Investigating the detection of the novel doping­relevant peptide kisspeptin­10 in urine using liquid chromatography high­resolution mass spectrometry.

Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle-stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography-mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a black-market vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.

Kisspeptin: a new marker for human pre-ovulation.

Kisspeptin is a polypeptide that plays an important role in reproductive endocrine regulation. The aim of present study was to investigate the dynamic trend of kisspeptin levels during the menstrual cycle and to elucidate the relationship between kisspeptin ovulation. First, 15 female volunteers with regular menstrual cycles were recruited to detect the change in serum and urine kisspeptin levels over one menstrual cycle within each individual. Subsequently, 114 serum samples and 79 urine samples from 114 individuals were randomly collected at the outpatient department to better ascertain the results. Kisspeptin levels showed a distinctive stage-specific pattern during the normal menstrual cycle in both serum and urine. It was low during the first 5 days, while the first surge appeared on the 11th day (the diameter of the dominant follicle was approximately 1.2 cm). Later, a second smaller surge appeared around the 14th day and the same changes were identified in serum and urine. Furthermore, serum kisspeptin levels were positively related to 17-ß estradiol (E2) level increase. Thus, kisspeptin surge in serum and urine may be used as a marker for dominant follicle development and pre-ovulation. Moreover, kisspeptin may also play a vital role in female reproduction through regulating hormonal state.

The identification of elevated urinary kisspeptin-immunoreactivity during pregnancy.

BACKGROUND: Kisspeptin is an arginine-phenylalanine amide peptide hormone critical for reproductive function. Kisspeptin is also abundantly expressed in the placenta, where it has an important physiological role in regulating placental invasion. Accordingly, plasma kisspeptin concentrations rise dramatically during normal pregnancy. However, lower plasma concentrations of kisspeptin are associated with obstetric complications such as pre-eclampsia. It is not currently known whether kisspeptin-immunoreactivity (IR) can be detected in bodily fluids not requiring invasive collection such as saliva or urine. AIM: To determine the clinical utility of urinary and salivary kisspeptin measurement in healthy pregnant women. METHODS: Forty-nine healthy third trimester pregnant women (gestational age 34 ± 0.6 w) from a single maternity unit and 50 healthy non-pregnant women were recruited. Urine, saliva and blood were simultaneously collected from all volunteers. Kisspeptin concentrations were determined by in-house manual radioimmunoassay. RESULTS: Mean concentrations of plasma kisspeptin-IR were over 200-fold greater in third trimester pregnant women compared with non-pregnant women (13,783 ± 864 pmol/L, pregnant; 65 ± 13 pmol/L, non-pregnant; p < 0.0001). The urine kisspeptin:creatinine ratio was greater in pregnant women when compared with non-pregnant women (urine kisspeptin:creatinine: 37 ± 6 pmol/µmol, pregnant; 7 ± 1 pmol/µmol, non-pregnant; p < 0.0001). Mean concentrations of salivary kisspeptin-IR were not statistically different between pregnant and non-pregnant women (123 ± 34 pmol/L, pregnant; 83 ± 33 pmol/L, non-pregnant; p = 0.2). CONCLUSION: We demonstrate for the first time that kisspeptin-IR is elevated in urine during pregnancy. Urinary measurement of kisspeptin-IR may, therefore, offer a non-invasive and simple method of screening for pregnancy and obstetric complications.