rindle
University of Washington

Darryl J. Holman
University of Washington

Jane Shofer
University of Washington

Nancy A. Klein
University of Washington

Michael R. Soules
University of Washington

Kathleen A. OConnor
University of Washington



UNIVERSITY OF WASHINGTON


CSDE Working Paper No. 04-02
Title:
Comparison of Specific Gravity and Creatinine Methods for Normalizing Urinary Reproductive
Hormone Concentrations.

Running Title: Methods for Normalizing Hormone Concentrations

Authors and Institutions:
Rebecca C. Miller
1

Eleanor Brindle
1
Darryl J. Holman
1

Jane Shofer
1

Nancy A. Klein
2

Michael R. Soules
2

Kathleen A. OConnor
1




1
Department of Anthropology and Center for Studies in Demography and Ecology,
University of Washington, Seattle WA 98195
2
Department of Obstetrics and Gynecology, University of Washington, Seattle WA 98195

Total Number of Pages:24
Number of Figures:4
Number of Tables:3

Keywords: Bangladesh, E1C, PDG, population variation

Corresponding Author:
Kathleen A. OConnor
Department of Anthropology
Box 353100
University of Washington
Seattle, Washington 98195
phone: (206) 616-9369
fax: (206) 543-3285
email:
oconnork@u.washington.edu

Date: 2/20/2004
Page 2 KA OConnor
ABSTRACT
Background: Specific gravity (SG) may perform as well as creatinine (CR) correction for adjusting
urinary hormone concentrations, as well as offer some advantages
.
We compared the two methods and
applied them to US and Bangladeshi specimens to evaluate their use in different populations.
Methods: Pearson correlations between serum concentrations and SG, CR, and uncorrected urinary
concentrations were compared using paired daily urine and serum specimens

from one menstrual cycle
from 30 US women. Corrected urinary estrone conjugate (E1C) and pregnanediol glucuronide (PDG)
concentrations were compared with serum estradiol (E2) and progesterone (P4). Urine specimens across
one menstrual cycle from 13 Bangladeshi women were used to evaluate the applicability of both methods
to a non-industrialized population. Linear mixed effects models were used to compare CR and SG
gravity values in the Bangladeshi vs. US specimens.
Results: There was no significant difference between SG corrected vs. serum and CR corrected vs. serum
correlations for either assay. Usable CR results were obtained for all US specimens, but 37% of the
Bangladeshi specimens were below the CR assay limit of detection. The Bangladeshi sample had
significantly lower CR, and higher inter- and intra-subject CR variability, compared to the US sample.
Conclusions: SG is a useful alternative to CR correction for normalizing urinary steroid hormone
concentrations, particularly in settings where CR values are highly variable or unusually low.
3
Nonstandard Abbreviations: SG, specific gravity; CR, creatinine; B, Bangladeshi sample; E2, estradiol;
IFMA, immunoflurometric assay; LH, luteinizing hormone; P4, progesterone; EIA, enzyme
immunoassay; PDG, pregnanediol glucuronide; E1C, estrone conjugates
Page 3 KA OConnor
Introduction
Urine specimens have several advantages over serum, saliva, and blood spots for monitoring
reproductive hormone patterns. Urine collection is non-invasive, poses minimal infectious
disease risk to participants and researchers, and provides sufficient volume for multiple assays
and future research. Urine specimens are ideally suited for large studies because they can be
collected and stored by participants, and compliance is high [1-3]. Urine also provides integrated
hormone measures without the confounding effects of pulsatile secretion [4]. Finally, urinary
levels of reproductive steroid hormone metabolites are generally higher than the circulating
serum levels, providing better quantification of the lower end of the physiological scale in
humans, which can be close to or below the limit of detection in serum RIAs [5]. Urinary
hormone results must, however, be adjusted to account for the concentration of the specimen,
which depends on a subjects hydration status and time since last urination.
We examined the performance of two methods of adjustment: specific gravity (SG)
3

correction and creatinine (CR) correction. Urine SG is the ratio of the density of a urine
specimen to the density of water [6]. Specific gravity increases with solute concentration and is
most accurately measured by refractometry [6]. Creatinine, a byproduct of muscle activity, is
cleared from the blood stream by the kidneys and excreted in urine [7]. Urinary creatinine
concentrations are determined by colorimetric assay [8], and analyte concentrations are usually
reported as a ratio of the analyte concentration to creatinine concentation.
Based on an early finding that daily individual creatinine excretion was fairly consistent [9],
urinary creatinine became a common method of assessing kidney function in clinical settings and
correcting for analyte concentrations, including reproductive hormones, in urine [4]. Using
creatinine to normalize urinary analyte concentrations can be problematic, however, because
Page 4 KA OConnor
there is evidence that creatinine excretion is not consistent: numerous studies have found
considerable inter- and intra-subject variability in creatinine values and dependence on sex, age,
activity, and diet [10-18]. Population variation in creatinine excretion may also exist but to the
best of our knowledge, this topic has not been investigated.
Specific gravity is an alternative method with several advantages over creatinine although it
is not widely used, perhaps in part because of a lack of data evaluating its performance relative to
creatinine. In this study we compared SG and CR correction methods on urinary hormone
metabolite concentrations from healthy US women using serum hormone measurements as the
standard. We then applied both methods to spot specimens from Bangladeshi women to evaluate
their applicability in a nonindustrialized population.
Materials and Methods
Samples and Specimens
A total of 799 daily urine and serum specimens were collected over one menstrual cycle from
30 US women in 1997-1998. Thirteen women aged 20-25 years and 17 women 40-45 years old
were recruited for a study on reproductive aging. Monetary compensation was provided,
participants provided written informed consent, and all procedures were approved by the
Institutional Review Board of the University of Washington. All participants were normally
cycling, in good health, had a mean body mass index of 22.6 kg/m
2
(SD = 2.36, range 18.9
27.7), and were not using medications or hormones. Blood specimens were obtained by
venipuncture, beginning with the first day of menstrual bleeding and continuing until the first
day of menstrual bleeding of the subsequent cycle. Serum specimens were immediately assayed
and all cycles were confirmed ovulatory by transvaginal ultrasound. Urine specimens were taken
daily in the clinic, usually before noon, at the same time as serum collection and immediately
Page 5 KA OConnor
stored at -20° C. Urine specimens remained frozen until thawing two years later for aliquoting,
assay, and measurement of specific gravity [19]. The specimens underwent 2 to 3 more freeze
thaws cycles prior to creatinine assay in 2003.
For the Bangladeshi sample (B), 13 cycling women were selected randomly from a sample of
subjects participating in a 9-month study of early pregnancy loss. All participants were married,
noncontracepting residents in the nonintervention demographic surveillance region of the rural
Matlab district in Bangladesh [20]. No monetary compensation was provided, all participants
provided written informed consent, and all procedures were approved by the Institutional Review
Boards of The Pennsylvania State University and the International Centre for Diarrhoeal Disease
and Research, Bangladesh [20]. Spot urine specimens were collected by community health care
workers every 3 or 4 days over the course of one menstrual cycle in 1993. The specimens were
stored at the health care workers home in a cooler with ice packs for 1 to 3 days until they were
transported to a field hospital and stored at 4° C. One to three days later, the specimens were
brought to room temperature and specific gravities were taken. The specimens were preserved
with 0.17 g/mL boric acid solution, stored at -20° C, and transported to the US by frozen air
freight. Specimens remained frozen until 1995 when they were thawed and assayed for
creatinine and steroid hormone metabolites.
Assays
All serum specimens were assayed by RIA for estradiol (E2) and by immunoflurometric
assay (IFMA) for luteinizing hormone (LH), but serum progesterone (P4) was measured only in
the luteal phase. The RIA for E2 (ICN Biomedicals) cross-reacts 20% with estrone, 1.5% with
estriol, and <1% with all other steroids. The inter- and intra-assay CVs were 16% and 7%,
respectively. The inter- and intra-assay CVs for the LH IFMA (Delphia) were 2.8% and 4.7%
Page 6 KA OConnor
respectively. The RIA for P4 (Diagnostic Systems Laboratories) cross-reacts <5% with all other
steroids and the inter- and intra-assay CVs were 13% and 11% respectively.
Urine specimens were analyzed with competitive enzyme immunoassays (EIAs) for urinary
steroid hormone metabolites. All US and Bangladeshi urine specimens were assayed for
pregnanediol glucuronide (PDG) and estrone conjugates (E1C). The PDG and E1C EIAs have
been described elsewhere [19].