Vitamin D Insufficiency during Pregnancy and Lactation: Functional Indicators of Vitamin D Adequacy

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Attainment and Maintenance of Nutritional Vitamin
D Status Health Implications Beyond Skeletal
Integrity
Bruce W. Hollis, Ph.D. Medical University of
South Carolina Charleston, SC
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Metabolism
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Metabolism of Vitamin D Under Conditions of
Adequate Vitamin D Supply
High/Normal Input of Cholecalciferol from diet or
UVB
METABOLITE COMPARTMENT
Vitamin D3
1
2
4
25(OH)D
5
3
Within Tissues Processing 1-OHase
1,25(OH)2D
In Plasma
24,25(OH)2D Catabolism
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Catabolism and excretion
When vitamin D supplies are adequate, flow of
25(OH)D through other potential pathways,
including its utilization by peripheral tissues
for paracrine regulation, is no longer
compromised.
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Metabolism of Vitamin D Under Conditions of Low
Vitamin D Supply
Low Input of Cholecalciferol from diet or UVB
METABOLITE COMPARTMENT
Vitamin D3
1
2
4
25(OH)D
5
3
Within Tissues Processing 1-OHase
1,25(OH)2D
In Plasma
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24,25(OH)2D Catabolism
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Catabolism and excretion
The vessels represent metabolic compartments,
stages in the metabolism of vitamin D. The height
of the shaded portion of each vessel represents
the relative concentration of each metabolite
indicated in the figure.
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Substrate Deprivation

• The vitamin D endocrine system is the ONLY
  steroid endocrine system in the body that is
  almost always limited by substrate availability
  due to latitude, lifestyle, race ect.
• Vitamin D conversion to 25(OH)D.
• 25(OH)D conversion to 1,25(OH)2D in extra-renal
  sites

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Vitamin D essentially does not occur in natural
foodstuffs.
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Cutaneous Generation of Vitamin D
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How much solar exposure is required to synthesize
endogenous vitamin D3?
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A single initial MED dose of UVB radiation to a
light-skinned individual will release
approximately 20,000 IU vitamin D3 into the
circulation within 24 hrs.However, if an
individual has very dark skin the exposure time
for a MED could increase by 10-fold.
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Number of Months that UVB from sunshine cannot
produce vitamin D3 in skin
Vit D all year
Vit D all year
Theoretical skin color
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World Distribution of Nonhuman Primates
Regions shaded white are the natural habitat of
non-human primates
from Primate Behavior Field studies of
monkeys and apes. I DeVore 1965
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Effect of UVB exposure time and skin colour on
Vitamin D production
White skin
Very Dark skin
Same capacity for Vit D, different exposure-time
requirements
Yield of vitamin D
20 min
120 min
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Childhood lack of vitamin D causes rickets
Contracted pelvis, in a case of osteomalacia
(adult rickets). Normal childbirth would be
impossible.
Vieth 2001. Nutritional Aspects of Osteoporosis,
Chapter 17, ed P Burckhardt, RP Heaney, B
Dawson-Hughes Academic Press
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Oral Supplementation of Vitamin D
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Based on Current Daily Reference Intakes (DRIs)
for Vitamin D

• Preterm infants receive 250-400 IU/kg/day
• Term infants receive 100-150 IU/kg/day
• Pregnant and lactating women receive 3-7
  IU/kg/day

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Circulating 25(OH)D as a Function of Oral
Vitamin D3 Intake
10,000 IU/d
5,000 IU/d
1,000 IU/d
400 IU/d
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How toxic is vitamin D?

• The U.S. Nutrition Guidelines state that the
  lowest observed adverse effect level (LOAEL) for
  humans is 2,000 IU vitamin D/day
• This statement is grossly in error and is an
  impediment to the health of humans

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Doses of vitamin D pertinent to the UL and LOAEL,
and their effects on serum calcium
MS Patients on 1200 mc Ca. EVERY MONTH THE
VITAMIN D3 DOSE WAS INCREASED IN A STUDY TO
CHARACTERIZE TOLERABILITY TO SPECIFIC SERUM
25(OH)D LEVELS
Serum 25(OH)D (nmol.L)
1000 MCG
Vitamin D3 Supplementation (IU/d)
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Serum Calcium (mmol.L)
80 120 150 180 190 340 420
?25(OH)D
Urine Calcium creatinine Ratio
Vitamin D3 Supplementation (IU/d)
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Optimal Circulating Concentrations of 25(OH)D
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Vitamin D Status in Primates and Early Humans
Winter 43o N Latitude Normal
160
Serum 25(OH)D nmol/L
120
80
80
?
Humans exposing full skin surface to
Sunshines UVB
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Blood Levels when taking 1000 IU/day
0
Old-World Primates
Northern People Taking 4000 IU/day
Physiological adult intake
Sources, include Cosman, Osteoporosis Int 2000
Fuleihan NEJM 1999 Scharla Osteoporosis Int
1998 Vieth AJCN 1999, 2000
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Haddad and Chyu JCEM 1971, 33 992-995
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Normal Vitamin D Status

• Should NEVER have been defined by Gaussian
  distribution.
• This is similar to defining normal estrogen
  levels by sampling a population of women whom are
  primarily postmenopausal.

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Is Vitamin D Multifunctional?

• We all know that vitamin Ds ONLY function is to
  maintain calcium homeostasis.
• We have known for 3 decades that the VDR exists
  in many tissues that have nothing to do with
  calcium homeostasis.
• We also know that functional CYP-27b1 is present
  and is functional in may tissues other than the
  kidney.

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Biomarkers for Vitamin D Sufficiency

• 25(OH)D
• Intact PTH
• Bone Mineral Density (BMD)
• Intestinal Calcium Absorption
• Mobility responsiveness
• Insulin sensitivity
• Beta cell function
• Immune function

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All By Itself, Vitamin D Increases Bone Density
NHANES Study of the US Population Each line shows
average bone densities for 2500 men and women lt
age 50 0 is set to cancel out density differences
among the groups of White, Black, Spanish
Americans) The Lines are local averages of BMD
for the 25(OH)D levels along the horizontal axis
5000 IU/day
W
MULTIVITAMIN
B S
0
Bischoff-Ferrari 2004 Am J Med 116634
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Activation of human TLR2/1 triggers a vitamin D
receptor-dependent antimicrobial
response   Philip T. Liu1, Steffen Stenger2,
Huiying Li3, Linda Wenzel2, Belinda H. Tan1,
Stephan Krutzik1, Maria Teresa Ochoa1, Jürgen
Schauber4, Kent Wu1, Christoph Meinken2, Manfred
Wagner5, Robert Bals6, Andreas Steinmeyer7,
Ulrich Zügel8, Richard L. Gallo4, David
Eisenberg3, Martin Hewison9, Bruce W. Hollis10,
John S. Adams7, Barry R. Bloom9 and Robert L.
Modlin1,11. 1Department of Microbiology,
Immunology and Molecular Genetics, University of
California at Los Angeles (UCLA), California,
USA. 2Institut für Klinische Mikrobiologie,
Immunologie, und Hygiene, Universität Erlangen,
91054 Erlangen, Germany. 3Department of Chemistry
and Biological Chemistry, Howard Hughes Medical
Institute, UCLA-Department of Energy Institute of
Genomics and Proteomics, UCLA.
4Division of Dermatology, University of
California San Diego, and VA San Diego Healthcare
Center, San Diego, California, USA. 5Klinikum
Nürnberg, Medizinische Klinik 3, Germany.
6Pneumologie, Universität Marburg, Germany.
7Medicinal Chemistry, Schering AG, Berlin,
Germany. 8CRBA Dermatology, Schering AG, Berlin,
Germany. 9Department of Medicine, Division of
Endocrinology, Burns and Allen Research
Institute, Cedars-Sinai Medical Center, Los
Angeles, California 90048. 10Departments of
Pediatrics, Biochemistry, and Molecular Biology,
Medical University of South Carolina, Charleston,
SC 29425. 9Office of the Dean, Harvard School of
Public Health, Boston, MA 02115, USA. 11Division
of Dermatology, Department of Medicine, David
Geffen School of Medicine at UCLA. These
authors contributed equally to this work.
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INNATE AND ADAPTIVE IMMUNITY
Innate Response
Adaptive Response

• Slow response
• Recognition - initially low affinity receptors
  Gene rearrangement Clonal
  expansion
• Response T- and B-cells with high affinity, very
  specific receptors and antibodies
• Memory

• Rapid response
• Pattern recognition receptors- germ-line encoded
• LPS, mannose and scavenger
• ? Cytokines, costimulatory molecules- instructive
  role for adaptive response
• Direct response for host defense
• Phagocytosis
• Antimicrobial activity

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Vitamin D and the Innate Immune System

• In 1903, Niels Ryberg Finsen was awarded the
  Nobel Prize for his work, demonstrating that UV
  light was beneficial to patients with Lupus
  vulgaris.
• The beneficial effects of UV exposure to
  tuberculosis patients is also known.

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What went wrong with sanatoriums?
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Rickets and Infection

• Rickets is not only associated with skeletal
  abnormalities but also respiratory infections.
• In 1994 a brief study demonstrated that
  respiratory infections in children with elevated
  alkaline phosphatase levels were eliminated by
  supplementing them with 60,000 IU vitamin D/wk
  for a period of 6 wks.

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Cathelicidin

• Cathelicidin (LL-37) is a endogenous
  antimicrobial peptide generated by the innate
  immune system in response to microbial invasion
  thru the Toll 2 surface receptor on monocytes and
  macrophages. A VDRE is also contained in its
  gene regulatory region in these cell types.

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Addition of 25D3 restores ability of sera from
African American individuals to support TLR2/1L
mediated induction of cathelicidin mRNA
Cath. mRNA
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7
6
5
Fold Change
4
3
2
1
25D3
--

--

TLR2/1L
--
--


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Results Clinical Attachment Level

• Regression analysis done on CAL shows significant
  reduction from control to 4000 IU treatment group
• Clinically significant attachment loss It
  appears that Vitamin D supplementation reduces
  the proportion of individuals with CAL 3

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Results Bleeding on Probing

• Trend analysis done on BOP shows a significant
  difference among both treatment groups (p0.01)

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Gingival inflammation (percent of sites with
bleeding on probing) by serum 25(OH)D levels at
delivery in non-smokers
Point of inflection 25(OH)D 37 µg/L R2 0.1,
plt0.0001
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0 25 50 75 100
125 150
(nmol/L)
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Future Implications

• Cancer
• Autoimmune diseases
• Immune dysfunction

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Serum 25-Hydroxyvitamin D Levels and Risk of
Multiple SclerosisKL Munger, LI Levin, BW
Hollis, NS Howard, A Ascherio

• Objective To evaluate whether serum levels of
  25(OH)D predict risk of MS among healthy adults
• Methods Prospective study among gt7 million US
  military personnel with 1 serum sample stored in
  Dept of Defense Serum Repository (DoDSR).
• MS cases identified from Army and Navy Physical
  Disability Agencies and medical records reviewed
  to confirm dx using Poser criteria.
• Each case was matched to 2 controls on age, sex,
  race, and dates of blood collection.
• 25(OH)D measured using RIA in up to 3 samples
  collected prior to onset of first MS s(x).
• Results 305 individuals with MS and 610 controls
  included in study.
• Average age at MS onset was 28 years 57 of
  cases and controls were White, 30 Black.
• Elevated 25(OH)D levels in Whites more protective
  at younger ages.
• Individuals lt20 years old with average 25(OH)D
  gt100 nmol/L had 91 reduction in MS risk compared
  to those with average 25(OH)D lt100 nmol/L
  (RR0.09, 95CI0.01-0.78, p0.03).
• JAMA 296, (2006)

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VITAMIN D CANCER

• 1179 healthy women
• aged 66.7 7.3
• four year trial
• 1032 finished (87.5)
• baseline 25(OH)D 71.8 nmol/L 20.3
• three treatment groups
• control
• Ca (14001500 mg/d)
• Ca plus D3 (1100 IU/d)

Lappe et al. AJCN 2007
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VITAMIN D CANCER
P lt 0.01
RR 0.402
Lappe et al. AJCN 2007
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VITAMIN D CANCER
RR 0.232
Lappe et al. AJCN 2007
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DiaSorin LIAISON 180 determinations/h
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Vitamin D External Quality Assessment Scheme
(DEQAS)

• Currently 322 worldwide participants in this QC
  survey
• 5 samples sent quarterly for 25(OH)D and /or
  1,25(OH)2D quantitation

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DEQAS
70
58.9
60
47.3
50
42.6
39.2
38.2
36.8
36.4
40
ng/ml
30
20
N4
N21
N53
N16
N16
N21
N9
10
0
HPLC
LC-MS
IDS RIA
IDS OCTEIA
DiaSorin RIA
DiaSorin LIAISON
Nichols Advantage
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DEQAS July 2005 Ability to measure 50.9 nmol/L
of added 25(OH)D2 or 25(OH)D3
120
25(OH)D3
25(OH)D2
100
97.1
112.2
111.5
118.1
80
81.4
88.6
83.3
82.1
Recovered
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78.8
56.4
54.2
29.1
46.3
43.2
40
20
N53
N16
N16
N21
N9
N4
N21
0
HPLC
LC-MS
IDS RIA
IDS OCTEIA
DiaSorin RIA
Nichols Advantage
DiaSorin LIAISON
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Accuracy of 25OHD methods used by DEQAS
participants
M
E
A
N



B
I
A
S
4
0
3
0
BIAS FROM ALTM
2
0
1
0
0
CPBA
HPLC
-
1
0
IDS OCTEIA (10/02-1/04)
IDS RIA
DiaSorin RIA
Nichols Advantage (7/01-1/04)
Mean deviation ( bias) of the method mean from
the target value (ALTM), over the period January
2000 to January 2004 unless stated otherwise.
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Comparison of hplc vrs. LC-MS/MS Lensmeyer et
al., Clin Chem 522006
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Method Comparison - 25(OH)D2 negative samples
Data from Dr. Ravinder Singh, Mayo Clinic
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LC-MS is clearly a superior analytical technique
by which to assess circulating 25(OH)D

• The data clearly do not support this statement !
• DEQAS results demonstrate that DiaSorin LIAISON
  and LC-MS are clinically equivalent IF LC-MS is
  properly performed.
• Further, a recent publication by Binkley et. al.
  (Clin Chem 2006) states that separate
  determination and reporting of 25(OH)D2 and
  25(OH)D3 does nothing more than confuse the
  physician.
• It is TOTAL 25(OH)D that is important !

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Data from Dr. Ravinder Singh, Mayo Clinic Clin
Chem 2005 51 (Suppl. 6)A114-5
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DEQAS Comparison of Low, Medium and High
Standards (n)
25(OH)D (nmol) CV

• LIAISON 29 19.4 29.8
• LC-MS 13 18.5 32.3
• LIAISON 37 34.1 18.9
• LC-MS 14 38.1 28.5
• LIAISON 38 96.6 16.1
• LC-MS 14 104.7 21.9

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DEQAS Linearity Study (1/07)25(OH)D nmol
1 2 3 4 5

• DS RIA 19.4 34.1 52.5 70.1 90.9
• (37.3) (55.2)
  (73.1)
• DS LS 21.3 39.6 57.4 75.3 91.0
• (38.8) (56.2)
  (73.6)
• LC-MS 18.5 38.1 63.4 80.8 104.7
• (40.1) (61.6)
  (83.2)
• ALL HAVE EXCELLENT LINEARITY

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Who Is Right? 25(OH)D2
25(OH)D3 25(OH)D

• LabCorp DS-LI
  15.6
• Quest LC-MS-MS lt4 45.0 45.0
• Hollis Lab DS-RIA
  21.3
• Mayo Labs LC-MS lt1 20.9 20.9
• All values in ng/ml

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Conclusions

• Based on biomarkers of nutritional vitamin D
  status (PTH, BMC, intestinal calcium absorption,
  insulin sensitivity, beta cell function, and
  innate immune function), circulating levels of
  25(OH)D lt32 ng/mL should be considered deficient.
• A 400 IU DRI for vitamin D is irrelevant with
  respect to the adult population in general.
• Guidelines stating that the lowest observed
  adverse effect level for humans is 2,000 IU
  vitamin D/day are incorrect. In actuality, the AI
  for adults may be 2,000 IU/day and in some cases
  such as pregnancy and lactationhigher.
• It is not unlikely that chronic nutritional
  vitamin D deficiency puts populations at risk for
  developing debilitating, long latency chronic
  diseases such as cancer and autoimmune disease.
• The physician will have to become familiar with
  vitamin Dnot simply as a dietary supplement.
  Active management of nutritional vitamin D status
  will become necessary.

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Thank you