Does hCG or hCGß play a role in cancer cell biology?

The role that hCG might play in the oncogenic process in cancer is certainly complex. We know that the expression of hCG and its beta subunit is a widespread phenomenon which has been described in many cancer subtypes. However, hCG's involvement in breast cancer has been antithetical: the detection of ectopically expressed hCG(ß) by breast tumors has been employed as a biomarker of malignancy, and hCG has been proposed as a ligand vehicle for toxic drugs, with the aim of targeting the LH/hCG receptor which is reported to be expressed by malignant breast tissue. However, it has also been proposed that hCG is a protective agent against the development of breast cancer, leading some to advocate hCG administration to non-pregnant women as a prophylactic measure against cancer. Nevertheless, suggestions that hCG is involved in the angiogenesis, metastasis and immune escape that are central to cancer progression - are phenomena which clearly apply to breast cancer. Indeed, a tumor vaccine based upon hCG has very recently been shown to protect against mammary tumors in mice. We propose that this apparent paradox is resolved if the free beta subunit of hCG produced by tumors acts as an autocrine anti-apoptotic and angiogenic growth factor, whilst intact heterodimeric hCG, as in pregnancy, is part of developmental signaling that initiates tissue differentiation (including breast ductal tissue development), and hence reduces the population of stem-like cells which are susceptible to oncogenic factors.

Capillary electrophoresis of human follicular fluid.

Some of the major serum proteins that are also found in follicular fluid, including transferrin, alpha-macroglobulin and albumin, are thought to play a role in oocyte maturation. This study set out to identify proteins in human follicular fluid by capillary zone electrophoresis and to investigate their relationship to follicular/oocyte maturity and fertility outcome. 176 individual follicular fluid samples, from 30 women undertaking in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), were run using an optimized capillary zone electrophoresis method that gave a good separation of sixteen peaks in most samples. Nine of the peaks were identified and quantified but seven remain unknown and require further proteomic identification. Of the identified protein peaks, levels of each were corrected for follicular volume and total content calculated. No significant difference in protein levels was found with regard to oocyte recovery and fertilization. Protein concentrations tended to decrease as the follicular sphere increased whilst total content in follicular fluid increased in proportion to size. This is consistent with simple transudation across a sphere surface area which does not increase in proportion to the follicular fluid. This is not true of the concentration and content pattern of other proteins/biomolecules which are produced by follicular cells locally. In conclusion, neither concentration nor absolute levels of nine major proteins identified in follicular fluids correlated with oocyte presence and fertility outcome. Future work to remove more concentrated proteins (e.g. albumin) would enhance separation of smaller peaks and identification of the unknown molecules.

hCG beta expression by cervical squamous carcinoma--in vivo histological association with tumour invasion and apoptosis.

AIMS: To investigate the correlation of beta-subunit of human chorionic gonadotrophin (hCG beta) expression by cervical carcinomas with measures of tumour apoptosis. METHODS AND RESULTS: Eighty-nine cervical carcinoma patients' samples were subject to hCG beta immunohistochemistry and scored with respect to intensity of immunopositivity and percentage of positive cells. Apoptosis was evaluated by three independent parameters: morphological characteristics [haematoxylin and eosin (H&E)], terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and poly (ADP-ribose) polymerase (PARP) immunopositivity. Of the 12 adenocarcinomas, only one (8%) was hCG beta+. However, 87% (61/70) of the squamous cell and 100% (7/7) of adenosquamous cell carcinomas were hCG beta+. hCG beta reactivity and intensity was predominantly confined to peripheral tumour cells at the stromal-epithelial interface. Correlation analysis showed that H&E and PARP apoptotic immunopositivity negatively correlated with hCG beta expression (P < 0.001 and P = 0.028 respectively), whereas TUNEL did not (P = 0.12). However, immunopositivity for apoptotic cells by TUNEL was significantly less in tumours where hCG beta expression was greater (scoring >or= 6) and vice versa. hCG beta immunopositivity was also observed in newly formed blood vessels, as well as tumour cells within lymphatic vessels. When tumour vascularization was taken into account, samples with noted vascularization positively correlated with hCG beta scoring. CONCLUSIONS: hCG beta expression correlates with reduced tumour cell apoptosis and may be involved in tumour vascularization and dissemination.

Designing a new generation of anti-hCG vaccines for cancer therapy.

The heterodimeric 'pregnancy-specific' hormone human chorionic gonadotropin (hCG) has been used as the basis for a contraceptive vaccine. More recently, the observation that hCG, particularly in the form of the beta-chain expressed in the absence of alpha-chain, is aberrantly expressed in a number of different tumors has opened up a second potential application for such vaccines. Drawbacks of the currently available vaccines are that they are either relatively weakly immunogenic or that they induce antibodies that cross-react with human leuteinizing hormone (hLH). We have explored the possibility of creating mutated versions of the hCG beta-chain with improved immunologic properties.

Ectopic hCGbeta expression by epithelial cancer: malignant behaviour, metastasis and inhibition of tumor cell apoptosis.

Ectopic expression of the beta-subunit of human chorionic gonadotropin (hCG) is now a recognized phenomenon in 20-40% of all common epithelial carcinoma arising from mucosal epithelia such as bladder, cervix, lung and naso-pharynx. Recent studies have shown that it acts as an autocrine growth factor by inhibiting apoptosis. Structural homology and in vitro studies suggest that it may achieve this by inhibition of the transforming growth factor beta (TGFbeta) receptor complex. Such a molecular mechanism would go some way to explaining ectopic hCGbeta's association with poor prognosis and tumors that will rapidly progress to metastasis.

Detection of human chorionic gonadotrophin-beta in serum or urine of prostate cancer patients is of no clinical significance.

The aim of this study was to prospectively evaluate the potential role of elevated urinary/serum human chorionic gonadotrophin-beta (hCGbeta) in prostate cancer prognosis. 104 patients with newly diagnosed prostate cancers were included; 68 patients had organ-confined, 18 had locally advanced and 18 had metastatic disease. A control group consisted of 115 patients presenting with benign prostatic disease. Serum and urinary total hCGbeta was measured prior to treatment and serum PSA was measured at diagnosis. The patients were treated along conventional lines and progression-free survival was assessed. Four patients had elevated serum and 10 had elevated urinary, total hCGbeta. There were no significant correlations between serum/urinary levels of hCGbeta and tumour stage, Gleason score or PSA. In contrast, serum PSA had significant linear correlations with both clinical tumour stage and Gleason score (p = 0.0001). At a median follow-up of 36 months, 22 (21.2%) patients had died while 17 (16.3%) others had progressed. Kaplan-Meier plots and log-rank test revealed no significant difference in progression-free survival between patients with elevated or normal levels of serum and/or urinary total hCGbeta. Clinical tumour stage, grade and PSA were statistically significant prognostic variables. Immunoassay measurement of serum or urinary hCGbeta has no significant role in the clinical management of prostate cancer.

Serum amino-terminal propeptide of type 1 procollagen (P1NP) in prostate cancer: a potential predictor of bone metastases and prognosticator for disease progression and survival.

INTRODUCTION: To determine if amino-terminal propeptide of type 1 procollagen (P1NP) is reliable as a predictor of prostate cancer bone metastases and assess its value as a prognostic indicator of disease progression and survival. MATERIALS AND METHODS: A cohort of patients with prostate cancer between January 1999 and July 2001 were recruited. Prostate-specific antigen (PSA) and P1NP levels were measured. Two years following completion of recruitment, patient notes were reviewed for symptoms of bone metastases and survival. RESULTS: 24 negative and 12 equivocal or positive bone scans were reported for 36 recruited patients. Mean PSA values for patients with negative, equivocal and positive scans were 18.3, 24.9 and 122.5 ng/ml while mean P1NP for the same groups were 38.2, 73.4 and 119.9 ng/ml. For patients with equivocal and positive scan, mean P1NP with and without bone symptoms were 111.5 and 65.7 ng/ml while for surviving and dead patients the values were 63.9 and 120.8 ng/ml, respectively. CONCLUSIONS: Though this study involved a small number of patients, it demonstrates P1NP's potential as a predictor of bone metastases and a prognosticator for disease progression and survival.

Characteristics of populations of granulosa cells from individual follicles in women undergoing 'coasting' during controlled ovarian stimulation (COS) for IVF.

BACKGROUND: The aim of this study was to evaluate the functional characteristics of granulosa cell populations of individual follicles of women undergoing controlled ovarian stimulation (COS) for IVF/ICSI in whom gonadotrophin had been withheld ('coasted') for the prevention of OHSS. METHODS: Follicular fluid and granulosa cells were isolated from 224 individual follicles in 41 women who had been coasted and from 257 individual follicles in 50 women who had a 'normal' response to COS. Cells were cultured at 10,000 cells per well, to evaluate progesterone secretion. Follicular fluid was assayed for progesterone and estradiol (E2). RESULTS: No significant differences were observed between the two groups with respect to granulosa cell number or follicular fluid progesterone and E2 and follicle size, the retrieval of an oocyte and the subsequent fertilization of the oocyte. However, the granulosa cells derived from the coasted group showed a higher rate of progesterone secretion per cell at 72 h which was sustained for longer. Differences were also seen at 72 and 120 h of culture with a loss of correlation between progesterone secretion and follicle diameter in the coasted group. CONCLUSIONS: Our findings suggest that coasting has an effect on the functional capacity of the granulosa cells and the duration of their function. It is likely that in women at risk of OHSS who are not coasted, the granulosa cells have the capacity to produce significantly more chemical mediators per cell and for a more prolonged period of time.

The effects of 'coasting' on follicular fluid concentrations of vascular endothelial growth factor in women at risk of developing ovarian hyperstimulation syndrome.

BACKGROUND: The aim of this study was to assess the effect of withholding gonadotrophins (coasting) during controlled ovarian stimulation (COS) on individual follicle concentrations of follicular fluid vascular endothelial growth factor (VEGF) in women at high risk of developing ovarian hyperstimulation syndrome (OHSS). METHODS: Twenty-two women who had been coasted and 26 optimally responding women (control group) undergoing COS for IVF were studied. At the time of oocyte retrieval, the follicular fluid from four to six individual follicles of different sizes was collected for VEGF analysis. RESULTS: A total of 118 follicles was analysed in the coasted group and 137 in the control group. A negative correlation was observed between the follicle size and VEGF concentration (r = -0.18, P = 0.03) in the control group, which was not seen in the coasted group. Similarly, the correlation between oestradiol (E(2)) and VEGF (r = 0.4, P < 0.0001) observed in the control group was not apparent in the coasted group. Significantly lower concentrations of VEGF were seen in the follicular fluid of the coasted patients. CONCLUSIONS: It is clear that there are differences in follicular fluid VEGF concentrations between the two groups. It is possible that coasting alters the capacity of the granulosa cells to produce VEGF and/or their response to hCG and in this way acts to reduce the severity and incidence of severe OHSS.

Identification of post-translational modifications resulting from LHbeta polymorphisms by matrix-assisted laser desorption time-of-flight mass spectrometric analysis of pituitary LHbeta core fragment.

Metabolism of the human chorionic gonadotrophin (hCG)- and LHbeta-subunits (hCGbeta, LHbeta) terminates with the urinary excretion of core fragment (hCGbetacf, LHbetacf) molecules that retain antigenic shape and constituent N-linked carbohydrate moieties. We have previously demonstrated the resolved mass spectra of hCGbetacf, from which the carbohydrate moieties present at two N-linked glycosylation sites were identified. LHbetacf was subjected to the same mass spectrometric analysis. As LHbeta shares 82% homology with hCGbeta but possesses only one glycosylation consensus site a simpler spectral fingerprint of LHbetacf glycoforms was expected. LHbetacf was reduced with dithiothreitol and analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Glycoforms were predicted by subtracting the peptide mass from the m/z values of the observed peaks and then sequentially subtracting the masses of the monosaccharide residues of hCGbeta N-linked carbohydrates reported in the literature. The mass spectra of LHbetacf revealed a broad single peak ranging from m/z 8700 to 10 700. Following reduction, this peak was replaced by a set of partially resolved peaks between m/z 4130 and 5205 corresponding to glycosylated forms of the peptide LHbeta6-40. A peak at m/z 4252.2 corresponded to the non-glycosylated peptide LHbeta55-93. Remaining peaks indicated that the pooled sample comprised a wide set of glycoforms, contained LHbetacf with two N-linked carbohydrate moieties and indicated evidence of further glycosylation due to amino acid substitution in polymorphic variants. This is evidence that a single nucleotide polymorphism alters the post-translational modification of a protein and hence its structural phenotype.

High concentrations of CA 125 in uterine flushings: influence of cause of infertility and menstrual cycle day.

Uterine flushings were obtained under transvaginal ultrasonographic control from 132 women presenting for investigation and treatment of infertility. Levels of CA 125 were measured by radioimmunoassay and results expressed in relation to the total protein concentration of the same flushings. CA 125 was detected in uterine fluid at levels higher than those previously reported in peripheral blood. Uterine fluid CA 125 concentrations varied throughout the menstrual cycle, being highest in the mid-follicular phase (days 6 to 10). Uterine fluid CA 125 concentrations may reflect endometrial secretion of this protein more directly than serum levels. CA 125 concentrations did not vary according to the cause of infertility but further work in larger numbers of women is required.

The analysis of human amniotic fluid using capillary electrophoresis.

This study has investigated the composition of amniotic fluid (AF) using capillary electrophoresis (CE). A detailed optimisation investigation was undertaken to obtain the best resolution of the major peaks in amniotic fluid. In the final method, capillary zone electrophoresis (CZE) of AF was performed on a Hewlett Packard3D CE instrument using a fused-silica capillary of 44 cm total length (36 cm to the detector) with in internal diameter of 50 microm. The background electrolyte was 20 mM sodium tetraborate containing 0.8 mM EDTA adjusted to pH 9.0. AF was diluted 1 plus 1 with deionised water prior to hydrodynamic injection for 3 s at 50 mbar. The separation was performed at +22.5 kV and resulted in a current of 65 microA. The capillary temperature was 28 degrees C. Using this CZE method, some eight peaks were consistently resolved in AF samples and several other more transient peaks have been separated from AF in less than 10 min. A scheme for the identification of peaks once they had been separated was also developed. Four peaks have been identified as proteins, i.e., gamma-globulin, alpha1-antitrypsin, transferrin and albumin. Surprisingly, one major peak was shown to be the purine catabolite, xanthine.

Regulation of endometrial cancer cell growth by luteinizing hormone (LH) and follicle stimulating hormone (FSH).

Gonadotrophin releasing hormone analogues (GnRHa) have been used to treat recurrent endometrial cancer. However, the mode of action is uncertain. Our previous studies showed no direct effect of GnRHa on endometrial cancer cell growth in vitro. We have now examined the effect of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on endometrial cancer cell growth. The aim was to determine whether suppression of pituitary LH and FSH by GnRHa could explain the tumour regression seen in up to 44% of patients treated with this drug. We show that recombinant human LH and FSH (rhLH and rhFSH) produce a concentration dependent stimulation of the endometrial cancer cell line HEC-1A, in serum-free medium (maximum increase of 62 and 50% respectively relative to untreated controls). This increase is equivalent to that obtained by addition of 10% newborn calf serum. Growth of the Ishikawa cell line in culture increases in the presence of rhLH (maximum increase of 67%) but not with rhFSH. Using RT-PCR, we show that the Ishikawa cell line intermittently expresses receptor mRNA of LH but not of FSH; there is no expression of either mRNA by HEC-1A. Classically, both LH and FSH act via cAMP linked membrane receptors. However, neither rhLH nor rhFSH elicit cAMP production in either of our endometrial cancer cell lines. Thus, although a growth response to LH and FSH can be shown, and some cells express the LH receptor, stimulation appears to be via a pathway separate from that of the classical gonadotrophin receptor.

Determination of the glycoforms of human chorionic gonadotropin beta-core fragment by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Metabolism of human chorionic gonadotropin (hCG) in the serum and kidney yields the terminal urinary product hCG beta-core fragment (hCGbetacf), comprising two disulfide-linked peptides (beta6-beta40 and beta55-beta92) of which one (beta6-beta40) retains truncated N-linked sugars. Hyperglycosylated hCGbetacf may indicate choriocarcinoma or Down syndrome, but the glycosylation profile of hCGbetacf has not been thoroughly evaluated. METHODS: hCGbetacf, purified from pregnancy urine, was reduced by "on-target" dithiothreitol (DTT) reduction and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass ([M+H](+)) of the primary sequence of the glycosylated peptide beta6-beta40 was subtracted from the m/z values of the discrete peaks observed to give the masses of the carbohydrate moieties. Carbohydrate structure was predicted by sequentially subtracting the masses of the monosaccharide residues corresponding to N-linked carbohydrates of the hCG beta-subunit reported in the literature. RESULTS: Mass spectra of hCGbetacf revealed a broad triple peak at m/z 8700-11300. After reduction, the triple peak was replaced by a discrete set of peaks between m/z 4156 and 6354. A peak at m/z 4156.8 corresponded to the nonglycosylated peptide (beta55-beta92). The remaining nine peaks indicated that urinary hCGbetacf comprises a set of glycoforms smaller and larger than the trimannosyl core. CONCLUSIONS: hCGbetacf comprises a wider set of glycoforms than reported previously. Peaks of highest mass indicate evidence of hyperglycosylated carbohydrate moieties. The data support previous reports that hCGbetacf oligosaccharides lack sialic acid and galactose residues. No indication was found of a beta6-beta40 peptide that was entirely devoid of carbohydrate.

The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation.

Ectopic production of free beta human chorionic gonadotrophin (hCGbeta) by bladder carcinoma is well described and occurs in approximately 35% of cases. hCGbeta secreting tumours are more aggressive, radioresistant and have a greater propensity to metastasize. We proposed that the ectopic production of hCGbeta was contributing in an autocrine fashion to the radioresistance and metastatic potential of such secreting tumours. Though we demonstrated that the addition of hCGbeta to the culture media of bladder, cervical and endometrial carcinoma cell lines brought about an increase in cell populations this was not accompanied by a significant increase in the rate of replication. Since a cell population size is a balance of mitosis and mortality, we proposed that hCGbeta was inhibiting apoptosis. Here we have demonstrated that following incubation with recombinant hCGbeta, bladder carcinoma cells refrain from undergoing apoptosis. Quantitation of apoptotic bodies was carried out by immunoassay and corrected to cell number as determined by MTT assay. In each cell line, addition of hCGbeta reduced the number of apoptotic bodies dose-dependently, indicating a diminished apoptotic rate. Furthermore, TGFbeta1-induced apoptosis could be dose-dependently inhibited by co-incubation with hCGbeta. We propose, therefore, that such a decline in apoptosis may account for the cell population increase previously reported. It may also explain the radioresistance and aggressive nature of hCGbeta-secreting tumours and the poor prognosis associated therein.

The beta-subunit of human chorionic gonadotrophin exists as a homodimer.

The free beta-subunit of human chorionic gonadotrophin (hCGbeta) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCGbeta may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo- and heterodimers, it is possible that the same may be true for hCGbeta. Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCGbeta contain hCGbetabeta homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCGbeta species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a |mf23 kDa monomer and a |mf46 kDa dimer were identified. Formation of hCGbetabeta homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCGbeta preparations in tumours.

Cross-reaction with luteinizing hormone beta-core is responsible for the age-dependent increase of immunoreactive beta-core fragment of human chorionic gonadotropin in women with nonmalignant conditions.

BACKGROUND: The beta-core fragment of human chorionic gonadotropin (hCGbetacf), also termed "beta-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 beta-LH-core. METHODS: We measured immunoreactive LH, hCG, free alpha-subunit, and free beta-subunit hCG (hCGbeta), as well as beta-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 beta-LH-core was determined. RESULTS: Cross-reaction with beta-LH-core was 100% in the LH and the S504 beta-core assay, 5% in the Triton UGP assay, and <0.1% in the hCG, free alpha-subunit, and free hCGbeta assays. Median urine concentrations of all analytes showed an age-dependent increase. LH and free alpha-subunit concentrations were approximately 10(3) pmol/mol creatinine; hCG and S504 beta-core were approximately 10(2) pmol/mol creatinine; free hCGbeta and Triton UGP beta-core were in the tens of pmol/mol creatinine. The S504 beta-core concentrations were 10% of those of LH. S504 beta-core was strongly correlated with LH, but not with hCG or with free hCGbeta (LH, r2 = 0.45; hCG, r2 = 0.26; free hCGbeta, r2 = 0.03). The concentrations of beta-core detected by the Triton UGP assay, which has a 5% cross-reaction with beta-LH-core, were 2% of LH and 5% of the S504 beta-core concentrations. Triton UGP values correlated strongly with LH concentrations, but less well with S504 beta-core, intact hCG, and free hCGbeta (LH, r2 = 0.44; S504 beta-core, r2 = 0.33; hCG, r2 = 0.32; free hCGbeta, r2 = 0.19). CONCLUSIONS: Immunoreactive beta-core in women free of malignancies reflects cross-reaction with concentrations of the metabolite of LH, beta-LH-core, within the health-related reference interval.

Dissociation of human chorionic gonadotropin into its free subunits is dependent on naturally occurring molecular structural variation, sample matrix and storage conditions.

Measurement of human chorionic gonadotrophin (hCG) is used in many areas of clinical medicine. Recent interest has focused on the stability of this molecule and its fragments during sample storage. In the body hCG is degraded and removed by specific metabolic processes which begin while the molecule is in the bloodstream. In particular, the receptor binding loop of the beta subunit is cut or 'nicked', initiating a catabolic cascade. Furthermore, the extent and nature of glycosylation is believed to have a significant influence on this process. In these studies we incubated seven glycoforms of hCG, each with different degrees of 'nicking', in phosphate-buffered saline, serum, defibrinated blood and urine from healthy non-pregnant women, under varying conditions. Degradation was expressed as the molar increase in free beta subunit. Under all conditions there was a steady dissociation of hCG over time, the process being more rapid at higher temperatures. 'Nicked' hCG dissociated more rapidly than did non-'nicked' hCG. Glycosylation reduced the rate of dissociation. Dissociation was most rapid in urine and buffer solutions, and slowest in serum and defibrinated blood.

Measures of urine concentration in maternal urine screening for Down syndrome.

A study was carried out to assess eight methods of normalizing the level of urinary beta-core human chorionic gonadotropin (hCG) for variable urine concentration. We compared the standard approach--creatinine determination by the Jaffe method--with high performance liquid chromatography (HPLC) measurement of creatinine, osmolarity and optical density at five wavelengths. Urine samples were included from a total of 472 women with unaffected singleton pregnancies at 15 weeks' gestation. The median beta-core hCG value was determined for each decile group when the results were ranked in turn according to the different measures of urine concentration. Creatinine using the Jaffe method had a much stronger relationship with median beta-core hCG than the other measures. Linear regression across the decile groups gave an R2 value for Jaffe of 0.85 compared with HPLC of 0.53, osmolarity of 0.52, optical density at 405 nm of 0.72, at 450 nm of 0.57, at 490 nm of 0.33, at 570 nm of 0.34 and at 630 nm of 0.33. We conclude that when screening with urinary beta-core hCG measuring creatinine appears to be an adequate method of allowing for variable urine concentration.