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Keywords:

  • vitamin D;
  • Crohn's disease;
  • bioavailability test;
  • IBD;
  • malabsorption;
  • 25-hydroxyvitamin D

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background:

Vitamin D deficiency is a common problem in patients with Crohn's disease (CD). The aim of this study was to determine the ability of normal subjects and patients with quiescent CD to absorb vitamin D2 using a novel vitamin D bioavailability test. In addition, we evaluated whether the location of disease or previous surgery had any influence on the bioavailability of vitamin D2 in CD patients.

Methods:

Ten normal subjects (50% female) and 37 CD patients with quiescent disease (51% female) were included in this study. Subjects who recently received any vitamin D2 were excluded. The vitamin D bioavailability test was performed in all subjects. After a baseline blood draw, all subjects were then given a single 50,000 IU oral dose of vitamin D2 in a capsule formulation and had their blood drawn 12 hours later to determine serum vitamin D2, which reflected their vitamin D2 absorption capacity.

Results:

Forty-two percent and 29% of CD patients were found to be either vitamin D-deficient (25-hydroxyvitamin D [25(OH)D] ≤20 ng/mL] or insufficient [25(OH)D 21–29 ng/mL], respectively. Twelve hours after ingesting 50,000 IU vitamin D2, vitamin D2 levels rose from a baseline of 0.7 ± 0.7 ng/mL (mean ± SEM) to 49.8 ± 3.0 ng/mL in normal subjects. In CD patients, baseline vitamin D2 levels rose from 0 ng/mL to 34.8 ± 2.8 ng/mL. CD patients had on average a 30% decrease in their ability to absorb vitamin D2 (P = 0.01). Moreover, we found a wide variability of vitamin D2 bioavailability in CD patients. Analysis of variance (ANOVA) revealed no statistical difference of vitamin D2 bioavailability between patients in the CD subgroup stratified by the location of disease, the type of surgery, and receiving or not receiving surgery.

Conclusions:

More than 70% of the patients with quiescent CD were vitamin D-deficient or insufficient. The ability to absorb vitamin D2 in CD patients is unpredictable and the only way to determine this is to perform a vitamin D bioavailability test. Use of this test may guide clinicians in administering the appropriate therapeutic dose of vitamin D for treating vitamin D deficiency in patients with CD. (Inflamm Bowel Dis 2011;)

Vitamin D is ingested in the diet, as well as synthesized in the skin from UVB irradiation from the sun. People living in areas that receive less sunlight have lower circulating 25-hydroxyvitamin D [25(OH)D] levels and have higher prevalence rates of inflammatory bowel disease (IBD).1 In addition, both children and adults with IBD have an increased incidence of vitamin D deficiency.2–4 In particular, patients with Crohn's disease (CD) have an increased incidence of vitamin D deficiency relative to both patients with ulcerative colitis (UC) and the general population.5

There is evidence that the presence or absence of vitamin D modifies inflammatory pathways in experimental models of IBD. In vitro, the activated form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D], has been shown to inhibit T-cell proliferation and to decrease the production of interleukin (IL)-2, interferon (IFN)-gamma, and tumor necrosis factor alpha (TNF-α).6 In animal models of IBD such as the IL-10 knockout (KO) mouse, vitamin D deficiency accelerated the development of disease symptoms such that 100% of the vitamin D-deficient IL-10 KO mice had symptoms (diarrhea, rectal bleeding) of IBD and 60% died due to a severe form of IBD before 9 weeks of age. In contrast, vitamin D-sufficient mice showed no outward symptoms of IBD in the same timeframe.7

Given that vitamin D is absorbed primarily in the proximal small intestine, it has been speculated that vitamin D deficiency in CD may be due to decreased absorption of dietary and supplemental vitamin D. A study by Lo et al8 reported in three patients with CD that two of the patients were unable to absorb vitamin D, whereas the third with ileocecal disease was able to absorb vitamin D to the same extent as normal controls. The ability to absorb vitamin D has not been studied in CD patients with quiescent disease. In this study, the primary endpoint was to evaluate the ability to absorb vitamin D2 in normal subjects and in patients with quiescent CD using a novel vitamin D bioavailability test. The secondary endpoint was to determine if the location of disease or the type of surgery had any influence on the bioavailability of vitamin D2 in CD patients.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The study was conducted from January 2007 through March 2008. Ten normal subjects (50% female) and 37 CD patients (51.4% female) were included in this study. All subjects were at least 18 years of age. The normal subjects were healthy adults. In the CD group, the diagnosis was established by radiologic, histologic, and/or endoscopic criteria without evidence of active disease at the time of inclusion in the study. A Crohn's Disease Activity Index (CDAI) or Harvey–Bradshaw Index (HBI) were not obtained. A complete blood count (CBC), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were measured in all patients. All female CD patients were postmenopausal at least 1 year or incapable of childbearing or were using contraception. Exclusion criteria for the CD patients included current pregnancy (as determined by a urine test), having a serum calcium level greater than 10.2 mg/dL, taking digoxin, another cardiac glycoside or thiazide, taking or had taken within 6 months 50,000 IU vitamin D2 for at least 8 weeks (the subjects would not be excluded if he/she were merely taking a multivitamin containing vitamin D3), having a history of renal, hepatic, hematologic, endocrine, pulmonary, cardiac, neurologic, or cerebral disease and having a history of an adverse reaction to vitamin D2.

A vitamin D2 bioavailability test was performed in both normal subjects and quiescent CD patients as previously described.8 In brief, subjects had baseline blood drawn. They were then given a single 50,000 IU oral dose of vitamin D2 in a capsule formulation and had their blood drawn 12 hours later to determine the serum vitamin D2 level as a measure of their vitamin D2 absorption capacity. All of blood samples were stored at −20°C and analyzed at the same time.

All serum samples for vitamin D2 determination were extracted and chromatographed by the method of Chen et al.9 Serum was extracted for its lipid followed by chromatography on a preparative silica column. The fraction that contained both vitamin D2 and vitamin D3 was chromatographed on a normal-phase high-performance liquid chromatograph (HPLC) and then a reverse-phase HPLC. The conditions for the normal phase HPLC were as follows: column, Econosphere silica column, 5 μ particle size, 250 × 4.6 mm (Alltech Associates, Deerfield, IL) mobile phase, hexane:isopropanol (99.2:0.8); and flow rate 1.1 mL/min. For the reverse-phase HPLC, samples were applied to a VYDAC-C-18 column (5 μ particle size, 250 × 4.6 mm, (Rainin Instrument, Woburn, MA.) equilibrated with 25% methanol in acetonitrile (mobile phase) and chromatographed with the same solvent system at a flow rate of 0.9 mL/min. The amount of vitamin D2 after the reverse-phase HPLC was calculated by comparing the area under a vitamin D2 peak against a calibration curve constructed from a serial injection of increasing concentrations of standard vitamin D2. The serum vitamin D2 concentration was obtained after a correction of its recovery.9 The definition of percent vitamin D2 bioavailability = the mean value of the vitamin D2 level 12 hours after ingesting 50,000 IU vitamin D2 in each subgroup divided by the mean value of the vitamin D2 level in normal subjects (49.8 ng/mL).

Vitamin D status was determined by serum levels of total 25-hydroxyvitamin D (25(OH)D) which was measured by liquid chromatography tandem mass spectrometry as previously described.10 Subjects who had 25(OH) D levels ≤20 ng/mL or 21–29 ng/mL were classified as vitamin D-deficient or -insufficient, respectively.11 To determine dietary vitamin D intake, a food frequency questionnaire was completed by all participants.12

On statistical analyses, all values are presented as means ± standard error mean (SEM) and ranges are as indicated. Student's t-test was performed to compare serum vitamin D2 levels between normal subjects and subjects with CD. Analysis of variance (ANOVA) was performed to compare value of vitamin D2 levels according to the location of disease and type of surgery. All statistical analyses were performed with SPSS v. 11.3 (Chicago, IL). A P-value of < 0.05 was considered statistically significant.

Ethical Considerations

The study was conducted at the General Clinical Research Unit (GCRU), Boston University Medical Campus. It was approved by the Institutional Review Board and all participants provided written informed consent prior to the study.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Clinical characteristics and biochemical data of 10 normal subjects and 37 CD patients are shown in Tables 1 and 2, respectively. Forty-two percent and 29% of CD patients were found to be either vitamin D-deficient, with 25(OH) D levels ≤20 ng/mL, or insufficient with 25(OH) D levels of 21–29 ng/mL, respectively.

Table 1. Baseline Characteristic of Normal Subjects
 Normal Subjects (N = 10)
  1. Data expressed as mean ± SE.

Gender (male/female) (n)5/5 (50%/50%)
Age (y)26.1 ± 0.8
BW (kg)62.2 ± 3.7
BMI (kg/m2)21.4 ± 1.1
Daily vitamin D intake (IU)120 ± 55.4
Ethnic: White4 (40%)
 Black
 Hispanic
 Other6 (60%)
Table 2. Baseline Characteristic of Patients with Crohn's Disease
 Crohn's Disease (N=37)
  1. Data expressed as mean ± SE.

Gender (male/female) (n)18/19 (48.6%/51.4%)
Ethnic: White30 (81.1%)
 Black4 (10.8%)
 Hispanic1 (2.7%)
 Other2 (5.4%)
Age (y)44.6 ± 2.1
BW (kg)75.5 ± 3.2
BMI (kg/m2)26.8 ± 1.0
Duration of disease (y)13.8 ± 2.1
ESR (mm/hr) (normal < 20 mm/hr)17.7 ± 2.6
CRP (mg/L) (normal < 1 mg/L)0.7 ± 0.2
Hematocrit (%) (normal ≥ 36% in female and 39% in male)40.9 ± 0.5
Daily vitamin D intake (IU)323.9 ± 36.2
25(OH)D (ng/mL)24.3 ± 2.2 (n = 31)
Calcium (mg/dL)9.3 ± 0.1

Twelve hours after ingesting 50,000 IU vitamin D2, vitamin D2 levels rose from a baseline of 0.7 ± 0.7 ng/mL (mean ± SEM) to 49.8 ± 3.0 ng/mL in normal subjects. In CD patients, baseline vitamin D2 levels rose from 0 ng/mL to 34.8 ± 2.8 ng/mL (range 0–76 ng/mL). CD patients had on average a significant 30% decrease in their ability to absorb vitamin D2 (P = 0.01) when compared with normal subjects, as shown in Figure 1. Moreover, we found a wide variability in vitamin D2 bioavailability in CD patients. One CD patient who had no gastrointestinal symptoms was found to be unable to absorb vitamin D2. Thirty-nine percent and 24.3% of the CD patients absorbed less than 50% and 50%–80% respectively, when compared with normal subjects, as shown in Figure 2.

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Figure 1. Vitamin D2 levels at baseline and 12 hours after ingesting 50,000 IU vitamin D2 in normal subjects and patients with quiescent CD. *P < 0.01 serum vitamin D2 levels in patients with CD compared with vitamin D2 levels in normal subjects.

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thumbnail image

Figure 2. Serum vitamin D2 levels in normal subjects and patients with quiescent CD. *P = 0.01 serum vitamin D2 levels in CD compared with vitamin D2 levels in normal subjects.

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For patients with CD diagnosed as colitis, ileocolitis, or isolated small bowel disease, the vitamin D2 levels at 12 hours after ingesting 50,000 IU vitamin D2 are shown in Table 3. ANOVA analysis revealed no statistically significant difference in the serum vitamin D2 levels between patients in all three groups (P = 0.83). Furthermore, patients with CD were stratified according to the type of surgery including no surgery, small bowel resection, ileocecectomy, and ileostomy. No statistically significance difference in the serum vitamin D2 levels between patients in each group was detected, as shown in Table 4. Vitamin D2 levels in CD subjects and normal subjects were in normal distribution.

Table 3. Vitamin D2 Levels 12 Hours After Ingesting 50,000 IU Vitamin D2 in Crohn's Disease Patients Stratified by Location of Disease
 Vitamin D2 Level (ng/mL)% Vitamin D2 Bioavailability Compared Crohn's Disease Patients with Normal Subjects
  1. Data expressed as mean ± SEM.

  2. *The P-value between groups = 0.83 by one-way ANOVA analysis.

  3. % vitamin D2 bioavailability = the mean value of the vitamin D2 level 12 hours after ingesting 50,000 IU vitamin D2 in each subgroup divided by the mean value of the vitamin D2 level in normal subjects (49.8 ng/mL).

Crohn's disease (n = 37)34.8 ± 2.869.9%
Colitis (n = 12)34.5 ± 5.069.3%
Ileocolitis (n = 15)33.1 ± 4.066.5%
Isolated small bowel disease (n = 10)37.5 ± 6.675.3%
Table 4. Vitamin D2 Levels 12 Hours After Ingesting 50,000 IU Vitamin D2 in Crohn's Disease Patients Stratified by Type of Surgery
 Vitamin D2 Level (ng/mL)% Vitamin D2 Bioavailability Comparing Crohn's Disease Patients with Normal Subjects
  1. Data expressed as mean ± SEM.

  2. *The P-value between groups = 0.35 by one-way ANOVA analysis.

  3. Percent vitamin D2 bioavailability = the mean value of the vitamin D2 level 12 hours after ingesting 50,000 IU vitamin D2 in each subgroup divided by the mean value of the vitamin D2 level normal subjects (49.8 ng/mL).

Crohn's disease (n = 37)34.8 ± 2.869.9%
No surgery (n = 17)39.6 ± 3.779.5%
Small bowel resection (n = 3)36.0 ± 8.072.7%
Ileocecectomy (n = 12)31.7 ± 6.163.7%
Ileostomy (n = 5)25.0 ± 5.550.2%

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In this study we used a novel vitamin D bioavailability test to demonstrate that a majority of patients with quiescent CD based on clinical features, ESR, and CRP had a reduced ability to absorb supplemental vitamin D2 compared with healthy, normal controls. In addition, our data show that the anatomic location of the disease or prior small or large intestinal resection does not appear to affect vitamin D2 bioavailability. To our knowledge, this is the first study to show that reduced vitamin D absorption in patients with quiescent CD may be the cause for their increased risk for vitamin D deficiency.

IBD patients have an increased incidence of vitamin D deficiency, especially patients with CD. Vitamin D deficiency can be identified in both children and adults with IBD. For example, the prevalence of vitamin D deficiency (≤10–20 ng/mL) in adults with IBD has been reported to be between 22%–83% in CD and up to 55% in UC.5, 13–16 In a study of recently diagnosed IBD patients, only a minority (22/101 or 21.8%) of patients had serum 25(OH)D levels greater than 30 ng/mL.2 In a study of pediatric IBD patients the prevalence of vitamin D deficiency was 34.6%.3 The consequence of vitamin D deficiency is low bone mineral density (BMD) and increased risk of fracture.17 Epidemiologic studies have demonstrated that the prevalence of low bone mass (defined by Z-score ≤ −2 SD) is ≈27% in patients with CD and 24.1%–27% in patients with UC.16, 18 When using the WHO criteria, the prevalence of osteopenia and osteoporosis in IBD patients is 35%–44% and 5%–14%, respectively.19–21 In addition to low BMD, vitamin D deficiency may affect immune function. A role for vitamin D supplementation in the regulation of the inflammatory process of CD has been suggested by studies with experimental IBD mouse models.7 Predictors of vitamin D deficiency in one study from Japan included disease duration and CDAI score.15 However, other studies have demonstrated that vitamin D deficiency can occur even in CD patients in remission.22, 23 As in our study, more than 70% of patients with quiescent CD were vitamin D-deficient or -insufficient. The food frequency questionnaire showed that CD patients in our study were consuming ≈300 IU of vitamin D daily. No subject took vitamin D2 or vitamin D3 from either supplementation or foods that would be equivalent to more than 700 IU/day. However, given the fact that every 100 IU of dietary vitamin D can only raise serum 25(OH)D levels by 1 ng/mL, it is not surprising that consuming 300 IU of vitamin D daily did little to raise serum levels and would not optimize vitamin D status.24 The pathogenesis of low 25(OH) D levels in these group of patients could be related to one or more possible mechanisms: decreased exposure to sunlight, inadequate oral intake of vitamin D in patients with CD versus healthy individuals, decreased intestinal absorption of vitamin D, disturbed enterohepatic circulation of vitamin D and 25(OH) D and drug induced destruction of 25(OH)D.3, 11

As shown in our study, vitamin D bioavailability was determined by measuring vitamin D2 levels 12 hours after ingesting 50,000 IU vitamin D2. Since we demonstrated previously that vitamin D2 and vitamin D3 are equally bioavailable and effective in maintaining serum 25(OH)D levels,24 we were able to use a vitamin D bioavailability test to determine the ability of patients with quiescent CD to absorb vitamin D2. We observed that a significant number of asymptomatic CD patients (39%) had only 50% of vitamin D2 absorption capability when compared with normal subjects, suggesting that the ability to absorb vitamin D and perhaps other fat-soluble vitamins may be compromised in this patient population. Overall, patients with CD had on average a 30% decrease in their ability to absorb vitamin D2. Although CDAI and HBI were not measured in these participants, the patients were felt to be in clinical remission based on symptoms, endoscopy, and radiologic imaging. Furthermore, CRP and ESR levels were normal.

Our study emphasized the important role of an oral vitamin D absorption test, which may be an excellent means to assess the malabsorption of fat-soluble vitamin D. In our study we demonstrated that neither disease activity nor prior surgery or location of disease predicts the ability to absorb vitamin D. In addition, in a pilot study we performed vitamin D absorption test on four patients with UC. The vitamin D2 levels at 12 hours were 36.8 ± 8.8 ng/mL (range = 16–59 ng/mL). We found a wide variability of vitamin D2 bioavailability in patients with UC as well as in patients with CD, which was unexpected since vitamin D is absorbed in the small intestine and not in the colon. Moreover, as noted above, vitamin D2 bioavailability did not differ among the CD patients who were classified by the location of disease and type of the previous surgery. In the other words, the ability to absorb vitamin D2 in quiescent CD patients is unpredictable and the only way to determine this is to perform a vitamin D2 bioavailability test. This test is convenient and can be performed with a single blood draw at 12 hours after taking a 50,000 IU capsule of vitamin D2. Typically, a blood draw at baseline is not necessary because there are few dietary sources of vitamin D2.11 Furthermore, none of our subjects took more than 700 IU/day of vitamin D in their diet or as a supplement. Consequently, if a patient has not recently received a pharmacologic dose of vitamin D2, the baseline vitamin D2 serum level should be 0 ng/mL. As demonstrated in our study, 14 of the 15 test subjects (nine healthy controls group and five patients in CD group) had baseline vitamin D2 level = 0 ng/mL. Our results, if confirmed by others, would merit the development of a vitamin D assay by reference laboratories as a clinical test. Our data support the use of an oral vitamin D absorption test in CD patients, especially in those patients who could not correct in the vitamin D deficiency by either dietary or pharmacologic means.

One of the limitations in our study is that we do not have blood levels of 25-hydroxyvitamin D in our normal subjects. We have previously reported in a similar group of healthy adults (at the same time of the year) in Boston that they have on average a blood level of 22 ng/mL.24 We have extensive experience in evaluating serum 25-hydroxyvitamin D levels in Caucasian, African American, and Hispanic patients and have not observed any difference in their ability to raise their blood level of 25-hydroxyvitamin D in response to vitamin D therapy.24–26 At the present, there is no evidence to suggest that a person's vitamin D status, i.e., serum 25(OH)D levels or ethnicity, have any influence on vitamin D absorption. Another limitation is that after ingesting vitamin D2, we measured only one timepoint of vitamin D2 levels, making it difficult to evaluate area under the curve for evaluating the pharmacokinetics of vitamin D2 absorption. It will be worthwhile in the future to obtain more data points to determine area under the curve to be sure that the decreased level of vitamin D at 12 hours is not due to loss of vitamin D that had been absorbed more efficiently at the earlier timepoint. Finally, we did not routinely measure CDAI or HBI in our patients. We did feel the participants were in clinical remission based on symptoms, endoscopy and imaging, ESR, and CRP. In an ongoing prospective study, we are measuring disease activity using the HBI.

In conclusion, vitamin D deficiency is common in CD patients. We demonstrated that even a majority of quiescent CD patients had a significantly reduced ability to absorb vitamin D2 when compared with normal subjects. Since the ability to absorb vitamin D in CD patients is unpredictable, the only way to determine absorption efficiency is to perform a vitamin D bioavailability test. Use of this test may guide clinicians in administering the appropriate therapeutic dose of vitamin D for treating vitamin D deficiency in patients with CD.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES