http://www.kidney-international.
org
& 2007 International Society of Nephrology
Prevalence of abnormal serum vitamin D, PTH,
calcium, and phosphorus in patients with chronic
kidney disease: Results of the study to evaluate early
kidney disease
A Levin1, GL Bakris2, M Molitch3, M Smulders4, J Tian5, LA Williams5 and DL Andress6
1
Department of Nephrology, University of British Columbia, Vancouver, BC, Canada; 2Rush Hypertension/Clinical Research Center, Rush
University Medical Center, Chicago, Illinois, USA; 3Division of Endocrinology, Northwestern University Feinberg School of Medicine,
Chicago, Illinois, USA; 4Quintiles Strategic Research Services, Falls Church, Virginia, USA; 5Renal Global Project Team, Abbott
Laboratories, Abbott Park, Illinois, USA and 6Division of Nephrology, University of Washington School of Medicine, Seattle, Washington,
USA
Abnormalities of mineral metabolism occur early in chronic
kidney disease. Quantification of the prevalence of these
abnormalities has not been described using current assays
nor in large unselected populations. This outpatient cohort
cross-sectional study was conducted in 153 centers, (71%
primary care practices). Blood for parathyroid hormone
(PTH), vitamin D metabolites, creatinine, calcium (Ca), and
phosphorus (P) were drawn between June and October 2004.
Low 1,25-dihydroxyvitamin D (1,25 OH2 D3) was defined as
o22 pg/ml. The 1814 patients had a mean age of 71.1 years
old; 48% had diabetes mellitus (DM). Low 1,25 OH2 D3 was
evident at all estimated glomerular filtration rate (eGFR)
levels: 13% in those with eGFR 480 ml/min, 460% in those
with eGFR o30 ml/min. High PTH (465pm/dl) occurred in
12% with eGFR 480 ml/min. Serum Ca and P were normal
until eGFR was o40 ml/min. Significant differences in the
mean and median values of 1,25 OH2 D3, PTH, but not
25(OH)D3 levels, were seen across deciles of eGFR (Po0.001).
Multivariate analysis revealed that DM, increased urinary
albumin/creatinine ratio and lower eGFR predicted lower
values of 1,25 OH2 D3. A high prevalence of mineral
metabolite abnormalities occurs in a large unreferred US
cohort. Low 1,25 OH2 D3 and elevated PTH are common at
higher eGFR than previously described. As bone,
cardiovascular disease, and mineral metabolite are
correlated; further studies are necessary to determine the
importance of these findings relative to outcomes.
Kidney International (2007) 71, 31–38. doi:10.1038/sj.ki.5002009;
published online 8 November 2006
KEYWORDS: kidney function, 1,25 dihydroxy vitamin D; 25 hydroxy vitamin
D; calcium, phosphorus, observational study; prevalence
Correspondence: A Levin, Department of Nephrology, University of British
Columbia, 1081 Burrard Street, Rm 6010A, Vancouver, Canada BC V6Z1Y8.
E-mail: alevin@providencehealth.bc.ca
Received 7 April 2006; revised 14 September 2006; accepted 3 October
2006; published online 8 November 2006
Kidney International (2007) 71, 31–38
original article
It is now accepted that the presence of chronic kidney disease
(CKD) is associated with poor outcomes.1–3 In particular,
cardiovascular events and mortality increase as the estimated
glomerular filtration rate (eGFR) declines below 60 ml/min.4
In dialysis patients, cardiovascular disease is 10- to 20-fold
higher than the general population, representing at least half
of the 15–25% per year mortality rate.5 The mechanisms
associating CKD with mortality have not been determined, in
part, owing to the lack of comprehensive clinical and
biochemical analyses.
Recently, increased attention has focused on endocrine
abnormalities in patients with CKD as a way to explain some
of these associations.6,7 For example, 1,25-dihydroxyvitamin
D (1,25 OH2 D3) deficiency is known to occur during the
progression of CKD because the final hydroxylation step of
25-hydroxyvitamin D (25(OH)D3) to 1,25 OH2 D3 (calci-
triol) is mediated by kidney 1á-hydroxylase.8 Calcitriol
deficiency plays a major role in the development of secondary
hyperparathyroidism (HPTH), as 1,25 OH2 D3 deficiency
promotes parathyroid gland growth (hyperplasia) and
increased parathyroid hormone (PTH) synthesis through
loss of the ability to upregulate vitamin D receptor expression
within parathyroid cells.9 The end result is elevated serum
PTH and abnormal calcium (Ca) and phosphorus (P)
balance.
Elevated PTH and hyperphosphatemia were recently
identified again as risk factors for mortality in dialysis
patients.10 However, additional data indicate that vitamin D
treatment is an important factor that may mitigate the effects
of HPTH and hyperphosphatemia on cardiovascular mortal-
ity.7 These findings not only have practical applications to
current dialysis treatment, but they may also have important
implications in the much larger population of CKD patients
who are not receiving dialysis. Currently, there are also
concerns regarding the potential side effects of vitamin D
therapy, such as hypercalcemia and vascular calcification. In
31
original article
order to better understand the role of vitamin D in this
setting, it is important to first establish the relationship of
eGFR to disordered vitamin D and mineral metabolism in
the untreated state.
The purpose of this cross-sectional analysis of baseline
data available from an observational, prospective study was
to determine the relationships of circulating vitamin D, PTH,
Ca, and P in CKD patients who were not receiving prescribed
vitamin D, as an initial attempt to better identify the role of
vitamin D deficiency as it relates to eGFR and the
development of HPTH.
RESULTS
Population baseline characteristics
Figure 1 describes the population screening and inclusion
process from which the final cohort for analysis was derived.
From June 2004 to October 2004, 5255 patients were
screened, and 1903 patients met the entry criteria. Screening
failures resulted primarily from the fact that the eGFR values
calculated using the most recent serum creatinine in the chart
were X60 ml/min/1.73 m2. Of the 1903 patients who were
enrolled in the Study for the Evaluation of Early Kidney
disease (SEEK) study, 89 patients were not included in the
analysis because of an inability to calculate an estimated
eGFR owing to missing laboratory values for serum
creatinine, and thus providing 1814 CKD patients (866 or
48% men and 948 women) with data available for analysis.
The general characteristics of the patient population are
shown in Table 1. The demographics are displayed by total
population, and by level of eGFR, as calculated from the
serum creatinine values taken at the baseline visit. Note that
the mean eGFR was 47 ml/min, 85% were hypertensive, the
majority of patients were 465 years of age (71%),
approximately 35% describe a history of some form of
coronary artery disease, and 47% were diabetic. The
representation of African Americans is similar to that of
the US population (12%). Patients were distributed across all
geographic regions in the United States with 71% being
enrolled from primary care practices, 19% from nephrolo-
gists, 7% from endocrinologists, and 3% from cardiologists.
Less than 25% of the population was taking Ca supplementa-
tion, and less than 10% were on hormone replacement
therapy or bisphosphonates.
Figure 2 describes the distribution of patients by eGFR
intervals. The majority of patients had stage 3 CKD, with an
5,255 subjects screened
99.6% failed due to eGFR > 60 mL / min
at screening
1,903 subjects enrolled
89 subjects have missing serum creatinine
data for eGFR calculation
1,814 subjects analyzed
Patients were enrolled between june to october 2004
Figure 1 | Screening, inclusion, and analytical sample flow.
32
A Levin et al.: Results of SEEK
eGFR between 30 and 60 ml/min/1.73 m2. Twenty-three
percent (405 patients) had an eGFR 460 ml/min/1.73 m2,
as evidenced in the figure, the percentages in each decile
above 60 ml/min/1.73 m2 were 3% 480, 7% ¼ 79–70
and 13% ¼ 69–60 ml/min/1.73 m2), which was outside of
the study entry criteria of o60 ml/min/1.73 m2. This was
the result of a discrepancy between the eGFR calculated
from the screening chart serum creatinine and the eGFR
calculated from the serum creatinine measured at baseline
(study visit 1).
Gender and ethnicity were not significantly different
between subjects with an eGFR of 30 and 60 ml/min/1.73 m2
when calculated from the screening chart serum creatinine
but who had an eGFR of 4 or o60 ml/min/1.73 m2 when
calculated from the serum creatinine measured at baseline.
Subjects with an eGFR of 30–60 ml/min/1.73 m2 when
calculated from the screening chart serum creatinine and
whose eGFR remained o60 ml/min/1.73 m2 when calculated
from the serum creatinine measured at baseline were
significantly older, had significantly lower 1,25 OH2 D3 and
25(OH)D3 levels and significantly higher intact PTH (iPTH)
levels compared to those with an eGFR of 30–60 ml/min/
1.73 m2 when calculated from the screening chart serum
creatinine and whose eGFR was 460 ml/min/1.73 m2
eGFR when calculated from the serum creatinine measured
at baseline.
iPTH, Ca, and P by eGFR intervals
Figure 3 describes the median values of Ca, P, and iPTH.
Note the relative stability of serum Ca and P over the
spectrum of eGFR values, until eGFR decreased to o20 ml/
min/1.73 m2. Median Ca and P values were within normal
ranges, and increases in iPTH (465 pg/dl) began to occur at
eGFR levels of approximately 45 ml/min/1.73 m2. Figure 4
describes the prevalence of abnormal values of Ca, P, and
iPTH levels. There is a increase in the prevalence of (HPTH,
iPTH 465 pg/ml) across declining eGFR levels. Interestingly,
HPTH was present in approximately 12% of those with eGFR
values 480 ml/min/1.73 m2, 17% of those with an eGFR of
70–79 ml/min/1.73 m2, 21% of those with an eGFR between
60 and 69 ml/min/1.73 m2, and 56% of those with an eGFR
o60 ml/min/1.73 m2.
Justification of definition for 25(OH)D3 and 1,25 OH2 D3
deficiency
For the purposes of analyses, deficiency of vitamin D
metabolites were defined as o15 ng/ml for 25(OH)D3 based
on current Kidney Disease Outcomes Quality Initiative
guidelines. Given the lack of published data regarding the
definition of 1,25 OH2 D3 deficiency, we determined, a priori,
a methodology to help define this value. We used the interim
baseline 1,25 OH2 D3 data available for this unselected,
community based, outpatient population, and selected the
lowest tertile of the underlying 1,25 OH2 D3 distribution as
the cutoff level for deficiency, rather than just the lower limit
of the reference range as reported by the laboratory. The
Kidney International (2007) 71, 31–38
A Levin et al.: Results of SEEK original article

Table 1 | Description of patient characteristics as a total group, and by eGFR levels (460, 59–30, and o30 ml/min)
Characteristics Patients total GFR460 ml/min GFR 59–30 ml/min GFRo30 ml/min P-value
N 1814 408 1109 297
Serum creatinine (mg/dl) 1.6 (0.86) 1.0 1.5 3.0 o0.0001
GFR (ml/min) 47.0 (17.7) 71.2 44.8 22.0 o0.0001

Age (mean years) 70.1 65.6 71.2 69.3 o0.0001

465 years 1283 (70.7%) 245 (60.1%) 827 (74.6%) 211 (71.0%) o0.0001

Ethnicitya 0.1028
AA 219 (12.1%) 42 (10.3%) 131 (11.8%) 46 (15.5%)
Other 1595 (87.9%) 366 (89.7%) 978 (88.2%) 251 (13.8%)
Male 866 (47.7%) 201 (49.3%) 524 (47.3%) 141 (47.5%) 0.7806

Geographic region 0.1900

Midwest 456 (25.1%) 92 (22.6%) 278 (25.1%) 86 (29.0%)
Northeast 374 (20.6%) 78 (19.1%) 228 (20.6%) 68 (229%)
Northwest 210 (11.6%) 58 (14.2%) 121 (10.9%) 31 (10.4%)
Southeast 514 (28.3%) 127 (31.1%) 313 (28.2%) 74 (24.9%)
Southwest 260 (14.3%) 53 (13.0%) 169 (15.2%) 38 (12.8%)
DMa 858 (47.3%) 137 (33.7%) 552 (50.1%) 169 (58.3%) o0.0001

CVD
Angina 326 (18.4%) 73 (18.1%) 201 (18.6%) 52 (18.4%) 0.9730
CAD 615 (34.9%) 109 (27.3%) 394 (36.5%) 112 (39.6%) 0.0008
CABG 265 (15.0%) 45 (11.2%) 170 (15.7%) 50 (17.7%) 0.0358
MI 278 (15.7%) 51 (12.7%) 171 (15.8%) 56 (19.9%) 0.0387
CHF 333 (18.9%) 50 (12.4%) 208 (19.3%) 75 (26.7%) o0.0001
PVD 299 (17.1%) 43 (10.8%) 203 (18.9%) 53 (18.9%) 0.0007
CVA 161 (9.1%) 28 (6.9%) 107 (9.9%) 26 (9.3%) 0.2070
HTN 1541 (86.7%) 296 (73.5%) 973 (89.3%) 272 (95.8%) o0.0001
Current smoking 345 (19.6%) 87 (21.6%) 212 (19.6%) 46 (16.5%) 0.2490
iPTH (pg/ml) 81.0 (89.88) 46.4 (28.11) 71.7(59.08) 185.5 (159.88) o0.0001
Serum albumin (g/dl) 4.2 (0.35) 4.3 4.3 4.1 o0.0001
Hemoglobin (g/dl) 13.1 (1.73) 13.8 13.0 12.1 o0.0001
UACR (mg/g) 475.4 (10896.37) 30.0 193.5 2151.3 0.0160
UACR430 mg/g 633 (35.6%) 62 (15.4%) 363 (33.4%) 208 (71.7%) o0.0001

Anti-hypertension medications
ACE inhibitor 678 (38.4%) 128 (32.1%) 444 (41.0%) 106 (37.2%) 0.0070
ARB 603 (33.9%) 105 (26.1%) 377 (34.6%) 121 (42.3%) o0.0001
CCB 625 (35.3%) 94 (23.3%) 388 (35.9%) 143 (50.2%) o0.0001
Beta blocker 785 (44.4%) 121 (30.0%) 510 (47.2%) 154 (54.2%) o0.0001
Diuretics 1142 (63.9%) 189 (46.8%) 735 (67.2%) 218 (75.7%) o0.0001

Other medications
Calcium supplement 442 (25.1%) 88 (21.9%) 282 (26.2%) 72 (25.3%) 0.2368
Multivitamin 726 (40.8%) 152 (37.6%) 458 (42.2%) 116 (40.6%) 0.2814
Statins 980 (55.2%) 190 (47.2%) 623 (57.4%) 167 (58.6%) 0.0009
Hormone RT 155 (8.7%) 42 (10.4%) 89 (8.2%) 24 (8.4%) 0.3958
Bisphosphonate 145 (8.2%) 37 (9.2%) 89 (8.2%) 19 (6.6%) 0.4848
NSAID 362 (20.4%) 101 (25.1%) 243 (22.4%) 18 (6.3%) o0.0001
Steroid 107 (5.9%) 31 (7.7%) 57 (5.3%) 19 (6.6%) 0.1917
ASA 984 (55.3%) 203 (50.5%) 634 (58.1%) 147 (51.2%) 0.0109
AA, African American; ACE, angiotensin converting enzyme; ARB, angiotensin II receptor blockade; ASA, aspirin; CAD, coronary artery disease; CABG, coronary artery bypass
graft; CCB, calcium channel blocker; CHF, congestive heart failure; CVA, cerebral vascular accident; CVD, cardiovascular disease; DM, diabetic mellitus; GFR, glomerular
filtration rate; HTN, hypertension; MI, myocardial infarction; NSAID, non-steroid anti-inflammation drugs; iPTH, intact parathyroid hormone; PVD, peripheral vascular disease;
RT, replacement therapy; UACR, urinary albumin to creatinine ratio.
Data are presented: mean (s.d.), or number (%). Percents may not add to 100% because of rounding.
a
Information on race and diabetes was reported by the subject.
Information on DM, CVD, smoking, UACR, and medications was not available for less than 5% of the subjects.
P-value alludes to the difference between the three groups.

level for 1,25 OH2 D3 deficiency based on this method and specificity of this 1,25 OH2 D3 deficiency level.
was 22.3 pg/ml, which, was just below the lower end of the The receiver operating characteristic curve described the
normal lab reference range. A receiver operating character- relationship between deficient/normal 1,25 OH2 D3
istic curve analysis was undertaken to evaluate the sensitivity (lower/higher than 24.4 pg/ml) and iPTH (lower/higher than

Kidney International (2007) 71, 31–38 33

original article A Levin et al.: Results of SEEK

Distribution of patients by GFR intervals Prevalence of abnormal serum calcium,

25 100 phosphorus, and intact PTH by GFR
22%
20% 20% Calcium < 8.4 mg/dl
20
Patients (%)

Phosphorus < 4.6 mg/dl

80
Intact PTH > 65 pg/ml
15 13%

Patients (%)
11%
10
60
7%
5%
5 3% 40

0
. 80 79 − 70 69 − 60 59 − 50 49 − 40 39 − 30 29 − 20 < 20 20
(n = 61) (n = 117) (n = 230) (n = 396) (n = 355) (n = 358) (n = 204) (n = 93)
GFR level (ml/min) 0
80 79 − 70 69 − 60 59 − 50 49 − 40 39 − 30 29 − 20 < 20
Figure 2 | Distribution of patients by eGFR intervals. The majority (n = 61) (n = 117) (n = 230) (n = 396) (n = 355) (n = 358) (n = 204) (n = 93)
of patients in the cohort had stage 3 CKD, with an eGFR between 30 GFR level (ml/min)
and 60 ml/min/1.73 m2. Twenty-two percent had an eGFR 460 ml/
min/1.73 m2. Figure 4 | The prevalence of HPTH, hypocalcemia, and
hyperphosphatemia by eGFR levels at 10 ml/min intervals.

Median values of serum calcium, phosphorus,

and intact PTH by GFR levels Median values of 1,25 dihydroxyvitamin D,
45 150
Serum calcium (mg/dl) 25 hydroxyvitamin D, and intact PTH by GFR levels
40 Serum phosphorus (mg/dl) 50
1,25 Dihydroxyvitamin D3 (pg/ml) 150
Intact PTH (mg/dl)

1,25 dihydroxyvitamin D (pg/ml),

Serum calcium (mg/dl),

35 45 25 Hydroxyvitamin D (ng/ml)
phosphorus (mg/dl)

25 hydroxyvitamin D (ng/ml)
Intact PTH (pg/ml)

Intact PTH (pg/ml)

30 100 40

Intact PTH (pg /ml)

25 35
100
30
20
25
15 50
20
10 50
15
5
10
0 0
79 − 70 69 − 60 59 − 50 49 − 40 39 − 30 29 − 20 5
> 80 < 20
0 0
GFR level (ml /min)
> 80 79 − 70 69 − 60 59 − 50 49 − 40 39 − 30 29 − 20 < 20
Figure 3 | Median values of serum Ca, P, and iPTH by eGFR levels. GFR level (ml/min)
Increases in iPTH preceded changes in serum Ca and P.
Figure 5 | Median values of 1,25 OH2 D3, 25(OH)D3 and iPTH by
GFR levels.

65 pg/ml). The sensitivity and specificity were 38.6% and

72.3%, respectively. The cutoff level for 1,25 OH2 D3 25(OH)D3 (P ¼ 0.8932). Although a significant interaction
deficiency was 22.3 pg/ml based on lowest tertile of 1,25 was found between levels of 1,25 OH2 D3 and 25(OH)D3
OH2 D3 distribution and 24.4 pg/ml based on receiver when all study participants were included in the analysis
operating characteristic curve. To be more conservative, (P ¼ 0.0182), the interaction disappeared when the analysis
we used the level o22 pg/ml as the cutoff to define 1,25 was limited to only those with an eGFR of o60 ml/min
OH2 D3 deficiency. (P ¼ 0.3433). Note that HPTH also begins to occur at eGFR
levels of approximately 45 ml/min/1.73 m2, similar to the
25(OH)D3, 1,25 OH2 D3, iPTH levels by eGFR intervals point where the median value of 1,25 OH2 D3 begins to
Our data confirmed the previous observations that approach values deemed ‘deficient’ (22 pg/ml) when using
25(OH)D3 was associated with iPTH levels, though the R2 the values in the lowest tertile for this population. This
value is relatively unimpressive (Adjusted R2 ¼ 0.0823; relationship was further explored by examining the pre-
Po0.01). Figure 5 describes the median values of valence of abnormalities within the population by eGFR
25(OH)D3, 1,25 OH2 D3 and iPTH by level of eGFR. The deciles (Figures 6 and 7). The prevalence of deficiency of
decrease in 1,25 OH2 D3 levels was over the spectrum of 25(OH)D3 remained relatively stable until eGFR fell below
eGFR values, whereas the slope of decline in 25(OH)D3 was 30 ml/min/1.73 m2 and appears to be dissociated from the
less steep over the same time period. By multiple regression HPTH prevalence. We then explored the prevalence of various
analyses, a model including eGFR as the dependent variable combinations of abnormalities, and found that 49% of those
and other mineral metabolites as the independent variable, with low 1,25 OH2 D3 levels had high iPTH, irrespective of
there was observed relationship between eGFR and 1,25 OH2 25(OH)D3 levels; whereas, only 35% of those with low
D3 (R2 ¼ 0.3827, Po0.0001) but not between eGFR and 25(OH)D3 levels had high iPTH levels (Po0.05, w2 test).

34 Kidney International (2007) 71, 31–38

A Levin et al.: Results of SEEK original article

Prevalence of 1,25(OH)2D3 and 25(OH)D3 deficiency eGFR, albumin, and UACR were significantly different. The
100
and intact PTH by GFR interesting finding that higher UACR was associated with
lower levels of 1,25 OH2 D3 at eGFR values of o60 ml/min/
25(OH)D3 <15 ng /ml
80 1.73 m2 is novel. Multivariate analysis revealed that only the
1,25(OH)2D3 < 22 pg /ml
presence of diabetes mellitus (DM) (odds ratio ¼ 1.727,
Patients (%)

Intact PTH > 65 pg/ml

60 confidence interval ¼ 1.385–2.152, Po0.0001), increased
UACR (odds ratio ¼ 1.429, confidence interval ¼ 1.129–1.808,
40 P ¼ 0.0029) and decreased eGFR (confidence interval ¼
0.427, confidence interval ¼ 0.350–0.519, Po0.0001) pre-
20
dicted lower values of 1,25 OH2 D3. Variables in the model
included age, gender, race, Ca, phosphorous, eGFR, Dm, and
0
. 80 79 − 70 69 − 60 59 − 50 49 − 40 39 − 30 29 − 20 < 20 UACR (Table 3).
(n = 61) (n = 117) (n = 230) (n = 396) (n = 355) (n = 358) (n = 204) (n = 93)
GFR level (ml/min) DISCUSSION
Figure 6 | The prevalence of deficiency of 1,25 OH2 D3, 25(OH)D3, We describe here, in the largest observational study of an
and HPTH by GFR intervals. unselected cohort of patients with varying levels of kidney
dysfunction, the prevalence of abnormalities of vitamin D
metabolites, iPTH, and Ca and P. This study demonstrates a
number of key findings, some of which corroborate previous
Prevalence of abnormalities
work, and some of which are novel. Within this cohort, we
Mutually exclusive categories Low 1,25(OH)2D3 combinations attempted to better define 1,25 OH2 D3 deficiency, or more
45
40
specifically, low values of 1,25 OH2 D3. Thus we describe a
35 high prevalence of low values of 1,25 OH2 D3 within this
Patients (%)

30 population, even at higher levels of eGFR than usually

25
20
15
10
5 the prevalence of elevated iPTH levels, which increased
0 significantly at eGFR values below 50 ml/min/1.73 m2.
Low 1,25 Low 25 High PTH
Normal PTH High PTH High PTH Normal PTH
(n = 536) (n = 223) (n = 694)
Figure 7 | Association between low 1,25 OH2 D3, low 25(OH)D3,
and high iPTH levels.
Differences in key variables between those with normal vs.
low values of 1,25 OH2 D3 by eGFR intervals
Univariate analysis between patients with normal versus low
values of 1,25 OH2 D3 revealed significant differences within
the total population for all variables except age (Table 2).
When examined by level of eGFR (460, 30–60, and o30 ml/
min/1.73 m2), different patterns emerged. For example, in
those with an eGFR 460 ml/min/1.73 m2, there were only
statistically significant differences between 25(OH)D3 levels
in relation to 1,25 OH2 D3 levels, and these were still in the
non-deficiency range. In those with an eGFR between 30 and
60 ml/min/1.73 m2, phosphorous was statistically signifi-
cantly different, though of questionable clinical significance
and within the normal range (3.7 vs 3.6 mg/dl, P ¼ 0.001).
Also within this group, values of 25(OH)D3 were different,
(as were the absolute values of eGFR, hemoglobin, and
urinary albumin-to-creatinine ratios (UACR)), based on
whether the 1,25 OH2 D3 levels were deficient or normal.
Finally, in those with an eGFR o30 ml/min/1.73 m2, iPTH
values were not substantially different, but most were well
above the normal range of 65 pg/dl. Values of 25(OH)D3,
expected or has not been reported previously. Note that the
low values are congruent with the low range of values as
reported by the laboratory reference range. We also describe
Low 1,25 Low 1,25 Low 1,25 Low 1,25
Low 25 Low 25 Normal 25 Normal 25 Importantly, neither of these changes was associated with
demonstrable changes in serum Ca, P, or major differences in
(n = 33) (n = 64) (n = 199) (n = 234)
values of 25(OH)D3. The dissociation of HPTH from any
significant changes in serum Ca and P in patients with a
lower eGFR, whereas physiologically expected, underscores
the need to assess this hormone level irrespective of these
mineral metabolites. The late occurrence of hyperpho-
sphatemia was not present in enough of the population to
explain the high prevalence of HPTH in stage 4 disease.
The univariate predictors of low 1,25 OH2 D3 levels
differed by eGFR values, but most interestingly, in the
multivariate analysis, we demonstrated that DM, elevated
UACR, and eGFR were independently associated with low
levels of 1,25 OH2 D3. There also seemed to be a strong
association between low 1,25 OH2 D3 levels and the
prevalence of HPTH. Although the Kidney Disease Outcomes
Quality Initiative guidelines recommend iPTH testing in
CKD patients at eGFR levels below 60 ml/min/1.73 m2, in
general, such testing only occurs in a small number of
patients, and these findings provide further support for those
testing guidelines. Although PTH testing is clearly more
practical at present, these findings also have implications for
1,25 OH2 D3 testing of individuals, given the biological
relevance of that deficiency to HPTH, and the demonstration
here that low levels of 1,25 OH2 D3 occur earlier in the course
of eGFR decline than does elevations in iPTH levels.
However, in the absence of longitudinal or outcome data

Kidney International (2007) 71, 31–38 35

original article A Levin et al.: Results of SEEK

Table 2 | Univariate analyses describing differences in key variables between those with low 1,25 OH2 D vs normal 1,25 OH2 D3
within the total group, and by levels of GFR460, 59–30, and o30 ml/min/1.73 m2
Total Total GFR460 GFR460 GFR 59–30 GFR 59–30 GFRo30 GFRo30
Low Normal Low 1,25 Normal Low 1,25 Normal Low 1,25 Normal
1,25 D 1,25 D P-value D 1,25 D P-value D 1,25 D P-value D 1,25 D P-value
N 536 1126 59 321 329 691 148 114
1,25 OH2 D3 (pg/ml) 14.4 34.9 o0.001 17.3 41.5 o0.001 14.9 33.7 o0.001 12.3 30.0 o0.001
Age (years) 71.0 71.0 0.934 67.0 68.0 0.830 71.0 72.0 0.398 73.0 72.5 0.784
GFR (ml/min/1.73 m2) 37.4 51.0 o0.001 66.4 67.7 0.405 41.0 47.2 o0.001 22.1 24.2 0.007
25(OH)D3 (ng/ml) 23.0 29.0 o0.001 24.5 31.0 o0.001 24.0 29.0 o0.001 21.0 25.0 0.003
Intact PTH (pg/ml) 65.0 53.0 o0.001 40.0 41.0 0.666 61.0 55.5 0.123 95.0 116.0 0.105
Calcium (mg/dl) 9.2 9.1 0.034 9.2 9.1 0.204 9.2 9.1 0.165 9.1 9.0 0.145
Phosphorus (mg/dl) 3.8 3.6 o0.001 3.4 3.5 0.312 3.7 3.6 0.001 4.2 4.0 0.049
Albumin (g/dl) 4.2 4.3 o0.001 4.2 4.3 0.075 4.2 4.3 o0.001 4.1 4.3 o0.001
Bicarbonate (Meq/l) 22.0 23.0 o0.001 23.0 23.0 0.487 22.0 23.0 0.041 20.0 21.0 0.111
Hgb (g/dl) 12.5 13.2 o0.001 13.7 13.8 0.691 12.6 13.2 o0.001 12.0 12.3 0.059
UACR (mg/g) 23.0 10.0 o0.001 7.0 7.0 0.594 15.0 10.0 0.002 215.0 57.0 o0.001
GFR, glomerular filtration rate; Hgb, hemoglobin; PTH, parathyroid hormone; UACR, urinary albumin to creatinine ratio.
All values represent median values.

Table 3 | Multivariable analysis: independent association with inadequate 25(OH)D3 levels, only 12% were frankly deficient
low 1,25 OH2 D3 in all subjects in 25(OH)D3 deficient (o15 ng/ml). In addition, our large
Odds ratio 95% CI P-values sample allowed us to determine that the relationship between
25(OH)D3 and 1,25 OH2 D3 was not significant (R2 ¼ 0.3666,
GFR 0.443 0.360–0.545 o0.0001
DM 1.757 1.404–2.197 o0.0001 P ¼ 0.3433 at eGFRo60 ml/min), which is consistent with
UACR 1.473 1.158–1.874 0.0016 Ishimura’s observation;15 and while statistically significantly
Age 1.001 0.990–1.012 0.881 different among the eGFR groups, the values were of
Sex 1.069 0.854–1.337 0.562
Race (AA or not) 0.736 0.514–1.053 0.093
questionable clinical significance (4–5 ng/ml). In this analysis,
Serum calcium 1.006 0.560–1.806 0.985 the relationship between serum 25(OH)D3 with elevated
Serum phosphorus 1.245 0.842–1.841 0.273 iPTH and decreased GFR levels was less relevant than that of
AA, African American; CI, confidence interval; DM, diabetic mellitus; GFR, glomerular 1,25 OH2 D3. This finding may have therapeutic ramifica-
filtration rate; UACR, urinary albumin to creatinine ratio. tions as recent Kidney Disease Outcomes Quality Initiative
Variables in the model include age, gender, AA, GFR by categories, diabetes, UACR
by categories 430 mg/g, o30 mg/g, calcium by categories o8.4, 48.4 mg/dl, and guidelines currently suggest the use of ergocalciferol (vitamin
phosphorus 44.6, o4.6 mg/dl; only GFR, DM, and UACR were independently D2) as the initial treatment for HPTH in CKD stage 3 and
associated with low 1,25 OH2 D3 levels.
4.16 However, the findings herein of an early low levels of 1,25
OH2 D3 and some preliminary data that show that this
that would provide clinical relevance, the use of 1,25 OH2 D3 treatment regimen may not effectively suppress PTH,17 may
levels as a surrogate for the development of HPTH is require that those recommendations are revisited.
premature, as this cross-sectional analysis of baseline data This study had several limitations. The patient ages were
cannot and does not address this question. Prospective weighted to older individuals (71% of the sample was over 65
descriptive and then, interventional clinical trials where 1,25 years old) and although a large proportion of CKD patients
OH2 D3 is repleted, with subsequent measurement of fall within this age range, these data may not be generalizable
clinically meaningful outcomes related to this treatment are to patients between 40 and 65 despite their inclusion in the
required to address this question. sample. The results are cross-sectional analyses, thus causal
Low serum 1,25 OH2 D3 occurs for a variety of reasons, relationships between dependent variables (kidney function)
and has been recently described as being more prevalent than and independent variables (vitamin D metabolites, iPTH, Ca,
previously thought in western populations. Although de- and P) cannot be defined. No longitudinal data has been
creased renal 1-a hydroxylase in CKD is largely responsible reported here, thus relationships between the variables and
for reduced circulating levels of 1,25 OH2 D3, other potential different eGFR levels may change over time in different
factors may exist, which also suppress this hydroxylating individuals. In addition, kidney disease was defined by a
enzyme.11 In addition, low levels of the 25(OH)D3 substrate single creatinine measure used to estimate the eGFR. In fact
may contribute to decreased levels of 1,25 OH2 D3 22% of the cohort changed ‘classification’ within the time
production, particularly in CKD patients with nephrotic period of screening and enrollment into the study, further
range proteinuria.12 One recent survey noted that 86% CKD underscoring the problems with defining populations based
patients (n ¼ 43) had inadequate 25(OH)D3 levels (o30 ng/ on single values without accompanying urine tests or clinical
ml), which has been previously defined by others.13,14 In data. Nonetheless, the majority (78%) of the population did
our study, we also found that although many subjects had have persistent decreases in their kidney function, within

36 Kidney International (2007) 71, 31–38

A Levin et al.: Results of SEEK
relatively stable categories. The population appears repre-
sentative of the general US population, but because the entry
criteria included the presence of a serum creatinine in the
patient record, this population, by default, may have less
healthy well being, as indicated by the underlying need to
order the test. Finally, the findings of this study represent
associations. Interventional studies are needed to define the
serum levels of 1,25 OH2 D3 that would provide adequate
suppression of iPTH and subsequent potential untoward
effects of mineral imbalances.
Despite these limitations, the study expands our knowl-
edge on the prevalence of abnormalities of key hormones at
different levels of eGFR, and raises questions regarding the
relative importance of 25(OH)D3 and 1,25 OH2 D3 in the
biology of abnormalities of PTH. Although we cannot
separate the potential effects of these derangements on
cardiovascular disease, we can speculate that 1,25 OH2 D3
and PTH may have independent roles in promoting vascular
calcification and cardiac events. For example, it has been
known for sometime that low 1,25 OH2 D3 is associated with
coronary artery calcifications in non-CKD populations.18 It is
also known that 1,25-dihyroxyvitamin D levels are indirectly
associated with serum renin levels in hypertensive subjects,19
a finding that was recently corroborated in vitamin D
receptor knockout mice.20 Elevated PTH is known to cause
cardiomyocyte damage in animals with CKD21 and para-
thyroidectomy in dialysis patients is associated with reduc-
tions in left ventricular hypertrophy22 and in cardiovascular
mortality.23 Thus, taken together we may speculate that low
1,25 OH2 D3 and elevated iPTH levels in CKD stage 3 and 4
may contribute to the high prevalence of cardiovascular and
advanced renal disease seen in these populations. In addition,
the independent association between low 1,25 OH2 D3 and
high UACR may support the observed anti-proteinuric effect
of active vitamin D in CKD patients.24 Further long
term data relating the levels to outcomes, and ulitmately
a randomized controlled trial examining the impact
of activated vitamin D on cardiovascular outcomes should
be undertaken.
In summary, we found, in a large, unselected population
that serum 1,25 OH2 D3 levels decline early in CKD before
significant elevations in PTH are observed. Hypocalcemia
and hyperphosphatemia, when and if they occur, are noted in
the later stages of CKD. Future research should be directed
toward attempting to further describe the relationship
between vitamin D levels, eGFR and other factors related to
clinically meaningful outcomes, by testing the impact of early
interventions with activated vitamin D therapy.
MATERIALS AND METHODS
Study design
SEEK is a prospective, community-based, non-interventional,
prospective cohort study designed to examine the prevalence of
abnormalities in serum iPTH, vitamin D, Ca, and P in patients with
CKD. To be eligible for the study, patients had to be older than 40
years of age and to be an established member of the investigator’s
Kidney International (2007) 71, 31–38
original article
practice. Investigators included primary care physicians, endocri-
nologists, cardiologists, and nephrologists. The required screening
data consisted of a signed informed consent form, a Health
Insurance Portability and Accountability Act (HIPPA) waiver, and
a serum creatinine value obtained in the calendar year 2003 or 2004.
One hundred fifty-three (153), US-based centers recruited patients
for the study, following approval from a central institutional review
board and or ethics committee. Patients enrolled in the study were
equally distributed across the United States (Table 1), and given that
sun exposure would affect serum 25(OH)D3 levels, study visits and
first blood sample collections were scheduled from June 2004 to
October 2004, when sunlight intensity was consistent across the US.
Patients enrolled were scheduled to visit the investigator’s practice
twice; one visit at baseline and one visit approximately 12 months
later. Results of baseline interim cross-sectional analyses are
described here.
Patient population
Following informed consent and screening, patients were enrolled if
they had an eGFR o60 ml/min, as determined by the simplified
Modification of Diet in Renal Disease (MDRD) equation (eGFR
(ml/min/1.73 m2) ¼ 186  (PCr)1.154  (Age)0.203  (0.742 if
female)  (1.210 if African American)). Exclusion criteria included
renal replacement therapy, a history of primary parathyroid disease,
or use of any prescription-based vitamin D therapy in the 12
months before screening. Dietary supplementation of vitamin D and
multivitamin intake up to 400 IU/day was not restricted, nor
considered an exclusion criteria. The use of Ca supplements,
diuretics, steroid, and anti-hypertensive medications was captured
during the first study visit.
Variables of interest and methods of measurement
Medical history, medications, blood and urine samples were
collected at study visit 1 (baseline). Blood was collected for serum
creatinine, iPTH, 25(OH)D3, 1,25 OH2 D3, Ca, P, albumin, and
bone alkaline phosphatase. Total Ca was corrected for serum
albumin using the equation: Calcium ¼ Ca þ 0.8 (4.0 albumin). All
samples were run at a central laboratory (Quintiles Laboratories,
Ltd., NJ, USA).
For the purposes of this study, the key variable of interest was the
distribution of 1,25 OH2 D3 values. Both 25(OH)D3 and 1,25 OH2
D3 were determined using the DiaSorin radioimmunoassay kit
(Stillwater, MN, USA). Calcium, P, and creatinine were analyzed
with a routine autoanalyzer. Serum iPTH was determined by a
chemiluminescence assay (DPC, Los Angeles, CA, USA). The lab
references are 10–65 pg/ml for iPTH, 8–60 ng/ml for 25(OH)D3 and
25–65 pg/ml for 1,25 OH2 D3.
Statistical analyses
Descriptive statistics of continuous variables are presented as means
with s.d. or medians, depending on the distribution of the variable.
Categorical variables are presented as distributions (i.e., frequencies
and percentages). Estimated GFR was used as both a continuous and
categorical variable, with intervals of 10 ml/min forming eight
categories. The statistical significance of differences in the measured
variables between subgroups was tested with the Pearson w2 test for
categorical analysis, and the t-test or the Wilcoxon rank sum test,
where appropriate, for continuous variables.
Multivariable regression analyses were performed to investigate
the predictors of levels of the different vitamin D metabolites and of
37
original article
iPTH in the context of this population. Variables included in the
model were age, gender, ethnicity, DM, UACR, eGFR, Ca, and P.
Data are presented in both continuous and categorical format, so as to
ensure interpretability for both clinical investigators and for clinicians.
Statistical analyses were performed using SAS software, version
8.01 (SAS Institute, Cary, NC, USA).
ACKNOWLEDGMENTS
SEEK Advisory Panel include Drs Dennis L Andress, George L Bakris,
Adeera Levin, and Mark Molitch. SEEK investigators include Drs Allen
Adolphe, Iftikhar Ahmad, Michael Alexander, Diego X Alvarez,
Gustavo A Alza, Dennis Andress, James Andrews, Abraham
Areephanthu, Carlos R Arguello, Carl Augustus, Dwight L Bailey,
Ronald E Ballek, Ernesto E Banalagay, Saad E Bedeir, Andrew J Behnke,
Sabrina Benjamin, Marializa V Bernardo, Jayantilal Bhimani, Edward
Braun, Bernard Buchanan, Mary Carol Greenlee, Laurence Carroll,
Chaim Charytan, Indira Chervu, William H Cleveland, Lou Constan,
Deena Craig, James Cuellar, Albert M Defabritus, Amar Desai, Rajnish
Dhingra, Douglass Domoto, Michael W Early Sr, Andrew E Edin, David
Evanko, Stephen Fadem, Timothy Fagan, Michael Fedak, Bradford T
Flagg, Brad Frandsen, Jeffery Freesemann, Linda Freilich, Stuart
Friedman, Susan Fullemann, Dinesh R Gandhi, Steven R Ginos, Daniel
Goodman, Burk Gossom, Catherine Grellet, Stephanie Gunnoe, Roger
J Haley, Rekha D Halligan, Daniel Halm, Maxine E Hamilton, Laila C
Hanna, Merfat Hassan, George Herron, Peck P Hsu, Yiling Huang,
Scott Isaacs, Shah Islam, Peter Jacobson, Albert F Johary, Ronald W
Johnson, Alan Jones, Regi Joseph, Lionel B Katchem, Bruce A Kauk,
Rebecca Knight, David Koeper, Lawrence Kramer, Gerald Kumin, Stella
S Kwong, David Lahasky, Vinodrai Lakhani, Rachel Langland, Chok
Lee, Irene Leech, Daniel J Legault, Lawrence Lehrner, April Liverman,
James Love, Joshua Lowentritt, Blanca Luna, Michael Macadams, Isam
Marar, Eric Marcotte, Steven Margolis, Eric Marshall, Carlos O
Martinez, Janus Maybee, Gela Mchedlishvili, Donald J Meyer, Kenneth
Miller, James Miller Oppy, Palghat V Mohan, Ramakant Mulay, Derek
Muse, Rhonda I Museums, Kent W Myers, Seymour Myers, Arif Nazir,
Patrick Ogilvie, Kevin W Ohara, Paul Pagnozzi, Meyappan
Palaniappan, Saradha Perinbasekar, Marvin Pietruszka, George E Platt,
John H Poehlman, Peterman Prosser, Alan D Purdy, Gary Raffel, David
Ramstad, Harvey Resnick, Wayne Reynolds, Louis Reznick, Matt
Rosenberg, Paul Rosenblit, Roset N Samuel, Rallabhandi Sankaram,
Deepak Santram, Neil Schwartzman, Peter Scuccimarri, Gladstone
Sellers, Chandrakant Shah, Jeffery G Shanes, Devendra K Sharma,
Michael Scheaffer, Raewyn Shell, Terry Sherraden, Anil Sheth, Thomas
Shetter, Phillip Shou, Haven Silver, George A Smith, Karen L Smith,
Donna Steadman, Helen Story, David C Subich, Robert M Taxin, Gary
Tigges, Robert W Tom, Thomas Tse, Umamaheswara Vejendla,
Samuel Verzosa, William C Ware, Robert Weiss, Lindsey White, Richard
Woronoff, Alberto Yataco, Raja Zabaneh, and Mayer Zayan.
Disclaimer: This study was initiated, developed and planned by the
investigators, funded by Abbott Laboratories, and executed by
Quintiles Strategic Research Services (SRS). All analyses presented in
this paper were developed by the principal investigators, and
executed by Quintiles (SRS).
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