Application of Analyte Harvesting Nanoparticle Technology to the Measurement of Urinary HGH in Healthy Individuals

Abstract

Urine represents a valuable biofluid for noninvasive measurement of Human Growth Hormone (HGH) secretion. Unfortunately, currently available commercial HGH immunoassays do not achieve the sensitivity needed for urinary HGH measurement in the low picogram per milliliter range, the expected normal concentration range of HGH in urine.A nanotechnology based sample preprocessing step was used to extract and concentrate HGH in urine so that urinary HGH could be measured with a clinical grade standard immunoassay designed for serum (Immulite 1000, Siemens). We applied the nanoparticle enhanced immunoassay to evaluate the baseline value of urinary HGH in a population of healthy young adults (age 18-30, N=33, median 21, M: F=39%:61%, with no reported medical therapies).Nanoparticle sample preprocessing effectively improved the lower limit of detection of the Immulite HGH assay by more than 50 fold, shifting the linear range of the assay to encompass the expected value of urinary HGH. The full process between run and within run CV% was 7.9 and 9.0%, respectively. On 33 healthy volunteers, the 95% reference values for hGH in spot urine normalized to specific gravity were 0.64 -16.85 pg/mL (0.05-5.82 ng/g creatinine).Nanoparticle preprocessing constitutes a reliable means of measuring urinary HGH with a clinical grade immunoassay, now establishing a normal baseline value for HGH in urine. Nanoparticles can be used to study the kinetics of HGH excretion in urine, and the factors that influence urinary HGH secretion and HGH isoform proportions.

Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: Preliminary results

“…The pharmacokinetics following rhGH administration was in line with what is known for this hormone [12,13] under similar administration regimes, with a maximum concentration at approximately 6–10 h, post-administration, and near baseline levels after 24 h. In urine, a very similar trend was observed, albeit that the maximum concentration appeared to be shifted slightly with respect to that encountered in serum. Serum measures correspond to a defined extraction time whereas the urine sample represents the total collected with respect to the previous collection point.…”

“…Serum samples of all three volunteers were analyzed following WADA specific guidelines for hGH analysis using isoform differential immunoassays [4,5,12] and urine samples as above described for the samples spiked with rhGh and phGH.…”

Current state of the art for enhancing urine biomarker discovery

“…Hydrogel nanoparticle protein harvesting has been used to concentrate human growth hormone in serum and urine specimens, allowing the first estimate of growth hormone levels in human urine [132, 144, 145]. Growth hormone levels are important during clinical evaluation of growth hormone insufficiency.…”

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

“…In one step, in solution, the nanoparticles perform a rapid (within minutes) sequestration and concentration of low molecular weight analytes 10,11,13,[15][16][17][18] . Proteins are subsequently eluted from the nanoparticles and detected using western blotting [19][20][21] , mass spectrometry 10,11,13,15,18,22,23 , immunoassays/ELISA 10,11,15,18 , or reverse phase protein microarray 16,24 assays. Nanoparticles functionalized with chemical bait, and presenting a core or core shell architecture, capture and concentrate proteins based on the bait/shell physicochemical properties.…”

“…The incorporation of the dye bait drives the uptake of molecules in solution and assures that captured molecules are preserved from degradation 13,15,18,25 . For this reason, protease inhibitors are not needed during sample pre-processing.…”