH-core was 100% in the LH and the S504 ß-core assay, 5% in the Triton UGP assay, and <0.1% in the hCG, free {alpha}-subunit, and free hCGß assays. Median urine concentrations of all analytes showed an age-dependent increase. LH and free {alpha}-subunit concentrations were ~103 pmol/mol creatinine; hCG and S504 ß-core were ~102 pmol/mol creatinine; free hCGß and Triton UGP ß-core were in the tens of pmol/mol creatinine. The S504 ß-core concentrations were 10% of those of LH. S504 ß-core was strongly correlated with LH, but not with hCG or with free hCGß (LH, r2 = 0.45; hCG, r2 = 0.26; free hCGß, r2 = 0.03). The concentrations of ß-core detected by the Triton UGP assay, which has a 5% cross-reaction with ß-LH-core, were 2% of LH and 5% of the S504 ß-core concentrations. Triton UGP values correlated strongly with LH concentrations, but less well with S504 ß-core, intact hCG, and free hCGß (LH, r2 = 0.44; S504 ß-core, r2 = 0.33; hCG, r2 = 0.32; free hCGß, r2 = 0.19).

Conclusions: Immunoreactive ß-core in women free of malignancies reflects cross-reaction with concentrations of the metabolite of LH, ß-LH-core, within the health-related reference interval.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Common epithelial tumors of the urogenital tract frequently express the free ß-subunit of human chorionic gonadotropin (hCGß)1 with no concomitant expression of its heterodimer partner, the common {alpha}-subunit of the glycoprotein hormone (1)(2)(3)(4). However, in vivo, this expression currently is detected by measurement of urinary ß-core fragment, termed hCGßcf by the IFCC, but also known as ß-core or urinary gonadotropin peptide (UGP). The rationale for this approach was that many more cancer patients were found to have increased urinary concentrations of hCGßcf than had increased, or even detectable, concentrations of hCG or hCGß in their serum. The physiological reasons for this have now been clarified. Free hCGß has a much shorter half-life [metabolic clearance rate (MCR), 19.0 mL · min-1 · m-2] than intact hCG (MCR, 1.9 mL · min-1 · m-2) Furthermore, aberrant glycosylation of the molecule could reduce its circulating blood half-life to a matter of seconds (MCR, 495 mL · min-1 · m-2) (5)(6)(7).

hCGßcf/ß-core is a major immunoreactive degradation product of hCG and its free ß-subunit and is often the only form of hCG present in the urine of cancer patients (8)(9). The process by which hCGßcf/ß-core arises starts in the circulation with cleavage or "nicking" of amide bonds between residues 47 and 48 and, less frequently, between residues 42 and 43 of hCGß, either as part of the intact heterodimer or as a free subunit (10)(11). This nicking occurs at the center of a major hCG/luteinizing hormone (LH) receptor binding loop (12)(13). These nicked molecules are then taken up by the kidneys and further degraded to hCGßcf/ß-core, which is excreted into the urine (14). Although missing 53 amino acids of the COOH terminus, 5 amino acids of the NH2 terminus, and residues 41–54, hCGßcf/ß-core retains many antigenic epitopes of its parent molecule because it retains a core, or central knot, of three sets of disulfide bond pairs, which are responsible for much of the three-dimensional shape of hCGß (8).

Increased urinary hCGßcf/ß-core was proposed originally as a general marker of gynecological cancer (vaginal, cervical, ovarian, and endometrial) (15)(16)(17). However, immunoreactive "ß-core" is detectable in urine of women with no malignant disease. Indeed, there is a general increase with age, especially after menopause (18). Thus, cutoff values between normal and abnormal must be adjusted according to menopausal status. Subsequent studies demonstrated that much of this immunoreactive ß-core could be attributed to cross-reaction with a metabolite of LH, which we termed ß-LH-core and which was almost certainly produced by the same degradation pathways that led to hCGßcf (19). Nevertheless, using a highly specific assay, Alfthan et al. (20) suggested that there was still an underlying increase in hCGßcf with age. Recently, ß-LH-core was purified by Birken et al. (21) at Columbia University, New York. We have assessed the molar cross-reactivity of ß-LH-core in our in-house ß-core, hCG, LH, free {alpha}-subunit, and free hCGß assays along with a recently developed ß-core assay, Triton UGP, which includes a pretreatment LH "scavenger" antibody step to remove cross-reacting LH metabolites.

We used these assays to measure immunoreactive ß-core in 254 women with nonmalignant conditions to determine whether the detected hCGßcf could be accounted for by cross-reactivity with ß-LH-core.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
urine samples
Urine samples were collected from 254 women attending St. Bartholomew's Hospital with nonmalignant pathology. Samples were stored frozen at -20 °C until assay. The creatinine concentration of each sample was measured by the Jaffé method on a Monarch 200 centrifugal analyzer.

hormone assays
Intact hCG was measured with an in-house IRMA with a polyclonal anti-{alpha}-subunit capture antibody conjugated to l,l'-carbonyldiimidazole (CDI)-activated cellulose and a 125I-radiolabeled monoclonal antibody 1/07 (Quantum Bioscience) to epitopes on the hCGß C-terminal peptide. The calibration curve ranged from 2.6 to 813 pmol/L.

LH was measured using an LH IRMA produced by North East Thames Immunoassay, St. Bartholomew's Hospital. This uses a polyclonal anti-LH antiserum for capture onto a CDI-activated cellulose solid phase and the same antibody, affinity purified and 125I-radiolabeled, for detection. The calibration curve ranged from 34 to 1333 pmol/L.

Free {alpha}-subunit was measured by use of an in-house RIA with an antiserum (S781) raised against the free {alpha}-subunit and affinity absorbed on a column of intact hCG conjugated to CNBr-activated Sepharose. The tracer was 125I-radiolabeled purified free {alpha}-subunit (NIH preparation CR123 {alpha}-subunit). The calibration curve ranged from 62.5 to 6250 pmol/L.

Free hCGß was measured using an in-house IRMA. A polyclonal antiserum (S752) raised in sheep against free hCGß was conjugated to CDI-activated cellulose. Free hCGß captured onto this solid phase was detected by 125I-radiolabeled monoclonal antibody 1/07, which recognizes epitopes on the hCGß C-terminal peptide. A calibration curve was constructed by plotting bound {gamma}-activity values against increasing concentration of hCGß (NIH preparation CR123). The cross-reactivity of this assay is detailed the Results, the calibration curve ranged from 22 to 1110 pmol/L.

The in-house ß-core RIA used a polyclonal antibody to hCGßcf (S504) in a late addition competition assay using 125I-radiolabeled purified hCGßcf (18). This assay is known to cross-react with ß-LH-core (19). The calibration curve ranged from 9 to 500 pmol/L.

The commercial Triton ELISA uses a scavenger anti-LH antibody in the initial diluent to bind LH-like material. The assay used an immobilized monoclonal antibody against hCGßcf and a peroxidase-conjugated polyclonal antibody to hCGß (and thus epitopes retained by hCGßcf). The calibration curve ranged from 2 to 16 pmol/L.

cross-reactivity studies
Hormones and subunits were NIH preparations (CR123) of intact hCG, free hCGß, and {alpha}-subunit of hCG; WHO International Reference Preparations of LH (LH-68/40), follicle-stimulating hormone (FSH; FSH-83/575), and thyrotropin (TSH; TSH-80/558; National Institute for Biological Calibrations and Control, Potters Bar, Herts, UK). Purified hCGßcf was donated by Drs. R. Wehmann and D. Blithe (NIH, Bethesda, MD). Recently purified ß-LH-core was kindly donated by Dr. S. Birken (Presbyterian Medical Center, Irving Center for Clinical Research, Columbia University, New York, NY). Cross-reactivity was determined on a molar basis using molecular weights calculated from the established primary structures: intact hCG, Mr 36 700; free hCGß, Mr 22 200; free {alpha}-subunit, Mr 14 500 (22)(23); hCGßcf and ß-LH-core, Mr 10 000 (21)(24); LH, Mr 29 000; FSH, Mr 33 000; and TSH, Mr 30 000 (19).

Cross-reactivities were calculated from the molar equivalents that produce 50% B/Bo displacement for the RIA and at concentrations in the IRMA and enzyme immunoassay at which calibrator and cross-reactant curves paralleled.

statistical analysis
Matched assay results for the samples were examined for correlations using the Spearman rank test (adjusted for ties). Calculations were performed using Astute (statistics add-in for Microsoft Excel; DDU software, University of Leeds, UK).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cross-reactivities of each assay are shown in Table 1 . Significantly, ß-LH-core yielded a 100% cross-reactivity with the LH IRMA and with the ß-core (S504) RIA. The Triton UGP ß-core assay showed a 5% cross-reaction with ß-LH-core (Fig. 1 ).


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  		Table 1. Molar cross-reaction of the glycoprotein hormones and their related fragments in the hormone- and subunit/fragment-specific assays used.



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  		Figure 1. Calibration and cross-reaction curves of hCGßcf ({bullet}) and LH ß-core fragment ({circ}) in the S504 ß-core RIA (A) and Triton UGP-II ELISA (B).

Age-dependent increases were seen (Fig. 2 ) in the median values of apparent immunoreactive LH, hCG, free hCGß, free {alpha}-subunit, S504 RIA ß-core, and Triton UGP ß-core.



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  		Figure 2. Concentrations of gonadotropin-associated analytes in the urine of nonpregnant women, grouped in decennial age ranges.

Comparison of immunoreactivity with antisera directed toward LH ({diamondsuit}), free {alpha}-subunit ({diamond}), ß-core [S504 ({blacksquare}), UGP II ({bullet}), and hCG ({square})], and free hCGß subunit ({circ}). Points represent median values for each decile.

LH and free {alpha}-subunit concentrations were ~103 pmol/mol creatinine, hCG and S504 ß-core concentrations were ~102 pmol/mol creatinine, and free hCGß and Triton UGP ß-core concentrations were ~10 pmol/mol creatinine (Table 2 ).


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  		Table 2. Immunoreactive concentrations1 (pmol/mol creatinine) of hCG, LH, free hCGß subunit, free {alpha}-subunit, and ß-core in the urine of women 20 to >80 years of age with benign gynecological conditions.

A correlation analysis between ß-core concentrations and all other hCG, LH, and free subunits analytes was conducted (Table 3 and Fig. 3 ). The S504 ß-core concentrations were 10% of those of LH, and there was a stronger correlation of S504 ß-core concentrations with LH than with hCG or free hCGß within individual sample (LH, r2 = 0.45; hCG, r2 = 0.26; free hCGß, r2 = 0.03; P = 0.60). The concentrations of ß-core detected by the Triton UGP assay were 2% of the LH and 5% of the S504 ß-core concentrations. Triton UGP ß-core values similarly correlated more strongly with LH and S504 ß-core concentrations than with intact hCG and free hCGß (LH, r2 = 0.44; S504 ß-core, r2 = 0.33; hCG, r2 = 0.32; free hCGß, r2 = 0.19).


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  		Table 3. Correlation statistics1 of hCG and related metabolites.



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  		Figure 3. Plots of the correlation between LH and the free {alpha}-subunit of the glycoprotein hormones (r2 = 0.68; P <0.0001; A), LH and ß-core measured using the S504 RIA (r2 = 0.45; P <0.0001; B), and ß-core measured using the S504 RIA and free hCGß (r2 = 0.03; P = 0.60; C) present in the urine of women with nonmalignant conditions.

r2 values are Spearman rank correlation coefficients.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of ß-core as a tumor marker is complicated by reports of variable urine concentration (2), changes during the menstrual cycle (25), and higher excretion in the postmenopausal age groups (18)(26). Our results suggest that cross-reacting ß-LH-core was responsible for the age-related increase in basal ß-core concentrations (19). The proposed ß-LH-core molecule, arising from the same metabolic processes, would share an 82% amino acid sequence homology, and the conserved cysteine knot disulfide would render the two molecules topologically identical. Assay systems developed to distinguish intact hCG and free hCGß from hCGßcf would, by definition, be directed to regions that are unique to the degraded core molecule. Because the same degradation process is likely to act on both hCGß and LHß (and most probably FSHß and TSHß) the resulting core-defining epitopes would be common to all glycoprotein hormone urinary ß-core molecules (19)(27). For this reason, the Triton UGP ELISA for hCGßcf incorporates a scavenger antibody for LH-like material.

Purified ß-LH-core did not cross-react in the free hCGß, free {alpha}-subunit, and intact hCG assays, but showed 100% cross-reaction in the LH and S-504 ß-core assays. However, ß-LH-core showed 5% cross-reaction in the Triton UGP ß-core, despite pretreatment of samples with the LH scavenger antibody (Fig. 1Up ).

On a molar basis, immunoreactive LH was the most abundant form of gonadotropic molecule in the urine of pre- and postmenopausal women. Both LH and free {alpha}-subunit concentrations were in the ~103 pmol/mol creatinine range; S504 RIA ß-core was in the ~102 pmol/mol creatinine range; free hCGß and intact hCG were in the ~10 pmol/mol creatinine range; and Triton UGP ß-core was in the ~1 pmol/mol creatinine range. Thus, cross-reactivity with LH and LH-related ß-LH-core is highly likely to influence the immunochemical measurement of other related gonadotropic peptides. For example, an hCG assay with 0.1% cross-reactivity with LH would measure apparent hCG concentrations in the urine of women >60 years of age as 2.7 pmol/mol creatinine, simply because of cross-reactivity.

Given the data on specificity for the various assays used in this study, all the apparent ß-core measured by the S504 RIA in these samples could be accounted for by cross-reaction with ß-LH-core. This interpretation is supported by the fact that there was a strong correlation between the S504 RIA concentrations of ß-core and LH concentrations (r2 = 0.45), but not with hCG and hCGß concentrations (r2 = 0.26 and 0.03, respectively). Thus, ß-LH-core could represent 10% of the LH immunoreactivity of the samples measured using the LH IRMA (this IRMA cross-reacts 100% with ß-LH-core). The concentrations of ß-core detected by the Triton UGP assay were 5% of those detected by the S504 ß-core RIA and 2% of the LH concentrations. Given that the UGP assay has a 5% cross-reactivity with authentic ß-LH-core, this is consistent with all of the ß-core immunoreactivity found in the samples being attributable to cross-reaction with ß-LH-core.

Alfthan et al. (20) claimed a true increase in urinary hCGßcf with age. However, there were no data on the cross-reactivity of authentic ß-LH-core in their assay system. The median concentration of immunoreactive ß-core found in urine by Alfthan et al. was 70 pmol/mol creatinine for those <50 years of age and 230 pmol/mol creatinine for those >50 years of age. This is consistent with the concentrations (molar ratios) detected by the S504 ß-core RIA. Furthermore, their studies of male urinary hCGßcf showed no dramatic increase with age. Thus, we believe it is possible that the assay developed by Alfthan et al. also cross-reacts 100% with ß-LH-core.

Free intact hCG and free hCGß concentrations also rose with age. However, hCG and free hCGß concentrations did not correlate significantly with hCGßcf (see Table 3Up ) and reactivity of LH and ß-LH-core in these assays was <0.1%. Intact hCG and free hCGß showed a weak correlation (r2 = 0.37), and hCGß concentrations were approximately 20% of those of intact hCG. The origin of these true hCG species is unknown, although hCG and free hCGß expression has been demonstrated in the nonmalignant pituitary (28)(29) and in testicular (30), prostatic (31), and urothelial epidermal tissues (32).

In conclusion, most, if not all, ß-core immunoreactivity found in the urine of healthy pre- and postmenopausal women and patients with nonmalignant gynecological conditions can be attributed to cross-reaction of the assays with the urinary metabolite of LH, ß-LH-core. Any true hCGßcf, derived from extremely low-level expression of the hCGß gene cluster by pituitary gonadotrophs, is likely to be extremely low and much smaller than immunoreactivity from cross-reacting ß-LH-core metabolite. If assays can be developed that clearly distinguish between urinary ß-LH-core and hCGßcf, the issue of the clinical utility of hCGßcf as a tumor marker should be reexamined.


   Acknowledgments
 
We thank Dr. Steve Birken for pure LHßcf. This study was supported by grants from the Cancer Research Committee and Joint Research Board of St. Bartholomew's Hospital, London, UK.


   Footnotes
 
Williamson Laboratory, Department of Obstetrics and Gynecology, St. Bartholomew's and The Royal London School of Medicine and Dentistry, St. Bartholomew's Hospital, London EC1A 7BE, UK.

1 Nonstandard abbreviations: hCGß, free ß-subunit of human chorionic gonadotropin; hCGßcf, ß-core fragment of human chorionic gonadotropin; UGP, urinary gonadotropin peptide; MCR, metabolic clearance rate; LH, luteinizing hormone; ß-LH-core, ß-core fragment of luteinizing hormone; CDI, l,l'-carbonyldiimidazole; FSH, follicle-stimulating hormone; and TSH, thyrotropin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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