Determination of the glycoforms of human chorionic gonadotropin b-core fragment by matrix-assisted laser desoption/ionisation time of flight mass spectrometry

Background: Metabolism of human chorionic gonadotropin (hCG) in the serum and kidney yields the terminal urinary product hCG ß-core fragment (hCGßcf), comprising two disulfide-linked peptides (ß6-ß40 and ß55-ß92) of which one (ß6-ß40) retains truncated N-linked sugars. Hyperglycosylated hCGßcf may indicate choriocarcinoma or Down syndrome, but the glycosylation profile of hCGßcf has not been thoroughly evaluated. Methods: hCGßcf, 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 ß6-ß40 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 ß-subunit reported in the literature. Results: Mass spectra of hCGßcf 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 (ß55-ß92). The remaining nine peaks indicated that urinary hCGßcf comprises a set of glycoforms smaller and larger than the trimannosyl core. Conclusions: hCGßcf 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 hCGßcf oligosaccharides lack sialic acid and galactose residues. No indication was found of a ß6-ß40 peptide that was entirely devoid of carbohydrate

Identification of placental transforming growth factor-beta and bikunin metabolites as contaminants of pharmaceutical human chorionic gonadotrophin preparations by proteomic techniques

This paper is the first to identify the novel bio-molecules found in pregnancy urine and commonly contaminate clinical grade hCG preparation extracted from urine reported to have anti-HIV/HIV Kaposis . Professor Iles was the Principal Investigator

Determination of the glycoforms of human chorionic gonadotropin b-core fragment by matrix-assisted laser desoption/ionisation time of flight mass spectrometry

Background: Metabolism of human chorionic gonadotropin (hCG) in the serum and kidney yields the terminal urinary product hCG ß-core fragment (hCGßcf), comprising two disulfide-linked peptides (ß6-ß40 and ß55-ß92) of which one (ß6-ß40) retains truncated N-linked sugars. Hyperglycosylated hCGßcf may indicate choriocarcinoma or Down syndrome, but the glycosylation profile of hCGßcf has not been thoroughly evaluated. Methods: hCGßcf, 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 ß6-ß40 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 ß-subunit reported in the literature. Results: Mass spectra of hCGßcf 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 (ß55-ß92). The remaining nine peaks indicated that urinary hCGßcf comprises a set of glycoforms smaller and larger than the trimannosyl core. Conclusions: hCGßcf 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 hCGßcf oligosaccharides lack sialic acid and galactose residues. No indication was found of a ß6-ß40 peptide that was entirely devoid of carbohydrate

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

Classical conditioning without verbal suggestions elicits placebo analgesia and nocebo hyperalgesia

The aim of this study was to examine the relationships among classical conditioning, expectancy, and fear in placebo analgesia and nocebo hyperalgesia. A total of 42 healthy volunteers were randomly assigned to three groups: placebo, nocebo, and control. They received 96 electrical stimuli, preceded by either orange or blue lights. A hidden conditioning procedure, in which participants were not informed about the meaning of coloured lights, was performed in the placebo and nocebo groups. Light of one colour was paired with pain stimuli of moderate intensity (control stimuli), and light of the other colour was paired with either nonpainful stimuli (in the placebo group) or painful stimuli of high intensity (in the nocebo group). In the control group, both colour lights were followed by control stimuli of moderate intensity without any conditioning procedure. Participants rated pain intensity, expectancy of pain intensity, and fear. In the testing phase, when both of the coloured lights were followed by identical moderate pain stimuli, we found a significant analgesic effect in the placebo group, and a significant hyperalgesic effect in the nocebo group. Neither expectancy nor fear ratings predicted placebo analgesia or nocebo hyperalgesia. It appears that a hidden conditioning procedure, without any explicit verbal suggestions, elicits placebo and nocebo effects, however we found no evidence that these effects are predicted by either expectancy or fear. These results suggest that classical conditioning may be a distinct mechanism for placebo and nocebo effects

Pharmacokinetics of Mephedrone Enantiomers in Whole Blood after a Controlled Intranasal Administration to Healthy Human Volunteers

Mephedrone, which is one of the most popular synthetic cathinones, has one chiral centre and thus exists as two enantiomers: R-(+)-mephedrone and S-(−)-mephedrone. There are some preliminary data suggesting that the enantiomers of mephedrone may display enantioselective pharmacokinetics and exhibit different neurological effects. In this study, enantiomers of mephedrone were resolved via chromatographic chiral recognition and the absolute configuration was unambiguously determined by a combination of elution order and chiroptical analysis (i.e., circular dichroism). A chiral liquid chromatography tandem mass spectrometry method was fully validated and was applied to the analysis of whole blood samples collected from a controlled intranasal administration of racemic mephedrone hydrochloride to healthy male volunteers. Both enantiomers showed similar kinetics, however, R-(+)-mephedrone had a greater mean Cmax of 48.5 ± 11.9 ng/mL and a longer mean half-life of 1.92 ± 0.27 h compared with 44.6 ± 11.8 ng/mL and 1.63 ± 0.23 h for S-(−)-mephedrone, respectively. Moreover, R-(+)-mephedrone had a lower mean clearance and roughly 1.3 times greater mean area under the curve than S-(−)-mephedrone. Significant changes in the enantiomeric ratio over time were observed, which suggest that the analytes exhibit enantioselective pharmacokinetics. Even though the clinical significance of this finding is not yet fully understood, the study confirms that the chiral nature, and consequently the enantiomeric purity of mephedrone, can be a crucial consideration when interpreting toxicological results