25 OH Vitamin D
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Introduction and
Overview
The discovery of vitamin D and the implementation of measures to reduce the inci-dence of vitamin D deficiency that occurred in the early part of the 20th century had a tremendous impact on the health of millions of people.1 Prior to this time, rickets, a bone deformity condition caused by vitamin D deficien-cy, was rampant, especially in the cities of industrialized nations throughout the world. Fortification of milk with vita-min D has all but elivita-minated this condition in the United States. We have only recently begun to understand the many other roles that vitamin D
plays in human physiology. A number of studies have shown that, today, vitamin D deficiency is very common, especially in certain high-risk populations. Moreover, the clinical thresholds of vitamin D levels required for optimal health may be significantly higher than once thought. In this article, we review the physiology of vitamin D metabolism and the role of vitamin D in supporting phys-ical well-being. We also con-sider the causes of the near epidemic of vitamin D defi-ciency in our society. Lastly, we cover the characteristics of a good assay for vitamin D and the problems with some of the assays available.
Vitamin D Deficiency
A number of clinical studies have shown that many people in the United States, both chil-dren and adults, suffer from vitamin D deficiency.1This sit-uation has occurred, in part, because the foods of the typi-cal American diet are very low in vitamin D. Fatty fish such as mackerel and salmon and fish liver oils are some of the few natural dietary sources of vita-min D.1-3Most people do not eat enough of these foods to maintain adequate vitamin D levels.
In the early part of the 20th century, when the role of vita-min D in maintaining bone health began to be understood, many manufacturers started
Clinical Significance
By Bruce Hollis, PhD; Gordon MacFarlane, PhD; and André Valcour, PhD
And Assessment of
25 OH Vitamin D
Learning
Objectives
Upon completion of this article readers should be able to:
•Characterize the veracity of statements regarding vitamin D. •Identify the factors that have
caused the near epidemic of vitamin D deficiency in the United States.
•Recall the disease states in which vitamin D is thought to have a role.
•Recognize the difference between observed reference ranges and a nutritional threshold below which an individual could be characterized as vitamin D-deficient.
•Review the principles employed by different assay
methodologies for the assessment of 25 OH vitamin D.
•Characterize the veracity of statements regarding 1,25 dihydroxy-vitamin D. •List the best sources of
vitamin D.
•Indicate the scientific basis for the clinical threshold (32 ng/dL) of vitamin D.
adding vitamin D to food products. Examples of foods that were fortified with vita-min D included such name brands as Bond Bread, Rickter’s Hot Dogs, Twang Soda and even Schlitz Beer.1 The unregulated supple-mentation with vitamin D, however, resulted in several cases of overdose; conse-quently, by the 1950s most manufacturers stopped adding vitamin D to their products.1
In the United States, vita-min D is still added to milk to prevent the occurrence of rickets in the pediatric pop-ulation. Unfortunately, too many children do not drink enough milk to raise their vitamin D levels to the opti-mum range. Also, recent studies have shown that the level of vitamin D in forti-fied milk is frequently much lower than that recom-mended by the FDA.1 Human milk contains very little vitamin D because many mothers are deficient, so children of mothers who choose to breast-feed are at risk of developing rickets if they are not given supple-mental vitamin D. The American Academy of Pediatrics recommends that infants that are exclusively breast-fed should be given a daily supplement of vitamin D.3
With the onset of the industrial revolution rickets became a problem of epi-demic proportions.1,4 In the early 1800s, Polish physi-cian Jedrzej Sniadecki observed a much higher incidence of rickets in chil-dren who lived in the urban environment of Warsaw compared to children who lived in the country.1Dr. Sniadecki found that the condition of the city
chil-dren with rickets improved dramatically when he took them into the countryside and had them spend time in the sun. He correctly sur-mised that the relative lack of sun exposure experi-enced by the city children was the cause of their con-dition. Despite the fact that the physiologic mechanism was not well understood, sun therapy became the standard of care for treating patients with rickets through the 1800s and into the early 1900s.1In fact, the Floating Hospital for Children, now part of Tufts University in Boston, was originally a large boat that was used to treat children with rickets by taking them out into Boston Harbor to increase their level of sun exposure.1
In 1919, Sir Edward Mellanby demonstrated that experimental rickets could be induced in dogs by restricting sunlight and feeding them oatmeal exclu-sively.5He also showed that
he could cure the rickets if he supplemented the oat-meal with cod-liver oil. In 1937, Windaus and Bock discovered that 7-dehydroc-holesterol in the skin is con-verted to vitamin D when irradiated with ultraviolet radiation in the UVB range (wavelength 290 nm to 315 nm).1,6
The extent of vitamin D formation is not tightly con-trolled and depends prima-rily on the duration and intensity of the UV irradia-tion. Levels typically reach a plateau within 30 minutes of exposure.6
Overproduction of vita-min D in the skin is pre-vented by the photosensi-tive conversion of vitamin D to tachysterol or
lumis-terol.6Vitamin D is not very water-soluble, so it must be delivered to and carried in the blood as a complex with vitamin D-binding pro-tein.2,6
Once in the circulation, vitamin D is metabolized to 25-hydroxy vitamin D (25-D) by the liver.2,6 The 25-D form of the hormone is the principle circulating reser-voir in the plasma and is generally the best indicator of overall vitamin D status.6 25-D is further metabolized
by the kidney to produce the biologically active form of vitamin D, 1, 25-dihy-droxy vitamin D (1,25-D).2,6 Renal production of 1,25-D is tightly controlled by parathyroid hormone and is important in the regulation of serum calcium homeosta-sis.6
Several factors are associ-ated with an increased risk of developing vitamin D deficiency (Table 1). Two selected populations are particularly vulnerable—
Table 1
Populations at Risk for Vitamin D Deficiency1,2,6
•Individuals with low dietary vitamin D levels: Infants fed only mother’s milk and children who do not drink fortified milk are at risk.
•Individuals with malabsorption syndromes: Patients with pancreatic enzyme deficiency, Crohn’s disease, cystic fibrosis, celiac disease and sur-gical resection of stomach or intestines are at risk.
•Individuals with severe liver disease: Hepatic disease can reduce the con-version of vitamin D to 25-D and can lead to malabsorption of vitamin D. •Individuals with kidney disease: Nephrotic syndrome can increase the
uri-nary loss of vitamin D.
•Individuals taking certain drugs: Several medications, including phenytoin, phenobarbitol and rifampin, accelerate the breakdown of vitamin D by the liver.
•Individuals who live at higher latitudes: Individuals who live in northern cli-mates are at increased risk of deficiency—especially in winter months due to diminished exposure to UVB radiation.
•Individuals who spend little time outside: Individuals who are homebound or simply choose to remain inside are at increased risk.
•Older adults: The skin becomes less efficient at producing vitamin D as one ages because of diminished levels of vitamin D precursors in the skin. •Individuals with decreased sun exposure for cultural reasons: Women in
some societies are required to cover themselves with heavy clothing, reducing exposure to the sun’s rays.
•Races with high skin melanin levels: Increased skin pigmentation can reduce the efficiency of vitamin D conversion in the skin as much as 50-fold. Individuals with dark complexions living at higher latitudes are at increased risk.
people with dark skin and the elderly.1,3 Melanin in the epithelial cells is the com-pound that gives skin its color.3This pigment acts as a natural sunscreen, protect-ing the skin from sunburn by absorbing UV radiation. Unfortunately, because this pigment absorbs the energy used to produce vitamin D, people with melanin-rich skin do not produce vitamin D efficiently. As a result, dark-complexioned individ-uals living in northern lati-tudes can very easily become vitamin D-deficient. Recent studies have indicat-ed that as many as 60 per-cent of black adults in the United States are vitamin D deficient.1
The ability of the skin to produce vitamin D on expo-sure to the sun decreases fourfold from age 20 to age 70.1This diminished vita-min D production efficien-cy—along with the fact the people tend to reduce their outdoor activities as they age—results in an increased risk of vitamin D deficiency in the elderly. Studies have indicated that more than 50 percent of elderly
Americans in general and as many as 80 percent of
elder-ly black Americans are vita-min D deficient.1
Many people do not spend enough time in the sun to allow for the produc-tion of adequate amounts of vitamin D.1The same fac-tors that have produced an epidemic of obesity in the United States have led to the increased rate of vitamin D deficiency. For many of us, a sedentary lifestyle results in decreased outdoor activity and reduced sun exposure. Also, health-conscious indi-viduals have become aware of the association between sun exposure and the devel-opment of wrinkles as well as melanoma and non-melanoma skin cancer. When they are exposed to the sun, many people use high SPF sunscreens to limit skin damage and prevent sunburn. Unfortunately, use of a sunscreen with SPF as low as 15 reduces the rate of vitamin D production by 99.9 percent.1
Clinical Manifestations
of Vitamin D
Deficiency
The hormonally active form of vitamin D, 1,25-D, plays an integral role in cal-cium homeostasis and in the maintenance of healthy bone.1-31,25-D stimulates the absorption of calcium at the level of the intestine and may also serve to increase calcium and phosphate resorption at the kidney level.2Vitamin D deficiency
leads to the mobilization of calcium from bone.6While it may appear that bones are inert structural elements, they are, in fact, dynamic organs. Human bones con-tinually undergo a tightly coupled process of degrada-tion mediated by osteoclasts and rebuilding mediated by
osteoblasts. Working in con-cert with parathyroid hor-mone, 1,25-D supports increased bone resorption by mobilizing calcium and phosphate.21,25-D also stim-ulates new bone formation by inducing osteoblast cells to synthesize alkaline phos-phatase and osteocalcin.2
In children and adoles-cents, the rate of new bone formation exceeds the rate of breakdown, resulting in a net increase in bone mass.6 Peak bone mass is achieved as individuals reach their mid 20s, after which the rate of bone degradation begins to exceed the rate of new bone formation. The rate of net bone loss then typically increases to between 1 per-cent and 2 perper-cent per year in older men and to between 2 percent and 4 percent per year in postmenopausal women. When this loss becomes excessive, bones become porous and brittle through a condition that is referred to as osteoporosis.
Osteoporotic fractures are a major cause of morbidity and mortality in both men and women. The bone mass of individuals reflects the amount of bone produced during their lifetime minus the amount lost.
Consequently, it is impor-tant that children and young adults build as much bone as possible to ensure that residual bone left as they get older is adequate to avoid the development of osteo-porosis.
Caucasians suffer from a significantly greater inci-dence of osteoporosis than blacks of African genetic lin-eage because their peak bone density is typically 7 percent to 9 percent lower.1 However, chronic excessive bone loss can still result in
osteoporosis in blacks. Individuals with more severe vitamin D deficiency can develop osteomalacia, a condition in which the bones fail to harden proper-ly.1Vitamin D deficiency is the most common cause of osteomalacia. Chronic osteo-malacia can cause bones to become weak, which in turn results in fractures—most commonly of the lower spine, hip or wrist. Unlike osteoporosis, in which patients are often asymptot-ic until a fracture occurs, patients with osteomalacia suffer from unrelenting deep bone pain and muscle aches. Because many clini-cians fail to consider vitamin D deficiency in their differ-ential diagnosis of patients with generalized bone or muscle pain, osteomalacia is often misdiagnosed as fibromyalgia or arthritis. It has been suggested that a significant portion of people who have been diagnosed with fibromyalgia may actu-ally have vitamin D defi-ciency-related osteomalacia.1
Osteomalacia-associated vitamin D deficiency may be playing a contributory role in the increased prevalence of obesity in the United States.1Since vitamin D is lipophilic, it tends to be absorbed by the excess fat of overweight patients, result-ing in diminished plasma levels. This can lead to the bone and muscle pain of osteomalacia, which can, in turn, inhibit patients from exercising and taking part in outdoor activities. These fac-tors simultaneously serve to exacerbate the weight gain by diminishing physical activity and reduce the per-son’s exposure to the sun, resulting in more profound vitamin D deficiency. This
Table 2
Conditions Associated With Vitamin D Deficiency Osteomalacia1 Osteoporosis1 Rickets1,4 Fibromyalgia1 Prostate Cancer1,7 Breast Cancer1,7 Colon Cancer1,7 Heart Disease8 Hypertension8 Multiple Sclerosis1 Type I Diabetes1,9
▲ cycle contributes to the
dif-ficulty that obese patients have in managing their weight.
Osteomalacia in children is referred to as rickets.1 Like adults, these children experience bone pain and muscle weakness; however, since their bones are active-ly growing, rickets also causes their bones to form improperly. A classic physi-cal manifestation of rickets is leg bones that are bent as the result of inability to withstand the weight of the child’s body. These children may have other skeletal deformities, including a sunken chest, rivet-like bone protrusions up and down the sides of the breast, and wider-than-nor-mal ends to the bones of the arms and legs.
The relationship between vitamin D levels and bone health is undisputed and has been understood by cli-nicians for a number of years. There is now evi-dence suggesting that sun exposure and vitamin D levels may also play a sig-nificant role in the health of other organ systems. The geographic distribution of the incidence rates for sev-eral cancers is strikingly similar to the distribution of the intensity of UV irradia-tion necessary for 25-D pro-duction.8Diminished aver-age serum concentrations of 25-D have been linked to increased risk of developing prostate, breast and colon cancer.1,7
Vitamin D deficiency also has been correlated with increased incidence of heart disease and hypertension. A recent study in which hypertensive patients were exposed to UVB radiation for just three days a week
for six weeks resulted in more than doubled 25-D levels and produced a 6 mmHg drop in blood pres-sure.8A similar course of treatment with UVA radia-tion had no effect on 25-D or blood pressure.
Epidemiological studies reveal that individuals liv-ing close to the equator have a lower incidence of several autoimmune dis-eases than people living at higher latitudes. For exam-ple, the incidence of multi-ple sclerosis is approximate-ly five times greater in North America than in areas near the equator.1 Type I diabetes, another autoimmune condition, is relatively rare in equatorial regions while Finland, a country with very little win-ter sun, has the world’s highest reported incidence of this disease.1When 12,000 Finnish babies were given vitamin D supple-ments, their likelihood of developing diabetes was decreased 80 percent rela-tive to a control group receiving no supplemental vitamin D.9
The new understanding about the relationship between vitamin D levels and multiple aspects of human health has led scien-tists to reconsider long-held perceptions about the way vitamin D works in the body.1,6 In the past, most scientists believed that the only clinically significant source of hormonally active 1,25-D came from the hydroxylation of circulating 25-D by the kidneys.2This classic model was devel-oped based on an under-standing of the relationship between plasma 1,25-D lev-els and parathyroid hor-mone in the maintenance of
bone health. Recent studies have found that, contrary to this previous understand-ing, cells from a large num-ber of tissues can produce 1,25-D.6 1,25-D is a potent inhibitor of normal and abnormal cell growth and an inducer of cell matura-tion.6 It has been proposed that 1,25-D produced remote from the kidneys exerts its effect directly at the tissue level and is then degraded.9This in situ tis-sue production and use may explain the fact that increased overall levels of 25-D have a positive effect on health without affecting the plasma levels of hor-monally active 1,25-D.1,6 Chronic vitamin D defi-ciency can be associated with a number of clinical conditions, including those listed in Table 2.
Developing a Clinical
Threshold
The diagnosis of vitamin D deficiency, or insufficien-cy, has received consider-able attention in recent years. Many investigators agree that subclinical 25-D deficiency is common, par-ticularly in Europe and the northern United States.1,4,6 The clinical community, however, is beginning to reach a consensus as to what concentration of 25-D should be considered suffi-cient.
Serum concentrations of 25-D are known to vary with age, sex, race, season and geographic location.1 This has led many to estab-lish seasonal expected ranges for their geographic location and local popula-tion. This approach pro-vides a “reference range,” but does not adequately determine health status
with regard to vitamin D levels if a significant portion of the reference population is, in fact, deficient. A more useful parameter in clinical practice would be a nutri-tional threshold, below which an individual could be characterized as vitamin D deficient. Several investi-gators have approached this problem by assessing the correlation of plasma 25-D concentration with various biological markers.10
For example, plasma 25-D levels have been shown to have an inverse relationship to serum parathyroid hor-mone levels.10Secondary hyperparathyroidism can be corrected when 25-D levels are increased to more than 32 ng/mL (80 nmol/L).10 Serum concentrations of less than 32 ng/mL have been shown to impair intestinal calcium absorption and sub-sequent skeletal density.10 Further studies have shown that 25-D levels of less than 32 ng/mL are associated with impaired insulin resist-ance and beta-cell func-tion.10Together, these data suggest that 32 ng/mL rep-resents the appropriate threshold for identifying individuals with clinical vitamin D deficiency.
Laboratory
Measurement of
Vitamin D
Laboratory assays are available for the measure-ment of both 25-D and 1,25-D. Assays for 25-D are gen-erally simpler to perform, in part because the levels of this metabolite are typically 500- to 1,000-fold higher than those of the more physiologically active hor-mone, 1,25-D. Since circu-lating levels of 25-D closely reflect the total amount of
vitamin D produced in the skin and absorbed from the diet, measurement of this analyte represents the best method for the assessment of overall vitamin D status and for the diagnoses of deficiency or toxicity.2,6In fact, levels of 1,25-D can often be normal in individu-als with overall vitamin D deficiency.6Measurement of 1,25-D levels is useful in the assessment of disorders of calcium metabolism and parathyroid disease.2
With the growing number of diseases associated with its deficiency, the accurate measurement of vitamin D levels has become increas-ingly important. For optimal clinical utility, the assays used must take into account the diverse chemical nature of vitamin D. The vitamin D produced in the skin and that found naturally in foods are both derived from the same cholecalciferol (vitamin D3) family. An alternate, synthetic form of vitamin D also can be man-ufactured by irradiation of ergosterol from yeast to pro-duce ergocalciferol, or vita-min D2. Both the D2 and D3 forms have similar biologi-cal activity.2
In the United States, vita-min D deficiency is com-monly treated with vitamin D2 in the form of Drisdol™, while both D2 and D3 are used to fortify foods.11 These two parent com-pounds contribute to the
overall vitamin D status of the individual, so it is important that metabolites of both forms be measured equally in clinical assays.11 Since the therapeutic vita-min D formulations used in the United States contain only the D2 form, a failure of the testing system to detect D2 in an equimolar manner may lead the clini-cian to the erroneous con-clusion that additional sup-plementation is required, resulting in vitamin D
intoxication and hypercal-cemia. It is important that clinicians interested in assessing both D2 and D3 be aware of the method used by their laboratory to measure vitamin D, as recent studies indicate that several commercially avail-able methods in the United States fail to measure both components in an equimo-lar manner.11
The measurement of 25-D has evolved from the cum-bersome and labor-intensive procedures developed in the 1970s to today’s highly automated methods per-formed on random-access analyzers.11Several early assays used the naturally occurring vitamin D-bind-ing protein (DBP) and required extraction or chro-matographic purification as pretreatment steps.11
Unfortunately, in the absence of extensive extrac-tion and/or chromatograph-ic sample preparation,
DBP-based assays were plagued with significant interference and cross-reactivity prob-lems.11These assays were generally replaced by the more reliable immunoassays that employ highly specific monoclonal antibodies to vitamin D.11Automated chemiluminescence-based assays have become com-mercially available, using either the DBP11,12 or a spe-cific antibody.11,13Recent studies indicate, however, that the DBP-based auto-mated assay may suffer from the same interference problems that caused the original to fall out of favor.11,14 ■
Dr. Hollis is with the Departments of Pediatrics, Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston; Dr. MacFarlane is director, Research and Development, DiaSorin Inc., Stillwater, MN; and Dr. Valcour is director, Esoteric Immunoassay, LabCorp Center for Esoteric Testing; Burlington, NC.
References
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3. National Institutes of Health, Office of Dietary Supplements. Dietary
Supplement Fact Sheet: Vitamin D. Available at:
http://ods/od/nih/ gov. Accessed 07/14/2004.
4. Holick MF. Vitamin D: A millennium perspective. J Cell
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5. [Mellanby E.] Nutrition Classics. The Lancet1:407-12, 1919. An experimental investiga-tion of rickets. Edward Mellanby. Nutr Rev. 1976; 34(11):338-340. Reprint.
6. Zittermann A. Vitamin D in preventive medicine: Are we ignoring the evidence? Br J Nutr.
2003;89(5):552-572. 7. Holick MF. Vitamin D: Importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am J Clin Nutr.2004;79(3):362-371.
8. Krause R, Buhring M, Hopfenmuller W, Holick MF, Sharma AM. Ultraviolet B and blood pressure.Lancet. 1998;352 (9129):709-710.
9. Hypponen E, Laara E, Reunanen A, Jarvelin M-R, Virtanen SM. Intake of vitamin D and risk of type 1 diabetes: A birth-cohort study.Lancet. 2001;358(9292):1500-1503.
10. Hollis BW. Circulating 25-OH D levels indicative of vita-min D sufficiency: Implication for establishing a new effective DRI for vitamin D. J Nutr.; In press.
11. Hollis BW. Editorial: The determination of circulating 25-hydroxyvitamin D: No easy task. J Clin Endocrinol Metab. 2004;89 (7):3149-3151.
12. Roth HJ, Zahn I, Alkier R, Schmidt H. Validation of the first automated chemilumines-cence protein-binding assay for the detection of 25-hydroxycal-ciferol. Clin Lab. 2001;47:357-365.
13. Ersfeld DL, Rao DS, Body J-J, et al. Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON® auto-mated analyzer. Clin Biochem. 2004;37(10):867-874.
14. Binkley N, Krueger D, Cowgill CS, et al. Assay varia-tion confounds the diagnosis of hypovitaminosis D: A call for standardization. J Clin Endocrinol Metab. 2004;89(7):3152-3157.
