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T
he prevalence of type 2 diabetes has importance of identifying individuals at many studies have assessed whether levels
reached epidemic levels, affecting risk to begin interventions as early as pos- of a few molecules might predict future di-
7% of the U.S. population, and sible and focus resources on those with abetes (1315), none have quantitatively
current epidemiological trends indicate the highest risk. measured a large number of molecules si-
that the prevalence will continue to in- The most commonly used method of multaneously in a sufficient number of
crease dramatically (1). Several long-term assessing risk of type 2 diabetes is mea- samples to robustly evaluate their utility
prospective clinical trials have shown that suring fasting plasma glucose (FPG); for risk assessment. We undertook a sys-
interventions can delay or possibly pre- however, the specificity of this test is poor tematic analysis of many candidates in
vent the onset of type 2 diabetes in high- (4,5). Although many individuals are pathways dysregulated in diabetes to
risk individuals (2,3), underscoring the identified as having impaired fasting glu- search for patterns of biomarkers with
more predictive power than individual bi-
From 1Tethys Bioscience, Emeryville, California; the 2Research Centre for Prevention and Health, Glostrup omarkers or previously examined bi-
Hospital, Copenhagen, Denmark; the 3Faculty of Health Science, University of Copenhagen, Copenha- omarker combinations. For this analysis,
gen, Denmark; the 4Steno Diabetes Center, Copenhagen, Denmark; and the 5Faculty of Health Science, we selected 632 baseline samples from
University of Aarhus, Aarhus, Denmark. the Inter99 Study and an ultrasensitive
Corresponding author: Janice A. Kolberg, jkolberg@tethysbio.com.
Received 25 October 2008 and accepted 21 February 2009.
immunoassay to measure many proteins
DOI: 10.2337/dc08-1935 in small amounts of serum.
2009 by the American Diabetes Association. Readers may use this article as long as the work is properly
cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.
org/licenses/by-nc-nd/3.0/ for details.
RESEARCH DESIGN AND
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby METHODS The Inter99 cohort
marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. consists of 61,301 subjects aged 30 60
years from the Danish Civil Registration Table 1Baseline characteristics of the analyzed subset of the Inter99 cohort
System. Although this was a lifestyle in-
tervention trial for cardiovascular disease Converters Nonconverters P
(14), the 5-year rate of progression to type
2 diabetes observed in this study (3.4%) Participants 160 472
was similar to other estimates of progres- Male sex 110 (68.8) 279 (59.1) 0.031
sion for this age-group (16). A sample of NFG and NGT 12 (7.6) 226 (49.7) 0.0001
13,016 was randomly selected; of these, IFG only 46 (29.1) 174 (38.2) 0.0433
12,934 were eligible and invited for an IGT only 25 (15.8) 19 (4.2) 0.0001
examination, and 6,784 (52.5%) at- Both IFG and IGT 75 (47.5) 36 (7.9) 0.0001
tended (17). Eligible individuals (n Family history 48 (30.0) 98 (20.8) 0.0223
6,536) were reinvited after 5 years and Age (years) 50.2 (45.255.0) 49.8 (44.854.8) 0.0001
4,511 (69%) attended. Fasting blood Height (cm) 172 (166179) 172 (166179) 0.9277
samples, lifestyle data, blood pressure, Weight (kg) 89 (80100) 84 (7793) 0.0001
waist circumference, plasma lipids, and BMI (kg/m2) 29.7 (27.532.9) 27.6 (26.130.1) 0.0001
OGTT results were collected at baseline Waist circumference (cm) 97 (91109) 93 (8699) 0.0001
and at 5-year time points. We defined an Hip circumference (cm) 106 (102113) 104 (100109) 0.004
at-risk subpopulation as those aged Systolic blood pressure (mmHg) 140 (130150) 130 (120144) 0.0001
39 years with BMI 25 kg/m2 and free Diastolic blood pressure (mmHg) 90 (8096) 85 (8090) 0.0008
of diabetes at baseline. Among these indi- Fasting serum total cholesterol (mmol/l) 5.8 (5.16.5) 5.7 (5.06.4) 0.2513
viduals, 174 progressed to type 2 diabetes Fasting serum HDL cholesterol (mmol/l) 1.2 (1.01.4) 1.3 (1.11.6) 0.0013
during the 5-year follow-up (converters), Fasting serum LDL cholesterol (mmol/l) 3.6 (3.14.4) 3.6 (3.14.3) 0.6898
and baseline samples were available for Fasting serum triglycerides (mmol/l) 1.6 (1.32.2) 1.3 (0.91.8) 0.0001
160, whereas 2,872 did not progress Fasting serum insulin (pmol/l) 58 (3781) 40 (2759) 0.0001
(nonconverters). Diagnosis of type 2 dia- 2-h serum insulin (pmol/l) 325 (210486) 186 (100298) 0.0001
betes was defined by a 2-h plasma glucose FPG (mmol/l) 6.1 (5.76.5) 5.6 (5.36.0) 0.0001
of 11.1 mmol/l in an OGTT or FPG of 2-h plasma glucose (mmol/l) 8.4 (7.19.5) 6.1 (5.17.0) 0.0001
7.0 mmol/l. Nonconverters (n 472) A1C (%) 6.1 (5.86.4) 5.9 (5.66.1) 0.0001
were randomly selected in an 3:1 ratio Adiponectin (g/ml) 19.5 (9.339.6) 22.2 (12.942.6) 0.0345
to converters. CRP (g/ml) 3.2 (1.57.9) 2.0 (0.85.3) 0.0001
Ferritin (ng/ml) 867 (2901749) 483 (1681045) 0.0001
Clinical and standard laboratory IL-2RA (pg/ml) 290 (230400) 270 (200350) 0.0049
measurements Data are n (%) or median (interquartile range) for continuous variables. Data are from 632 subjects in the
Blood pressure was obtained and anthro- subsample of 3,032 at-risk individuals with BMI 25 kg/m2 and age 39 years from the Inter99 cohort.
Converters are individuals who developed epidemiologically defined diabetes within 5 years. Nonconverters
pometric measurements, routine labora- were randomly selected from the Inter99 cohort in an approximately 3:1 ratio to converters. IFG was defined
tory measures (FPG, insulin, and lipids), as FPG of 5.6 6.9 mmol/l. Impaired glucose tolerance (IGT) was defined as 2-h postload glucose of
and the OGTT were performed as de- 7.8 11.1 mmol/l. At baseline, 92% of the converters had IFG, IGT, or both, whereas 50% of nonconverters
scribed previously (17). Serum was had IFG, IGT, or both. For categorical descriptors, values are counts (percentage of total for that cohort).
stored at 19C. Differences in frequency between converters and nonconverters were evaluated with a Monte Carlo estima-
tion of the 2 statistic (2,000 replicates). Differences in medians were evaluated with a Wilcoxon test. NFG,
normal fasting glucose; NGT, normal glucose tolerance.
Candidate biomarker selection
Potential biomarkers were identified by
searching the PubMed database using sensitive molecular counting technology (Swampscott, MA) (C-reactive protein
search terms relevant to the development platform (Singulex, St. Louis, MO). De- [CRP] and ferritin heavy chain 1
of diabetes. Of 260 candidate biomarkers tails regarding assay reagents have been [FTH1]). Detection antibodies for
identified as being involved in pathways described previously (18). In brief, la- ADIPOQ, CRP, and FTH1 were conju-
associated with metabolic or cardiovascu- beled antibodies were detected with the gated with Alexa Fluor 647 (Invitrogen,
lar disorders, obesity, cell death, or in- ZeptX system, in which liquid from each Carlsbad, CA) according to the manu-
flammatory response, we successfully well is pumped through an interrogation facturers instructions and purified by
obtained assay reagents for 89. Data from space within a capillary flow cell. Laser ultrafiltration with Microcon YM-30
58 candidate biomarkers met our quality light (wavelength 650 nm) is directed from Millipore (Billerica, MA). Analytes
control criteria, which required that re- into the interrogation space, and the re- detected using DuoSet kits used biotin-
sults from 66% of the samples had to sulting emission from each labeled anti- ylated detection antibodies and Alexa
fall within the assays linear dynamic body (wavelength 668 nm) is measured Fluor 647 conjugated streptavidin
range. via a confocal microscope with a photon (Invitrogen).
detector. One biomarker was measured per
Molecular assays For biomarkers in the model, re- 384 microwell plates, using an average of
Sandwich immunoassays developed for agents were obtained from R&D Sys- 1.3 l serum in a total assay volume of 10
the 58 proteins typically used a monoclo- tems (Minneapolis, MN) individually l/well. Biomarker concentrations were
nal capture antibody and a fluorescently (monomeric adiponectin [ADIPOQ]) or calculated as the mean of three replicates.
labeled detection antibody. Biomarker as DuoSet kits (interleukin-2 receptor A Assays had dynamic ranges of 102103,
candidates were measured using an ultra- [IL-2RA]) and from U.S. Biological intraplate coefficients of variation of
Jrgensen (PI), K. Borch-Johnsen (co-PI), 743 tion study for prevention of ischaemic
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