Study populations
A prospective observational study of urine collected from a population of healthy adult volunteers who agreed to participate in validation of urine analyses was conducted. This first study population included 26 individuals who provided data on hormonal measures (cortisol and cortisol metabolites, reproductive hormones, and 6-hydroxymelatoninsulfate) to compare samples from the 4-spot urine collection method to a 24-hour urine collection. A subset of these individuals (n = 18) had data available to compare measures from dried versus liquid urine. As cortisol and cortisol metabolites15 and a and b-pregnanediol12 were validated in previous analyses, only the following hormones were included in this analysis: estrone (E1), estradiol (E2), estriol (E3), 2-hydroxyestrone (2OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestrone (4OHE1), 16-hydroxyestrone (16OHE1), 2-methoxyestrone (2-methoxyE1), testosterone (T), epitestosterone (EpiT), 5a- dihydrotestosterone (DHT), androsterone, etiocholanolone, 5a-androstanediol, 5b-androstanediol, dehydroepiandrosterone (DHEA), and 6-hydroxymelatoninsulfate. The data were collected between February and November of 2015 and informed consent was obtained from all participants.
The second analysis included 20 individuals whose deidentified data was pulled from the larger databank of 144,561 laboratory visits. Each of these samples included a single first-morning urine collection to compare results of dried versus liquid urine for the following organic acids: homovanillic acid (HVA), vanillylmandelic acid (VMA), kynurenic acid, xanthurenic acid, methymalonic acid (MMA), pyroglutamic acid, 5-hydroxyindoleacetic acid (5-HIAA), and b-hydroxyisovaleric (Hiv) acid. All data reports were deidentified during extraction so the IRB determined that written informed consent could be waived for this study population. Both studies (Clinical Trials ID: NCT04305093) were approved by the National University of Natural Medicine Institutional Review Board.
All volunteers in both study populations reported no medical problems and were not pregnant. Individuals were not excluded based on current or recent use of any hormonal medications, as the goal was only to compare measurement values for differing methodologies. Eighty percent of women in the first study population and all women in the second study population were premenopausal.
Sample Collection
The 4-spot method involves urine samples collected at home at four times during the day: 1) the first urine of the day, 2) two hours after awakening, 3) in the afternoon (approximately 4 PM), and 4) before bed (10PM). Participants collected samples by completely saturating 2 × 3 inches of filter paper (Whatman Body Fluid Collection Paper) with urine. The paper was left exposed at room temperature for 24 hours to dry. The stability of analytes in dried urine at room temperature for as long as 84 days has previously been demonstrated by this laboratory12. Dried samples were stored at -80°C until analyzed. Reproductive hormones were assessed in all four samples collected, while only the first morning sample was used for 6-hydroxymelatoninsulfate and the organic acid tests.
During the same day, all liquid urine samples for the 24-hour collection were added to a low-density polyethylene plastic container (ES Robbins, USA) container with approximately 1 g of boric acid and kept refrigerated for the duration of the collection. The four dried urine samples removed a total of about 8 mL of urine from the 24-hour collection. This was considered negligible and was not accounted for. The total volume of urine from 24-hour collections was measured, and an aliquot was frozen and stored at -80°C until tested.
Urine Reproductive Hormone Analysis
The urinary steroid hormones were analyzed using proprietary in-house CLIA (Clinical Laboratory Improvement Amendments) approved assays on the Agilent 7890/7000B GC-MS/MS (Agilent Technologies, Santa Clara, CA, USA). A 600 ul aliquot of liquid urine was taken from the sample collection and the equivalent of approximately 600 ul of urine was extracted from the filter paper using 2 mL of 100 mM ammonium acetate adjusted to a pH of 5.9. These aliquots of the conjugated hormones were transferred to a C18 solid phase extraction (SPE) column (UCT LLC, Briston, PA, USA), eluted using methanol, and the eluate was dried under nitrogen at 40 °C.
The conjugated hormones were then hydrolyzed from their glucuronide and sulfate forms to free forms using enzymes from Helix pomatia (Sigma-Aldrich, St. Louis, MO, USA) in acetate buffer (55 °C, 90 minutes). The enzymatic reaction was quenched with sodium hydroxide and the hormones extracted with ethyl acetate. The ethyl acetate extracts were dried under nitrogen at 40 °C. The analytes were derivatized using a mixture of 100 ul acetonitrile (ACN) and 50 ul bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 70 °C. Internal standards (Steraloids, Newport, RI, USA) were added prior to ethyl acetate extraction, and the percentage recovery from all assays was greater than 90%. Derivatized extract (1.6 ul) was injected into the GC-MS/MS. Samples and controls were analyzed along with a standard curve spanning the expected range of concentrations. Instrument conditions for the oven were an initial temperature of 130 °C increasing to 200 °C at 25 °C /min, then to 230 °C at 4.3 °C/min, and finally to 290 °C at 25 °C /min. Multiple reaction monitoring transitions for ion mass > ion product of fragmentation were 342.1 > 257.1 for E1, 416.2 > 285.1 for E2, 504.3 > 296.3 for E3, 430.2 > 345.0 for 2OHE1, 462.2.2 > 195.2 for 2OHE2, 430.2 > 354.0 for 4OHE1, 430.2 > 286.1 for 16OHE1, 372.2-342.1 for 2-methoxyE1, 360.2 > 174.1 for T, 360.2 > 174.1 for EpiT, 347.2 > 271.2 for DHT, 347.2 > 271.2 for androsterone, 347.2 > 253 for etiocholanolone, 421.2 > 331.2 for 5α-androstanediol, 241.2 > 185.2 for 5β-androstanediol, and 303.7 > 199.1 for DHEA.
Creatinine was measured using a conventional colorimetric (Jaffe) method, after initial extraction from the filter paper. The average inter-assay coefficient of variation was 6.7% for creatinine. In addition to expressing the measures per mg of creatinine to correct for variations in filter paper saturation and hydration status, a secondary equation was applied to reduce bias related to the effects of age, sex, weight, and height on creatinine excretion55.
Urine 6-hydroxymelatoninsulfate And Organic Acid Analysis
The hydrophilic analytes were assessed by LC-MS/MS using proprietary in-house CLIA approved assays. For the 6-hydroxymelatoninsulfate assay, a 30 ul aliquot was taken from the methanol elution of both the liquid urine collection and the waking sample dried urine collected on filter paper. This extract was then reconstituted in 130 ul of deionized water. For the organic acids, a 100 ul aliquot of liquid urine was taken and an equivalent amount was extracted from the waking sample dried urine filter paper using 250 ul of water with the addition of 50 ul 100 mM ammonium acetate (Sigma-Aldrich, St. Louis, MO, USA) adjusted to a pH of 5.9 and 2% formic acid.
For 6-hydroxymelatoninsulfate, 20 ul was injected into an ultra-performance liquid chromatography (UPLC) (Waters Corporation, Milford, MA, USA) column with a Waters™ tandem quadrupole mass spectrometer detector (TQD). The sample was eluted from a 1.8u 2.1 × 50 mm pentafluorophenyl (PFP) column (Agilent Technologies, Santa Clara, CA, USA) using a gradient of 95% 0.001% formic acid in 5% ACN to 45% 0.001% formic acid in 55% ACN. For the organic acids, 5 ul was injected into the Waters™ UPLC column with TQD. These analytes were eluted from a 1.6 um 2.1 × 50 mm Luna Omega PS C18 column (Phenomenex, Torrance, CA, USA) using a gradient 99.9% 0.2% formic acid in 0.1% ACN to 73% 0.2% formic acid in 27% ACN. Multiple reaction monitoring transitions for ion mass > ion product of fragmentation were 372.5 > 176.2 for 6-hydroxymelatoninsulfate, 331.5 > 179.2 for d4-6-hydroxymelatoninsulfate, 183.0 > 137.0 for HVA, 185.0 > 139.0 for d2-HVA, 197.0 > 138.0 for VMA, 199.8 > 140.9 for d3-VMA, 190.0 > 144.0 for kynurenic acid, 192.0 > 149.0 for d5-kynurenic acid, 206.0 > 160.0 for xanthurenic acid, 210.0 > 164.0 for d3-xanthurenic acid, 117.0 > 73.0 for MMA, 120.0 > 76.0 for d2-MMA, 130.0 > 84.0 for pyroglutamic acid, 135.0 > 89.0 for d5-pyroglutamic acid, and 192.0 > 146.0 for 5-HIAA. The same creatinine corrections of the measures used for the reproductive hormones were also used for 6-hydroxymelatoninsulfate and the organic acids.
Statistical Methods
A sample size of 18 individuals provides a power of greater than 80% to detect an intraclass correlation coefficient (ICC) of at least 0.6 with an alpha of 0.0556. The statistical analyses were performed using SAS/STAT® software, Version 9.3 (SAS Institute Inc., Cary, NC, USA) and generated 2-sided p-values.
Variables are described as means ± standard deviation if normally distributed and median (interquartile range (IQR)) if the distribution was skewed. Student’s t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for skewed variables) were used to determine differences between men and women. Spearman correlation coefficients (ρ) were used to determine interclass associations between variables.
Consistency between liquid versus dried urine measures and 4-spot versus 24-h collection methodology was assessed using intraclass correlation coefficients (ICC). ICCs, which range from 0 to 1 with proximity to 1 indicating better agreement, assess for agreement of a measure between two differing methodologies within individuals57. Skewed variables were log transformed to approximate a normal distribution prior to assessing ICCs. As 4-spot (ng/mg-Cr) and 24-h (ug/d) measures were expressed in differing units, sex-specific Z-scores ([individual measurement-mean]/standard deviation) were created to standardize the measures and allow for direct comparison. Comparisons of differences between measures within an individual were assessed using signed-rank tests (for skewed variables) or paired t-tests (for normally distributed variables). Because the hypotheses of this paper were intrinsically correlated, no adjustments were made for multiple comparisons.